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Sample records for ccr5 coreceptor antagonist

  1. Computational modeling of human coreceptor CCR5 antagonist as a HIV-1 entry inhibitor: using an integrated homology modeling, docking, and membrane molecular dynamics simulation analysis approach.

    PubMed

    Gadhe, Changdev G; Kothandan, Gugan; Cho, Seung Joo

    2013-01-01

    Chemokine receptor 5 (CCR5) is an integral membrane protein that is utilized during human immunodeficiency virus type-1 entry into host cells. CCR5 is a G-protein coupled receptor that contains seven transmembrane (TM) helices. However, the crystal structure of CCR5 has not been reported. A homology model of CCR5 was developed based on the recently reported CXCR4 structure as template. Automated docking of the most potent (14), medium potent (37), and least potent (25) CCR5 antagonists was performed using the CCR5 model. To characterize the mechanism responsible for the interactions between ligands (14, 25, and 37) and CCR5, membrane molecular dynamic (MD) simulations were performed. The position and orientation of ligands (14, 25, and 37) were found to be changed after MD simulations, which demonstrated the ability of this technique to identify binding modes. Furthermore, at the end of simulation, it was found that residues identified by docking were changed and some new residues were introduced in the proximity of ligands. Our results are in line with the majority of previous mutational reports. These results show that hydrophobicity is the determining factor of CCR5 antagonism. In addition, salt bridging and hydrogen bond contacts between ligands (14, 25, and 37) and CCR5 are also crucial for inhibitory activity. The residues newly identified by MD simulation are Ser160, Phe166, Ser180, His181, and Trp190, and so far no site-directed mutagenesis studies have been reported. To determine the contributions made by these residues, additional mutational studies are suggested. We propose a general binding mode for these derivatives based on the MD simulation results of higher (14), medium (37), and lower (25) potent inhibitors. Interestingly, we found some trend for these inhibitors such as, salt bridge interaction between basic nitrogen of ligand and acidic Glu283 seemed necessary for inhibitory activity. Also, two aromatic pockets (pocket I - TM1-3 and pocket II

  2. Analysis of co-receptor usage of circulating viral and proviral HIV genome quasispecies by ultra-deep pyrosequencing in patients who are candidates for CCR5 antagonist treatment.

    PubMed

    Abbate, I; Rozera, G; Tommasi, C; Bruselles, A; Bartolini, B; Chillemi, G; Nicastri, E; Narciso, P; Ippolito, G; Capobianchi, M R

    2011-05-01

    UDPS combined with genotypic algorithms for prediction of HIV-1 co-receptor usage may provide quantitative data about the tropism of each variant present in the viral quasispecies. The aim of the present study was to assess co-receptor usage by ultra-deep pyrosequencing (UDPS), in comparison with the reference phenotypic test (Trofile), in patients who are candidates for CCR5 antagonist treatment, in both circulating and proviral HIV-1. Seventeen patients who were tested by Trofile were enrolled. UDPS of the V3 loop region was carried out on both plasma RNA and proviral DNA. Genotypic prediction of co-receptor usage was established by position-specific score matrices (PSSM) and confirmed, in discordant cases, with geno2pheno. Genetic heterogeneity of the RNA and DNA quasispecies was assessed as well. A total of 196,729 V3 sequences were considered (mean coverage per site, 6346). Concordance between phenotypic test and UDPS with PSSM was 0.82. Geno2pheno results were in line with those obtained with PSSM. Proviral quasispecies were more heterogeneous than those found in circulating HIV. In most patients eligible for CCR5 antagonist treatment, X4 variants were detected in proviral DNA, ranging from 1.0% to 52.7%. UDPS combined with genotypic algorithms for co-receptor usage prediction highlighted the presence of minority variants, with a discordant tropism with respect to the predominant population, in both circulating viral and proviral HIV. In most patients treated with Maraviroc the virological response was independent of the presence of X4 in proviral DNA. The clinical impact of minority X4 variants present in patients who are candidates for anti-CCR5 antagonists remains a crucial point to be addressed.

  3. Molecular Gymnastics: Mechanisms of HIV-1 Resistance to CCR5 Antagonists and Impact on Virus Phenotypes.

    PubMed

    Roche, Michael; Borm, Katharina; Flynn, Jacqueline K; Lewin, Sharon R; Churchill, Melissa J; Gorry, Paul R

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) enters host cells through the binding of its envelope glycoproteins (Env) to the host cell receptor CD4 and then subsequent binding to a chemokine coreceptor, either CCR5 or CXCR4. CCR5 antagonists are a relatively recent class addition to the armamentarium of anti-HIV-1 drugs. These compounds act by binding to a hydrophobic pocket formed by the transmembrane helices of CCR5 and altering the conformation of the extracellular domains, such that they are no longer recognized by Env. Maraviroc is the first drug within this class to be licenced for use in HIV-1 therapy regimens. HIV resistance to CCR5 antagonists occurs either through outgrowth of pre-existing CXCR4-using viruses, or through acquisition of the ability of CCR5-using HIV-1 to use the antagonist bound form of CCR5. In the latter scenario, the mechanism underlying resistance is through complex alterations in the way that resistant Envs engage CCR5. These significant changes are unlikely to occur without consequence to the viral entry phenotype and may also open up new avenues to target CCR5 antagonist resistant viruses. This review discusses the mechanism of action of CCR5 antagonists, how HIV resistance to CCR5 antagonists occurs, and the subsequent effects on Env function.

  4. Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc.

    PubMed

    Latinovic, Olga; Schneider, Kate; Szmacinski, Henryk; Lakowicz, Joseph R; Heredia, Alonso; Redfield, Robert R

    2014-12-01

    The CCR5 chemokine receptor is crucial for human immunodeficiency virus type 1 (HIV-1) infection, acting as the principal coreceptor for HIV-1 entry and transmission and is thus an attractive target for antiviral therapy. Studies have suggested that CCR5 surface density and its conformational changes subsequent to virion engagement are rate limiting for entry, and consequently, infection. Not all CCR5 antibodies inhibit HIV-1 infection, suggesting a need for more potent reagents. Here we evaluated full length single chain (FLSC) IgG1, a novel IgG-CD4-gp120(BAL) fusion protein with several characteristics that make it an attractive candidate for treatment of HIV-1 infections, including bivalency and a potentially increased serum half-life over FLSC, the parental molecule. FLSC IgG1 binds two domains on CCR5, the N-terminus and the second extracellular loop, lowering the levels of available CCR5 viral attachment sites. Furthermore, FLSC IgG1 synergizes with Maraviroc (MVC), the only licensed CCR5 antagonist. In this study, we used both microscopy and functional assays to address the mechanistic aspects of the interactions of FLSC IgG1 and MVC in the context of CCR5 conformational changes and viral infection. We used a novel stochastic optical reconstruction microscopy (STORM), based on high resolution localization of photoswitchable dyes to visualize direct contacts between FLSC IgG1 and CCR5. We compared viral entry inhibition by FLSC IgG1 with that of other CCR5 blockers and showed FLSC IgG1 to be the most potent. We also showed that lower CCR5 surface densities in HIV-1 infected primary cells result in lower FLSC IgG1 EC50 values. In addition, CCR5 binding by FLSC IgG1, but not CCR5 Ab 2D7, was significantly increased when cells were treated with MVC, suggesting MVC allosterically increases exposure of the FLSC IgG1 binding site. These data have implications for future antiviral therapy development.

  5. An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immunodeficiency virus type 1 entry

    SciTech Connect

    Safarian, Diana; Carnec, Xavier; Tsamis, Fotini; Kajumo, Francis; Dragic, Tatjana . E-mail: tdragic@aecom.yu.edu

    2006-09-01

    HIV-1 coreceptors are attractive targets for novel antivirals. Here, inhibition of entry by two classes of CCR5 antagonists was investigated. We confirmed previous findings that HIV-1 isolates vary greatly in their sensitivity to small molecule inhibitors of CCR5-mediated entry, SCH-C and TAK-779. In contrast, an anti-CCR5 monoclonal antibody (PA14) similarly inhibited entry of diverse viral isolates. Sensitivity to small molecules was V3 loop-dependent and inversely proportional to the level of gp120 binding to CCR5. Moreover, combinations of the MAb and small molecules were highly synergistic in blocking HIV-1 entry, suggesting different mechanisms of action. This was confirmed by time course of inhibition experiments wherein the PA14 MAb and small molecules were shown to inhibit temporally distinct stages of CCR5 usage. We propose that small molecules inhibit V3 binding to the second extracellular loop of CCR5, whereas PA14 preferentially inhibits subsequent events such as CCR5 recruitment into the fusion complex or conformational changes in the gp120-CCR5 complex that trigger fusion. Importantly, our findings suggest that combinations of CCR5 inhibitors with different mechanisms of action will be central to controlling HIV-1 infection and slowing the emergence of resistant strains.

  6. Chemokine Receptor CCR5 Antagonist Maraviroc: Medicinal Chemistry and Clinical Applications

    PubMed Central

    Xu, Guoyan G.; Guo, Jia; Wu, Yuntao

    2015-01-01

    The human immunodeficiency virus (HIV) causes acquired immumodeficiency syndrome (AIDS), one of the worst global pandemic. The virus infects human CD4 T cells and macrophages, and causes CD4 depletion. HIV enters target cells through the binding of the viral envelope glycoprotein to CD4 and the chemokine coreceptor, CXCR4 or CCR5. In particular, the CCR5-utilizing viruses predominate in the blood during the disease course. CCR5 is expressed on the surface of various immune cells including macrophages, monocytes, microglia, dendric cells, and active memory CD4 T cells. In the human population, the CCR5 genomic mutation, CCR5Δ32, is associated with relative resistance to HIV. These findings paved the way for the discovery and development of CCR5 inhibitors to block HIV transmission and replication. Maraviroc, discovered as a CCR5 antagonist, is the only CCR5 inhibitor that has been approved by both US FDA and the European Medicines Agency (EMA) for treating HIV/AIDS patients. In this review, we summarize the medicinal chemistry and clinical studies of Maraviroc. PMID:25159165

  7. Chemokine receptor CCR5 antagonist maraviroc: medicinal chemistry and clinical applications.

    PubMed

    Xu, Guoyan G; Guo, Jia; Wu, Yuntao

    2014-01-01

    The human immunodeficiency virus (HIV) causes acquired immumodeficiency syndrome (AIDS), one of the worst global pandemic. The virus infects human CD4 T cells and macrophages, and causes CD4 depletion. HIV enters target cells through the binding of the viral envelope glycoprotein to CD4 and the chemokine coreceptor, CXCR4 or CCR5. In particular, the CCR5-utilizing viruses predominate in the blood during the disease course. CCR5 is expressed on the surface of various immune cells including macrophages, monocytes, microglia, dendric cells, and active memory CD4 T cells. In the human population, the CCR5 genomic mutation, CCR5Δ32, is associated with relative resistance to HIV. These findings paved the way for the discovery and development of CCR5 inhibitors to block HIV transmission and replication. Maraviroc, discovered as a CCR5 antagonist, is the only CCR5 inhibitor that has been approved by both US FDA and the European Medicines Agency (EMA) for treating HIV/AIDS patients. In this review, we summarize the medicinal chemistry and clinical studies of Maraviroc.

  8. Lack of protection from HIV infection by the mutant HIV coreceptor CCR5 in intravenously HIV infected hemophilia patients.

    PubMed

    Malo, A; Rommel, F; Bogner, J; Gruber, R; Schramm, W; Goebel, F D; Riethmüller, G; Wank, R

    1998-02-01

    The CCR5 chemokine receptor is an important coreceptor for macrophage-tropic HIV strains. Homozygous carriers of the mutated CCR5 receptor with a 32 bp deletion (delta 32-CCR5) are highly protected against HIV infection. A protective effect has also been described for heterozygous individuals carrying both mutated and wildtype CCR5 receptors. We compared the frequency of the mutated delta 32-CCR5 HIV coreceptor in HIV positive patients infected by sexual contact (N = 160) with intravenously HIV infected hemophilic patients (N = 84) and HIV negative individuals (N = 421). We found no protective effect of delta 32-CCR5 HIV coreceptor in hemophilic patients (p = 0.0134). If proteins of plasma concentrates would be responsible for facilitating the entry of HIV macrophages by upregulation of the CCR5 wildtype receptor it would be of therapeutical interest to identify the responsible plasma proteins.

  9. Relative concordance of human immunodeficiency virus oligomeric and monomeric envelope in CCR5 coreceptor usage

    SciTech Connect

    Teeravechyan, Samaporn; Suphaphiphat, Pirada; Essex, Max; Lee, Tun-Hou

    2008-01-20

    A major difference between binding and fusion assays commonly used to study the human immunodeficiency virus (HIV) envelope is the use of monomeric envelope for the former assay and oligomeric envelope for the latter. Due to discrepancies in their readouts for some mutants, envelope regions involved in CCR5 coreceptor usage were systematically studied to determine whether the discordance is due to inherent differences between the two assays or whether it genuinely reflects functional differences at each entry step. By adding the binding inhibitor TAK-779 to delay coreceptor binding kinetics in the fusion assay, the readouts were found comparable between the assays for the mutants analysed in this study. Our finding indicates that monomeric binding reflects oligomeric envelope-CCR5 interaction, thus discordant results between binding and fusion assays do not necessarily indicate differences in coreceptor usage by oligomeric envelope and monomeric gp120.

  10. New approaches in the treatment of HIV/AIDS – focus on maraviroc and other CCR5 antagonists

    PubMed Central

    Schlecht, Hans P; Schellhorn, Sarah; Dezube, Bruce J; Jacobson, Jeffrey M

    2008-01-01

    Treatment of HIV-1 infection has produced dramatic success for many patients. Nevertheless, viral resistance continues to limit the efficacy of currently available agents in many patients. The CCR5 antagonists are a new class of antiretroviral agents that target a necessary coreceptor for viral entry of many strains of HIV-1. Recently, the first agent within this class, maraviroc, was approved by a number of regulatory agencies, including the Food and Drug Administration. Herein we review the role of the CCR5 receptor in HIV-1 infection and potential methods to target it in anti-HIV-1 therapy. We review the various categories of agents and discuss specific agents that have progressed to clinical study. We discuss in detail the recently approved, first in class CCR5 antagonist, maraviroc, and discuss aspects of resistance to CCR5 antagonism and the potential role of CCR5 antagonism in the management of HIV-1 infection. PMID:18728830

  11. HIV type 1 coreceptor tropism, CCR5 genotype, and integrase inhibitor resistance profiles in Vietnam: implications for the introduction of new antiretroviral regimens.

    PubMed

    Luu, Quynh Phuong; Dean, Jonathan; Do, Trinh Thi Diem; Carr, Michael J; Dunford, Linda; Coughlan, Suzie; Connell, Jeff; Nguyen, Hien Tran; Hall, William W; Nguyen Thi, Lan Anh

    2012-10-01

    In Vietnam, where an estimated 280,000 people will be HIV-positive by 2012, recommended antiretroviral regimens do not include more recently developed therapeutics, such as Integrase inhibitors (INI) and coreceptor antagonists. This study examined HIV-1 coreceptor tropism and INI drug resistance profiles, in parallel with CCR5 genotypes, in a cohort of 60 HIV-positive individuals from different regions of Vietnam. No evidence of INI resistance was detected. Some 40% of individuals had X4-tropic HIV-1, making them unsuitable for treatment with CCR5 antagonists. We identified a novel CCR5 variant-S272P-along with other, previously reported variants: G106R, C178R, W153C, R223Q, and S336I. Interestingly, CCR5 variants known to affect HIV-1 infectivity were observed only in individuals harboring X4-tropic virus. Together, this study presents valuable baseline information on HIV-1 INI resistance, coreceptor tropism, and CCR5 variants in HIV-positive individuals in Vietnam. This should help inform policy on the future use of novel antiretrovirals in Vietnam.

  12. [Frequency of the coreceptor CCR5 gene delta 32 mutation in different French regions].

    PubMed

    Lucotte, G; Mercier, G

    1998-05-01

    We studied the frequency of the coreceptor CCR5 gene delta 32 mutation on 1,836 DNA samples originating from ten French regions. This mutation confers, in the homozygous state, resistance to HIV-1 infection. For the whole territory, the mean percentage presence of the delta 32 mutation is 9.2%. The mutation is statistically more frequent in the north (11.2%) than in the south (6.3%) of the country; this differentiation corresponds probably to a gradient of decreasing frequencies of the delta 32 mutation in Europe.

  13. Simian Immunodeficiency Virus SIVagm Efficiently Utilizes Non-CCR5 Entry Pathways in African Green Monkey Lymphocytes: Potential Role for GPR15 and CXCR6 as Viral Coreceptors

    PubMed Central

    Riddick, Nadeene E.; Wu, Fan; Matsuda, Kenta; Whitted, Sonya; Ourmanov, Ilnour; Goldstein, Simoy; Goeken, Robert M.; Plishka, Ronald J.; Buckler-White, Alicia; Brenchley, Jason M.

    2015-01-01

    ABSTRACT African green monkeys (AGM) are natural hosts of simian immunodeficiency virus (SIV), and infection in these animals is generally nonpathogenic, whereas infection of nonnatural hosts, such as rhesus macaques (RM), is commonly pathogenic. CCR5 has been described as the primary entry coreceptor for SIV in vivo, while human-derived CXCR6 and GPR15 also appear to be used in vitro. However, sooty mangabeys that are genetically deficient in CCR5 due to an out-of-frame deletion are infectible with SIVsmm, indicating that SIVsmm can use alternative coreceptors in vivo. In this study, we examined the CCR5 dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer replicated efficiently in AGM and RM peripheral blood mononuclear cells (PBMC) in the presence of the CCR5 antagonist maraviroc, despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3, and simian-human immunodeficiency virus SHIV-AD8 in RM PBMC. We also found that AGM CXCR6 and AGM GPR15, to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15, and CXCR6 mRNAs were detected in AGM and RM memory CD4+ T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4+ T cells and are potential alternative coreceptors for SIVagm use in vivo. These data suggest that the use of non-CCR5 entry pathways may be a common feature of SIV replication in natural host species, with the potential to contribute to nonpathogenicity in these animals. IMPORTANCE African green monkeys (AGM) are natural hosts of SIV, and infection in these animals generally does not cause AIDS, whereas SIV-infected rhesus macaques (RM) typically develop AIDS. Although it has been reported that SIV generally uses CD4 and CCR5 to enter target cells in vivo, other

  14. Inheritance pattern of mutant human immunodeficiency virus type 1 coreceptor gene CCR5 in an Indian family.

    PubMed

    Husain, S; Goila, R; Shahi, S; Banerja, A C

    1998-01-01

    The most common form of mutation found in the CCR5 gene has been the precise 32-base pair (bp) deletion in the region corresponding to second extracellular loop of the chemokine receptor CCR5. Individuals homozygous for the delta 32 allele of CCR5 usually remain uninfected despite multiple exposures to HIV, whereas heterozygous individuals support less virus replication and show slower progression of the disease. This mutant allele in either homozygous or heterozygous form is quite common in white people of European heritage. Earlier work involving large populations of Asians and Africans failed to detect the presence of this mutant allele. We screened 145 normal unrelated healthy Indians and found one person who was heterozygous for the delta 32 allele of CCR5. We studied the inheritance of this deleted allele in this person's family. One parent, one of two sons, and the only daughter possessed this mutant allele. We cloned the entire coding region of wild-type and mutant alleles of CCR5 gene from the heterozygous individual mentioned and studied its coreceptor functions. The mutant allele had only a moderate interfering effect on coreceptor activity of the wild-type CCR5 allele in a cell fusion assay. We also report an improved method of genotyping CCR5 gene in this communication.

  15. Viremic Control and Viral Coreceptor Usage in Two HIV-1-Infected Persons Homozygous for CCR5 Δ32

    PubMed Central

    Henrich, Timothy J.; Hanhauser, Emily; Hu, Zixin; Stellbrink, Hans-Jürgen; Noah, Christian; Martin, Jeffrey N.; Deeks, Steven G.; Kuritzkes, Daniel R.; Pereyra, Florencia

    2015-01-01

    Objectives To determine viral and immune factors involved in transmission and control of HIV-1 infection in persons without functional CCR5 Design Understanding transmission and control of HIV-1 in persons homozygous for CCR5Δ32 is important given efforts to develop HIV-1 curative therapies aimed at modifying or disrupting CCR5 expression. Methods We identified two HIV-infected CCR5Δ32/Δ32 individuals among a cohort of patients with spontaneous control of HIV-1 infection without antiretroviral therapy and determined co-receptor usage of the infecting viruses. We assessed genetic evolution of full-length HIV-1 envelope sequences by single-genome analysis from one participant and his sexual partner, and explored HIV-1 immune responses and HIV-1 mutations following virologic escape and disease progression. Results Both participants experienced viremia of less than 4,000 RNA copies/ml with preserved CD4+ T cell counts off ART for at least 3.3 and 4.6 years after diagnosis, respectively. One participant had phenotypic evidence of X4 virus, had no known favorable HLA alleles, and appeared to be infected by minority X4 virus from a pool that predominately used CCR5 for entry. The second participant had virus that was unable to use CXCR4 for entry in phenotypic assay but was able to engage alternative viral coreceptors (e.g. CXCR6) in vitro. Conclusions Our study demonstrates that individuals may be infected by minority X4 viruses from a population that predominately uses CCR5 for entry, and that viruses may bypass traditional HIV-1 coreceptors (CCR5 and CXCR4) completely by engaging alternative coreceptors to establish and propagate HIV-1 infection. PMID:25730507

  16. [Secondary effects of treatment with maraviroc and other CCR5 antagonists. Potential impact of the CCR5 blocker].

    PubMed

    Arribas López, José R

    2008-10-01

    Maraviroc is the first inhibitor of CCR5 co-receptors to be marketed as an antiretroviral. The pre-clinical studies and phase III trials have shown that it has a very favourable safety profile. No characteristic adverse effect of maraviroc has been identified. Unlike with aplaviroc, where its clinical development was stopped due to serious hepatoxicity, no increase in liver toxicity has been demonstrated in patients treated with maraviroc even if they are co-infected by hepatotropic virus. Nor was there any evidence of an increase in the incidence of neoplasms or serious infections in patients treated with maraviroc. In a study on naive patients, maraviroc produced nonsignificant changes in total cholesterol, LDL, HDL and triglycerides. Although CCR5 co-receptors play a role in the immune response of the body, it has not been shown whether individuals homozygote for its deletion (delta-32 mutation) have an increased risk of serious infections, with the possible exception of encephalitis due to the West Nile virus. However, long-term follow up is required on patients treated with to be able to rule out any increased susceptibility to infections or neoplasms.

  17. Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes

    PubMed Central

    Kuzmina, Alona; Vaknin, Karin; Gdalevsky, Garik; Vyazmensky, Maria; Marks, Robert S.; Taube, Ran

    2015-01-01

    Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential. PMID:26629902

  18. Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes.

    PubMed

    Kuzmina, Alona; Vaknin, Karin; Gdalevsky, Garik; Vyazmensky, Maria; Marks, Robert S; Taube, Ran; Engel, Stanislav

    2015-01-01

    Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.

  19. The GHS-R blocker D-[Lys3] GHRP-6 serves as CCR5 chemokine receptor antagonist.

    PubMed

    Patel, Kalpesh; Dixit, Vishwa Deep; Lee, Jun Ho; Kim, Jie Wan; Schaffer, Eric M; Nguyen, Dzung; Taub, Dennis D

    2012-01-01

    [D-Lys3]-Growth Hormone Releasing Peptide-6 (DLS) is widely utilized in vivo and in vitro as a selective ghrelin receptor (GHS-R) antagonist. This antagonist is one of the most common antagonists utilized in vivo to block GHS-R function and activity. Here, we found that DLS also has the ability to modestly block chemokine function and ligand binding to the chemokine receptor CCR5. The DLS effects on RANTES binding and Erk signaling as well as calcium mobilization appears to be much stronger than its effects on MIP-1α and MIP-1β. CCR5 have been shown to act as major co-receptor for HIV-1 entry into the CD4 positive host cells. To this end, we also found that DLS blocks M-tropic HIV-1 propagation in activated human PBMCs. These data demonstrate that DLS may not be a highly selective GHS-R1a inhibitor and may also effects on other G-protein coupled receptor (GPCR) family members. Moreover, DLS may have some potential clinical applications in blocking HIV infectivity and CCR5-mediated migration and function in various inflammatory disease states.

  20. Discovery of a Potent and Orally Bioavailable CCR2 and CCR5 Dual Antagonist

    PubMed Central

    2009-01-01

    This report describes the discovery of a potent, orally bioavailable CC chemokine receptor 2 (CCR2) antagonist which, while optimized for CCR2 potency, also had potent CC chemokine receptor 5 (CCR5) activity.

  1. High intrapatient HIV-1 evolutionary rate is associated with CCR5-to-CXCR4 coreceptor switch.

    PubMed

    Mild, Mattias; Gray, Rebecca R; Kvist, Anders; Lemey, Philippe; Goodenow, Maureen M; Fenyö, Eva Maria; Albert, Jan; Salemi, Marco; Esbjörnsson, Joakim; Medstrand, Patrik

    2013-10-01

    In approximately 70% of individuals infected with HIV-1 subtype B, the virus switches coreceptor use from exclusively CCR5 use (R5 virus) to either inclusion of or exclusively CXCR4 use (X4 virus) during infection. This switch is associated with an accelerated loss of CD4(+) T-cells and a faster progression to AIDS. Despite intensive research, the mechanisms responsible for coreceptor switch remains elusive. In the present study, we investigated associations between viral evolutionary rate and selection pressure versus viral coreceptor use and rate of disease progression in eight patients with longitudinally sampled HIV-1 env V1-V3 sequences. By employing a Bayesian hierarchical phylogenetic model, we found that the HIV-1 evolutionary rate was more strongly associated with coreceptor switch than with rate of disease progression in terms of CD4(+)T-cell decline. Phylogenetic analyses showed that X4 variants evolved from R5 populations. In addition, coreceptor switch was associated with higher evolutionary rates on both the synonymous and non-synonymous substitution level, but not with dN/dS ratio rates. Our findings suggest that X4 viruses evolved from pre-existing R5 viral populations and that the evolution of coreceptor switch is governed by high replication rates rather than by selective pressure. Furthermore, the association of viral evolutionary rate was more strongly associated with coreceptor switch than disease progression. This adds to the understanding of the complex virus-host interplay that influences the evolutionary dynamics of HIV-1 coreceptor use.

  2. HIV-1 entry inhibition by small-molecule CCR5 antagonists: A combined molecular modeling and mutant study using a high-throughput assay

    SciTech Connect

    Labrecque, Jean; Metz, Markus; Lau, Gloria; Darkes, Marilyn C.; Wong, Rebecca S.Y.; Bogucki, David; Carpenter, Bryon; Chen Gang; Li Tongshuang; Nan, Susan; Schols, Dominique; Bridger, Gary J.; Fricker, Simon P.; Skerlj, Renato T.

    2011-05-10

    Based on the attrition rate of CCR5 small molecule antagonists in the clinic the discovery and development of next generation antagonists with an improved pharmacology and safety profile is necessary. Herein, we describe a combined molecular modeling, CCR5-mediated cell fusion, and receptor site-directed mutagenesis approach to study the molecular interactions of six structurally diverse compounds (aplaviroc, maraviroc, vicriviroc, TAK-779, SCH-C and a benzyloxycarbonyl-aminopiperidin-1-yl-butane derivative) with CCR5, a coreceptor for CCR5-tropic HIV-1 strains. This is the first study using an antifusogenic assay, a model of the interaction of the gp120 envelope protein with CCR5. This assay avoids the use of radioactivity and HIV infection assays, and can be used in a high throughput mode. The assay was validated by comparison with other established CCR5 assays. Given the hydrophobic nature of the binding pocket several binding models are suggested which could prove useful in the rational drug design of new lead compounds.

  3. Development of maraviroc, a CCR5 antagonist for treatment of HIV, using a novel tropism assay.

    PubMed

    van der Ryst, Elna; Heera, Jayvant; Demarest, James; Knirsch, Charles

    2015-06-01

    Assays to identify infectious organisms are critical for diagnosis and enabling the development of therapeutic agents. The demonstration that individuals with a 32-bp deletion within the CCR5 locus were resistant to human immunodeficiency virus (HIV) infection, while those heterozygous for the mutation progressed more slowly, led to the discovery of maraviroc (MVC), a CCR5 antagonist. As MVC is only active against CCR5-tropic strains of HIV, it was critical to develop a diagnostic assay to identify appropriate patients. Trofile™, a novel phenotypic tropism assay, was used to identify patients with CCR5-tropic virus for the MVC development program. Results of these clinical studies demonstrated that the assay correctly identified patients likely to respond to MVC. Over time, the performance characteristics of the phenotypic assay were enhanced, necessitating retesting of study samples. Genotypic tropism tests that have the potential to allow for local use and more rapid turnaround times are also being developed.

  4. Epigenetic mechanisms, T-cell activation, and CCR5 genetics interact to regulate T-cell expression of CCR5, the major HIV-1 coreceptor.

    PubMed

    Gornalusse, German G; Mummidi, Srinivas; Gaitan, Alvaro A; Jimenez, Fabio; Ramsuran, Veron; Picton, Anabela; Rogers, Kristen; Manoharan, Muthu Saravanan; Avadhanam, Nymisha; Murthy, Krishna K; Martinez, Hernan; Molano Murillo, Angela; Chykarenko, Zoya A; Hutt, Richard; Daskalakis, Demetre; Shostakovich-Koretskaya, Ludmila; Abdool Karim, Salim; Martin, Jeffrey N; Deeks, Steven G; Hecht, Frederick; Sinclair, Elizabeth; Clark, Robert A; Okulicz, Jason; Valentine, Fred T; Martinson, Neil; Tiemessen, Caroline Tanya; Ndung'u, Thumbi; Hunt, Peter W; He, Weijing; Ahuja, Sunil K

    2015-08-25

    T-cell expression levels of CC chemokine receptor 5 (CCR5) are a critical determinant of HIV/AIDS susceptibility, and manifest wide variations (i) between T-cell subsets and among individuals and (ii) in T-cell activation-induced increases in expression levels. We demonstrate that a unifying mechanism for this variation is differences in constitutive and T-cell activation-induced DNA methylation status of CCR5 cis-regulatory regions (cis-regions). Commencing at an evolutionarily conserved CpG (CpG -41), CCR5 cis-regions manifest lower vs. higher methylation in T cells with higher vs. lower CCR5 levels (memory vs. naïve T cells) and in memory T cells with higher vs. lower CCR5 levels. HIV-related and in vitro induced T-cell activation is associated with demethylation of these cis-regions. CCR5 haplotypes associated with increased vs. decreased gene/surface expression levels and HIV/AIDS susceptibility magnify vs. dampen T-cell activation-associated demethylation. Methylation status of CCR5 intron 2 explains a larger proportion of the variation in CCR5 levels than genotype or T-cell activation. The ancestral, protective CCR5-HHA haplotype bears a polymorphism at CpG -41 that is (i) specific to southern Africa, (ii) abrogates binding of the transcription factor CREB1 to this cis-region, and (iii) exhibits a trend for overrepresentation in persons with reduced susceptibility to HIV and disease progression. Genotypes lacking the CCR5-Δ32 mutation but with hypermethylated cis-regions have CCR5 levels similar to genotypes heterozygous for CCR5-Δ32. In HIV-infected individuals, CCR5 cis-regions remain demethylated, despite restoration of CD4+ counts (≥800 cells per mm(3)) with antiretroviral therapy. Thus, methylation content of CCR5 cis-regions is a central epigenetic determinant of T-cell CCR5 levels, and possibly HIV-related outcomes.

  5. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

    PubMed

    Mock, Ulrike; Machowicz, Rafał; Hauber, Ilona; Horn, Stefan; Abramowski, Pierre; Berdien, Belinda; Hauber, Joachim; Fehse, Boris

    2015-06-23

    Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

  6. Pharmacotherapy of HIV-1 Infection: Focus on CCR5 Antagonist Maraviroc

    PubMed Central

    Latinovic, Olga; Kuruppu, Janaki; Davis, Charles; Le, Nhut; Heredia, Alonso

    2009-01-01

    Sustained inhibition of HIV-1, the goal of antiretroviral therapy, is often impeded by the emergence of viral drug resistance. For patients infected with HIV-1 resistant to conventional drugs from the viral reverse transcriptase and protease inhibitor classes, the recently approved entry and integration inhibitors effectively suppress HIV-1 and offer additional therapeutic options. Entry inhibitors are particularly attractive because, unlike conventional antiretrovirals, they target HIV-1 extracellularly, thereby sparing cells from both viral- and drug-induced toxicities. The fusion inhibitor enfuvirtide and the CCR5 antagonist maraviroc are the first entry inhibitors licensed for patients with drug-resistant HIV-1, with maraviroc restricted to those infected with CCR5-tropic HIV-1 (R5 HIV-1) only. Vicriviroc (another CCR5 antagonist) is in Phase III clinical trials, whereas the CCR5 antibodies PRO 140 and HGS 004 are in early stages of clinical development. Potent antiviral synergy between maraviroc and CCR5 antibodies, coupled with distinct patterns of resistance, suggest their combinations might be particularly effective in patients. In addition, given that oral administration of maraviroc achieves high drug levels in cervicovaginal fluid, combinations of maraviroc and other CCR5 inhibitors could be effective in preventing HIV-1 transmission. Moreover, since CCR5 antagonists prevent rejection of transplanted organs, maraviroc could both suppress HIV-1 and prolong organ survival for the growing number of HIV-1 patients with kidney or liver failure necessitating organ transplantation. Thus, maraviroc offers an important treatment option for patients with drug-resistant R5 HIV-1, who presently account for >50% of drug-resistance cases. PMID:19920876

  7. The isolation of novel phage display-derived human recombinant antibodies against CCR5, the major co-receptor of HIV.

    PubMed

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai; Hizi, Amnon

    2013-08-01

    Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest.

  8. Usage of the coreceptors CCR-5, CCR-3, and CXCR-4 by primary and cell line-adapted human immunodeficiency virus type 2.

    PubMed Central

    Sol, N; Ferchal, F; Braun, J; Pleskoff, O; Tréboute, C; Ansart, I; Alizon, M

    1997-01-01

    The chemokine receptors CCR-5 and CXCR-4, and possibly CCR-3, are the principal human immunodeficiency virus type 1 (HIV-1) coreceptors, apparently interacting with HIV-1 envelope, in association with CD4. Cell lines coexpressing CD4 and these chemokine receptors were infected with a panel of seven primary HIV-2 isolates passaged in peripheral blood mononuclear cells (PBMC) and three laboratory HIV-2 strains passaged in T-cell lines. The CCR-5, CCR-3, and CXCR-4 coreceptors could all be used by HIV-2. The ability to use CXCR-4 represents a major difference between HIV-2 and the closely related simian immunodeficiency viruses. Most HIV-2 strains using CCR-5 could also use CCR-3, sometimes with similar efficiencies. As observed for HIV-1, the usage of CCR-5 or CCR-3 was observed principally for HIV-2 strains derived from asymptomatic individuals, while HIV-2 strains derived from AIDS patients used CXCR-4. However, there were several exceptions, and the patterns of coreceptor usage seemed more complex for HIV-2 than for HIV-1. The two T-tropic HIV-2 strains tested used CXCR-4 and not CCR-5, while T-tropic HIV-1 can generally use both. Moreover, among five primary HIV-2 strains all unable to use CXCR-4, three could replicate in CCR-5-negative PBMC, which has not been reported for HIV-1. These observations suggest that the CCR-5 coreceptor is less important for HIV-2 than for HIV-1 and indicate that HIV-2 can use other cell entry pathways and probably other coreceptors. One HIV-2 isolate replicating in normal or CCR-5-negative PBMC failed to infect CXCR-4+ cells or the U87MG-CD4 and sMAGI cell lines, which are permissive to infection by HIV-2 but not by HIV-1. This suggests the existence of several HIV-2-specific coreceptors, which are differentially expressed in cell lines and PBMC. PMID:9343175

  9. I-TAC/CXCL11 is a natural antagonist for CCR5.

    PubMed

    Petkovic, Vibor; Moghini, Christian; Paoletti, Samantha; Uguccioni, Mariagrazia; Gerber, Basil

    2004-09-01

    The selective CXC chemokine receptor 3 (CXCR3) agonists, monokine induced by interferon-gamma (IFN- gamma)/CXC chemokine ligand 9 (CXCL9), IFN-inducible protein 10/CXCL10, and IFN-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, attract CXCR3+ cells such as CD45RO+ T lymphocytes, B cells, and natural killer cells. Further, all three chemokines are potent, natural antagonists for chemokine receptor 3 (CCR3) and feature defensin-like, antimicrobial activities. In this study, we show that I-TAC, in addition to these effects, acts as an antagonist for CCR5. I-TAC inhibited the binding of macrophage-inflammatory protein-1alpha (MIP-1alpha)/CC chemokine ligand 3 (CCL3) to cells transfected with CCR5 and to monocytes. Furthermore, cell migration evoked by regulated on activation, normal T expressed and secreted (RANTES)/CCL5 and MIP-1beta/CCL4, the selective agonist of CCR5, was inhibited in transfected cells and monocytes, respectively. In two other functional assays, namely the release of free intracellular calcium and actin polymerization, I-TAC reduced CCR5 activities to minimal levels. Sequence and structure analyses indicate a potential role for K17, K49, and Q51 of I-TAC in CCR5 binding. Our results expand on the potential role of I-TAC as a negative modulator in leukocyte migration and activation, as I-TAC would specifically counteract the responses mediated by many "classical," inflammatory chemokines that act not only via CCR3 but via CCR5 as well.

  10. In vivo effect of statins on the expression of the HIV co-receptors CCR5 and CXCR4

    PubMed Central

    2013-01-01

    Background During the HIV-1 replication cycle, several molecules including chemokine receptors and cholesterol are crucial, and are therefore potential targets for therapeutic intervention. Indeed statins, compounds that inhibit cellular synthesis of cholesterol and have anti-inflammatory and immunomodulatory properties were shown to inhibit HIV-1 infection by R5 tropic strains but not by X4 strains in vitro, mainly by altering the chemokine receptor/ligands axes. Therefore, the objective of this study was to characterize in vivo, the capacity of statins to modulate in HIV seronegative and chronically HIV-1-infected adults the expression of CCR5 and CXCR4, of their ligands and the tropism of circulating HIV-1 strains. Methods Samples from asymptomatic HIV-1-infected adults enrolled in a clinical trial aimed at evaluating the antiretroviral activity of lovastatin were used to evaluate in vivo the modulation by lovastatin of CCR5, CXCR4, their ligands, and the shift in plasma viral tropism over one year of intervention. In addition, ten HIV negative adults received a daily oral dose of 40 mg of lovastatin or 20 mg of atorvastatin; seven other HIV negative individuals who received no treatment were followed as controls. The frequency and phenotype of immune cells were determined by flow-cytometry; mRNA levels of chemokine receptors and their ligands were determined by real-time PCR. Viral tropism was determined by PCR and sequencing, applying the clonal and clinical model of analyses. Results Our study shows that long-term administration of lovastatin in HIV-infected individuals does not induce a shift in viral tropism, or induce a significant modulation of CCR5 and CXCR4 on immune cells in HIV-infected patients. Similar results were found in HIV seronegative control subjects, treated with lovastatin or atorvastatin, but a significant increase in CCL3 and CCL4 transcription was observed in these individuals. Conclusions These findings suggest that long

  11. Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry.

    PubMed

    Westby, Mike; Smith-Burchnell, Caroline; Mori, Julie; Lewis, Marilyn; Mosley, Michael; Stockdale, Mark; Dorr, Patrick; Ciaramella, Giuseppe; Perros, Manos

    2007-03-01

    Maraviroc is a CCR5 antagonist in clinical development as one of a new class of antiretrovirals targeting human immunodeficiency virus type 1 (HIV-1) coreceptor binding. We investigated the mechanism of HIV resistance to maraviroc by using in vitro sequential passage and site-directed mutagenesis. Serial passage through increasing maraviroc concentrations failed to select maraviroc-resistant variants from some laboratory-adapted and clinical isolates of HIV-1. However, high-level resistance to maraviroc was selected from three of six primary isolates passaged in peripheral blood lymphocytes (PBL). The SF162 strain acquired resistance to maraviroc in both treated and control cultures; all resistant variants were able to use CXCR4 as a coreceptor. In contrast, maraviroc-resistant virus derived from isolates CC1/85 and RU570 remained CCR5 tropic, as evidenced by susceptibility to the CCR5 antagonist SCH-C, resistance to the CXCR4 antagonist AMD3100, and an inability to replicate in CCR5 Delta32/Delta32 PBL. Strain-specific mutations were identified in the V3 loop of maraviroc-resistant CC1/85 and RU570. The envelope-encoding region of maraviroc-resistant CC1/85 was inserted into an NL4-3 background. This recombinant virus was completely resistant to maraviroc but retained susceptibility to aplaviroc. Reverse mutation of gp120 residues 316 and 323 in the V3 loop (numbering from HXB2) to their original sequence restored wild-type susceptibility to maraviroc, while reversion of either mutation resulted in a partially sensitive virus with reduced maximal inhibition (plateau). The plateaus are consistent with the virus having acquired the ability to utilize maraviroc-bound receptor for entry. This hypothesis was further corroborated by the observation that a high concentration of maraviroc blocks the activity of aplaviroc against maraviroc-resistant virus.

  12. HIV-1 tropism testing and clinical management of CCR5 antagonists: Quebec review and recommendations.

    PubMed

    Tremblay, Cécile; Hardy, Isabelle; Lalonde, Richard; Trottier, Benoit; Tsarevsky, Irina; Vézina, Louis-Philippe; Roger, Michel; Wainberg, Mark; Baril, Jean-Guy

    2013-01-01

    HIV-1 tropism assays play a crucial role in determining the response to CCR5 receptor antagonists. Initially, phenotypic tests were used, but limited access to these tests prompted the development of alternative strategies. Recently, genotyping tropism has been validated using a Canadian technology in clinical trials investigating the use of maraviroc in both experienced and treatment-naive patients. The present guidelines review the evidence supporting the use of genotypic assays and provide recommendations regarding tropism testing in daily clinical management.

  13. Evolution of CCR5 Antagonist Resistance in an HIV-1 Subtype C Clinical Isolate

    PubMed Central

    Henrich, Timothy J.; Tsibris, Athe M.N.; Lewine, Nicolas R.P.; Konstantinidis, Ioannis; Leopold, Kay E.; Sagar, Manish; Kuritzkes, Daniel R.

    2011-01-01

    Objectives We previously reported vicriviroc (VCV) resistance in an HIV-infected subject and used deep sequencing and clonal analyses to track the evolution of V3 sequence forms over 28 weeks of therapy. Here, we test the contribution of gp120 mutations to CCR5 antagonist resistance and investigate why certain minority V3 variants emerged as the dominant species under drug pressure. Methods 19 site-directed HIV-1 mutants were generated that contained gp120 VCV-resistance mutations. Viral sensitivities to VCV, maraviroc, TAK-779 and HGS004 were determined. Results Three patterns of susceptibilities were observed: sigmoid inhibition curves with IC50s similar to pre-treatment virus (07J-week 0 [W0]), single mutants with decreased IC50s compared to 07J-W0, and mutants that contained ≥5 of 7 VCV-resistance mutations with flattened inhibition curves and decreased or negative percent maximal inhibition. Substitutions such as S306P, which sensitized virus to CCR5 antagonists when present as single mutations, were not detected in the baseline virus population but were necessary for maximal resistance when incorporated into V3 backbones that included pre-existing VCV resistance mutations. Conclusion CCR5 antagonist resistance was reproduced only when a majority of V3 mutations were present. Minority V3 loop variants may serve as a scaffold upon which additional mutations lead to complete VCV resistance. PMID:20856130

  14. CCR5 and HIV infection.

    PubMed

    Blanpain, Cédric; Libert, Frédérick; Vassart, Gilbert; Parmentier, Marc

    2002-01-01

    Chemokines and chemokine receptors play a crucial role in the trafficking of leukocyte populations across the body, and are involved in the development of a large variety of human diseases. CCR5 is the main coreceptor used by macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2, which are responsible for viral transmission. CCR5 therefore plays an essential role in HIV pathogenesis. A number of inflammatory CC-chemokines, including MIP-1 alpha, MIP-1 beta, RANTES, MCP-2, and HCC-1[9-74] act as CCR5 agonists, while MCP-3 is a natural antagonist of the receptor. CCR5 is mainly expressed in memory T-cells, macrophages, and immature dendritic cells, and is upregulated by proinflammatory cytokines. It is coupled to the Gi class of heterotrimeric G-proteins, and inhibits cAMP production, stimulates Ca2+ release, and activates PI3-kinase and MAP kinases, as well as other tyrosine kinase cascades. A mutant allele of CCR5, CCR5 delta 32 is frequent in populations of European origin, and encodes a nonfunctional truncated protein that is not transported to the cell surface. Homozygotes for the delta 32 allele exhibit a strong, although incomplete, resistance to HIV infection, whereas heterozygotes display delayed progression to acquired immunodeficiency syndrome (AIDS). Many other alleles, affecting the primary structure of CCR5 or its promoter have been described, some of which lead to nonfunctional receptors or otherwise influence AIDS progression. CCR5 is considered as a drug target in the field of HIV, but also in a growing number of inflammatory diseases. Modified chemokines, monoclonal antibodies and small chemical antagonists, as well as a number of gene therapy approaches have been developed in this frame.

  15. Medicinal chemistry of small molecule CCR5 antagonists for blocking HIV-1 entry: a review of structural evolution.

    PubMed

    Tian, Ye; Zhang, Dujuan; Zhan, Peng; Liu, Xinyong

    2014-01-01

    CCR5, a member of G protein-coupled receptors superfamily, plays an important role in the HIV-1 entry process. Antagonism of this receptor finally leads to the inhibition of R5 strains of HIV entry into the human cells. The identification of CCR5 antagonists as antiviral agents will provide more option for HAART. Now, more than a decade after the first small molecule CCR5 inhibitor was discovered, great achievements have been made. In this article, we will give a brief introduction of several series of small molecule CCR5 antagonists, focused on their appealing structure evolution, essential SAR information and thereof the enlightenment of strategies on CCR5 inhibitors design.

  16. T-Cell surface CCR5 density is not correlated with hepatitis severity in hepatitis C virus/HIV-coinfected individuals: implications for the therapeutic use of CCR5 antagonists.

    PubMed

    Vincent, Thierry; Portales, Pierre; Baillat, Vincent; de Boever, Corinne Merle; Le Moing, Vincent; Vidal, Michèle; Ducos, Jacques; Clot, Jacques; Reynes, Jacques; Corbeau, Pierre

    2005-03-01

    CCR5 antagonists represent promising anti-HIV agents. Yet, if the CCR5 chemokine receptor plays a positive role in hepatitis C virus (HCV) infection, CCR5 antagonists might be contraindicated in HCV/HIV-coinfected subjects. Therefore, we tested the hypothesis that the level of T-cell surface CCR5 expression, which might determine the intensity of HCV-specific T-cell recruitment into the liver, and thereby the efficiency of the anti-HCV response, could determine HCV disease evolution. For this purpose, we compared CCR5 density, measured by quantitative flow cytometry at the surface of nonactivated (human leukocyte antigen-D-related [HLA-DR]-) T cells of 51 HCV/HIV patients, with HCV load, serum aminotransferase levels, and liver histology (inflammatory activity, fibrosis, and rate of fibrosis progression). DR-CD4+ T-cell surface CCR5 density, which correlated with DR-CD8+ T-cell surface CCR5 density and was stable over time in HCV/HIV-coinfected individuals, did not correlate with any of the biologic parameters of HCV infection analyzed and was not linked to the capacity to clear the virus. In conclusion, we failed to demonstrate any impact of interindividual variability in T-cell surface CCR5 density on HCV infection, which would have argued against the use of CCR5 antagonists in HIV/HCV-coinfected subjects.

  17. Protection against human immunodeficiency virus type 1 infection in persons with repeated exposure: evidence for T cell immunity in the absence of inherited CCR5 coreceptor defects.

    PubMed

    Goh, W C; Markee, J; Akridge, R E; Meldorf, M; Musey, L; Karchmer, T; Krone, M; Collier, A; Corey, L; Emerman, M; McElrath, M J

    1999-03-01

    It has been hypothesized that protection against human immunodeficiency virus (HIV)-1 infection may result from either acquired host immunity, inheritance of a dysfunctional CCR5 HIV-1 coreceptor, or a low or attenuated virus inoculum. Thirty-seven HIV-1-uninfected persons engaging in repeated high-risk sexual activity with an HIV-1-infected partner were prospectively studied to determine the contribution of these factors in protecting against HIV-1 transmission. More than one-third (13/36) demonstrated HIV-1-specific cytotoxicity, and this activity significantly correlated with the wild type CCR5 genotype (P=.03). Only 1 subject (3%) demonstrated the homozygous CCR5 32-bp deletion (Delta32/Delta32). Median plasma HIV-1 RNA levels from 18 HIV-1-infected sex partners were not statistically different from those of matched infected control patients. These results indicate that inheritance of the Delta32 CCR5 mutation does not account for the majority of persistently HIV-1-resistant cases, and the presence of cellular immunity in these persons suggests either undetected infection or protective immunity.

  18. CCR5 receptor antagonists block metastasis to bone of v-Src-oncogene-transformed metastatic prostate cancer cell lines

    PubMed Central

    Sicoli, Daniela; Jiao, Xuanmao; Ju, Xiaoming; Velasco-Velazquez, Marco; Ertel, Adam; Addya, Sankar; Li, Zhiping; Ando, Sebastiano; Fatatis, Alessandro; Paudyal, Bishnuhari; Cristofanilli, Massimo; Thakur, Mathew L.; Lisanti, Michael P; Pestell, Richard G.

    2014-01-01

    Src family kinases (SFKs) integrate signal transduction for multiple receptors, regulating cellular proliferation invasion and metastasis in human cancer. Although Src is rarely mutated in human prostate cancer, SFK activity is increased in the majority of human prostate cancers. In order to determine the molecular mechanisms governing prostate cancer bone metastasis, FVB murine prostate epithelium was transduced with oncogenic v-Src. The prostate cancer cell lines metastasized in FVB mice to brain and bone. Gene expression profiling of the tumors identified activation of a CCR5 signaling module when the prostate epithelial cells (PEC) lines were grown in vivo vs. tissue cultures. The whole body, bone and brain metastatic prostate cancer burden was reduced by oral CCR5 antagonist. Clinical trials of CCR5 inhibitors may warrant consideration in patients with CCR5 activation in their tumors. PMID:25452256

  19. Combined 3D-QSAR modeling and molecular docking study on azacycles CCR5 antagonists

    NASA Astrophysics Data System (ADS)

    Ji, Yongjun; Shu, Mao; Lin, Yong; Wang, Yuanqiang; Wang, Rui; Hu, Yong; Lin, Zhihua

    2013-08-01

    The beta chemokine receptor 5 (CCR5) is an attractive target for pharmaceutical industry in the HIV-1, inflammation and cancer therapeutic areas. In this study, we have developed quantitative structure activity relationship (QSAR) models for a series of 41 azacycles CCR5 antagonists using comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), and Topomer CoMFA methods. The cross-validated coefficient q2 values of 3D-QASR (CoMFA, CoMSIA, and Topomer CoMFA) methods were 0.630, 0.758, and 0.852, respectively, the non-cross-validated R2 values were 0.979, 0.978, and 0.990, respectively. Docking studies were also employed to determine the most probable binding mode. 3D contour maps and docking results suggested that bulky groups and electron-withdrawing groups on the core part would decrease antiviral activity. Furthermore, docking results indicated that H-bonds and π bonds were favorable for antiviral activities. Finally, a set of novel derivatives with predicted activities were designed.

  20. Complexity in human immunodeficiency virus type 1 (HIV-1) co-receptor usage: roles of CCR3 and CCR5 in HIV-1 infection of monocyte-derived macrophages and brain microglia.

    PubMed

    Agrawal, Lokesh; Maxwell, Christina R; Peters, Paul J; Clapham, Paul R; Liu, Sue M; Mackay, Charles R; Strayer, David S

    2009-03-01

    CCR3 has been implicated as a co-receptor for human immunodeficiency virus type 1 (HIV-1), particularly in brain microglia cells. We sought to clarify the comparative roles of CCR3 and CCR5 in the central nervous system (CNS) HIV-1 infection and the potential utility of CCR3 as a target for manipulation via gene transfer. To target CCR3, we developed a single-chain antibody (SFv) and an interfering RNA (RNAi), R3-526. Coding sequences for both were cloned into Tag-deleted SV40-dervied vectors, as these vectors transduce brain microglia and monocyte-derived macrophages (MDM) highly efficiently. These anti-CCR3 transgenes were compared to SFv-CCR5, an SFv against CCR5, and RNAi-R5, an RNAi that targets CCR5, for the ability to protect primary human brain microglia and MDM from infection with peripheral and neurotropic strains of HIV-1. Downregulation of CCR3 and CCR5 by these transgenes was independent from one another. Confocal microscopy showed that CCR3 and CCR5 co-localized at the plasma membrane with each other and with CD4. Targeting either CCR5 or CCR3 largely protected both microglia and MDM from infection by many strains of HIV-1. That is, some HIV-1 strains, isolated from either the CNS or periphery, required both CCR3 and CCR5 for optimal productive infection of microglia and MDM. Some HIV-1 strains were relatively purely CCR5-tropic. None was purely CCR3-tropic. Thus, some CNS-tropic strains of HIV-1 utilize CCR5 as a co-receptor but do not need CCR3, while for other isolates both CCR3 and CCR5 may be required.

  1. Resistance to human immunodeficiency virus type 1 (HIV-1) generated by lentivirus vector-mediated delivery of the CCR5{Delta}32 gene despite detectable expression of the HIV-1 co-receptors.

    PubMed

    Jin, Qingwen; Marsh, Jon; Cornetta, Kenneth; Alkhatib, Ghalib

    2008-10-01

    It has previously been demonstrated that there are two distinct mechanisms for genetic resistance to human immunodeficiency virus type 1 (HIV-1) conferred by the CCR5Delta32 gene: the loss of wild-type CCR5 surface expression and the generation of CCR5Delta32 protein, which interacts with CXCR4. To analyse the protective effects of long-term expression of the CCR5Delta32 protein, recombinant lentiviral vectors were used to deliver the CCR5Delta32 gene into human cell lines and primary peripheral blood mononuclear cells that had been immortalized by human T-cell leukemia virus type 1. Blasticidin S-resistant cell lines expressing the lentivirus-encoded CCR5Delta32 showed a significant reduction in HIV-1 Env-mediated fusion assays. It was shown that CD4(+) T lymphocytes expressing the lentivirus-encoded CCR5Delta32 gene were highly resistant to infection by a primary but not by a laboratory-adapted X4 strain, suggesting different infectivity requirements. In contrast to previous studies that analysed the CCR5Delta32 protective effects in a transient expression system, this study showed that long-term expression of CCR5Delta32 conferred resistance to HIV-1 despite cell-surface expression of the HIV co-receptors. The results suggest an additional unknown mechanism for generating the CCR5Delta32 resistance phenotype and support the hypothesis that the CCR5Delta32 protein acts as an HIV-suppressive factor by altering the stoichiometry of the molecules involved in HIV-1 entry. The lentiviral-CCR5Delta32 vectors offer a method of generating HIV-resistant cells by delivery of the CCR5Delta32 gene that may be useful for stem cell- or T-cell-based gene therapy for HIV-1 infection.

  2. Characterizing the Diverse Mutational Pathways Associated with R5-Tropic Maraviroc Resistance: HIV-1 That Uses the Drug-Bound CCR5 Coreceptor

    PubMed Central

    Jiang, Xiaowei; Feyertag, Felix; Meehan, Conor J.; McCormack, Grace P.; Travers, Simon A.; Craig, Charles; Westby, Mike; Lewis, Marilyn

    2015-01-01

    ABSTRACT Entry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant's phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable. IMPORTANCE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5

  3. Mutations in the V3 stem versus the V3 crown and C4 region have different effects on the binding and fusion steps of human immunodeficiency virus type 1 gp120 interaction with the CCR5 coreceptor

    SciTech Connect

    Suphaphiphat, Pirada; Essex, Max; Lee, Tun-Hou . E-mail: tunhoule@hsph.harvard.edu

    2007-03-30

    The current model for HIV-1 envelope-coreceptor interaction depicts the V3 stem and bridging sheet binding to the CCR5 N-terminus while the V3 crown interacts with the second extracellular loop, which is the coreceptor domain that appears to be relatively more important for fusion and infection. Our prediction based on this model is that mutations in the V3 crown might consequently have more effects on cell-cell fusion and virus entry than mutations introduced in the V3 stem and C4 region. We performed alanine-scanning of the V3 loop and selected C4 residues in the JRFL envelope and tested the capacity of the resulting mutants for CCR5 binding, cell-cell fusion, and virus infection. Our cross comparison analysis revealed that residues in C4 and in both the V3 stem and crown were important for CCR5 binding of gp120 subunits. Contrary to our prediction, mutations in the V3 crown had less effect on membrane fusion than mutations in the V3 stem. The V3 stem thus appears to be the most important region for CCR5 utilization since it affected both coreceptor binding and subsequent fusion and viral entry. Our data raises the possibility that some residues in the V3 crown and in C4 may play distinct roles in the binding and fusion steps of envelope-coreceptor interaction.

  4. Maraviroc, a CCR5 Antagonist, Prevents Development of Hepatocellular Carcinoma in a Mouse Model

    PubMed Central

    Ochoa-Callejero, Laura; Pérez-Martínez, Laura; Rubio-Mediavilla, Susana; Oteo, José A.; Martínez, Alfredo; Blanco, José R.

    2013-01-01

    Chronic liver disease may result in a sequential progression through fibrosis, cirrhosis and lead, eventually, to hepatocellular carcinoma (HCC). Hepatic stellate cells (HSC) seem to be responsible for the fibrogenic response through the activation of an autocrine loop involving the chemokine receptor, CCR5. However, the role of CCR5 in HCC remains poorly understood. Since this receptor is also one of the main ports of entry for the human immunodeficiency virus (HIV), several CCR5 inhibitors are being used in the clinic to reduce viral load. We used one of these inhibitors, maraviroc (MVC), in a mouse model of diet-induced HCC to investigate whether this intervention would reduce disease progression. Animals treated with MVC on top of a normal control diet did not present any evidence of toxicity or any morphological change when compared with non-treated mice. Animals treated with MVC presented higher survival, less liver fibrosis, lower levels of liver injury markers and chemokines, less apoptosis, lower proliferation index, and lower tumor burden than their counterparts receiving only the hepatotoxic diet. In addition, MVC inhibits HSC activation markers such as phosphorylation of p38 and ERK, and increases hepatocyte survival. This study suggests that MVC, a well tolerated and clinically characterized drug, may be used as a preventative treatment for HCC. Clinical studies are needed to demonstrate the efficacy of this drug, or other CCR5 inhibitors, in patients with high risk of developing HCC. PMID:23326556

  5. The strength of the chemotactic response to a CCR5 binding chemokine is determined by the level of cell surface CCR5 density.

    PubMed

    Desmetz, Caroline; Lin, Yea-Lih; Mettling, Clément; Portalès, Pierre; Rabesandratana, Herisoa; Clot, Jacques; Corbeau, Pierre

    2006-12-01

    We have shown that the intensity of expression of the C-C chemokine receptor CCR5 at the single CD4(+) cell level strongly determines the efficiency of its function as a coreceptor for human immunodeficiency virus type 1. By analogy, we examined if the number of CCR5 molecules at the cell surface might determine its chemotactic response to CCR5 ligands. To test this hypothesis, we measured by flow cytometry the migration of primary human T cells towards the CCR5-binding chemokine CCL5 in vitro. First, we observed a dose-dependent blockage of this migration exerted by an anti-CCR5 monoclonal antibody. Second, we sorted peripheral blood mononuclear cells into five subpopulations expressing various cell surface CCR5 densities, and observed a correlation between the intensity of migration towards CCL5 and the level of CCR5 expression on these subpopulations. Third, we transduced CCR5(+) peripheral blood mononuclear cells with the CCR5 gene, and observed that the CCR5 over-expression induced an over-migration towards CCL5. Finally, we observed in healthy donors a correlation between the chemotactic response of peripheral blood CD8(+) T cell to CCL5 and their level of surface CCR5 expression. T-cell surface CCR5 density, which is constant over time for a given individual, but varies drastically among individuals, might therefore be an important personal determinant of T-cell migration in many biological situations where CCR5-binding chemokines play a role, such as graft rejection, T helper 1-mediated auto-immune diseases, and infectious diseases involving CCR5. Moreover, our data highlight the therapeutic potential of CCR5 antagonists in these situations.

  6. Frequencies of 32 base pair deletion of the (Delta 32) allele of the CCR5 HIV-1 co-receptor gene in Caucasians: a comparative analysis.

    PubMed

    Lucotte, Gérard

    2002-05-01

    The CCR5 gene encodes for the co-receptor for the major macrophage-tropics strains of human immunodeficiency virus (HIV-1), and a mutant allele of this gene (Delta 32) provide to homozygotes a strong resistance against infection by HIV. The frequency of the Delta 32 allele was investigated in 40 populations of 8842 non-infected subjects coming from Europe, the Middle-East and North Africa. A clear north-south decreasing gradient was evident for Delta 32 frequencies, with a significant correlation coefficient (r=0.83). The main frequency value of Delta 32 for Sweden, Norway, Denmark, Finland and Iceland (0.134) is significantly (chi(2)=63.818, P<0.001) highest than the Delta 32 mean value, indicating that probably the Vikings might have been instrumental in disseminating the Delta 32 allele during the eighth to the tenth centuries during historical times. Possibly variola virus has discriminated the Delta 32 carriers in Europe since the eighth century AD, explaining the high frequency of the Delta 32 allele in Europe today.

  7. Specificity for a CCR5 Inhibitor Is Conferred by a Single Amino Acid Residue: ROLE OF ILE198.

    PubMed

    Lau, Gloria; Labrecque, Jean; Metz, Markus; Vaz, Roy; Fricker, Simon P

    2015-04-24

    The chemokine receptors CCR5 and CCR2b share 89% amino acid homology. CCR5 is a co-receptor for HIV and CCR5 antagonists have been investigated as inhibitors of HIV infection. We describe the use of two CCR5 antagonists, Schering-C (SCH-C), which is specific for CCR5, and TAK-779, a dual inhibitor of CCR5 and CCR2b, to probe the CCR5 inhibitor binding site using CCR5/CCR2b chimeric receptors. Compound inhibition in the different chimeras was assessed by inhibition of chemokine-induced calcium flux. SCH-C inhibited RANTES (regulated on activation, normal T cell expressed and secreted) (CCL5)-mediated calcium flux on CCR5 with an IC50 of 22.8 nM but was inactive against monocyte chemoattractant protein-1 (CCL2)-mediated calcium flux on CCR2b. However, SCH-C inhibited CCL2-induced calcium flux against a CCR5/CCR2b chimera consisting of transmembrane domains IV-VI of CCR5 with an IC50 of 55 nM. A sequence comparison of CCR5 and CCR2b identified a divergent amino acid sequence located at the junction of transmembrane domain V and second extracellular loop. Transfer of the CCR5 sequence KNFQTLKIV into CCR2b conferred SCH-C inhibition (IC50 of 122 nM) into the predominantly CCR2b chimera. Furthermore, a single substitution, R206I, conferred partial but significant inhibition (IC50 of 1023 nM) by SCH-C. These results show that a limited amino acid sequence is responsible for SCH-C specificity to CCR5, and we propose a model showing the interaction with CCR5 Ile(198).

  8. Incompatible Natures of the HIV-1 Envelope in Resistance to the CCR5 Antagonist Cenicriviroc and to Neutralizing Antibodies

    PubMed Central

    Enomoto, Ikumi; Baba, Masanori

    2015-01-01

    Cenicriviroc is a CCR5 antagonist which prevents human immunodeficiency virus type 1 (HIV-1) from cellular entry. The CCR5-binding regions of the HIV-1 envelope glycoprotein are important targets for neutralizing antibodies (NAbs), and mutations conferring cenicriviroc resistance may therefore affect sensitivity to NAbs. Here, we used the in vitro induction of HIV-1 variants resistant to cenicriviroc or NAbs to examine the relationship between resistance to cenicriviroc and resistance to NAbs. The cenicriviroc-resistant variant KK652-67 (strain KK passaged 67 times in the presence of increasing concentrations of cenicriviroc) was sensitive to neutralization by NAbs against the V3 loop, the CD4-induced (CD4i) region, and the CD4-binding site (CD4bs), whereas the wild-type (WT) parental HIV-1 strain KKWT from which cenicriviroc-resistant strain KK652-67 was obtained was resistant to these NAbs. The V3 region of KK652-67 was important for cenicriviroc resistance and critical to the high sensitivity of the V3, CD4i, and CD4bs epitopes to NAbs. Moreover, induction of variants resistant to anti-V3 NAb 0.5γ and anti-CD4i NAb 4E9C from cenicriviroc-resistant strain KK652-67 resulted in reversion to the cenicriviroc-sensitive phenotype comparable to that of the parental strain, KKWT. Resistance to 0.5γ and 4E9C was caused by the novel substitutions R315K, G324R, and E381K in the V3 and C3 regions near the substitutions conferring cenicriviroc resistance. Importantly, these amino acid changes in the CCR5-binding region were also responsible for reversion to the cenicriviroc-sensitive phenotype. These results suggest the presence of key amino acid residues where resistance to cenicriviroc is incompatible with resistance to NAbs. This implies that cenicriviroc and neutralizing antibodies may restrict the emergence of variants resistant to each other. PMID:26525792

  9. Incompatible Natures of the HIV-1 Envelope in Resistance to the CCR5 Antagonist Cenicriviroc and to Neutralizing Antibodies.

    PubMed

    Kuwata, Takeo; Enomoto, Ikumi; Baba, Masanori; Matsushita, Shuzo

    2015-11-02

    Cenicriviroc is a CCR5 antagonist which prevents human immunodeficiency virus type 1 (HIV-1) from cellular entry. The CCR5-binding regions of the HIV-1 envelope glycoprotein are important targets for neutralizing antibodies (NAbs), and mutations conferring cenicriviroc resistance may therefore affect sensitivity to NAbs. Here, we used the in vitro induction of HIV-1 variants resistant to cenicriviroc or NAbs to examine the relationship between resistance to cenicriviroc and resistance to NAbs. The cenicriviroc-resistant variant KK652-67 (strain KK passaged 67 times in the presence of increasing concentrations of cenicriviroc) was sensitive to neutralization by NAbs against the V3 loop, the CD4-induced (CD4i) region, and the CD4-binding site (CD4bs), whereas the wild-type (WT) parental HIV-1 strain KKWT from which cenicriviroc-resistant strain KK652-67 was obtained was resistant to these NAbs. The V3 region of KK652-67 was important for cenicriviroc resistance and critical to the high sensitivity of the V3, CD4i, and CD4bs epitopes to NAbs. Moreover, induction of variants resistant to anti-V3 NAb 0.5γ and anti-CD4i NAb 4E9C from cenicriviroc-resistant strain KK652-67 resulted in reversion to the cenicriviroc-sensitive phenotype comparable to that of the parental strain, KKWT. Resistance to 0.5γ and 4E9C was caused by the novel substitutions R315K, G324R, and E381K in the V3 and C3 regions near the substitutions conferring cenicriviroc resistance. Importantly, these amino acid changes in the CCR5-binding region were also responsible for reversion to the cenicriviroc-sensitive phenotype. These results suggest the presence of key amino acid residues where resistance to cenicriviroc is incompatible with resistance to NAbs. This implies that cenicriviroc and neutralizing antibodies may restrict the emergence of variants resistant to each other.

  10. Antifibrotic Effects of the Dual CCR2/CCR5 Antagonist Cenicriviroc in Animal Models of Liver and Kidney Fibrosis

    PubMed Central

    Lefebvre, Eric; Moyle, Graeme; Reshef, Ran; Richman, Lee P.; Thompson, Melanie; Hong, Feng; Chou, Hsin-l; Hashiguchi, Taishi; Plato, Craig; Poulin, Dominic; Richards, Toni; Yoneyama, Hiroyuki; Jenkins, Helen; Wolfgang, Grushenka; Friedman, Scott L.

    2016-01-01

    Background & Aims Interactions between C-C chemokine receptor types 2 (CCR2) and 5 (CCR5) and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC) is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC’s anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis. Methods Monocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC’s antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH) and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses. Results CVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05). At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05), and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls). CVC treatment had no notable effect on body or liver/kidney weight. Conclusions CVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC’s antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475). PMID:27347680

  11. CCR5 inhibitors: Emerging promising HIV therapeutic strategy.

    PubMed

    Rao, Padmasri Kutikuppala Surya

    2009-01-01

    Though potent anti-HIV therapy has spectacularly reduced the morbidity and mortality of human immunodeficiency virus (HIV)-1 infection in the advanced countries, it continues to be associated with substantial toxicity, drug-drug interactions, difficulties in adherence, and abnormal cost. As a result, better effective, safe antiretroviral drugs and treatment strategies keep on to be pursued. In this process, CCR5 (chemokine receptor 5) inhibitors are a new class of antiretroviral drug used in the treatment of HIV. They are designed to prevent HIV infection of CD4 T-cells by blocking the CCR5. When the CCR5 receptor is unavailable, 'R5-tropic' HIV (the variant of the virus that is common in earlier HIV infection) cannot engage with a CD4 T-cell to infect the cell. In August 2007, the FDA approved the first chemokine (C-C motif) CCR5 inhibitor, maraviroc, for treatment-experienced patients infected with R5-using virus. Studies from different cohort in regions, affected by clad B HIV-1, demonstrate that 81-88% of HIV-1 variants in treatment naïve patients are CCR5 tropic and that virtually all the remaining variants are dual/mixed tropic i.e., are able to utilize both CCR5 and CXCR4 coreceptors. In treatment experienced patients, 49-78% of the variants are purely CCR5 tropic, 22-48% are dual/mixed tropic, and 2-5% exclusively utilize CXCR4. A 32 bp deletion in the CCR5 gene, which results in a frame shift and truncation of the normal CCR5 protein, was identified in a few persons who had remained uninfected after exposure to CCR5 tropic HIV-1 virus. This allele is common in white of European origin, with prevalence near to 10%, but is absent among East Asian, American Indian, Tamil Indian, and African ethnic groups. HIV-infected individuals, who are heterozygous for CCR5 delta 32, have slower rates of disease progression. The currently available data supports the continuation of the development of CCR5 antagonists in different settings related to HIV-1 infection. If

  12. Interaction of small molecule inhibitors of HIV-1 entry with CCR5

    SciTech Connect

    Seibert, Christoph . E-mail: seiberc@mail.rockefeller.edu; Ying Weiwen; Gavrilov, Svetlana; Tsamis, Fotini; Kuhmann, Shawn E.; Palani, Anandan; Tagat, Jayaram R.; Clader, John W.; McCombie, Stuart W.; Baroudy, Bahige M.; Smith, Steven O.; Dragic, Tatjana; Moore, John P.; Sakmar, Thomas P.

    2006-05-25

    The CC-chemokine receptor 5 (CCR5) is the major coreceptor for macrophage-tropic (R5) HIV-1 strains. Several small molecule inhibitors of CCR5 that block chemokine binding and HIV-1 entry are being evaluated as drug candidates. Here we define how CCR5 antagonists TAK-779, AD101 (SCH-350581) and SCH-C (SCH-351125), which inhibit HIV-1 entry, interact with CCR5. Using a mutagenesis approach in combination with a viral entry assay to provide a direct functional read out, we tested predictions based on a homology model of CCR5 and analyzed the functions of more than 30 amino acid residues. We find that a key set of aromatic and aliphatic residues serves as a hydrophobic core for the ligand binding pocket, while E283 is critical for high affinity interaction, most likely by acting as the counterion for a positively charged nitrogen atom common to all three inhibitors. These results provide a structural basis for understanding how specific antagonists interact with CCR5, and may be useful for the rational design of new, improved CCR5 ligands.

  13. Exploration on natural product anibamine side chain modification toward development of novel CCR5 antagonists and potential anti-prostate cancer agents.

    PubMed

    Xu, Guoyan G; Zaidi, Saheem A; Zhang, Feng; Singh, Shilpa; Raborg, Thomas J; Yuan, Yunyun; Zhang, Yan

    2015-09-01

    Prostate cancer is one of the leading causes of death among males in the world. Prostate cancer cells have been shown to express upregulated chemokine receptor CCR5, a G protein-coupled receptor (GPCR) that relates to the inflammation process. Anibamine, a natural product containing a pyridine ring and two aliphatic side chains, was shown to carry a binding affinity of 1 μM at CCR5 as an antagonist with potential anti-cancer activity. However, it is not drug-like according to the Lipinski's rule of five mainly due to its two long aliphatic side chains. In our effort to improve its drug-like property, a series of anibamine derivatives were designed and synthesized by placement of aromatic side chains through an amide linkage to the pyridine ring. The newly synthesized compounds were tested for their CCR5 affinity and antagonism, and potential anti-proliferation activity against prostate cancer cell lines. Basal cytotoxicity was finally studied for compounds showing potent anti-proliferation activity. It was found that compounds with hydrophobic substitutions on the aromatic systems seemed to carry more promising CCR5 binding and prostate cancer cell proliferation inhibition activities.

  14. Virological Response after Short-Term CCR5 Antagonist Exposure in HIV-Infected Patients: Frequency of Subjects with Virological Response and Associated Factors▿

    PubMed Central

    Ruiz-Mateos, Ezequiel; González-Serna, Alejandro; Genebat, Miguel; Machmach, Kawthar; Vidal, Francesc; Muñoz-Fernández, Ángeles; Ferrando-Martinez, Sara; Leal, Manuel

    2011-01-01

    The virological response after an 8-day maraviroc monotherapy has been proposed to be an alternative method to determine whether an CCR5 antagonist should be prescribed to HIV-infected patients. The frequency of patients eligible for a combined antiretroviral therapy which includes maraviroc on the basis of the result of this clinical test is not well-known at the moment. In the same way, clinical and immunovirological factors associated with the virological response after antagonist exposure need to be determined. Ninety consecutive HIV-infected patients were exposed to an 8-day maraviroc monotherapy. The virological response was considered positive if either a reduction of ≥1-log10 HIV RNA copies/ml or an undetectable viral load (<40 HIV RNA copies/ml) was achieved. CXCR4- and CCR5-tropic virus levels were determined by using patients' viral isolates and multiple rounds of infection of indicator cell lines (U87-CXCR4 and U87-CCR5). The frequency of patients with a positive virological response was 72.2% (94.7% and 66.2% for treatment-naïve and pretreated patients, respectively). The positive response rates dramatically decreased in patients with lower CD4+ T-cell counts. The CXCR4-tropic virus level was the only variable independently associated with the virological response after short-term maraviroc exposure. Lower CD4+ T-cell strata were associated with higher CXCR4-tropic virus levels. These results support the suggestion that CCR5 antagonists should be an early treatment option before the expansion of CXCR4-tropic strains. PMID:21807977

  15. Biased small-molecule ligands for selective inhibition of HIV-1 cell entry via CCR5.

    PubMed

    Berg, Christian; Spiess, Katja; Lüttichau, Hans R; Rosenkilde, Mette M

    2016-12-01

    Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small-molecule receptor antagonists for inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small-molecule CCR5 agonists as HIV-1 fusion inhibitors. A virus-free cell-based fusion reporter assay, based on mixing "effector cells" (expressing HIV Env and luciferase activator) with "target cells" (expressing CD4, CCR5 wild type or a selection of well-described mutations, and luciferase reporter), was used as fusion readout. Receptor expression was evaluated by ELISA and fluorescence microscopy. On CCR5 WT, Maraviroc and Aplaviroc inhibited fusion with high potencies (EC 50 values of 91 and 501 nM, respectively), whereas removal of key residues for both antagonists (Glu283Ala) or Maraviroc alone (Tyr251Ala) prevented fusion inhibition, establishing this assay as suitable for screening of HIV entry inhibitors. Both ligands inhibited HIV fusion on signaling-deficient CCR5 mutations (Tyr244Ala and Trp248Ala). Moreover, the steric hindrance CCR5 mutation (Gly286Phe) impaired fusion, presumably by a direct hindrance of gp120 interaction. Finally, the efficacy switch mutation (Leu203Phe) - converting small-molecule antagonists/inverse agonists to full agonists biased toward G-protein activation - uncovered that also small-molecule agonists can function as direct HIV-1 cell entry inhibitors. Importantly, no agonist-induced receptor internalization was observed for this mutation. Our studies of the pharmacodynamic requirements for HIV-1 fusion inhibitors highlight the possibility of future development of biased ligands with selective targeting of the HIV-CCR5 interaction without interfering with the normal functionality of CCR5.

  16. CCR5 antibodies HGS004 and HGS101 preferentially inhibit drug-bound CCR5 infection and restore drug sensitivity of Maraviroc-resistant HIV-1 in primary cells

    SciTech Connect

    Latinovic, Olga; Reitz, Marvin; Le, Nhut M.; Foulke, James S.; Faetkenheuer, Gerd; Lehmann, Clara; Redfield, Robert R.; Heredia, Alonso

    2011-03-01

    R5 HIV-1 strains resistant to the CCR5 antagonist Maraviroc (MVC) can use drug-bound CCR5. We demonstrate that MVC-resistant HIV-1 exhibits delayed kinetics of coreceptor engagement and fusion during drug-bound versus free CCR5 infection of cell lines. Antibodies directed against the second extracellular loop (ECL2) of CCR5 had greater antiviral activity against MVC-bound compared to MVC-free CCR5 infection. However, in PBMCs, only ECL2 CCR5 antibodies HGS004 and HGS101, but not 2D7, inhibited infection by MVC resistant HIV-1 more potently with MVC-bound than with free CCR5. In addition, HGS004 and HGS101, but not 2D7, restored the antiviral activity of MVC against resistant virus in PBMCs. In flow cytometric studies, CCR5 binding by the HGS mAbs, but not by 2D7, was increased when PBMCs were treated with MVC, suggesting MVC increases exposure of the relevant epitope. Thus, HGS004 and HGS101 have antiviral mechanisms distinct from 2D7 and could help overcome MVC resistance.

  17. A Simplified Technique for Evaluating Human "CCR5" Genetic Polymorphism

    ERIC Educational Resources Information Center

    Falteisek, Lukáš; Cerný, Jan; Janštová, Vanda

    2013-01-01

    To involve students in thinking about the problem of AIDS (which is important in the view of nondecreasing infection rates), we established a practical lab using a simplified adaptation of Thomas's (2004) method to determine the polymorphism of HIV co-receptor CCR5 from students' own epithelial cells. CCR5 is a receptor involved in inflammatory…

  18. Maraviroc Clinical Test (MCT) as an alternative tool to decide CCR5-antagonists prescription in naïve HIV-infected patients.

    PubMed

    Genebat, Miguel; de Pablo-Bernal, Rebeca S; Pulido, Ildefonso; Jiménez-Mejías, Manuel E; Martínez, Onofre; Pacheco, Yolanda M; Raffi-El-Idrissi Benhia, Mohammed; Abad, María Antonia; Ruiz-Mateos, Ezequiel; Leal, Manuel

    2015-09-01

    Our aim was to analyze the virological response to a combined antiretroviral therapy started after Maraviroc Clinical Test (MCT) in naïve HIV-infected patients. Forty-one patients were exposed to MCT, based on an 8-day MVC monotherapy. If undetectability or a viral load reduction >1 log10 HIV-RNA copies/ml was achieved, a MVC-containing cART was prescribed. Forty patients showed a positive MCT; undetectability after 48weeks on cART was achieved in 34/41 (82.9%) patients. The result of MCT was compared with a genotypic tropism method and with Trofile®, showing 10.7% and 18.75% discordance rates, respectively. MCT is a reliable tool to decide CCR5-antagonists prescription, also in the naïve scenario where most patients show a virological response to MVC independently the tropism result reported by genotypic or phenotypic methods.

  19. Cenicriviroc, a Novel CCR5 (R5) and CCR2 Antagonist, Shows In Vitro Activity against R5 Tropic HIV-2 Clinical Isolates

    PubMed Central

    Visseaux, Benoit; Charpentier, Charlotte; Collin, Gilles; Bertine, Mélanie; Peytavin, Gilles; Damond, Florence; Matheron, Sophie; Lefebvre, Eric; Brun-Vézinet, Françoise; Descamps, Diane

    2015-01-01

    Background Maraviroc activity against HIV-2, a virus naturally resistant to different HIV-1 antiretroviral drugs, has been recently demonstrated. The aim of this study was to assess HIV-2 susceptibility to cenicriviroc, a novel, once-daily, dual CCR5 and CCR2 antagonist that has completed Phase 2b development in HIV-1 infection. Methods Cenicriviroc phenotypic activity has been tested using a PBMC phenotypic susceptibility assay against four R5-, one X4- and one dual-tropic HIV-2 clinical primary isolates. All isolates were obtained by co-cultivation of PHA-activated PBMC from distinct HIV-2-infected CCR5-antagonist-naïve patients included in the French HIV-2 cohort and were previously tested for maraviroc susceptibility using the same protocol. HIV-2 tropism was determined by phenotypic assay using Ghost(3) cell lines. Results Regarding the 4 R5 HIV-2 clinical isolates tested, effective concentration 50% EC50 for cenicriviroc were 0.03, 0.33, 0.45 and 0.98 nM, similar to those observed with maraviroc: 1.13, 0.58, 0.48 and 0.68 nM, respectively. Maximum percentages of inhibition (MPI) of cenicriviroc were 94, 94, 93 and 98%, similar to those observed with maraviroc (93, 90, 82, 100%, respectively). The dual- and X4-tropic HIV-2 strains were resistant to cenicriviroc with EC50 >1000 nM and MPI at 33% and 4%, respectively. Conclusions In this first study assessing HIV-2 susceptibility to cenicriviroc, we observed an in vitro activity against HIV-2 R5-tropic strains similar to that observed with maraviroc. Thus, cenicriviroc may offer a once-daily treatment opportunity in the limited therapeutic arsenal for HIV-2. Clinical studies are warranted. PMID:26247470

  20. Development of Tetravalent, Bispecific CCR5 Antibodies with Antiviral Activity against CCR5 Monoclonal Antibody-Resistant HIV-1 Strains▿

    PubMed Central

    Schanzer, Jürgen; Jekle, Andreas; Nezu, Junichi; Lochner, Adriane; Croasdale, Rebecca; Dioszegi, Marianna; Zhang, Jun; Hoffmann, Eike; Dormeyer, Wilma; Stracke, Jan; Schäfer, Wolfgang; Ji, Changhua; Heilek, Gabrielle; Cammack, Nick; Brandt, Michael; Umana, Pablo; Brinkmann, Ulrich

    2011-01-01

    In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly4Ser)n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18- to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibody-resistant HIV-1 strains. PMID:21300827

  1. Study on CCR5 analogs and affinity peptides.

    PubMed

    Wu, Yingping; Deng, Riqiang; Wu, Wenyan

    2012-03-01

    The G protein-coupled receptor of human chemokine receptor 5 (CCR5) is a key target in the human immunodeficiency virus (HIV) infection process due to its major involvement in binding to the HIV type 1 (HIV-1) envelope glycoprotein gp120 and facilitating virus entry into the cells. The identification of naturally occurring CCR5 mutations (especially CCR5 delta-32) has allowed us to address the CCR5 molecule as a promising target to prevent or resist HIV infection in vivo. To obtain high-affinity peptides that can be used to block CCR5, CCR5 analogs with high conformational similarity are required. In this study, two recombinant proteins named CCR5 N-Linker-E2 and CCR5 mN-E1-E2 containing the fragments of the CCR5 N-terminal, the first extracellular loop or the second extracellular loop are cloned from a full-length human CCR5 cDNA. The recombinant human CCR5 analogs with self-cleavage activity of the intein Mxe or Ssp in the vector pTwinI were then produced with a high-yield expression and purification system in Escherichia coli. Experiments of extracellular epitope-activity identification (such as immunoprecipitation and indirective/competitive enzyme-linked immunosorbent assay) confirmed the close similarity between the epitope activity of the CCR5 analogs and that of the natural CCR5, suggesting the applicability of the recombinant CCR5 analogs as antagonists of the chemokine ligands. Subsequent screening of high-affinity peptides from the phage random-peptides library acquired nine polypeptides, which could be used as CCR5 peptide antagonists. The CCR5 analogs and affinity peptides elucidated in this paper provide us with a basis for further study of the mechanism of inhibition of HIV-1 infection.

  2. Rapamycin causes down-regulation of CCR5 and accumulation of anti-HIV beta-chemokines: an approach to suppress R5 strains of HIV-1.

    PubMed

    Heredia, A; Amoroso, A; Davis, C; Le, N; Reardon, E; Dominique, J K; Klingebiel, E; Gallo, R C; Redfield, R R

    2003-09-02

    Propagation of R5 strains of HIV-1 on CD4 lymphocytes and macrophages requires expression of the CCR5 coreceptor on the cell surface. Individuals lacking CCR5 (CCR5 Delta 32 homozygous genotype) are phenotypically normal and resistant to infection with HIV-1. CCR5 expression on lymphocytes depends on signaling through the IL-2 receptor. By FACS analysis we demonstrate that rapamycin (RAPA), a drug that disrupts IL-2 receptor signaling, reduces CCR5 surface expression on T cells at concentrations as low as 1 nM. In addition, lower concentrations of RAPA (0.01 nM) were sufficient to reduce CCR5 surface expression on maturing monocytes. PCR analysis on peripheral blood mononuclear cells (PBMCs) showed that RAPA interfered with CCR5 expression at the transcriptional level. Reduced expression of CCR5 on PBMCs cultured in the presence of RAPA was associated with increased extracellular levels of macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta. In infectivity assays, RAPA suppressed the replication of R5 strains of HIV-1 both in PBMC and macrophage cultures. In total PBMC cultures, RAPA-mediated inhibition of CCR5-using strains of HIV-1 occurred at 0.01 nM, a concentration of drug that is approximately 103 times lower than therapeutic through levels of drug in renal transplant recipients. In addition, RAPA enhanced the antiviral activity of the CCR5 antagonist TAK-779. These results suggest that low concentrations of RAPA may have a role in both the treatment and prevention of HIV-1 infection.

  3. Neurological Response to cART vs. cART plus Integrase Inhibitor and CCR5 Antagonist Initiated during Acute HIV

    PubMed Central

    Valcour, Victor G.; Spudich, Serena S.; Sailasuta, Napapon; Phanuphak, Nittaya; Lerdlum, Sukalaya; Fletcher, James L. K.; Kroon, Eugene D. M. B.; Jagodzinski, Linda L.; Allen, Isabel E.; Adams, Collin L.; Prueksakaew, Peeriya; Slike, Bonnie M.; Hellmuth, Joanna M.; Kim, Jerome H.; Ananworanich, Jintanat

    2015-01-01

    Objective To compare central nervous system (CNS) outcomes in participants treated during acute HIV infection with standard combination antiretroviral therapy (cART) vs. cART plus integrase inhibitor and CCR5 antagonist (cART+). Design 24-week randomized open-label prospective evaluation. Method Participants were evaluated then randomized to initiate cART (efavirenz, tenofovir, and either emtricitabine or lamivudine) vs. cART+ (cART plus raltegravir and maraviroc) during acute HIV and re-evaluated at 4, 12 and 24 weeks. We examined plasma and CSF cytokines, HIV RNA levels, neurological and neuropsychological findings, and brain MRS across groups and compared to healthy controls. Results At baseline, 62 participants were in Fiebig stages I-V. Randomized groups were similar for mean age (27 vs. 25, p = 0.137), gender (each 94% male), plasma log10 HIV RNA (5.4 vs. 5.6, p = 0.382), CSF log10 HIV RNA (2.35 vs. 3.31, p = 0.561), and estimated duration of HIV (18 vs. 17 days, p = 0.546). Randomized arms did not differ at 24 weeks by any CNS outcome. Combining arms, all measures concurrent with antiretroviral treatment improved, for example, neuropsychological testing (mean NPZ-4 of -0.408 vs. 0.245, p<0.001) and inflammatory markers by MRS (e.g. mean frontal white matter (FWM) choline of 2.92 vs. 2.84, p = 0.045) at baseline and week 24, respectively. Plasma neopterin (p<0.001) and interferon gamma-induced protein 10 (IP-10) (p = 0.007) remained elevated in participants compared to controls but no statistically significant differences were seen in CSF cytokines compared to controls, despite individual variability among the HIV-infected group. Conclusions A 24-week course of cART+ improved CNS related outcomes, but was not associated with measurable differences compared to standard cART. PMID:26555069

  4. Short Communication: HIV-1 Variants That Use Mouse CCR5 Reveal Critical Interactions of gp120's V3 Crown with CCR5 Extracellular Loop 1.

    PubMed

    Platt, Emily J; Durnin, James P; Kabat, David

    2015-10-01

    The CCR5 coreceptor amino terminus and extracellular (ECL) loops 1 and 2 have been implicated in HIV-1 infections, with species differences in these regions inhibiting zoonoses. Interactions of gp120 with CD4 and CCR5 reduce constraints on metastable envelope subunit gp41, enabling gp41 conformational changes needed for infection. We previously selected HIV-1JRCSF variants that efficiently use CCR5(Δ18) with a deleted amino terminus or CCR5(HHMH) with ECL2 from an NIH/Swiss mouse. Unexpectedly, the adaptive gp120 mutations were nearly identical, suggesting that they function by weakening gp120's grip on gp41 and/or by increasing interactions with ECL1. To analyze this and further wean HIV-1 from human CCR5, we selected variants using CCR5(HMMH) with murine ECL1 and 2 sequences. HIV-1JRCSF mutations adaptive for CCR5(Δ18) and CCR5(HHMH) were generally maladaptive for CCR5(HMMH), whereas the converse was true for CCR5(HMMH) adaptations. The HIV-1JRCSF variant adapted to CCR5(HMMH) also weakly used intact NIH/Swiss mouse CCR5. Our results strongly suggest that HIV-1JRCSF makes functionally critical contacts with human ECL1 and that adaptation to murine ECL1 requires multiple mutations in the crown of gp120's V3 loop.

  5. Editing CCR5: a novel approach to HIV gene therapy.

    PubMed

    Cornu, Tatjana I; Mussolino, Claudio; Bloom, Kristie; Cathomen, Toni

    2015-01-01

    Acquired immunodeficiency syndrome (AIDS) is a life-threatening disorder caused by infection of individuals with the human immunodeficiency virus (HIV). Entry of HIV-1 into target cells depends on the presence of two surface proteins on the cell membrane: CD4, which serves as the main receptor, and either CCR5 or CXCR4 as a co-receptor. A limited number of people harbor a genomic 32-bp deletion in the CCR5 gene (CCR5∆32), leading to expression of a truncated gene product that provides resistance to HIV-1 infection in individuals homozygous for this mutation. Moreover, allogeneic hematopoietic stem cell (HSC) transplantation with CCR5∆32 donor cells seems to confer HIV-1 resistance to the recipient as well. However, since Δ32 donors are scarce and allogeneic HSC transplantation is not exempt from risks, the development of gene editing tools to knockout CCR5 in the genome of autologous cells is highly warranted. Targeted gene editing can be accomplished with designer nucleases, which essentially are engineered restriction enzymes that can be designed to cleave DNA at specific sites. During repair of these breaks, the cellular repair pathway often introduces small mutations at the break site, which makes it possible to disrupt the ability of the targeted locus to express a functional protein, in this case CCR5. Here, we review the current promise and limitations of CCR5 gene editing with engineered nucleases, including factors affecting the efficiency of gene disruption and potential off-target effects.

  6. Inefficient entry of vicriviroc-resistant HIV-1 via the inhibitor-CCR5 complex at low cell surface CCR5 densities

    PubMed Central

    Pugach, Pavel; Ray, Neelanjana; Klasse, Per Johan; Ketas, Thomas J.; Michael, Elizabeth; Doms, Robert W.; Lee, Benhur; Moore, John P.

    2009-01-01

    HIV-1 variants resistant to small molecule CCR5 inhibitors such as vicriviroc (VVC) have modified Env complexes that can use both the inhibitor-bound and -free forms of the CCR5 co-receptor to enter target cells. However, entry via the inhibitor-CCR5 complex is inefficient in some, but not all, cell types, particularly cell lines engineered to express CCR5. We investigated the effect of increasing CCR5 expression, and hence the density of the inhibitor-CCR5 complex when a saturating inhibitor (VVC) concentration was present, by using 293-Affinofile cells, in which CCR5 expression is up-regulated by the transcriptional activator, ponasterone. When CCR5 expression was low, the resistant virus entered the target cells to a lesser extent when VVC was present than absent. However, at a higher CCR5 level, there was much less entry inhibition at a constant, saturating VVC concentration. We conclude that the relative decrease in entry of a VVC-resistant virus in some cell types results from its less efficient use of the VVC-CCR5 complex, and that increasing the CCR5 expression level can compensate for this inefficiency. PMID:19303620

  7. The CCL5/CCR5 axis promotes metastasis in basal breast cancer

    PubMed Central

    Velasco-Velázquez, Marco; Pestell, Richard G.

    2013-01-01

    Recently, we have shown that the CCL5/CCR5 axis is active in patients affected by an aggressive basal subtype of breast cancer. Using preclinical models, we have demonstrated that CCR5 promotes breast cancer invasiveness and metastatic potential, while CCR5 inhibition abrogates them. Thus, CCR5 antagonists may constitute an alternative therapeutic approach for patients affected by metastatic basal breast cancer. PMID:23734321

  8. The simultaneous blockade of chemokine receptors CCR2, CCR5 and CXCR3 by a non-peptide chemokine receptor antagonist protects mice from dextran sodium sulfate-mediated colitis.

    PubMed

    Tokuyama, Hirotake; Ueha, Satoshi; Kurachi, Makoto; Matsushima, Kouji; Moriyasu, Fuminori; Blumberg, Richard S; Kakimi, Kazuhiro

    2005-08-01

    Chemokine receptors CCR2, CCR5 and CXCR3 are involved in the regulation of macrophage- and T cell-mediated immune responses and in the migration and activation of these cells. In order to determine whether blockade of these chemokine receptors modulates intestinal inflammation, we investigated here the effect of a non-peptide chemokine receptor antagonist, TAK-779 (N,N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]carbonyl]amino]benzyl]-tetrahydro-2H-pyran-4-aminium chloride), in mice with dextran sodium sulfate (DSS)-induced experimental colitis. C57BL/6 mice were fed 5% DSS in their drinking water for up to 7 days with or without the administration of TAK-779. The severity of inflammation in the colon was assessed by clinical signs and histological examination. Infiltration of inflammatory cells into the mucosa was analyzed by immunohistochemistry, and the expression of cytokine and chemokine mRNAs in tissues was quantitated by reverse transcription-PCR. During DSS-induced colitis, the recruitment of monocytes/macrophages into the colonic mucosa and the induction of proinflammatory cytokines correlated with the severity of intestinal inflammation. The onset of clinical signs and histopathologic features were delayed in animals treated with TAK-779. The expression of CCR2, CCR5 and CXCR3 mRNAs was inhibited in the TAK-779-treated mice. Consistent with these results, infiltration of monocytes/macrophages into the lamina propria was almost completely inhibited and the expression of colonic IL-1beta and IL-6 was significantly decreased in the TAK-779-treated mice. The blockade of CCR2, CCR5 and CXCR3 prevents murine experimental colitis by inhibiting the recruitment of inflammatory cells into the mucosa. Therefore, chemokines and their receptors may be therapeutic targets for the treatment of inflammatory bowel disease.

  9. Common promoter deletion is associated with 3.9-fold differential transcription of ovine CCR5 and reduced proviral level of ovine progressive pneumonia virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CCR5 is a chemokine receptor that regulates immune cell recruitment in inflammation and serves as a coreceptor for human immunodeficiency virus (HIV). A human CCR5 coding deletion (termed delta-32) results in strong resistance to HIV infection, and polymorphisms in CCR5 regulatory regions have been ...

  10. CCR5 susceptibility to ligand-mediated down-modulation differs between human T lymphocytes and myeloid cells.

    PubMed

    Fox, James M; Kasprowicz, Richard; Hartley, Oliver; Signoret, Nathalie

    2015-07-01

    CCR5 is a chemokine receptor expressed on leukocytes and a coreceptor used by HIV-1 to enter CD4(+) T lymphocytes and macrophages. Stimulation of CCR5 by chemokines triggers internalization of chemokine-bound CCR5 molecules in a process called down-modulation, which contributes to the anti-HIV activity of chemokines. Recent studies have shown that CCR5 conformational heterogeneity influences chemokine-CCR5 interactions and HIV-1 entry in transfected cells or activated CD4(+) T lymphocytes. However, the effect of CCR5 conformations on other cell types and on the process of down-modulation remains unclear. We used mAbs, some already shown to detect distinct CCR5 conformations, to compare the behavior of CCR5 on in vitro generated human T cell blasts, monocytes and MDMs and CHO-CCR5 transfectants. All human cells express distinct antigenic forms of CCR5 not detected on CHO-CCR5 cells. The recognizable populations of CCR5 receptors exhibit different patterns of down-modulation on T lymphocytes compared with myeloid cells. On T cell blasts, CCR5 is recognized by all antibodies and undergoes rapid chemokine-mediated internalization, whereas on monocytes and MDMs, a pool of CCR5 molecules is recognized by a subset of antibodies and is not removed from the cell surface. We demonstrate that this cell surface-retained form of CCR5 responds to prolonged treatment with more-potent chemokine analogs and acts as an HIV-1 coreceptor. Our findings indicate that the regulation of CCR5 is highly specific to cell type and provide a potential explanation for the observation that native chemokines are less-effective HIV-entry inhibitors on macrophages compared with T lymphocytes.

  11. CCR5 promoter activity correlates with HIV disease progression by regulating CCR5 cell surface expression and CD4 T cell apoptosis.

    PubMed

    Joshi, Anjali; Punke, Erin B; Sedano, Melina; Beauchamp, Bethany; Patel, Rima; Hossenlopp, Cassady; Alozie, Ogechika K; Gupta, Jayanta; Mukherjee, Debabrata; Garg, Himanshu

    2017-03-22

    CCR5 is the major co-receptor for HIV and polymorphisms in the CCR5 gene as well as promoter region that alter cell surface expression have been associated with disease progression. We determined the relationship between CCR5 promoter polymorphisms and CD4 decline and other immunopathological features like immune activation and CD4+ T cell apoptosis in HIV patients. CCR5 promoter haplotype HHC was significantly associated with higher CD4 counts in patients. The relative promoter activity (RPA) of each haplotype was determined in vitro and combined promoter activity based on both alleles (CRPA) was assigned to each patients. Interestingly, CCR5 CRPA correlated inversely with CD4 counts and CD4:CD8 ratio specifically in viremic patients. In normal individuals, the CRPA correlated with the number of CCR5+ CD4+ T cells in the peripheral blood suggesting an effect on CCR5 expression. In a subset of high viremic patients harboring R5 tropic HIV, there was a strong correlation between CCR5 CRPA and both CD4 counts and CD4 T cell apoptosis. Our study demonstrates that, CCR5 promoter polymorphisms correlate with CD4 T cell loss possibly by regulating CD4 T cell apoptosis in HIV patients. Furthermore, assigning CRPAs to each patient is a new method of translating genotype to phenotype.

  12. Allosteric Model of Maraviroc Binding to CC Chemokine Receptor 5 (CCR5)*

    PubMed Central

    Garcia-Perez, Javier; Rueda, Patricia; Alcami, Jose; Rognan, Didier; Arenzana-Seisdedos, Fernando; Lagane, Bernard; Kellenberger, Esther

    2011-01-01

    Maraviroc is a nonpeptidic small molecule human immunodeficiency virus type 1 (HIV-1) entry inhibitor that has just entered the therapeutic arsenal for the treatment of patients. We recently demonstrated that maraviroc binding to the HIV-1 coreceptor, CC chemokine receptor 5 (CCR5), prevents it from binding the chemokine CCL3 and the viral envelope glycoprotein gp120 by an allosteric mechanism. However, incomplete knowledge of ligand-binding sites and the lack of CCR5 crystal structures have hampered an in-depth molecular understanding of how the inhibitor works. Here, we addressed these issues by combining site-directed mutagenesis (SDM) with homology modeling and docking. Six crystal structures of G-protein-coupled receptors were compared for their suitability for CCR5 modeling. All CCR5 models had equally good geometry, but that built from the recently reported dimeric structure of the other HIV-1 coreceptor CXCR4 bound to the peptide CVX15 (Protein Data Bank code 3OE0) best agreed with the SDM data and discriminated CCR5 from non-CCR5 binders in a virtual screening approach. SDM and automated docking predicted that maraviroc inserts deeply in CCR5 transmembrane cavity where it can occupy three different binding sites, whereas CCL3 and gp120 lie on distinct yet overlapped regions of the CCR5 extracellular loop 2. Data suggesting that the transmembrane cavity remains accessible for maraviroc in CCL3-bound and gp120-bound CCR5 help explain our previous observation that the inhibitor enhances dissociation of preformed ligand-CCR5 complexes. Finally, we identified residues in the predicted CCR5 dimer interface that are mandatory for gp120 binding, suggesting that receptor dimerization might represent a target for new CCR5 entry inhibitors. PMID:21775441

  13. Identification of New Agonists and Antagonists of the Insect Odorant Receptor Co-Receptor Subunit

    PubMed Central

    Chen, Sisi; Luetje, Charles W.

    2012-01-01

    Background Insects detect attractive and aversive chemicals using several families of chemosensory receptors, including the OR family of olfactory receptors, making these receptors appealing targets for the control of insects. Insect ORs are odorant-gated ion channels, comprised of at least one common subunit (the odorant receptor co-receptor subunit, Orco) and at least one variable odorant specificity subunit. Each of the many ORs of an insect species is activated or inhibited by an unique set of odorants that interact with the variable odorant specificity subunits, making the development of OR directed insect control agents complex and laborious. However, several N-,2-substituted triazolothioacetamide compounds (VUAA1, VU0450667 and VU0183254) were recently shown to act directly on the highly conserved Orco subunit, suggesting that broadly active compounds can be developed. We have explored the chemical space around the VUAA1 structure in order to identify new Orco ligands. Principal Findings We screened ORs from several insect species, using heterologous expression in Xenopus oocytes and an electrophysiological assay, with a panel of 22 compounds structurally related to VUAA1. By varying the nitrogen position in the pyridine ring and altering the moieties decorating the phenyl ring, we identified two new agonists and a series of competitive antagonists. Screening smaller compounds, similar to portions of the VUAA1 structure, also yielded competitive antagonists. Importantly, we show that Orco antagonists inhibit odorant activation of ORs from several insect species. Detailed examination of one antagonist demonstrated inhibition to be through a non-competitive mechanism. Conclusions A similar pattern of agonist and antagonist sensitivity displayed by Orco subunits from different species suggests a highly conserved binding site structure. The susceptibility to inhibition of odorant activation by Orco antagonism is conserved across disparate insect species

  14. A topical microbicide gel formulation of CCR5 antagonist maraviroc prevents HIV-1 vaginal transmission in humanized RAG-hu mice.

    PubMed

    Neff, C Preston; Kurisu, Theresa; Ndolo, Thomas; Fox, Kami; Akkina, Ramesh

    2011-01-01

    For prevention of HIV infection many currently licensed anti-HIV drugs and new ones in the pipeline show potential as topically applied microbicides. While macaque models have been the gold standard for in vivo microbicide testing, they are expensive and sufficient numbers are not available. Therefore, a small animal model that facilitates rapid evaluation of potential candidates for their preliminary efficacy is urgently needed in the microbicide field. We previously demonstrated that RAG-hu humanized mouse model permits HIV-1 mucosal transmission via both vaginal and rectal routes and that oral pre-exposure chemo-prophylactic strategies could be tested in this system. Here in these proof-of-concept studies, we extended this system for topical microbicide testing using HIV-1 as the challenge virus. Maraviroc, a clinically approved CCR5 inhibitor drug for HIV treatment, was formulated as a microbicide gel at 5 mM concentration in 2.2% hydroxyl ethyl cellulose. Female RAG-hu mice were challenged vaginally with HIV-1 an hour after intravaginal application of the maraviroc gel. Our results showed that maraviroc gel treated mice were fully protected against vaginal HIV-1 challenge in contrast to placebo gel treated mice which all became infected. These findings highlight the utility of the humanized mouse models for microbicide testing and, together with the recent data from macaque studies, suggest that maraviroc is a promising candidate for future microbicide clinical trials in the field.

  15. Structure of the CCR5 Chemokine Receptor-HIV Entry Inhibitor Maraviroc Complex

    SciTech Connect

    Tan, Qiuxiang; Zhu, Ya; Li, Jian; Chen, Zhuxi; Han, Gye Won; Kufareva, Irina; Li, Tingting; Ma, Limin; Fenalti, Gustavo; Li, Jing; Zhang, Wenru; Xie, Xin; Yang, Huaiyu; Jiang, Hualiang; Cherezov, Vadim; Liu, Hong; Stevens, Raymond C.; Zhao, Qiang; Wu, Beili

    2013-10-21

    The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here we report the 2.7 angstrom–resolution crystal structure of human CCR5 bound to the marketed HIV drug maraviroc. The structure reveals a ligand-binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor–gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity. These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.

  16. ERK1-Based Pathway as a New Selective Mechanism To Modulate CCR5 with Natural Antibodies.

    PubMed

    Venuti, Assunta; Pastori, Claudia; Siracusano, Gabriel; Riva, Agostino; Sciortino, Maria Teresa; Lopalco, Lucia

    2015-10-01

    Natural human Abs, recognizing an epitope within the first extramembrane loop of CCR5 (the main HIV coreceptor), induce a long-lasting internalization (48 h) of the protein, whereas all known CCR5 modulating molecules show a short-term kinetics (60-90 min). Despite extensive studies on the regulation of CCR5 signaling cascades, which are the effect of concomitant CCR5 internalization by exogenous stimuli such as Abs, downstream signaling continues to be poorly understood. In this article, we report a hitherto unrecognized mechanism of CCR5 modulation mediated by G protein-dependent ERK1 activity. We further demonstrate that ERK1 is localized mainly in the cytoplasmic compartment and that it interacts directly with the CCR5 protein, thus provoking possible CCR5 degradation with a subsequent de novo synthesis, and that re-expression of CCR5 on the cell membrane required several days. In contrast, the RANTES treatment induces a recovery of the receptor on the cell membrane in short-term kinetics without the involvement of de novo protein synthesis. The said new pathway could be relevant not only to better understand the molecular basis of all pathologic conditions in which CCR5 is involved but also to generate new tools to block viral infections, such as the use of recombinant Abs.

  17. The first report of CCR5 delta 32 mutant in Thai injecting drug users.

    PubMed

    Ruchusatsawat, N; Vongsheree, S; Thaisri, H; Phutiprawan, T

    2000-06-01

    CCR5, a chemokine receptor, is the principal coreceptor for macrophage-tropic HIV-1 which is the most important variant for viral transmission. It has been demonstrated that a homozygous genotype of a 32-bp deletion in CCR5 gene (delta32CCR5) shows a high degree of resistance to HIV-1 infection. To demonstrate that delta32CCR5 does exist in Thai natives, the CCR5 genotypes and allelic frequencies in 860 Thai injecting drug users (IDUs) were determined by PCR and DNA sequencing. Of these, six (0.7%) were CCR5/delta32CCR5 heterozygotes and no homozygote was found. The overall delta32CCR5 allelic frequency was 0.0035 and in HIV-1 seronegative (n = 490) and seropositive (n = 370) IDUs were 0.0051 and 0.0004, respectively, which were not significantly different (p = 0.3776). Here we report that the delta32CCR5 does exist in Thai IDUs as it is present in other human races. Such low allelic frequency may indicate that this mutation does not attribute a significant role in HIV-1 transmission in Thai IDUs.

  18. Highly specific blockade of CCR5 inhibits leukocyte trafficking and reduces mucosal inflammation in murine colitis.

    PubMed

    Mencarelli, Andrea; Cipriani, Sabrina; Francisci, Daniela; Santucci, Luca; Baldelli, Franco; Distrutti, Eleonora; Fiorucci, Stefano

    2016-08-05

    Targeted disruption of leukocyte trafficking to the gut represents a promising approach for the treatment of inflammatory bowel diseases (IBDs). CCR5, the shared receptor for MIP1α and β and RANTES, is expressed by multiple leukocytes. Here, we aimed to determine the role of CCR5 in mediating leukocyte trafficking in models of colitis, and evaluate the therapeutic potential of maraviroc, an orally active CCR5 antagonist used in the treatment of CCR5-tropic HIV. Acute and chronic colitis were induced by administration of DSS or TNBS to wild-type and CCR5(-/-) mice or adoptive transfer of splenic naïve CD4(+) T-cells from wild type or CCR5(-/-) mice into RAG-1(-/-). CCR5 gene ablation reduced the mucosal recruitment and activation of CCR5-bearing CD4(+) and CD11b(+) leukocytes, resulting in profound attenuation of signs and symptoms of inflammation in the TNBS and transfer models of colitis. In the DSS/TNBS colitis and in the transfer model, maraviroc attenuated development of intestinal inflammation by selectively reducing the recruitment of CCR5 bearing leukocytes. In summary, CCR5 regulates recruitment of blood leukocytes into the colon indicating that targeting CCR5 may offer therapeutic options in IBDs.

  19. Highly specific blockade of CCR5 inhibits leukocyte trafficking and reduces mucosal inflammation in murine colitis

    PubMed Central

    Mencarelli, Andrea; Cipriani, Sabrina; Francisci, Daniela; Santucci, Luca; Baldelli, Franco; Distrutti, Eleonora; Fiorucci, Stefano

    2016-01-01

    Targeted disruption of leukocyte trafficking to the gut represents a promising approach for the treatment of inflammatory bowel diseases (IBDs). CCR5, the shared receptor for MIP1α and β and RANTES, is expressed by multiple leukocytes. Here, we aimed to determine the role of CCR5 in mediating leukocyte trafficking in models of colitis, and evaluate the therapeutic potential of maraviroc, an orally active CCR5 antagonist used in the treatment of CCR5-tropic HIV. Acute and chronic colitis were induced by administration of DSS or TNBS to wild-type and CCR5−/− mice or adoptive transfer of splenic naïve CD4+ T-cells from wild type or CCR5−/− mice into RAG-1−/−. CCR5 gene ablation reduced the mucosal recruitment and activation of CCR5-bearing CD4+ and CD11b+ leukocytes, resulting in profound attenuation of signs and symptoms of inflammation in the TNBS and transfer models of colitis. In the DSS/TNBS colitis and in the transfer model, maraviroc attenuated development of intestinal inflammation by selectively reducing the recruitment of CCR5 bearing leukocytes. In summary, CCR5 regulates recruitment of blood leukocytes into the colon indicating that targeting CCR5 may offer therapeutic options in IBDs. PMID:27492684

  20. Reduced Frequencies and Activation of Regulatory T Cells After the Treatment of HIV-1-Infected Individuals with the CCR5 Antagonist Maraviroc Are Associated with a Reduction in Viral Loads Rather Than a Direct Effect of the Drug on Regulatory T Cells.

    PubMed

    Joedicke, Jara J; Dirks, Miriam; Esser, Stefan; Verheyen, Jens; Dittmer, Ulf

    2016-04-01

    Regulatory T cells (Tregs) play an important role in the pathogenesis of HIV-1 infection and they frequently express the chemokine receptor CCR5. We therefore investigated whether antiretroviral treatment with the CCR5 antagonist Maraviroc affected Tregs in chronically HIV-1-infected individuals. HIV-1-infected patients with high viral loads had elevated frequencies of activated Tregs in the peripheral blood compared with healthy controls. In patients successfully treated with antiretroviral drugs (undetectable viral loads), the frequency and the activation status of Tregs were comparable with healthy controls without any specific effect related to the treatment with Maraviroc. These results indicate that the control of viral replication in general rather than a direct binding of Maraviroc to CCR5-positive Tregs influences Treg responses in successfully treated chronically HIV-1-infected individuals.

  1. CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus.

    PubMed

    Kang, HyunJun; Minder, Petra; Park, Mi Ae; Mesquitta, Walatta-Tseyon; Torbett, Bruce E; Slukvin, Igor I

    2015-12-15

    The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.

  2. CCR5 deficiency accelerates lipopolysaccharide-induced astrogliosis, amyloid-beta deposit and impaired memory function

    PubMed Central

    Hwang, Jae Yeon; Kim, Ju Hwan; Yun, Na Young; Oh, Sang Yeon; Song, Ju Kyung; Seo, Hyun Ok; Kim, Yun-Bae; Hwang, Dae Yeon; Oh, Ki-Wan; Han, Sang-Bae; Hong, Jin Tae

    2016-01-01

    Chemokine receptors are implicated in inflammation and immune responses. Neuro-inflammation is associated with activation of astrocyte and amyloid-beta (Aβ) generations that lead to pathogenesis of Alzheimer disease (AD). Previous our study showed that deficiency of CC chemokine receptor 5 (CCR5) results in activation of astrocytes and Aβ deposit, and thus memory dysfunction through increase of CC chemokine receptor 2 (CCR2) expression. CCR5 knockout mice were used as an animal model with memory dysfunction. For the purpose LPS was injected i.p. daily (0.25 mg/kg/day). The memory dysfunctions were much higher in LPS-injected CCR5 knockout mice compared to CCR5 wild type mice as well as non-injected CCR5 knockout mice. Associated with severe memory dysfuction in LPS injected CCR5 knockout mice, LPS injection significant increase expression of inflammatory proteins, astrocyte activation, expressions of β-secretase as well as Aβ deposition in the brain of CCR5 knockout mice as compared with that of CCR5 wild type mice. In CCR5 knockout mice, CCR2 expressions were high and co-localized with GFAP which was significantly elevated by LPS. Expression of monocyte chemoattractant protein-1 (MCP-1) which ligands of CCR2 also increased by LPS injection, and increment of MCP-1 expression is much higher in CCR5 knockout mice. BV-2 cells treated with CCR5 antagonist, D-ala-peptide T-amide (DAPTA) and cultured astrocytes isolated from CCR5 knockout mice treated with LPS (1 μg/ml) and CCR2 antagonist, decreased the NF-ĸB activation and Aβ level. These findings suggest that the deficiency of CCR5 enhances response of LPS, which accelerates to neuro-inflammation and memory impairment. PMID:26910914

  3. CCR5 deficiency accelerates lipopolysaccharide-induced astrogliosis, amyloid-beta deposit and impaired memory function.

    PubMed

    Hwang, Chul Ju; Park, Mi Hee; Hwang, Jae Yeon; Kim, Ju Hwan; Yun, Na Young; Oh, Sang Yeon; Song, Ju Kyung; Seo, Hyun Ok; Kim, Yun-Bae; Hwang, Dae Yeon; Oh, Ki-Wan; Han, Sang-Bae; Hong, Jin Tae

    2016-03-15

    Chemokine receptors are implicated in inflammation and immune responses. Neuro-inflammation is associated with activation of astrocyte and amyloid-beta (Aβ) generations that lead to pathogenesis of Alzheimer disease (AD). Previous our study showed that deficiency of CC chemokine receptor 5 (CCR5) results in activation of astrocytes and Aβ deposit, and thus memory dysfunction through increase of CC chemokine receptor 2 (CCR2) expression. CCR5 knockout mice were used as an animal model with memory dysfunction. For the purpose LPS was injected i.p. daily (0.25 mg/kg/day). The memory dysfunctions were much higher in LPS-injected CCR5 knockout mice compared to CCR5 wild type mice as well as non-injected CCR5 knockout mice. Associated with severe memory dysfuction in LPS injected CCR5 knockout mice, LPS injection significant increase expression of inflammatory proteins, astrocyte activation, expressions of β-secretase as well as Aβ deposition in the brain of CCR5 knockout mice as compared with that of CCR5 wild type mice. In CCR5 knockout mice, CCR2 expressions were high and co-localized with GFAP which was significantly elevated by LPS. Expression of monocyte chemoattractant protein-1 (MCP-1) which ligands of CCR2 also increased by LPS injection, and increment of MCP-1 expression is much higher in CCR5 knockout mice. BV-2 cells treated with CCR5 antagonist, D-ala-peptide T-amide (DAPTA) and cultured astrocytes isolated from CCR5 knockout mice treated with LPS (1 μg/ml) and CCR2 antagonist, decreased the NF-ĸB activation and Aβ level. These findings suggest that the deficiency of CCR5 enhances response of LPS, which accelerates to neuro-inflammation and memory impairment.

  4. A Novel Role for the Receptor of the Complement Cleavage Fragment C5a, C5aR1, in CCR5-Mediated Entry of HIV into Macrophages.

    PubMed

    Moreno-Fernandez, Maria E; Aliberti, Julio; Groeneweg, Sander; Köhl, Jörg; Chougnet, Claire A

    2016-04-01

    The complement system is an ancient pattern recognition system that becomes activated during all stages of HIV infection. Previous studies have shown that C5a can enhance the infection of monocyte-derived macrophages and T cells indirectly through the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and the attraction of dendritic cells. C5a exerts its multiple biologic functions mainly through activation of C5a receptor 1 (C5aR1). Here, we assessed the role of C5aR1 as an enhancer of CCR5-mediated HIV infection. We determined CCR5 and C5aR1 heterodimer formation in myeloid cells and the impact of C5aR1 blockade on HIV entry and genomic integration. C5aR1/CCR5 heterodimer formation was identified by immunoprecipitation and western blotting. THP-1 cells and monocyte-derived macrophages (MDM) were infected by R5 laboratory strains or HIV pseudotyped for the vesicular stomatitis virus (VSV) envelope. Levels of integrated HIV were measured by quantitative PCR after targeting of C5aR1 by a C5aR antagonist, neutralizing C5aR1 monoclonal antibody (mAb) or hC5a. C5aR1 was also silenced by specific siRNA prior to viral entry. We found that C5aR1 forms heterodimers with the HIV coreceptor CCR5 in myeloid cells. Targeting C5aR1 significantly decreased integration by R5 viruses but not by VSV-pseudotyped viruses, suggesting that C5aR1 is critical for viral entry. The level of inhibition achieved with C5aR1-blocking reagents was comparable to that of CCR5 antagonists. Mechanistically, C5aR1 targeting decreased CCR5 expression. MDM from CCR5Δ32 homozygous subjects expressed levels of C5aR1 similar to CCR5 WT individuals, suggesting that mere C5aR1 expression is not sufficient for HIV infection. HIV appeared to preferentially enter THP-1 cells expressing high levels of both C5aR1 and CCR5. Targeted reduction of C5aR1 expression in such cells reduced HIV infection by ~50%. Our data thus suggest that C5aR1 acts as an enhancer of CCR5-mediated HIV entry into

  5. TALEN-Mediated Knockout of CCR5 Confers Protection Against Infection of Human Immunodeficiency Virus.

    PubMed

    Shi, Bingjie; Li, Juan; Shi, Xuanling; Jia, Wenxu; Wen, Yi; Hu, Xiongbing; Zhuang, Fengfeng; Xi, Jianzhong; Zhang, Linqi

    2017-02-01

    Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity, and lower cytotoxicity compared with zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials. Sequence analysis of target cell line GHOST-CCR5-CXCR4 and human primary CD4 T cells showed that the double-strand breaks at the TALEN targeted sites resulted in truncated or nonfunctional CCR5 proteins thereby conferring protection against HIV-1 infection in vitro. None of the CCR5-TALENs had detectable levels of off-target nuclease activity against the homologous region in CCR2 although substantial level was identified for CCR5-ZFN in the primary CD4 T cells. Our results suggest that the CCR5-TALENs identified here are highly functional nucleases that produce protective genetic alterations to human CCR5. Application of these TALENs directly to the primary CD4 T cells and CD34 hematopoietic stem cells (HSCs) of infected individuals could help to create an immune system resistant to HIV-1 infection, recapitulating the success of "Berlin patient" and serving as an essential first step towards a "functional" cure of AIDS.

  6. Linkage of the CCR5 Delta 32 mutation with a functional polymorphism of CD45RA.

    PubMed

    Liao, H X; Montefiori, D C; Patel, D D; Lee, D M; Scott, W K; Pericak-Vance, M; Haynes, B F

    2000-07-01

    A 32-bp deletion in CCR5 (CCR5 Delta 32) confers to PBMC resistance to HIV-1 isolates that use CCR5 as a coreceptor. To study this mutation in T cell development, we have screened 571 human thymus tissues for the mutation. We identified 72 thymuses (12.6%) that were heterozygous and 2 (0.35%) that were homozygous for the CCR5 Delta 32 mutation. We found that thymocyte development was normal in both CCR5 Delta 32 heterozygous and homozygous thymuses. In 3% of thymuses we identified a functional polymorphism of CD45RA, in which cortical and medullary thymocytes failed to down-regulate the 200- and 220-kDa CD45RA isoforms during T cell development. Moreover, we found an association of this CD45 functional polymorphism in thymuses with the CCR5 Delta 32 mutation (p = 0.00258). In vitro HIV-1 infection assays with CCR5-using primary isolates demonstrated that thymocytes with the heterozygous CCR5 Delta 32 mutation produced less p24 than did CCR5 wild-type thymocytes. However, the functional CD45RA polymorphism did not alter the susceptibility of thymocytes to HIV-1 infection. Taken together, these data demonstrate association of the CCR5 Delta 32 mutation with a polymorphism in an as yet unknown gene that is responsible for the ability to down-regulate the expression of high m.w. CD45RA isoforms. Although the presence of the CCR5 Delta 32 mutation down-regulates HIV-1 infection of thymocytes, the functional CD45RA polymorphism does not alter the susceptibility of thymocytes to HIV-1 infection in vitro.

  7. Autologous Hematopoietic Stem Cells transplantation and genetic modification of CCR5 m303/m303 mutant patient for HIV/AIDS.

    PubMed

    Esmaeilzadeh, Abdolreza; Farshbaf, Alieh; Erfanmanesh, Maryam

    2015-03-01

    HIV and AIDS is one of the biggest challenges all over the world. There are an approximately 34 million people living with the virus, and a large number of them become infected each year. Although there are some antiviral drugs for HIV viral load reduction, they are not sufficient. There is no cure for AIDS. Nowadays natural resistance or immunity has absorbed attentions. Because in some HIV positive patients progression trend is slow or even they indicate resistance to AIDS. One of the most interesting approaches in this category is CCR5 gene. CCR5 is a main cc-chemokine co-receptor that facilitates HIV-1 entry to macrophage and CD4(+) T cells. To now, many polymorphisms have been known by CCR5 gene that produces a truncated protein with no function. So, HIV-1 could not entry to immune-cells and the body resistant to HIV/AIDS. Δ32/Δ32 and m303/m303 homozygotes are example of mutations that could create this resistance mechanism. There is a new treatment, such as Hematopoietic Stem Cell transplantation (HSCT) in Berlin and Boston patients for Δ32/Δ32 mutation. It could eliminate co-receptor antagonist and highly-active-anti retroviral therapy (HAART) drugs problems such as toxicity, low safety and side-effects. Now there, the aim of this hypothesis will be evaluation of a new mutation CCR5 m303/m303 as autologous HSCT. This novel hypothesis indicates that autologous HSCT for m303/m303 could be effective treatment for anyone HIV/AIDS affected patient worldwide.

  8. Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization.

    PubMed

    Romano, J W; Tetali, S; Lee, E M; Shurtliff, R N; Wang, X P; Pahwa, S; Kaplan, M H; Ginocchio, C C

    1999-11-01

    The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

  9. Genotyping of the CCR5 Chemokine Receptor by Isothermal NASBA Amplification and Differential Probe Hybridization

    PubMed Central

    Romano, Joseph W.; Tetali, Surya; Lee, Eun Mi; Shurtliff, Roxanne N.; Wang, Xue Ping; Pahwa, Savita; Kaplan, Mark H.; Ginocchio, Christine C.

    1999-01-01

    The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectibility of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems. PMID:10548593

  10. Quantifying CD4/CCR5 Usage Efficiency of HIV-1 Env Using the Affinofile System.

    PubMed

    Webb, Nicholas E; Lee, Benhur

    2016-01-01

    Entry of HIV-1 into target cells involves the interaction of the HIV envelope (Env) with both a primary receptor (CD4) and a coreceptor (CXCR4 or CCR5). The relative efficiency with which a particular Env uses these receptors is a major component of cellular tropism in the context of entry and is related to a variety of pathological Env phenotypes (Chikere et al. Virology 435:81-91, 2013). The protocols outlined in this chapter describe the use of the Affinofile system, a 293-based dual-inducible cell line that expresses up to 25 distinct combinations of CD4 and CCR5, as well as the associated Viral Entry Receptor Sensitivity Assay (VERSA) metrics used to summarize the CD4/CCR5-dependent infectivity results. This system allows for high-resolution profiling of CD4 and CCR5 usage efficiency in the context of unique viral phenotypes.

  11. CCR5 Expression Levels in HIV-Uninfected Women Receiving Hormonal Contraception.

    PubMed

    Sciaranghella, Gaia; Wang, Cuiwei; Hu, Haihong; Anastos, Kathryn; Merhi, Zaher; Nowicki, Marek; Stanczyk, Frank Z; Greenblatt, Ruth M; Cohen, Mardge; Golub, Elizabeth T; Watts, D Heather; Alter, Galit; Young, Mary A; Tsibris, Athe M N

    2015-11-01

    Human immunodeficiency virus (HIV) infectivity increases as receptor/coreceptor expression levels increase. We determined peripheral CD4, CCR5, and CXCR4 expression levels in HIV-uninfected women who used depot medroxyprogesterone acetate (DMPA; n = 32), the levonorgestrel-releasing intrauterine device (LNG-IUD; n = 27), oral contraceptive pills (n = 32), or no hormonal contraception (n = 33). The use of LNG-IUD increased the proportion of CD4(+) and CD8(+) T cells that expressed CCR5; increases in the magnitude of T-cell subset CCR5 expression were observed with DMPA and LNG-IUD use (P < .01 for all comparisons). LNG-IUD and, to a lesser extent, DMPA use were associated with increased peripheral T-cell CCR5 expression.

  12. CCR5 Expression Levels in HIV-Uninfected Women Receiving Hormonal Contraception

    PubMed Central

    Sciaranghella, Gaia; Wang, Cuiwei; Hu, Haihong; Anastos, Kathryn; Merhi, Zaher; Nowicki, Marek; Stanczyk, Frank Z.; Greenblatt, Ruth M.; Cohen, Mardge; Golub, Elizabeth T.; Watts, D. Heather; Alter, Galit; Young, Mary A.; Tsibris, Athe M. N.

    2015-01-01

    Human immunodeficiency virus (HIV) infectivity increases as receptor/coreceptor expression levels increase. We determined peripheral CD4, CCR5, and CXCR4 expression levels in HIV-uninfected women who used depot medroxyprogesterone acetate (DMPA; n = 32), the levonorgestrel-releasing intrauterine device (LNG-IUD; n = 27), oral contraceptive pills (n = 32), or no hormonal contraception (n = 33). The use of LNG-IUD increased the proportion of CD4+ and CD8+ T cells that expressed CCR5; increases in the magnitude of T-cell subset CCR5 expression were observed with DMPA and LNG-IUD use (P < .01 for all comparisons). LNG-IUD and, to a lesser extent, DMPA use were associated with increased peripheral T-cell CCR5 expression. PMID:25895986

  13. HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation.

    PubMed

    Qi, Chunxia; Jia, Xiaopeng; Lu, Lingling; Ma, Ping; Wei, Min

    2016-01-01

    C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.

  14. HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

    PubMed Central

    Qi, Chunxia; Jia, Xiaopeng; Lu, Lingling; Ma, Ping; Wei, Min

    2016-01-01

    C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis. PMID:27042825

  15. Distribution of the mutated delta 32 allele of the CCR5 gene in a Sicilian population.

    PubMed

    Sidoti, A; D'Angelo, R; Rinaldi, C; De Luca, G; Pino, F; Salpietro, C; Giunta, D E; Saltalamacchia, F; Amato, A

    2005-06-01

    The CCR5 gene encodes a cell-surface chemokine receptor molecule that serves as a co-receptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). A mutation in this gene may alter the expression or the function of the protein product, thereby altering chemokine binding and/or signalling or HIV-1 infection of cells that normally express CCR5 protein. Individuals homozygous for a 32-bp deletion allele of CCR5 (CCR5 delta32), heritable as a Mendelian trait, are relatively resistant to HIV-1 infection. The CCR5 delta32 mutation is present in the Caucasian population at different frequencies. The aim of this study was to investigate the frequency of truncated alleles of the CCR5 delta32 gene in a Sicilian population, as the interpopulation variation in CCR5 delta32 frequency may be a significant factor in the prediction of AIDS endemicity in future studies. We examined 901 healthy individuals from several Sicilian provinces. We found a mean (+/- standard deviation) delta32 allele frequency (fr) of 0.04 +/- 0.012. The highest value was observed in the province of Messina, with a mean delta32 allele frequency of 0.06 +/- 0.024, where we collected samples from a cohort of 114 HIV-1-infected individuals. The observed frequency amongst these patients was quite low (fr = 0.03 +/- 0.031) compared to the healthy population, although the difference was not statistically significant.

  16. Analysis of CCR5 and SDF-1 genetic variants and HIV infection in Indian population.

    PubMed

    Gupta, A; Padh, Harish

    2015-08-01

    HIV-1 infection and progression exhibits interindividual variation. The polymorphism in the chemokine receptors CCR5 and CXCR4, the principal coreceptors for HIV-1 and their ligands like SDF-1 have a profound effect in altering the HIV-1 disease progression rate. A single nucleotide polymorphism designated SDF1-3'UTR-801G-A has been associated with resistance to HIV-1 infection or delayed progression to AIDS. In this study, the SDF1-3'A polymorphism, CCR5∆32 polymorphism and CCR5 promoter polymorphism at positions 58934 G/T, 59029 G/A, 59353 T/C, 59356 C/T, 59402 A/G and 59653 C/T were analysed in Indian population. The polymorphisms in HIV-1 patients and healthy individuals were evaluated by conventional PCR, RFLP-PCR and direct sequencing techniques. The CCR5∆32 mutant allele was found to be almost absent in Indian population. The analysis of the CCR5-59356C/T polymorphism revealed a trend towards an association of the C allele with an increased risk of HIV-1 infection. The frequency of allele CCR5-59356C was higher in HIV-1 patients (100%) as compared to healthy control subjects (89%, P = 0.003). The correlation of SDF1-3'A and CCR5 promoter CCR5-58934G/T, CCR5-59029G/A, CCR5-59353T/C, CCR5-59402 A/G and CCR5-59653C/T polymorphisms and protection to HIV-1 infection and progression to AIDS was found to be nonsignificant. Nine haplotypes with more than 1% frequency were detected but were not significant in their protective role against HIV. Comparative analysis with global populations showed a noteworthy difference in CCR5 and SDF-1 polymorphisms' frequency distribution, indicating the ethnic variability of Indians. Although susceptibility to infections cannot be completely dependent on one or few genetic variants, it is important to remember that SDF-1 and CCR5 variants have been correlated globally with HIV-1 infection and disease progression. In the light of that, higher frequency of SDF-1 variants in the Indian population is noteworthy.

  17. Dengue virus requires the CC-chemokine receptor CCR5 for replication and infection development.

    PubMed

    Marques, Rafael E; Guabiraba, Rodrigo; Del Sarto, Juliana L; Rocha, Rebeca F; Queiroz, Ana Luiza; Cisalpino, Daniel; Marques, Pedro E; Pacca, Carolina C; Fagundes, Caio T; Menezes, Gustavo B; Nogueira, Maurício L; Souza, Danielle G; Teixeira, Mauro M

    2015-08-01

    Dengue is a mosquito-borne disease that affects millions of people worldwide yearly. Currently, there is no vaccine or specific treatment available. Further investigation on dengue pathogenesis is required to better understand the disease and to identify potential therapeutic targets. The chemokine system has been implicated in dengue pathogenesis, although the specific role of chemokines and their receptors remains elusive. Here we describe the role of the CC-chemokine receptor CCR5 in Dengue virus (DENV-2) infection. In vitro experiments showed that CCR5 is a host factor required for DENV-2 replication in human and mouse macrophages. DENV-2 infection induces the expression of CCR5 ligands. Incubation with an antagonist prevents CCR5 activation and reduces DENV-2 positive-stranded (+) RNA inside macrophages. Using an immunocompetent mouse model of DENV-2 infection we found that CCR5(-/-) mice were resistant to lethal infection, presenting at least 100-fold reduction of viral load in target organs and significant reduction in disease severity. This phenotype was reproduced in wild-type mice treated with CCR5-blocking compounds. Therefore, CCR5 is a host factor required for DENV-2 replication and disease development. Targeting CCR5 might represent a therapeutic strategy for dengue fever. These data bring new insights on the association between viral infections and the chemokine receptor CCR5.

  18. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection.

    PubMed

    Wang, Weiming; Ye, Chaobaihui; Liu, Jingjing; Zhang, Di; Kimata, Jason T; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.

  19. CCR2, CCR5, and CXCL12 variation and HIV/AIDS in Papua New Guinea.

    PubMed

    Mehlotra, Rajeev K; Hall, Noemi B; Bruse, Shannon E; John, Bangan; Blood Zikursh, Melinda J; Stein, Catherine M; Siba, Peter M; Zimmerman, Peter A

    2015-12-01

    Polymorphisms in chemokine receptors, serving as HIV co-receptors, and their ligands are among the well-known host genetic factors associated with susceptibility to HIV infection and/or disease progression. Papua New Guinea (PNG) has one of the highest adult HIV prevalences in the Asia-Pacific region. However, information regarding the distribution of polymorphisms in chemokine receptor (CCR5, CCR2) and chemokine (CXCL12) genes in PNG is very limited. In this study, we genotyped a total of nine CCR2-CCR5 polymorphisms, including CCR2 190G >A, CCR5 -2459G >A and Δ32, and CXCL12 801G >A in PNG (n=258), North America (n=184), and five countries in West Africa (n=178). Using this data, we determined previously characterized CCR5 haplotypes. In addition, based on the previously reported associations of CCR2 190, CCR5 -2459, CCR5 open reading frame, and CXCL12 801 genotypes with HIV acquisition and/or disease progression, we calculated composite full risk scores, considering both protective as well as susceptibility effects of the CXCL12 801 AA genotype. We observed a very high frequency of the CCR5 -2459A allele (0.98) in the PNG population, which together with the absence of Δ32 resulted in a very high frequency of the HHE haplotype (0.92). These frequencies were significantly higher than in any other population (all P-values<0.001). Regardless of whether we considered the CXCL12 801 AA genotype protective or susceptible, the risk scores were significantly higher in the PNG population compared with any other population (all P-values<0.001). The results of this study provide new insights regarding CCR5 variation in the PNG population, and suggest that the collective variation in CCR2, CCR5, and CXCL12 may increase the risk of HIV/AIDS in a large majority of Papua New Guineans.

  20. Persistence of dual-tropic HIV-1 in an individual homozygous for the CCR5 Delta 32 allele.

    PubMed

    Gorry, Paul R; Zhang, Chengsheng; Wu, Sam; Kunstman, Kevin; Trachtenberg, Elizabeth; Phair, John; Wolinsky, Steven; Gabuzda, Dana

    2002-05-25

    Entry of HIV-1 into a cell happens only after viral envelope glycoproteins have bound to CD4 and a chemokine receptor. Generally, macrophage-tropic strains use CCR5, and T cell-line-tropic strains use CXCR4 as coreceptors for virus entry. Dual-tropic viruses can use both CCR5 and CXCR4. About 1% of white people are homozygous for a non-functional CCR5 allele, containing a 32 base pair deletion (CCR5 Delta 32). We studied the persistence of dual-tropic HIV-1 in an individual homozygous for this deletion. Our results suggest that structural features of the HIV-1 envelope linked to CCR5 tropism could confer a selective advantage in vivo.

  1. Staphylococcus aureus Leukocidin LukED and HIV-1 gp120 Target Different Sequence Determinants on CCR5

    PubMed Central

    Tam, Kayan; Schultz, Megan; Reyes-Robles, Tamara; Vanwalscappel, Bénédicte; Horton, Joshua; Alonzo, Francis; Wu, Beili

    2016-01-01

    ABSTRACT Leukocidin ED (LukED) is a bicomponent pore-forming toxin produced by Staphylococcus aureus that lyses host cells by targeting the chemokine receptors CC chemokine receptor type 5 (CCR5), CXCR1, CXCR2, and DARC. In addition to its role as a receptor for LukED, CCR5 is the major coreceptor for primary isolates of human immunodeficiency virus type 1 (HIV-1) and has been extensively studied. To compare how LukED and HIV-1 target CCR5, we analyzed their respective abilities to use CCR5/CCR2b chimeras to mediate cytotoxicity and virus entry. These analyses showed that the second and third extracellular loops (ECL) of CCR5 are necessary and sufficient for LukED to target the receptor and promote cell lysis. In contrast, the second ECL of CCR5 is necessary but not sufficient for HIV-1 infectivity. The analysis of CCR5 point mutations showed that glycine-163 is critical for HIV-1 infectivity, while arginine-274 and aspartic acid-276 are critical for LukED cytotoxicity. Point mutations in ECL2 diminished both HIV-1 infectivity and LukED cytotoxicity. Treatment of cells with LukED did not interfere with CCR5-tropic HIV-1 infectivity, demonstrating that LukED and the viral envelope glycoprotein use nonoverlapping sites on CCR5. Analysis of point mutations in LukE showed that amino acids 64 to 69 in the rim domain are required for CCR5 targeting and cytotoxicity. Taking the results together, this study identified the molecular basis by which LukED targets CCR5, highlighting the divergent molecular interactions evolved by HIV-1 and LukED to interact with CCR5. PMID:27965453

  2. Staphylococcus aureus Leukocidin LukED and HIV-1 gp120 Target Different Sequence Determinants on CCR5.

    PubMed

    Tam, Kayan; Schultz, Megan; Reyes-Robles, Tamara; Vanwalscappel, Bénédicte; Horton, Joshua; Alonzo, Francis; Wu, Beili; Landau, Nathaniel R; Torres, Victor J

    2016-12-13

    Leukocidin ED (LukED) is a bicomponent pore-forming toxin produced by Staphylococcus aureus that lyses host cells by targeting the chemokine receptors CC chemokine receptor type 5 (CCR5), CXCR1, CXCR2, and DARC. In addition to its role as a receptor for LukED, CCR5 is the major coreceptor for primary isolates of human immunodeficiency virus type 1 (HIV-1) and has been extensively studied. To compare how LukED and HIV-1 target CCR5, we analyzed their respective abilities to use CCR5/CCR2b chimeras to mediate cytotoxicity and virus entry. These analyses showed that the second and third extracellular loops (ECL) of CCR5 are necessary and sufficient for LukED to target the receptor and promote cell lysis. In contrast, the second ECL of CCR5 is necessary but not sufficient for HIV-1 infectivity. The analysis of CCR5 point mutations showed that glycine-163 is critical for HIV-1 infectivity, while arginine-274 and aspartic acid-276 are critical for LukED cytotoxicity. Point mutations in ECL2 diminished both HIV-1 infectivity and LukED cytotoxicity. Treatment of cells with LukED did not interfere with CCR5-tropic HIV-1 infectivity, demonstrating that LukED and the viral envelope glycoprotein use nonoverlapping sites on CCR5. Analysis of point mutations in LukE showed that amino acids 64 to 69 in the rim domain are required for CCR5 targeting and cytotoxicity. Taking the results together, this study identified the molecular basis by which LukED targets CCR5, highlighting the divergent molecular interactions evolved by HIV-1 and LukED to interact with CCR5.

  3. Protection of macaques from vaginal SHIV challenge by an orally delivered CCR5 inhibitor.

    PubMed

    Veazey, Ronald S; Springer, Martin S; Marx, Preston A; Dufour, Jason; Klasse, Per Johan; Moore, John P

    2005-12-01

    Pre-exposure oral prophylaxis with antiviral drugs is a potential method for preventing transmission of human immunodeficiency virus type 1 (HIV-1). We show that oral delivery of CMPD167, a small molecule that binds to the CCR5 coreceptor, for 10-14 d can protect a substantial proportion of macaques from vaginal infection with a CCR5-using virus (SHIV-162P3). The macaques that became infected despite receiving CMPD167 had reduced plasma viremia levels during the earliest stages of infection.

  4. CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5

    PubMed Central

    Chou, Szu-Yi; Ajoy, Reni; Changou, Chun Austin; Hsieh, Ya-Ting; Wang, Yang-Kao; Hoffer, Barry

    2016-01-01

    Many neurodegenerative diseases are accompanied by metabolic disorders. CCL5/RANTES, and its receptor CCR5 are known to contribute to neuronal function as well as to metabolic disorders such as type 2 diabetes mellitus, obesity, atherosclerosis and metabolic changes after HIV infection. Herein, we found that the lack of CCR5 or CCL5 in mice impaired regulation of energy metabolism in hypothalamus. Immunostaining and co-immunoprecipitation revealed the specific expression of CCR5, associated with insulin receptors, in the hypothalamic arcuate nucleus (ARC). Both ex vivo stimulation and in vitro tissue culture studies demonstrated that the activation of insulin, and PI3K-Akt pathways were impaired in CCR5 and CCL5 deficient hypothalamus. The inhibitory phosphorylation of insulin response substrate-1 at Ser302 (IRS-1S302) but not IRS-2, by insulin was markedly increased in CCR5 and CCL5 deficient animals. Elevating CCR5/CCL5 activity induced GLUT4 membrane translocation and reduced phospho-IRS-1S302 through AMPKα-S6 Kinase. Blocking CCR5 using the antagonist, MetCCL5, abolished the de-phosphorylation of IRS-1S302 and insulin signal activation. In addition, intracerebroventricular delivery of MetCCL5 interrupted hypothalamic insulin signaling and elicited peripheral insulin responsiveness and glucose intolerance. Taken together, our data suggest that CCR5 regulates insulin signaling in hypothalamus which contributes to systemic insulin sensitivity and glucose metabolism. PMID:27898058

  5. CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5.

    PubMed

    Chou, Szu-Yi; Ajoy, Reni; Changou, Chun Austin; Hsieh, Ya-Ting; Wang, Yang-Kao; Hoffer, Barry

    2016-11-29

    Many neurodegenerative diseases are accompanied by metabolic disorders. CCL5/RANTES, and its receptor CCR5 are known to contribute to neuronal function as well as to metabolic disorders such as type 2 diabetes mellitus, obesity, atherosclerosis and metabolic changes after HIV infection. Herein, we found that the lack of CCR5 or CCL5 in mice impaired regulation of energy metabolism in hypothalamus. Immunostaining and co-immunoprecipitation revealed the specific expression of CCR5, associated with insulin receptors, in the hypothalamic arcuate nucleus (ARC). Both ex vivo stimulation and in vitro tissue culture studies demonstrated that the activation of insulin, and PI3K-Akt pathways were impaired in CCR5 and CCL5 deficient hypothalamus. The inhibitory phosphorylation of insulin response substrate-1 at Ser302 (IRS-1(S302)) but not IRS-2, by insulin was markedly increased in CCR5 and CCL5 deficient animals. Elevating CCR5/CCL5 activity induced GLUT4 membrane translocation and reduced phospho-IRS-1(S302) through AMPKα-S6 Kinase. Blocking CCR5 using the antagonist, (Met)CCL5, abolished the de-phosphorylation of IRS-1(S302) and insulin signal activation. In addition, intracerebroventricular delivery of (Met)CCL5 interrupted hypothalamic insulin signaling and elicited peripheral insulin responsiveness and glucose intolerance. Taken together, our data suggest that CCR5 regulates insulin signaling in hypothalamus which contributes to systemic insulin sensitivity and glucose metabolism.

  6. The frequency of CCR5 promoter polymorphisms and CCR5 Δ 32 mutation in Iranian populations.

    PubMed

    Zare-Bidaki, Mohammad; Karimi-Googheri, Masoud; Hassanshahi, Gholamhossein; Zainodini, Nahid; Arababadi, Mohammad Kazemi

    2015-04-01

    Evidence showed that chemokines serve as pro-migratory factors for immune cells. CCL3, CCL4 and CCL5, as the main CC chemokines subfamily members, activate immune cells through binding to CC chemokine receptor 5 or CCR5. Macrophages, NK cells and T lymphocytes express CCR5 and thus, affected CCR5 expression or functions could be associated with altered immune responses. Deletion of 32 base pairs (Δ 32) in the exon 1 of the CCR5 gene, which is known as CCR5 Δ 32 mutation causes down regulation and malfunction of the molecule. Furthermore, it has been evidenced that three polymorphisms in the promoter region of CCR5 modulate its expression. Altered CCR5 expression in microbial infection and immune related diseases have been reported by several researchers but the role of CCR5 promoter polymorphisms and CCR5 Δ 32 mutation in Iranian patients suffering from these diseases are controversial. Due to the fact that Iranian people have different genetic backgrounds compared to other ethnics, hence, CCR5 promoter polymorphisms and CCR5 32 mutation association with the diseases may be different in Iranian patients. Therefore, this review addresses the most recent information regarding the prevalence as well as association of the mutation and polymorphisms in Iranian patients with microbial infection and immune related diseases as along with normal population.

  7. B-1 cells promote immunosurveillance against murine melanoma in host absence of CCR5: new perspective in autologous vaccination therapy.

    PubMed

    Vivanco, Bruno C; Viana, Jacqueline D; Perez, Elisabeth C; Konno, Fabiana T C; Guereschi, Marcia G; Xander, Patricia; Keller, Alexandre C; Lopes, José D

    2014-11-01

    Autologous vaccination with tumor-primed dendritic cells increases immune response against tumor, which seems to be improved in host absence of CCR5. Because B-1 lymphocytes modulate the activity of different immune cells, we decided to study their influence in the resistance against murine B16F10 melanoma in a CCR5 deprived environment. Adoptive transfer of peritoneal B-1 CCR5(+/+) lymphocytes to CCR5(-/-) animals inhibited the establishment of lung metastasis and melanoma cell growth, in comparison to saline-treated CCR5(-/-) mice. In loco cell analysis demonstrated that the adoptive transfer of B-1 CCR5(+/+) lymphocytes to CCR5 deficient host was associated with a more intense influx of T CD8(+) to tumor site, indicating that the presence of CCR5(+/+) B-1 cells in the tumor environment induces the migration of T CD8 CCR5(-/-) cells to the implantation site. To corroborate this idea, CCR5(-/-) mice were injected with non B-1 peritoneal cells from wild type (WT) mice before B16F10 inoculation. In this regimen, CCR5(-/-) mice were not protected from tumor growth reinforcing the idea that, in host absence of CCR5, B-1 cells are essential to confer tumor resistance. This work indicates that, in the host absence of CCR5, naive B-1 cells may activate CD8T lymphocytes thereby promoting tumor resistance. Our results strongly suggest that autologous vaccination with B-1 lymphocytes in combination with CCR5 antagonists can be an alternative approach to tumor therapy.

  8. Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells.

    PubMed

    DiGiusto, David L; Cannon, Paula M; Holmes, Michael C; Li, Lijing; Rao, Anitha; Wang, Jianbin; Lee, Gary; Gregory, Philip D; Kim, Kenneth A; Hayward, Samuel B; Meyer, Kathleen; Exline, Colin; Lopez, Evan; Henley, Jill; Gonzalez, Nancy; Bedell, Victoria; Stan, Rodica; Zaia, John A

    2016-01-01

    Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov).

  9. Preclinical development and qualification of ZFN-mediated CCR5 disruption in human hematopoietic stem/progenitor cells

    PubMed Central

    DiGiusto, David L; Cannon, Paula M; Holmes, Michael C; Li, Lijing; Rao, Anitha; Wang, Jianbin; Lee, Gary; Gregory, Philip D.; Kim, Kenneth A; Hayward, Samuel B; Meyer, Kathleen; Exline, Colin; Lopez, Evan; Henley, Jill; Gonzalez, Nancy; Bedell, Victoria; Stan, Rodica; Zaia, John A

    2016-01-01

    Gene therapy for HIV-1 infection is a promising alternative to lifelong combination antiviral drug treatment. Chemokine receptor 5 (CCR5) is the coreceptor required for R5-tropic HIV-1 infection of human cells. Deletion of CCR5 renders cells resistant to R5-tropic HIV-1 infection, and the potential for cure has been shown through allogeneic stem cell transplantation with naturally occurring homozygous deletion of CCR5 in donor hematopoietic stem/progenitor cells (HSPC). The requirement for HLA-matched HSPC bearing homozygous CCR5 deletions prohibits widespread application of this approach. Thus, a strategy to disrupt CCR5 genomic sequences in HSPC using zinc finger nucleases was developed. Following discussions with regulatory agencies, we conducted IND-enabling preclinical in vitro and in vivo testing to demonstrate the feasibility and (preclinical) safety of zinc finger nucleases-based CCR5 disruption in HSPC. We report here the clinical-scale manufacturing process necessary to deliver CCR5-specific zinc finger nucleases mRNA to HSPC using electroporation and the preclinical safety data. Our results demonstrate effective biallelic CCR5 disruption in up to 72.9% of modified colony forming units from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. Tumorigenicity studies demonstrated initial product safety; further safety and feasibility studies are ongoing in subjects infected with HIV-1 (NCT02500849@clinicaltrials.gov). PMID:27900346

  10. CCL3L1 gene-containing segmental duplications and polymorphisms in CCR5 affect risk of systemic lupus erythaematosus

    PubMed Central

    Mamtani, M; Rovin, B; Brey, R; Camargo, J F; Kulkarni, H; Herrera, M; Correa, P; Holliday, S; Anaya, J-M; Ahuja, S K

    2013-01-01

    Objectives There is an enrichment of immune response genes that are subject to copy number variations (CNVs). However, there is limited understanding of their impact on susceptibility to human diseases. CC chemokine ligand 3 like-1 (CCL3L1) is a potent ligand for the HIV coreceptor, CC chemokine receptor 5 (CCR5), and we have demonstrated previously an association between CCL3L1- gene containing segmental duplications and polymorphisms in CCR5 and HIV/AIDS susceptibility. Here, we determined the association between these genetic variations and risk of developing systemic lupus erythaematosus (SLE), differential recruitment of CD3+ and CD68+ leukocytes to the kidney, clinical severity of SLE reflected by autoantibody titres and the risk of renal complications in SLE. Methods We genotyped 1084 subjects (469 cases of SLE and 615 matched controls with no autoimmune disease) from three geographically distinct cohorts for variations in CCL3L1 and CCR5. Results Deviation from the average copy number of CCL3L1 found in European populations increased the risk of SLE and modified the SLE-influencing effects of CCR5 haplotypes. The CCR5 human haplogroup (HH)E and CCR5-Δ32-bearing HHG*2 haplotypes were associated with an increased risk of developing SLE. An individual’s CCL3L1–CCR5 genotype strongly predicted the overall risk of SLE, high autoantibody titres, and lupus nephritis as well as the differential recruitment of leukocytes in subjects with lupus nephritis. The CCR5 HHE/HHG*2 genotype was associated with the maximal risk of developing SLE. Conclusion CCR5 haplotypes HHE and HHG*2 strongly influence the risk of SLE. The copy number of CCL3L1 influences risk of SLE and modifies the SLE-influencing effects associated with CCR5 genotypes. These findings implicate a key role of the CCL3L1–CCR5 axis in the pathogenesis of SLE. PMID:17971457

  11. HIV-1 CCR5 gene therapy will fail unless it is combined with a suicide gene.

    PubMed

    Pandit, Aridaman; de Boer, Rob J

    2015-12-17

    Highly active antiretroviral therapy (ART) has successfully turned Human immunodeficiency virus type 1 (HIV-1) from a deadly pathogen into a manageable chronic infection. ART is a lifelong therapy which is both expensive and toxic, and HIV can become resistant to it. An alternative to lifelong ART is gene therapy that targets the CCR5 co-receptor and creates a population of genetically modified host cells that are less susceptible to viral infection. With generic mathematical models we show that gene therapy that only targets the CCR5 co-receptor fails to suppress HIV-1 (which is in agreement with current data). We predict that the same gene therapy can be markedly improved if it is combined with a suicide gene that is only expressed upon HIV-1 infection.

  12. HIV-1 CCR5 gene therapy will fail unless it is combined with a suicide gene

    PubMed Central

    Pandit, Aridaman; de Boer, Rob J.

    2015-01-01

    Highly active antiretroviral therapy (ART) has successfully turned Human immunodeficiency virus type 1 (HIV-1) from a deadly pathogen into a manageable chronic infection. ART is a lifelong therapy which is both expensive and toxic, and HIV can become resistant to it. An alternative to lifelong ART is gene therapy that targets the CCR5 co-receptor and creates a population of genetically modified host cells that are less susceptible to viral infection. With generic mathematical models we show that gene therapy that only targets the CCR5 co-receptor fails to suppress HIV-1 (which is in agreement with current data). We predict that the same gene therapy can be markedly improved if it is combined with a suicide gene that is only expressed upon HIV-1 infection. PMID:26674113

  13. Exploration of Bivalent Ligands Targeting Putative Mu Opioid Receptor and Chemokine Receptor CCR5 Dimerization

    PubMed Central

    Arnatt, Christopher K.; Falls, Bethany A.; Yuan, Yunyun; Raborg, Thomas J.; Masvekar, Ruturaj R.; El-Hage, Nazira; Selley, Dana E.; Nicola, Anthony V.; Knapp, Pamela E.; Hauser, Kurt F.; Zhang, Yan

    2016-01-01

    Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR-CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5-MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5-MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation. PMID:27720326

  14. Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

    SciTech Connect

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika; E-mail: hiokada@med.nagoya-cu.ac.jp

    2007-02-23

    HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1{sub IIIB} infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.

  15. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    SciTech Connect

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-08-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo.

  16. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    PubMed Central

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-01-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO 140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo. PMID:18519143

  17. First report of a healthy Indian heterozygous for delta 32 mutant of HIV-1 co-receptor-CCR5 gene.

    PubMed

    Husain, S; Goila, R; Shahi, S; Banerjea, A

    1998-01-30

    The beta-chemokine receptor, CCR5, is a major co-receptor for macrophage tropic non-syncytia-inducing isolates of HIV-1. Recently a 32 bp homozygous deletion in the coding region of CCR5 has been reported in a very small percentage (< 1%) of Caucasian individuals who remain uninfected, despite multiple exposure to the wild-type virus. This mutant allele in the heterozygous form (CCR5/32 ccr5) was readily detected in a normal unrelated Caucasian population of European heritage with varying frequencies (13-20%). However, when a large number of the non-Caucasian population (261 Africans and 423 Asians) were screened for the presence of this deleted allele, not a single case of either homozygous or heterozygous mutant for delta 32 allele of CCR5 was detected. We screened 100 normal individuals and found a single heterozygous case with an identical 32 bp deletion in CCR5 gene reported earlier, the rest possessed wild-type alleles. This deleted gene was inherited in Mendelian fashion among the family members of this individual. Thus, the frequency of this deleted allele in India among unrelated normal individuals is likely to be very low (< 1%). We observed a moderate transdominant effect of this mutant allele in a fusion assay. Finally, we show a significant inhibition of fusion of cell membranes when the 176-bp region of CCR5 was used as an antisense.

  18. Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

    PubMed Central

    Stephens, J C; Reich, D E; Goldstein, D B; Shin, H D; Smith, M W; Carrington, M; Winkler, C; Huttley, G A; Allikmets, R; Schriml, L; Gerrard, B; Malasky, M; Ramos, M D; Morlot, S; Tzetis, M; Oddoux, C; di Giovine, F S; Nasioulas, G; Chandler, D; Aseev, M; Hanson, M; Kalaydjieva, L; Glavac, D; Gasparini, P; Kanavakis, E; Claustres, M; Kambouris, M; Ostrer, H; Duff, G; Baranov, V; Sibul, H; Metspalu, A; Goldman, D; Martin, N; Duffy, D; Schmidtke, J; Estivill, X; O'Brien, S J; Dean, M

    1998-01-01

    The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations. PMID:9585595

  19. Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.

    PubMed

    Sather, Blythe D; Romano Ibarra, Guillermo S; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A; Scharenberg, Andrew M; Rawlings, David J

    2015-09-30

    Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties.

  20. Chemokine receptor CCR5 and CXCR4 might influence virus replication during IBDV infection.

    PubMed

    Ou, Changbo; Wang, Qiuxia; Yu, Yan; Zhang, Yanhong; Ma, Jinyou; Kong, Xianghui; Liu, Xingyou

    2017-03-27

    Both CCR5 and CXCR4 are important chemokine receptors and take vital role in migration, development and distribution of T cells, however, whether they will influence the process of T cell infiltration into bursa of Fabricius during infectious bursal disease virus (IBDV) infection is unclear. In the current study, CCR5 and CXCR4 antagonists, Maraviroc and AMD3100, were administrated into chickens inoculated with IBDV, and the gene levels of IBDV VP2, CCR5, CXCR4 and related cytokines were determined by real-time PCR. The results showed that large number of T cells began to migrate into the bursae on Day 3 post infection with IBDV and the mRNA of chemokine receptors CCR5 and CXCR4 began to increase on Day 1. Moreover, antagonist treatments have increased the VP2, CCR5 and CXCR4 gene transcriptions and influenced on the gene levels of IL-2, IL-6, IL-8, IFN-γ, TGF-β4, MHC-I and MDA5. In conclusion, the chemokine receptors CCR5 and CXCR4 might influence virus replication during IBDV infection and further study would focus on the interaction between chemokine receptors and their ligands.

  1. CCR5 blockade for neuroinflammatory diseases--beyond control of HIV.

    PubMed

    Martin-Blondel, Guillaume; Brassat, David; Bauer, Jan; Lassmann, Hans; Liblau, Roland S

    2016-02-01

    Chemokine receptors have been implicated in a wide range of CNS inflammatory diseases and have important roles in the recruitment and positioning of immune cells within tissues. Among them, the chemokine (C-C motif) receptor 5 (CCR5) can be targeted by maraviroc, a readily available and well-tolerated drug that was developed for the treatment of HIV. Correlative evidence implicates the CCR5-chemokine axis in multiple sclerosis, Rasmussen encephalitis, progressive multifocal leukoencephalopathy-associated immune reconstitution inflammatory syndrome, and infectious diseases, such as cerebral malaria and HIV-associated neurocognitive disorders. On the basis of this evidence, we postulate in this Review that CCR5 antagonists, such as maraviroc, offer neuroprotective benefits in settings in which CCR5 promotes deleterious neuroinflammation, particularly in diseases in which CD8(+) T cells seem to play a pivotal role.

  2. Functional impact of HIV coreceptor-binding site mutations

    SciTech Connect

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Reeves, Jacqueline D. . E-mail: jreeves@MonogramBio.com

    2006-07-20

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.

  3. Cytokine-induced killer cells interact with tumor lysate-pulsed dendritic cells via CCR5 signaling.

    PubMed

    Lee, Hong Kyung; Kim, Yong Guk; Kim, Ji Sung; Park, Eun Jae; Kim, Boyeong; Park, Ki Hwan; Kang, Jong Soon; Hong, Jin Tae; Kim, Youngsoo; Han, Sang-Bae

    2016-08-10

    The antitumor activity of cytokine-induced killer (CIK) cells can be increased by co-culturing them with tumor lysate-pulsed dendritic cells (tDCs); this phenomenon has been studied mainly at the population level. Using time-lapse imaging, we examined how CIK cells gather information from tDCs at the single-cell level. tDCs highly expressed CCL5, which bound CCR5 expressed on CIK cells. tDCs strongly induced migration of Ccr5(+/+) CIK cells, but not that of Ccr5(-/-) CIK cells or Ccr5(+/+) CIK cells treated with the CCR5 antagonist Maraviroc. Individual tDCs contacted Ccr5(+/+) CIK cells more frequently and lengthily than with Ccr5(-/-) CIK cells. Consequently, tDCs increased the antitumor activity of Ccr5(+/+) CIK cells in vitro and in vivo, but did not increase that of Ccr5(-/-) CIK cells. Taken together, our data provide insight into the mechanism of CIK cell activation by tDCs at the single-cell level.

  4. Genotypic Prediction of Co-receptor Tropism of HIV-1 Subtypes A and C.

    PubMed

    Riemenschneider, Mona; Cashin, Kieran Y; Budeus, Bettina; Sierra, Saleta; Shirvani-Dastgerdi, Elham; Bayanolhagh, Saeed; Kaiser, Rolf; Gorry, Paul R; Heider, Dominik

    2016-04-29

    Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients.

  5. Genotypic Prediction of Co-receptor Tropism of HIV-1 Subtypes A and C

    PubMed Central

    Riemenschneider, Mona; Cashin, Kieran Y.; Budeus, Bettina; Sierra, Saleta; Shirvani-Dastgerdi, Elham; Bayanolhagh, Saeed; Kaiser, Rolf; Gorry, Paul R.; Heider, Dominik

    2016-01-01

    Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients. PMID:27126912

  6. A Randomized, Double-Blind, Placebo-Controlled Clinical Trial of a Chemokine Receptor 5 (CCR5) Antagonist to Decrease the Occurrence of Immune Reconstitution Inflammatory Syndrome in HIV-Infection: The CADIRIS Study

    PubMed Central

    Sierra-Madero, Juan G.; Ellenberg, Susan; Rassool, Mohammed S.; Tierney, Ann; Belaunzarán-Zamudio, Pablo F.; López-Martínez, Alondra; Piñeirúa-Menéndez, Alicia; Montaner, Luis J.; Azzoni, Livio; Benítez, César Rivera; Sereti, Irini; Andrade-Villanueva, Jaime; Mosqueda-Gómez, Juan L.; Rodriguez, Benigno; Sanne, Ian; Lederman, Michael M.

    2015-01-01

    Background Immune Reconstitution Inflammatory Syndrome (IRIS) is a common complication of antiretroviral therapy (ART) in HIV-infected patients. IRIS is associated with an increased risk of hospitalization and death. We ascertained whether CCR5 blockade using maraviroc reduces the risk of IRIS. Methods The CADIRIS study was a randomized, double-blind, placebo-controlled, clinical trial that accrued subjects from five clinical sites in Mexico and one in South Africa between November 2009 and January 2012, and followed them for one year. The primary outcome was occurrence of IRIS by 24 weeks. HIV-infected adults, naïve to ART, with CD4 cells <100/μL, and HIVRNA >1,000 copies/mL were eligible. We screened 362 subjects; 279 met inclusion criteria, 3 refused participation, and 276 were randomized. Participants received maraviroc 600 mg twice daily or placebo added to an ART regimen that included tenofovir, emtricitabine, and efavirenz for 48 weeks. Findings There were 276 patients randomized (140 received maraviroc and 136 placebo). There was no difference in the time to IRIS events between treatment arms (HR 1·08, 95% CI (0·66, 1·77), log-rank test p=0·743). In total, 64 (23%) patients had IRIS events, 33 (24%) in the maraviroc arm and 31 (23%) in the placebo arm (p=0·88). Interpretation Maraviroc had no significant effect on frequency, time or severity of IRIS events after ART initiation. Including a CCR5 inhibitor in an initial treatment regimen does not confer a meaningful protection from the occurrence of IRIS in persons with advanced HIV infection. Funding The trial was funded as investigator initiated research by Pfizer Inc, New York, NY, USA. Trial Registration ClinicalTrials.gov. ID: NCT00988780 (http://clinicaltrials.gov/ct2/show/NCT00988780) PMID:26366430

  7. Chemokine receptor CCR5 genotype influences the kinetics of human immunodeficiency virus type 1 infection in human PBL-SCID mice.

    PubMed

    Picchio, G R; Gulizia, R J; Mosier, D E

    1997-09-01

    Individuals homozygous for a 32-bp deletion (delta 32) in the CCR5 gene encoding the coreceptor for macrophage-tropic human immunodeficiency virus type 1 (HIV-1) are resistant to virus infection, and heterozygous individuals show some slowing of disease progression. The impact of the CCR5 genotype on HIV-1 infection was assessed in vitro and in the human PBL-SCID (hu-PBL-SCID) model. Cells and hu-PBL-SCID mice from CCR5 delta 32/delta 32 donors were resistant to infection with macrophage-tropic HIV-1 and showed slower replication of dual-tropic HIV-1. hu-PBL-SCID mice derived from CCR5 delta 32/+ heterozygotes showed delayed replication of macrophage-tropic HIV-1 despite a small and variable effect of heterozygosity on viral replication in vitro. The level of CCR5 expression appears to limit replication of macrophage-tropic and dual-tropic HIV-1 strains in vivo.

  8. CCR5/CD4/CXCR4 oligomerization prevents HIV-1 gp120IIIB binding to the cell surface

    PubMed Central

    Martínez-Muñoz, Laura; Barroso, Rubén; Dyrhaug, Sunniva Y.; Navarro, Gemma; Lucas, Pilar; Soriano, Silvia F.; Vega, Beatriz; Costas, Coloma; Muñoz-Fernández, M. Ángeles; Santiago, César; Frade, José Miguel Rodríguez; Franco, Rafael; Mellado, Mario

    2014-01-01

    CCR5 and CXCR4, the respective cell surface coreceptors of R5 and X4 HIV-1 strains, both form heterodimers with CD4, the principal HIV-1 receptor. Using several resonance energy transfer techniques, we determined that CD4, CXCR4, and CCR5 formed heterotrimers, and that CCR5 coexpression altered the conformation of both CXCR4/CXCR4 homodimers and CD4/CXCR4 heterodimers. As a result, binding of the HIV-1 envelope protein gp120IIIB to the CD4/CXCR4/CCR5 heterooligomer was negligible, and the gp120-induced cytoskeletal rearrangements necessary for HIV-1 entry were prevented. CCR5 reduced HIV-1 envelope-induced CD4/CXCR4-mediated cell-cell fusion. In nucleofected Jurkat CD4 cells and primary human CD4+ T cells, CCR5 expression led to a reduction in X4 HIV-1 infectivity. These findings can help to understand why X4 HIV-1 strains infection affect T-cell types differently during AIDS development and indicate that receptor oligomerization might be a target for previously unidentified therapeutic approaches for AIDS intervention. PMID:24778234

  9. Involvement of Spinal CCR5/PKCγ Signaling Pathway in the Maintenance of Cancer-Induced Bone Pain.

    PubMed

    Hang, Li-Hua; Li, Shu-Na; Dan, Xiang; Shu, Wei-Wei; Luo, Hong; Shao, Dong-Hua

    2017-02-01

    Cancer-induced bone pain (CIBP) is a challenging medical problem that considerably influences cancer patients' quality of life. Currently, few treatments have been developed to conquer CIBP because of a poor understanding of the potential mechanisms. Our previous work has proved that spinal RANTES (a major ligand for CCR5) was involved in the maintenance of CIBP. In this study, we attempted to investigate whether spinal CCR5 and its downstream PKCγ pathway is involved in the maintenance of CIBP. Inoculation of Walker 256 cells into the tibia could induce a marked mechanical allodynia with concomitant upregulation of spinal CCR5 and p-PKCγ expression from day 6 to day 15 after inoculation. Spinal CCR5 was prominently expressed in microglia, and mechanical allodynia was attenuated by intrathecal injection of DAPTA (a specific antagonist of CCR5) with downregulation of spinal CCR5 and p-PKCγ expression levels at day 15 in inoculated rats. Pre-intrathecal injection of RANTES could reverse the anti-allodynia effects of DAPTA. Intrathecal administration of GF109203X (an inhibitor of PKC) could alleviate mechanical allodynia as well as decrease of spinal p-PKCγ expression level, but no influence on spinal CCR5 level. Our findings suggest that CCR5/PKCγ signaling pathway in microglia may contribute to the maintenance of CIBP in rats.

  10. Role of CCR5, CCR2 and SDF-1 gene polymorphisms in a population of HIV-1 infected individuals.

    PubMed

    Mazzucchelli, R; Corvasce, S; Violin, M; Riva, C; Bianchi, R; Dehò, L; Velleca, R; Cibella, J; Bada, M; Moroni, M; Galli, M; Balotta, C

    2001-01-01

    The finding that in addition to CD4 molecule HIV-1 uses, CCR5 or CXCR4 receptors to enter target cells prompted the research to identify polymorphisms in coreceptor genes affecting disease progression. In this study we analyzed the prevalence of CCR5-delta32, CCR2-641 and SDF1-3'A alleles in a highly selected group of 42 Long-Term Nonprogressors (LTNPs) compared to 112 subjects with a typical course of HIV-1 infection (TPs) and 117 healthy controls (HCs). In addition, we correlated CCR5, CCR2 and SDF-1 genotypes with molecular indexes of HIV-1 replication, cell-free RNA and both unspliced (US) and multiply spliced (MS) intracellular transcripts, to investigate the role of the mutant alleles in determining a long-term nonprogressive course of HIV-1 disease. Our results indicate a significantly higher prevalence of CCR5-delta32 allele in LTNPs compared to TPs (p=0.0434), while the proportions of CCR2-64I and SDF1-3'A alleles were comparable between the two groups. However, SDF-1 wild type LTNP subjects showed significantly lower levels of HIV-1 genomic RNA, US and MS transcripts than SDF1-3'A heterozygous ones (p=0.0021, 0.016, 0.0031, respectively), whereas both CCR5 and CCR2 wild type individuals had similar rates of viral replication compared to CCR5-delta32 and CCR2-64I heterozygous ones. CCR5, CCR2 and SDF-1 combined genotypes were also studied and this analysis did not identify a specific protective cluster of alleles in LTNPs. Taken together, our results indicate that genetic background involving CCR5, CCR2 and SDF-1 alleles may play a limited role in the natural history of HIV-1 infection.

  11. Response to Vicriviroc in Treatment-Experienced Subjects Using an Enhanced Sensitivity Co-receptor Tropism Assay: Reanalysis of AIDS Clinical Trials Group A5211

    PubMed Central

    Su, Zhaohui; Gulick, Roy M.; Krambrink, Amy; Coakley, Eoin; Hughes, Michael D.; Han, Dong; Flexner, Charles; Wilkin, Timothy J.; Skolnik, Paul R.; Greaves, Wayne L.; Kuritzkes, Daniel R.; Reeves, Jacqueline D.

    2009-01-01

    The enhanced sensitivity Trofile assay was used to re-test co-receptor usage at study screening and entry for the 118 ACTG A5211 treatment-experienced subjects who had CCR5-tropic (R5) virus by the original Trofile assay at study screening. Among 90 vicriviroc recipients, a significantly (P<0.001) greater mean reduction in HIV-1 RNA was observed in 72 subjects with R5 virus versus 15 subjects reclassified with dual/mixed-tropic viruses at screening: −1.11 vs. −0.09 (day 14), −1.91 vs. −0.57 (week 24) log10 copies/mL, respectively. Results suggest that the enhanced sensitivity assay is a better screening tool for determining patient eligibility for CCR5 antagonist therapy. PMID:19874179

  12. Identification of potential CCR5 inhibitors through pharmacophore-based virtual screening, molecular dynamics simulation and binding free energy analysis.

    PubMed

    Wang, Juan; Shu, Mao; Wang, Yuanqiang; Hu, Yong; Wang, Yuanliang; Luo, Yanfeng; Lin, Zhihua

    2016-10-18

    CC chemokine receptor 5 (CCR5), a member of G protein-coupled receptors (GPCRs), plays a vital role in inflammatory responses to infection. Alterations in the expression of CCR5 have been correlated with disease progression in many types of cancers. The idea of using CCR5 as a target for therapeutic intervention has been demonstrated to prevent disease progression. To date, only a few compounds have been reported as CCR5 inhibitors. In this study, a series of CCR5 antagonists were used to construct pharmacophore models. Then the optimal model was utilized as a 3D query to identify novel chemical entities from structural databases. After refinement by molecular docking, drug-likeness analysis, molecular dynamics simulations (MDS) and binding free energy analysis, three potential inhibitors (25, 29 and 45) were identified. MD simulations suggested that the screened compounds retained the important common binding mode known for CCR5 inhibitors (maraviroc and nifeviroc), which occupied the bottom of a pocket and stabilized the conformation of CCR5. During the binding process, van der Waals interactions provided the substantial driving force. The most favorable contributions were from Tyr37, Trp86, Tyr89, Tyr108, Phe109, Phe112, Gln194, Thr195, Ile198, Trp248, Tyr251, Leu255, Thr259, Met279, Glu283 and Met287. The above results suggest that the hybrid strategy would provide a basis for rational drug design.

  13. Infection of macrophages and dendritic cells with primary R5-tropic human immunodeficiency virus type 1 inhibited by natural polyreactive anti-CCR5 antibodies purified from cervicovaginal secretions.

    PubMed

    Eslahpazir, Jobin; Jenabian, Mohammad-Ali; Bouhlal, Hicham; Hocini, Hakim; Carbonneil, Cédric; Grésenguet, Gérard; Mbopi Kéou, François-Xavier; LeGoff, Jérôme; Saïdi, Héla; Requena, Mary; Nasreddine, Nadine; de Dieu Longo, Jean; Kaveri, Srinivas V; Bélec, Laurent

    2008-05-01

    Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection.

  14. Infection of Macrophages and Dendritic Cells with Primary R5-Tropic Human Immunodeficiency Virus Type 1 Inhibited by Natural Polyreactive Anti-CCR5 Antibodies Purified from Cervicovaginal Secretions▿

    PubMed Central

    Eslahpazir, Jobin; Jenabian, Mohammad-Ali; Bouhlal, Hicham; Hocini, Hakim; Carbonneil, Cédric; Grésenguet, Gérard; Kéou, François-Xavier Mbopi; LeGoff, Jérôme; Saïdi, Héla; Requena, Mary; Nasreddine, Nadine; de Dieu Longo, Jean; Kaveri, Srinivas V.; Bélec, Laurent

    2008-01-01

    Heterosexual contact is the primary mode of human immunodeficiency virus (HIV) type 1 (HIV-1) transmission worldwide. The chemokine receptor CCR5 is the major coreceptor that is associated with the mucosal transmission of R5-tropic HIV-1 during sexual intercourse. The CCR5 molecule is thus a target for antibody-based therapeutic strategies aimed at blocking HIV-1 entry into cells. We have previously demonstrated that polyreactive natural antibodies (NAbs) from therapeutic preparations of immunoglobulin G and from human breast milk contain NAbs directed against CCR5. Such antibodies inhibit the infection of human macrophages and T lymphocytes by R5-tropic isolates of HIV in vitro. In the present study, we demonstrate that human immunoglobulins from the cervicovaginal secretions of HIV-seronegative or HIV-seropositive women contain NAbs directed against the HIV-1 coreceptor CCR5. Natural affinity-purified anti-CCR5 antibodies bound to CCR5 expressed on macrophages and dendritic cells and further inhibited the infection of macrophages and dendritic cells with primary and laboratory-adapted R5-tropic HIV but not with X4-tropic HIV. Natural anti-CCR5 antibodies moderately inhibited R5-tropic HIV transfer from monocyte-derived dendritic cells to autologous T cells. Our results suggest that mucosal anti-CCR5 antibodies from healthy immunocompetent donors may hamper the penetration of HIV and may be suitable for use in the development of novel passive immunotherapy regimens in specific clinical settings of HIV infection. PMID:18353923

  15. CXCR6-Mediated Simian Immunodeficiency Virus SIVagmSab Entry into Sabaeus African Green Monkey Lymphocytes Implicates Widespread Use of Non-CCR5 Pathways in Natural Host Infections.

    PubMed

    Wetzel, Katherine S; Yi, Yanjie; Elliott, Sarah T C; Romero, Dino; Jacquelin, Beatrice; Hahn, Beatrice H; Muller-Trutwin, Michaela; Apetrei, Cristian; Pandrea, Ivona; Collman, Ronald G

    2017-02-15

    African green monkeys (AGM) and sooty mangabeys (SM) are well-studied natural hosts of simian immunodeficiency virus (SIV) that do not progress to AIDS when infected with their species-specific viruses. Natural hosts of SIV express very low levels of the canonical entry coreceptor CCR5, and recent studies have shown that CCR5 is dispensable for SIV infection of SM in vivo and that blocking of CCR5 does not prevent ex vivo infection of peripheral blood mononuclear cells (PBMC) from SM or vervet AGM. In both hosts, CXCR6 is an efficient entry pathway in vitro Here we investigated the use of species-matched CXCR6 and other alternative coreceptors by SIVagmSab, which infects sabaeus AGM. We cloned sabaeus CD4 and 10 candidate coreceptors. Species-matched CXCR6, CCR5, and GPR15 mediated robust entry into transfected cells by pseudotypes carrying SIVagmSab92018ivTF Env, with lower-level entry through GPR1 and APJ. We cloned genetically divergent env genes from the plasma of two wild-infected sabaeus AGM and found similar patterns of coreceptor use. Titration experiments showed that CXCR6 and CCR5 were more efficient than other coreceptors when tested at limiting CD4/coreceptor levels. Finally, blocking of CXCR6 with its ligand CXCL16 significantly inhibited SIVagmSab replication in sabaeus PBMC and had a greater impact than did the CCR5 blocker maraviroc, confirming the use of CXCR6 in primary lymphocyte infection. These data suggest a new paradigm for SIV infection of natural host species, whereby a shared outcome of virus-host coevolution is the use of CXCR6 or other alternative coreceptors for entry, which may direct SIV toward CD4(+) T cell subsets and anatomical sites that support viral replication without disrupting immune homeostasis and function.

  16. Protective effect of CCR5 Delta-32 allele against HIV-1 in Mexican women.

    PubMed

    Estrada-Aguirre, Jesús A; Cázarez-Salazar, Silvestre G; Ochoa-Ramírez, Luis A; Acosta-Cota, Selene de J; Zamora-Gómez, Román; Najar-Reyes, Guilermi M; Villarreal-Escamilla, Perla; Osuna-Ramírez, Ignacio; Díaz-Camacho, Sylvia P; Sánchez-Zazueta, Jorge G; Ríos-Tostado, Juan J; Velarde-Félix, Jesús S

    2013-09-01

    C-C chemokine receptor type 5 (CCR5) is known for its role as a co-receptor for HIV-1 infection. Some individuals possess a 32 bp deletion, known as Delta-32 allele which has been reported to confer resistance to HIV-1 infection. In order to estimate the distribution of Delta-32 allele of CCR5 gene, 1034 mestizo individuals from the Northwest of Mexico, including 385 HIV-1-infected individuals, 472 healthy controls and 177 uninfected female sex workers; were examined by allele-specific PCR. There was no statistically significant difference in the frequency of Delta-32 allele between HIV-1 positive and healthy individuals (OR= 1.1, p= 0.6). However, we found a significantly reduced prevalence of CCR5 Delta-32 heterozygous genotype in female patients (OR= 0.084, 95% CI= 0.011 - 0.630, p= 0.002), as well as in allele frequency, compared to male patients. Furthermore, we observed an inverse relationship between allele frequency and the risk of HIV-1 transmission and AIDS progression among female healthy controls, sex workers and HIV-1 infected groups. Our findings support previous data showing Delta-32 as a genetic protective factor against HIV-1 infection in Mexican women, as well as in women from other populations.

  17. Class B β-arrestin2-dependent CCR5 signalosome retention with natural antibodies to CCR5.

    PubMed

    Venuti, Assunta; Pastori, Claudia; Pennisi, Rosamaria; Riva, Agostino; Sciortino, Maria Teresa; Lopalco, Lucia

    2016-12-23

    CCR5 stimulation with natural ligands, such as RANTES, classically induces short-term internalization with transient activation of β-arrestins and rapidly recycling on the cell surface. Here we discovered that, in T cells, natural CCR5 antibodies induce a CCR5-negative phenotype with the involvement of β-arrestin2, which leads to the formation of a stable CCR5 signalosome with both β-arrestin2 and ERK1. The activation of β-arrestin2 is necessary to CCR5 signaling for the signalosome formation and stabilization. When all stimuli were washed out, β-arrestin1 silencing favors the activity of β-arrestin2 for the CCR5 signalosome retention. Interestingly, CCR5 turn from Class A trafficking pattern, normally used for its internalization with natural modulating molecules (i.e. RANTES), into a long lasting Class B type specifically induced by stimulation with natural anti-CCR5 antibodies. This new CCR5 pathway is relevant not only to study in depth the molecular basis of all pathologies where CCR5 is involved but also to generate new antidody-based therapeutics.

  18. Class B β-arrestin2-dependent CCR5 signalosome retention with natural antibodies to CCR5

    PubMed Central

    Venuti, Assunta; Pastori, Claudia; Pennisi, Rosamaria; Riva, Agostino; Sciortino, Maria Teresa; Lopalco, Lucia

    2016-01-01

    CCR5 stimulation with natural ligands, such as RANTES, classically induces short-term internalization with transient activation of β-arrestins and rapidly recycling on the cell surface. Here we discovered that, in T cells, natural CCR5 antibodies induce a CCR5-negative phenotype with the involvement of β-arrestin2, which leads to the formation of a stable CCR5 signalosome with both β-arrestin2 and ERK1. The activation of β-arrestin2 is necessary to CCR5 signaling for the signalosome formation and stabilization. When all stimuli were washed out, β-arrestin1 silencing favors the activity of β-arrestin2 for the CCR5 signalosome retention. Interestingly, CCR5 turn from Class A trafficking pattern, normally used for its internalization with natural modulating molecules (i.e. RANTES), into a long lasting Class B type specifically induced by stimulation with natural anti-CCR5 antibodies. This new CCR5 pathway is relevant not only to study in depth the molecular basis of all pathologies where CCR5 is involved but also to generate new antidody-based therapeutics. PMID:28008933

  19. Predicted HIV-1 coreceptor usage among Kenya patients shows a high tendency for subtype d to be cxcr4 tropic

    PubMed Central

    2012-01-01

    Background CCR5 antagonists have clinically been approved for prevention or treatment of HIV/AIDS. Countries in Sub-Saharan Africa with the highest burden of HIV/AIDS are due to adopt these regimens. However, HIV-1 can also use CXCR4 as a co-receptor. There is hence an urgent need to map out cellular tropism of a country’s circulating HIV strains to guide the impending use of CCR5 antagonists. Objectives To determine HIV-1 coreceptor usage among patients attending a comprehensive care centre in Nairobi, Kenya. Methods Blood samples were obtained from HIV infected patients attending the comprehensive care centre, Kenyatta National Hospital in years 2008 and 2009. The samples were separated into plasma and peripheral blood mononuclear cells (PBMCs). Proviral DNA was extracted from PBMCs and Polymerase Chain reaction (PCR) done to amplify the HIV env fragment spanning the C2-V3 region. The resultant fragment was directly sequenced on an automated sequencer (ABI, 3100). Co-receptor prediction of the env sequences was done using Geno2pheno [co-receptor], and phylogenetic relationships determined using CLUSTALW and Neighbor Joining method. Results A total of 67 samples (46 treatment experienced and 21 treatment naive) were successfully amplified and sequenced. Forty nine (73%) sequences showed a prediction for R5 tropism while 18(27%) were X4 tropic. Phylogenetic analysis showed that 46(69%) were subtype A, 11(16%) subtype C, and 10(15%) subtype D. No statistical significant associations were observed between cell tropism and CD4+ status, patient gender, age, or treatment option. There was a tendency for more X4 tropic strains being in the treatment experienced group than the naive group: Of 46 treatment experiencing participants, 14(30%) harboured X4, compared with 4(19%) of 21 of the treatment-naïve participants, the association is however not statistically significant (p = 0.31). However, a strong association was observed between subtype D and CXCR4 co

  20. Prediction of HIV-1 coreceptor usage (tropism) by sequence analysis using a genotypic approach.

    PubMed

    Sierra, Saleta; Kaiser, Rolf; Lübke, Nadine; Thielen, Alexander; Schuelter, Eugen; Heger, Eva; Däumer, Martin; Reuter, Stefan; Esser, Stefan; Fätkenheuer, Gerd; Pfister, Herbert; Oette, Mark; Lengauer, Thomas

    2011-12-01

    Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 2011. For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3). This region carries enough information to perform a reliable prediction. The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL < 1000 copies/μl roughly increased in the last years (Fig. 3). The main steps for tropism testing (Fig. 4) demonstrated in

  1. Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9.

    PubMed

    Li, Chang; Guan, Xinmeng; Du, Tao; Jin, Wei; Wu, Biao; Liu, Yalan; Wang, Ping; Hu, Bodan; Griffin, George E; Shattock, Robin J; Hu, Qinxue

    2015-08-01

    CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5(Δ32) variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4(+) T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4(+) T-lymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4(+) T-cells utilizing adenovirus-delivered CRISPR/Cas9.

  2. An extended CCR5-ECL2 peptide forms a helix that binds HIV-1 gp120 through non-specific hydrophobic interactions

    PubMed Central

    Kessler, Naama; Arshava, Boris; Naider, Fred; Scherf, Tali; Anglister, Jacob

    2015-01-01

    The chemokine receptor CCR5 serves as a co-receptor for the Human Immunodefficiency Virus type-1, HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing the ECL1 and ECL3 were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study we determined that in aqueous buffer the segment Q188-Q194 in an elongated ECL2 peptide (R168 to K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two dimensional Saturation Transfer Difference NMR spectroscopy and dynamic filtering studies revealed the involvement of Y187, F189, W190 and F193 of the helical segment, in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule and therefore, could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. PMID:25703038

  3. Frequency of the CCR5 delta 32 mutant allele in HIV-1-positive patients, female sex workers, and a normal population in Taiwan.

    PubMed

    Li, C; Yan, Y P; Shieh, B; Lee, C M; Lin, R Y; Chen, Y M

    1997-12-01

    A specific 32-nucleotide deletion mutant of the CCR5 gene (Accr5), the coreceptor gene for human immunodeficiency virus type 1 (HIV-1), can effectively suppress the transmission and pathogenesis of the virus. Individuals homozygous for the delta ccr5 allele resist primary macrophage-tropic HIV-1 infection, despite multiple high-risk sexual exposures. This gene deletion is relatively common among Caucasians but uncommon among Africans, Asians, and South Americans. We used polymerase chain reaction (PCR) technology to determine the frequency of the delta ccr5 allele in a Taiwanese population with diverse health status and social backgrounds. Subjects included 24 HIV-1-infected persons in the northern and southern parts of Taiwan; 131 HIV-1 high-risk, licensed female sex workers in the northern part of the island (21% of whom were aborigines); and 187 unrelated, healthy, HIV-1-negative individuals in southern Taiwan. PCR with primers encompassing the entire CCR5 gene was used to explore possible deletions at regions other than the 32-nucleotide area in the female sex workers. No ccr5 deletions were detected, indicating that they are rare or absent in the Taiwanese population. This finding implies that delta ccr5 is not likely to be part of the defense against the spread of HIV-1-infection in Taiwanese.

  4. Functional analysis of naturally occurring mutations in the open reading frame of CCR5 in HIV-infected Chinese patients and healthy controls.

    PubMed

    Zhao, Xiu-Ying; Lee, Shui-Shan; Wong, Ka-Hing; Chan, Kenny C W; Ng, Fai; Chan, Chris C S; Han, Dan; Yam, Wing-Cheong; Yuen, Kwok-Yung; Ng, Mun-Hon; Zheng, Bo-Jian

    2005-04-15

    We studied polymorphism of the HIV coreceptor CC chemokine receptor (CCR) 5 in 1099 Chinese adults residing in Hong Kong, including 785 HIV-negative healthy donors and 314 HIV-positive patients. Ten mutants in the CCR5 open reading frame were identified, 7 of which were nonsynonymous. The frequencies of these alleles did not show a significant difference between HIV patients and healthy controls. G106R, Delta32, R223Q, 299(FS), and S336I were cloned from prevalent mutant genes, and their effects on HIV infection were analyzed by a series of in vitro experiments to determine their transcription levels, expression levels, conformational changes, and HIV coreceptor function. R223Q is the most prevalent CCR5 mutant in ethnic Chinese, with a frequency of 0.046, which does not affect HIV infection in vitro, however. The S336I mutant also does not affect its transcription, expression, or HIV coreceptor function. Similar to 299(FS), the mutant G106R located in the third transmembrane domain results in diminished HIV coreceptor function in vitro through conformation changes in ECL2.

  5. Attenuation of rodent neuropathic pain by an orally active peptide, RAP-103, which potently blocks CCR2- and CCR5-mediated monocyte chemotaxis and inflammation.

    PubMed

    Padi, Satyanarayana S V; Shi, Xiang Q; Zhao, Yuan Q; Ruff, Michael R; Baichoo, Noel; Pert, Candace B; Zhang, Ji

    2012-01-01

    Chemokine signaling is important in neuropathic pain, with microglial cells expressing CCR2 playing a well-established key role. DAPTA, a HIV gp120-derived CCR5 entry inhibitor, has been shown to inhibit CCR5-mediated monocyte migration and to attenuate neuroinflammation. We report here that as a stabilized analog of DAPTA, the short peptide RAP-103 exhibits potent antagonism for both CCR2 (half maximal inhibitory concentration [IC50] 4.2 pM) and CCR5 (IC50 0.18 pM) in monocyte chemotaxis. Oral administration of RAP-103 (0.05-1 mg/kg) for 7 days fully prevents mechanical allodynia and inhibits the development of thermal hyperalgesia after partial ligation of the sciatic nerve in rats. Administered from days 8 to 12, RAP-103 (0.2-1 mg/kg) reverses already established hypersensitivity. RAP-103 relieves behavioral hypersensitivity, probably through either or both CCR2 and CCR5 blockade, because by using genetically deficient animals, we demonstrated that in addition to CCR2, CCR5 is also required for the development of neuropathic pain. Moreover, RAP-103 is able to reduce spinal microglial activation and monocyte infiltration, and to inhibit inflammatory responses evoked by peripheral nerve injury that cause chronic pain. Our findings suggest that targeting CCR2/CCR5 should provide greater efficacy than targeting CCR2 or CCR5 alone, and that dual CCR2/CCR5 antagonist RAP-103 has the potential for broad clinical use in neuropathic pain treatment.

  6. Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV

    PubMed Central

    Tebas, Pablo; Stein, David; Tang, Winson W.; Frank, Ian; Wang, Shelley Q.; Lee, Gary; Spratt, S. Kaye; Surosky, Richard T.; Giedlin, Martin A.; Nichol, Geoff; Holmes, Michael C.; Gregory, Philip D.; Ando, Dale G.; Kalos, Michael; Collman, Ronald G.; Binder-Scholl, Gwendolyn; Plesa, Gabriela; Hwang, Wei-Ting; Levine, Bruce L.; June, Carl H.

    2014-01-01

    BACKGROUND CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene (“gene editing”) — in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN) — is safe. METHODS We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (−1.81 cells per day) was significantly less than the decline in unmodified cells (−7.25 cells per day) (P = 0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National

  7. Common promoter deletion is associated with 3.9-fold differential transcription of ovine CCR5 and reduced proviral level of ovine progressive pneumonia virus.

    PubMed

    White, S N; Mousel, M R; Reynolds, J O; Lewis, G S; Herrmann-Hoesing, L M

    2009-10-01

    Chemokine (C-C motif) Receptor 5 (CCR5) is a chemokine receptor that regulates immune cell recruitment in inflammation and serves as a coreceptor for human immunodeficiency virus (HIV). A human CCR5 coding deletion (termed delta-32) results in strong resistance to HIV infection, and sequence variants in CCR5 regulatory regions have been implicated in delayed progression to acquired immune deficiency syndrome. Both ovine progressive pneumonia virus (OPPV), also known as maedi-visna, and HIV are macrophage-tropic lentiviruses, have similar genomic structures, and cause lifelong persistent host infection, suggesting CCR5 may have a role in regulating OPPV provirus levels. Therefore, the ovine CCR5 genomic sequence was determined, and sequence variants were obtained from the open reading frame and surrounding regulatory sites. One CCR5 variant contained a 4-base deletion within a binding site for octamer transcription factors in the promoter region. A test for differential transcription from each allele in heterozygous animals showed a 3.9-fold transcription difference (P < 0.0001). OPPV proviral levels were also measured in 351 naturally exposed Rambouillet, Polypay and Columbia sheep. Deletion homozygotes showed reduced OPPV proviral levels among these animals (P < 0.01). The association of this CCR5 promoter deletion with OPPV levels will need to be validated in additional populations before the deletion can be recommended for widespread use in marker-assisted selection. However, because of the large impact on transcription and because CCR5 has roles in inflammation, recruitment of effector cells, and cell-mediated immunity, this deletion may play a role in the control of infections of many diverse pathogens of sheep.

  8. Analysis of the CCR5 gene coding region diversity in five South American populations reveals two new non-synonymous alleles in Amerindians and high CCR5*D32 frequency in Euro-Brazilians

    PubMed Central

    2009-01-01

    The CC chemokine receptor 5 (CCR5) molecule is an important co-receptor for HIV. The effect of the CCR5*D32 allele in susceptibility to HIV infection and AIDS disease is well known. Other alleles than CCR5*D32 have not been analysed before, neither in Amerindians nor in the majority of the populations all over the world. We investigated the distribution of the CCR5 coding region alleles in South Brazil and noticed a high CCR5*D32 frequency in the Euro-Brazilian population of the Paraná State (9.3%), which is the highest thus far reported for Latin America. The D32 frequency is even higher among the Euro-Brazilian Mennonites (14.2%). This allele is uncommon in Afro-Brazilians (2.0%), rare in the Guarani Amerindians (0.4%) and absent in the Kaingang Amerindians and the Oriental-Brazilians. R223Q is common in the Oriental-Brazilians (7.7%) and R60S in the Afro-Brazilians (5.0%). A29S and L55Q present an impaired response to β-chemokines and occurred in Afro- and Euro-Brazilians with cumulative frequencies of 4.4% and 2.7%, respectively. Two new non-synonymous alleles were found in Amerindians: C323F (g.3729G > T) in Guarani (1.4%) and Y68C (g.2964A > G) in Kaingang (10.3%). The functional characteristics of these alleles should be defined and considered in epidemiological investigations about HIV-1 infection and AIDS incidence in Amerindian populations. PMID:21637640

  9. The CCL3L1-CCR5 genotype influences the development of AIDS, but not HIV susceptibility or the response to HAART

    SciTech Connect

    Bhattacharya, Tanmoy; Stanton, Jennifer; Kim, Eun - Young; Kunstman, Kevin; Phair, John; Jacobson, Lisa P; Wolinsky, Steven M

    2008-01-01

    A selective advantage against infectious diseases such as HIV/AIDS is associated with differences in the genes relevant to immunity and virus replication. The CC chemokine receptor 5 (CCR5), the principal coreceptor for HIV, and its chemokine ligands, including CCL3L1, influences the CD4+ target cells susceptibility to infection. The CCL3L1 gene is in a region of segmental duplication on the q-arm of human chromosome 17. Increased numbers of CCL3L1 gene copies that affect the gene expression phenotype might have substantial protective effects. Here we show that the population-specific CCL3L1 gene copy number and the CCR5 {Delta}32 protein-inactivating deletion that categorizes the CCL3L1-CCR5 genotype do not influence HIV/AIDS susceptibility or the robustness of immune recovery after the initiation of highly active antiretroviral therapy (HAART).

  10. Crucial involvement of the CCL3-CCR5 axis-mediated fibroblast accumulation in colitis-associated carcinogenesis in mice.

    PubMed

    Sasaki, Soichiro; Baba, Tomohisa; Shinagawa, Kei; Matsushima, Kouji; Mukaida, Naofumi

    2014-09-15

    Patients with inflammatory bowel diseases often develop colon carcinoma. Combined treatment of azoxymethane (AOM) and dextran sulfate sodium (DSS) recapitulates colitis-associated cancer in mice. AOM/DSS-induced tumor formation was reduced in CCL3- or its specific receptor, CCR5-deficient mice despite the presence of a massive infiltration of inflammatory cells. However, AOM/DSS-induced type I collagen-positive fibroblast accumulation in the colon was reduced in CCL3- or CCR5-deficient mice. This was associated with depressed expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which is expressed mainly by fibroblasts. Moreover in vitro, CCL3 induced fibroblasts to proliferate and to enhance HB-EGF expression. Furthermore, CCR5 blockade reduced tumor formation together with reduced fibroblast accumulation and HB-EGF expression, even when administered after the development of multiple colon tumors. Thus, CCL3-CCR5-mediated fibroblast accumulation may be required, in addition to leukocyte infiltration, to induce full-blown colitis-associated carcinogenesis. Our studies shed light on a therapeutic potential of CCR5 antagonist for patients with colitis-associated cancer.

  11. Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

    PubMed Central

    Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D; Younan, Patrick M; Sommer, Karen; Kowalski, John P; Hale, Malika; Stoddard, Barry; Jarjour, Jordan; Astrakhan, Alexander; Kiem, Hans-Peter; Rawlings, David J

    2016-01-01

    A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection. PMID:27741222

  12. Increasing the efficacy of radiotherapy by modulating the CCR2/CCR5 chemokine axes

    PubMed Central

    Connolly, Kelli A.; Belt, Brian A.; Figueroa, Nathania M.; Murthy, Aditi; Patel, Ankit; Kim, Minsoo; Lord, Edith M.; Linehan, David C.; Gerber, Scott A.

    2016-01-01

    Although radiotherapy (RT) is widely used to control tumor growth across many cancer types, there is a relatively high incidence of RT failure exhibited by tumor recurrence, therefore a clear need exists to achieve improved effectiveness of RT. The RT-elicited immune response largely impacts the efficacy of RT and includes immune cells that kill tumor cells, but also immunosuppressive cells, which dampen anti-tumor immunity. Using murine models in which syngeneic tumor cell lines (Colon38, Glioma261, Line1) are grown intramuscularly and treated with 15 Gy local RT, we assessed the effects of RT on both the systemic and intratumoral immune response. Here we demonstrate that RT stimulates increased production of two chemokines, CCL2 and CCL5, at the tumor site. Further, that this leads to increased CCR2+ CCR5+ monocytes in circulation and subsequently alters the intratumoral immune infiltrate favoring the largely immunosuppressive CCR2+ CCR5+ monocytes. Importantly, a CCR2/CCR5 antagonist administered daily (15 mg/kg subcutaneously) starting two days prior to RT reduces both circulating and intratumoral monocytes resulting in increased efficacy of RT in radioresponsive tumors. Overall, these data have important implications for the mechanism of RT and present a means to improve RT efficacy across many cancer types. PMID:27852031

  13. Could FIV zoonosis responsible of the breakdown of the pathocenosis which has reduced the European CCR5-Delta32 allele frequencies?

    PubMed Central

    Faure, Eric

    2008-01-01

    Background In Europe, the north-south downhill cline frequency of the chemokine receptor CCR5 allele with a 32-bp deletion (CCR5-Δ32) raises interesting questions for evolutionary biologists. We had suggested first that, in the past, the European colonizers, principally Romans, might have been instrumental of a progressively decrease of the frequencies southwards. Indeed, statistical analyses suggested strong negative correlations between the allele frequency and historical parameters including the colonization dates by Mediterranean civilisations. The gene flows from colonizers to native populations were extremely low but colonizers are responsible of the spread of several diseases suggesting that the dissemination of parasites in naive populations could have induced a breakdown rupture of the fragile pathocenosis changing the balance among diseases. The new equilibrium state has been reached through a negative selection of the null allele. Results Most of the human diseases are zoonoses and cat might have been instrumental in the decrease of the allele frequency, because its diffusion through Europe was a gradual process, due principally to Romans; and that several cat zoonoses could be transmitted to man. The possible implication of a feline lentivirus (FIV) which does not use CCR5 as co-receptor is discussed. This virus can infect primate cells in vitro and induces clinical signs in macaque. Moreover, most of the historical regions with null or low frequency of CCR5-Δ32 allele coincide with historical range of the wild felid species which harbor species-specific FIVs. Conclusion We proposed the hypothesis that the actual European CCR5 allelic frequencies are the result of a negative selection due to a disease spreading. A cat zoonosis, could be the most plausible hypothesis. Future studies could provide if CCR5 can play an antimicrobial role in FIV pathogenesis. Moreover, studies of ancient DNA could provide more evidences regarding the implications of

  14. Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway

    PubMed Central

    Belew, Ashton Trey; Meskauskas, Arturas; Musalgaonkar, Sharmishtha; Advani, Vivek M.; Sulima, Sergey O.; Kasprzak, Wojciech K.; Shapiro, Bruce A.; Dinman, Jonathan D.

    2015-01-01

    Programmed –1 ribosomal frameshift (–1 PRF) signals redirect translating ribosomes to slip back one base on messenger RNAs. Although well characterized in viruses, how these elements may regulate cellular gene expression is not understood. Here we describe a –1 PRF signal in the human mRNA encoding CCR5, the HIV-1 co-receptor. CCR5 mRNA-mediated –1 PRF is directed by an mRNA pseudoknot, and is stimulated by at least two microRNAs. Mapping the mRNA–miRNA interaction suggests that formation of a triplex RNA structure stimulates –1 PRF. A –1 PRF event on the CCR5 mRNA directs translating ribosomes to a premature termination codon, destabilizing it through the nonsense-mediated mRNA decay pathway. At least one additional mRNA decay pathway is also involved. Functional –1 PRF signals that seem to be regulated by miRNAs are also demonstrated in mRNAs encoding six other cytokine receptors, suggesting a novel mode through which immune responses may be fine-tuned in mammalian cells. PMID:25043019

  15. Performance comparison of next-generation sequencing platforms for determining HIV-1 coreceptor use

    PubMed Central

    Raymond, Stéphanie; Nicot, Florence; Jeanne, Nicolas; Delfour, Olivier; Carcenac, Romain; Lefebvre, Caroline; Cazabat, Michelle; Sauné, Karine; Delobel, Pierre; Izopet, Jacques

    2017-01-01

    The coreceptor used by HIV-1 must be determined before a CCR5 antagonist, part of the arsenal of antiretroviral drugs, is prescribed because viruses that enter cells using the CXCR4 coreceptor are responsible for treatment failure. HIV-1 tropism is also correlated with disease progression and so must be determined for virological studies. Tropism can be determined by next-generation sequencing (NGS), but not all of these new technologies have been fully validated for use in clinical practice. The Illumina NGS technology is used in many laboratories but its ability to predict HIV-1 tropism has not been evaluated while the 454 GS-Junior (Roche) is used for routine diagnosis. The genotypic prediction of HIV-1 tropism is based on sequencing the V3 region and interpreting the results with an appropriate algorithm. We compared the performances of the MiSeq (Illumina) and 454 GS-Junior (Roche) systems with a reference phenotypic assay. We used clinical samples for the NGS tropism predictions and assessed their ability to quantify CXCR4-using variants. The data show that the Illumina platform can be used to detect minor CXCR4-using variants in clinical practice but technical optimization are needed to improve quantification. PMID:28186189

  16. Global distribution of the CCR5 gene 32-basepair deletion.

    PubMed

    Martinson, J J; Chapman, N H; Rees, D C; Liu, Y T; Clegg, J B

    1997-05-01

    A mutant allele of the beta-chemokine receptor gene CCR5 bearing a 32-basepair (bp) deletion (denoted delta ccr5) which prevents cell invasion by the primary transmitting strain of HIV-1 has recently been characterized. Homozygotes for the mutation are resistant to infection, even after repeated high-risk exposures, but this resistance appears not to be total, as isolated cases of HIV-positive deletion homozygotes are now emerging. The consequence of the heterozygous state is not clear, but it may delay the progression to AIDS in infected individuals. A gene frequency of approximately 10% was found for delta ccr5 in populations of European descent, but no mutant alleles were reported in indigenous non-European populations. As the total number of non-European samples surveyed was small in comparison with the Europeans the global distribution of this mutation is far from clear. We have devised a rapid PCR assay for delta ccr5 and used it to screen 3,342 individuals from a globally-distributed range of populations. We find that delta ccr5 is not confined to people of European descent but is found at frequencies of 2-5% throughout Europe, the Middle East and the Indian subcontinent (Fig. 1). Isolated occurrences are seen elsewhere throughout the world, but these most likely represent recent European gene flow into the indigenous populations. The inter-population differences in delta ccr5 frequency may influence the pattern of HIV transmission and so will need to be incorporated into future predictions of HIV levels.

  17. Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5)

    PubMed Central

    Steen, A; Sparre-Ulrich, A H; Thiele, S; Guo, D; Frimurer, T M; Rosenkilde, M M

    2014-01-01

    Background and Purpose A conserved amino acid within a protein family indicates a significance of the residue. In the centre of transmembrane helix (TM)-5, position V:13/5.47, an aromatic amino acid is conserved among class A 7TM receptors. However, in 37% of chemokine receptors – a subgroup of 7TM receptors – it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity. Experimental Approach The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F;G286F]-CCR5 (V:13/5.47;VII:09/7.42) were determined in G-protein-and β-arrestin-coupled signalling. Computational modelling monitored changes in amino acid conformation. Key Results [L203F]-CCR5 increased the basal level of G-protein coupling (20–70% of Emax) and β-arrestin recruitment (50% of Emax) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr244 in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5 activation. On [L203F;G286F]-CCR5 the antagonist aplaviroc was converted to a superagonist. Conclusions and Implications The results imply that an aromatic amino acid in the centre of TM-5 controls the level of receptor activity. Furthermore, Ile116 acts as a gate for the movement of Tyr244 towards TM-5 in the active state, a mechanism proposed previously for the β2-adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists. PMID:24328926

  18. Partial Agonist and Biased Signaling Properties of the Synthetic Enantiomers J113863/UCB35625 at Chemokine Receptors CCR2 and CCR5.

    PubMed

    Corbisier, Jenny; Huszagh, Alexandre; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

    2017-01-13

    Biased agonism at G protein-coupled receptors constitutes a promising area of research for the identification of new therapeutic molecules. In this study we identified two novel biased ligands for the chemokine receptors CCR2 and CCR5 and characterized their functional properties. We showed that J113863 and its enantiomer UCB35625, initially identified as high affinity antagonists for CCR1 and CCR3, also bind with low affinity to the closely related receptors CCR2 and CCR5. Binding of J113863 and UCB35625 to CCR2 or CCR5 resulted in the full or partial activation of the three Gi proteins and the two Go isoforms. Unlike chemokines, the compounds did not activate G12 Binding of J113863 to CCR2 or CCR5 also induced the recruitment of β-arrestin 2, whereas UCB35625 did not. UCB35625 induced the chemotaxis of L1.2 cells expressing CCR2 or CCR5. In contrast, J113863 induced the migration of L1.2-CCR2 cells but antagonized the chemokine-induced migration of L1.2-CCR5 cells. We also showed that replacing the phenylalanine 3.33 in CCR5 TM3 by the corresponding histidine of CCR2 converts J113863 from an antagonist for cell migration and a partial agonist in other assays to a full agonist in all assays. Further analyses indicated that F3.33H substitution strongly increased the activation of G proteins and β-arrestin 2 by J113863. These results highlight the biased nature of the J113863 and UCB35625 that act either as antagonist, partial agonist, or full agonist according to the receptor, the enantiomer, and the signaling pathway investigated.

  19. Characterization of the "CCR5" Chemokine Receptor Gene

    ERIC Educational Resources Information Center

    Thomas, John C.

    2004-01-01

    The life cycle of retroviruses is an essential topic of modern cell biology instruction. Furthermore, the process of HIV viral entry into the cell is a question of great interest in basic and clinical biology. This paper describes how students can easily recover their own DNA, amplify a portion of the "CCR5" chemokine receptor gene, characterize…

  20. Blockade of the chemokine receptor, CCR5, reduces the growth of orthotopically injected colon cancer cells via limiting cancerassociated fibroblast accumulation

    PubMed Central

    Tanabe, Yamato; Sasaki, Soichiro; Mukaida, Naofumi; Baba, Tomohisa

    2016-01-01

    We previously demonstrated that cancer-associated fibroblasts (CAFs) accumulate at tumor sites through the interaction between a chemokine, CCL3, and its receptor, CCR5, in the late phase of colitis-associated colon carcinogenesis. Here we examined the effect of a CCR5 antagonist, maraviroc, on tumor growth arising from the orthotopic injection of mouse or human colon cancer cell lines into the cecal wall by focusing on CAFs. Orthotopic injection of either cell line caused tumor formation together with leukocyte infiltration and fibroblast accumulation. Concomitant oral administration of maraviroc reduced tumor formation with few effects on leukocyte infiltration. In contrast, maraviroc reduced the intratumor number of α-smooth muscle actin-positive fibroblasts, which express epidermal growth factor, a crucial growth factor for colon cancer cell growth. These observations suggest that maraviroc or other CCR5 antagonists might act as novel anti-CRC drugs to dampen CAFs, an essential cell component for tumor progression. PMID:27340784

  1. Use of four next-generation sequencing platforms to determine HIV-1 coreceptor tropism.

    PubMed

    Archer, John; Weber, Jan; Henry, Kenneth; Winner, Dane; Gibson, Richard; Lee, Lawrence; Paxinos, Ellen; Arts, Eric J; Robertson, David L; Mimms, Larry; Quiñones-Mateu, Miguel E

    2012-01-01

    HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.

  2. Combination of the CCL5-Derived Peptide R4.0 with Different HIV-1 Blockers Reveals Wide Target Compatibility and Synergic Cobinding to CCR5

    PubMed Central

    Secchi, Massimiliano; Vassena, Lia; Morin, Sébastien; Schols, Dominique

    2014-01-01

    R4.0, a synthetic CCL5/RANTES-derived peptide, exerts potent anti-HIV-1 activity via its nonactivating interaction with CCR5, the major HIV-1 coreceptor. CCR5 chronic activation may promote undesirable inflammatory effects and enhance viral infection; thus, receptor antagonism is a necessary requisite. HIV-1 gp120, CCL5, and maraviroc dock on CCR5 by sharing two receptor sites: the N terminus and the second extracellular loop. In combination studies, R4.0, CCL5, and maraviroc exhibited concomitant interactions with CCR5 and promoted synergic inhibition of HIV-1 in acute-infection assays. Furthermore, various degrees of additive/synergic HIV-1 inhibition were observed when R4.0 was tested in combination with drugs and lead compounds directed toward different viral targets (gp120, gp41, reverse transcriptase, and protease). In combination with tenofovir, R4.0 provides cross-clade synergic inhibition of primary HIV-1 isolates. Remarkably, an in vitro-generated maraviroc-resistant R5 HIV-1 strain was inhibited by R4.0 comparably to the wild-type strain, suggesting the presence of viral resistance barriers similar to those reported for CCL5. Overall, R4.0 appears to be a promising lead peptide with potential for combination in anti-HIV-1 therapy and in microbicide development to prevent sexual HIV-1 transmission. PMID:25114130

  3. Impact of Delta 32-CCR5 heterozygosity on HIV-1 genetic evolution and variability--a study of 4 individuals infected with closely related HIV-1 strains.

    PubMed

    Chalmet, Kristen; Van Wanzeele, Filip; Demecheleer, Els; Dauwe, Kenny; Pelgrom, Jolanda; Van Der Gucht, Bea; Vogelaers, Dirk; Plum, Jean; Stuyver, Lieven; Vandekerckhove, Linos; Verhofstede, Chris

    2008-09-30

    A cluster of four patients acutely infected with a genetically almost identical virus, allowed us to investigate genetic variability and disease progression in early HIV-1 infection with minimal interference of virus specific factors. Two of the patients were heterozygous for the 32-bp deletion in the CCR5 coreceptor gene. Both showed a slower disease progression with lower viral load levels and a reduced rate of genetic evolution compared to the patients with normal CCR5 alleles. During 3 years of treatment-free follow-up, the mean pairwise genetic distance increased with 1.45% and 1.58% in the two patients with a 32-bp deletion allele compared to 3.05% and 3.57% in the two patients with normal CCR5 alleles. The observed relation between slower disease progression and a reduced evolutionary rate illustrates the influence of the virus replicative capacity, here most possibly hampered by the CCR5 heterozygosity in two of the four individuals, on the genetic evolution of the virus in the host.

  4. Combination of the CCL5-derived peptide R4.0 with different HIV-1 blockers reveals wide target compatibility and synergic cobinding to CCR5.

    PubMed

    Secchi, Massimiliano; Vassena, Lia; Morin, Sébastien; Schols, Dominique; Vangelista, Luca

    2014-10-01

    R4.0, a synthetic CCL5/RANTES-derived peptide, exerts potent anti-HIV-1 activity via its nonactivating interaction with CCR5, the major HIV-1 coreceptor. CCR5 chronic activation may promote undesirable inflammatory effects and enhance viral infection; thus, receptor antagonism is a necessary requisite. HIV-1 gp120, CCL5, and maraviroc dock on CCR5 by sharing two receptor sites: the N terminus and the second extracellular loop. In combination studies, R4.0, CCL5, and maraviroc exhibited concomitant interactions with CCR5 and promoted synergic inhibition of HIV-1 in acute-infection assays. Furthermore, various degrees of additive/synergic HIV-1 inhibition were observed when R4.0 was tested in combination with drugs and lead compounds directed toward different viral targets (gp120, gp41, reverse transcriptase, and protease). In combination with tenofovir, R4.0 provides cross-clade synergic inhibition of primary HIV-1 isolates. Remarkably, an in vitro-generated maraviroc-resistant R5 HIV-1 strain was inhibited by R4.0 comparably to the wild-type strain, suggesting the presence of viral resistance barriers similar to those reported for CCL5. Overall, R4.0 appears to be a promising lead peptide with potential for combination in anti-HIV-1 therapy and in microbicide development to prevent sexual HIV-1 transmission.

  5. CCR5 knockout suppresses experimental autoimmune encephalomyelitis in C57BL/6 mice.

    PubMed

    Gu, Sun Mi; Park, Mi Hee; Yun, Hyung Mun; Han, Sang Bae; Oh, Ki Wan; Son, Dong Ju; Yun, Jae Suk; Hong, Jin Tae

    2016-03-29

    Multiple sclerosis (MS) is an inflammatory disease in which myelin in the spinal cord is damaged. C-C chemokine receptor type 5 (CCR5) is implicated in immune cell migration and cytokine release in central nervous system (CNS). We investigated whether CCR5 plays a role in MS progression using a murine model, experimental autoimmune encephalomyelitis (EAE), in CCR5 deficient (CCR5-/-) mice. CCR5-/- and CCR5+/+ (wild-type) mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) followed by pertussis toxin, after which EAE paralysis was scored for 28 days. We found that clinical scoring and EAE neuropathology were lower in CCR5-/- mice than CCR5+/+ mice. Immune cells (CD3+, CD4+, CD8+, B cell, NK cell and macrophages) infiltration and astrocytes/microglial activation were attenuated in CCR5-/- mice. Moreover, levels of IL-1β, TNF-α, IFN-γ and MCP-1 cytokine levels were decreased in CCR5-/- mice spinal cord. Myelin basic protein (MBP) and CNPase were increased while NG2 and O4 were decreased in CCR5-/- mice, indicating that demyelination was suppressed by CCR5 gene deletion. These findings suggest that CCR5 is likely participating in demyelination in the spinal cord the MS development, and that it could serve as an effective therapeutic target for the treatment of MS.

  6. Frequency of the delta ccr5 deletion allele in the urban Brazilian population.

    PubMed

    Passos, G A; Picanço, V P

    1998-04-01

    Studies on screening genes conferring resistance to HIV-1 and AIDS onset have shown a direct relationship between a 32 base pair (bp) deletion in the CCR5 beta-chemokine receptor gene (delta ccr5 mutant allele) and long survival of HIV-1 infected individuals bearing this mutation. These findings led to an interest in studies of delta ccr5 allele distribution in human populations. In the present study, polymerase chain reactions (PCR) in genomic DNA samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 193-bp product from the normal CCR5 allele and a 161-bp product from the 32-bp deletion allele. In an investigation of the urban Brazilian population we detected a 93% frequency of normal CCR5/CCR5 homozygous individuals and a 7% frequency of CCR5/delta ccr5 heterozygous individuals. The frequency of the delta ccr5 mutant allele in this population is 0.035; however, no homozygous delta ccr5 individual has been detected thus far. This is the first evidence for the contribution of the delta ccr5 allele to the genetic background of the urban Brazilian population, which is characterized by intense ethnic admixture. These findings open perspectives for further studies on the relationship between delta ccr5 allele frequency and AIDS onset in high-risk HIV-1 exposures individuals.

  7. CCR5 knockout suppresses experimental autoimmune encephalomyelitis in C57BL/6 mice

    PubMed Central

    Yun, Hyung Mun; Han, Sang Bae; Oh, Ki Wan; Son, Dong Ju; Yun, Jae Suk; Hong, Jin Tae

    2016-01-01

    Multiple sclerosis (MS) is an inflammatory disease in which myelin in the spinal cord is damaged. C-C chemokine receptor type 5 (CCR5) is implicated in immune cell migration and cytokine release in central nervous system (CNS). We investigated whether CCR5 plays a role in MS progression using a murine model, experimental autoimmune encephalomyelitis (EAE), in CCR5 deficient (CCR5−/−) mice. CCR5−/− and CCR5+/+ (wild-type) mice were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) followed by pertussis toxin, after which EAE paralysis was scored for 28 days. We found that clinical scoring and EAE neuropathology were lower in CCR5−/− mice than CCR5+/+ mice. Immune cells (CD3+, CD4+, CD8+, B cell, NK cell and macrophages) infiltration and astrocytes/microglial activation were attenuated in CCR5−/− mice. Moreover, levels of IL-1β, TNF-α, IFN-γ and MCP-1 cytokine levels were decreased in CCR5−/− mice spinal cord. Myelin basic protein (MBP) and CNPase were increased while NG2 and O4 were decreased in CCR5−/− mice, indicating that demyelination was suppressed by CCR5 gene deletion. These findings suggest that CCR5 is likely participating in demyelination in the spinal cord the MS development, and that it could serve as an effective therapeutic target for the treatment of MS. PMID:26985768

  8. Inhibition of Inflammatory and Neuropathic Pain by Targeting a Mu Opioid Receptor/Chemokine Receptor5 Heteromer (MOR-CCR5).

    PubMed

    Akgün, Eyup; Javed, Muhammad I; Lunzer, Mary M; Powers, Michael D; Sham, Yuk Y; Watanabe, Yoshikazu; Portoghese, Philip S

    2015-11-12

    Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR5 heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR5 antagonist pharmacophores linked through homologous spacers (14-24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (MCC22; 3e) with a 22-atom spacer exhibited profound antinociception (i.t. ED50 = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, MCC22 was ~3500× more potent than a mixture of mu agonist and CCR5 antagonist monovalent ligands. These data strongly suggest that MCC22 acts by bridging the protomers of a MOR-CCR5 heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR5 heteromer as a novel target for the treatment of chronic pain.

  9. CCR5 in recruitment and activation of myeloid-derived suppressor cells in melanoma.

    PubMed

    Umansky, Viktor; Blattner, Carolin; Gebhardt, Christoffer; Utikal, Jochen

    2017-04-05

    Malignant melanoma is characterized by the development of chronic inflammation in the tumor microenvironment, leading to the accumulation of myeloid-derived suppressor cells (MDSCs). Using ret transgenic mouse melanoma model, we found a significant migration of MDSCs expressing C-C chemokine receptor (CCR)5 into primary tumors and metastatic lymph nodes, which was correlated with tumor progression. An increased CCR5 expression on MDSCs was associated with elevated concentrations of CCR5 ligands in melanoma microenvironment. In vitro experiments showed that the upregulation of CCR5 expression on CD11b(+)Gr1(+) immature myeloid cells was induced by CCR5 ligands, IL-6, GM-CSF, and other inflammatory factors. Furthermore, CCR5(+) MDSCs infiltrating melanoma lesions displayed a stronger immunosuppressive pattern than their CCR5(-) counterparts. Targeting CCR5/CCR5 ligand signaling via a fusion protein mCCR5-Ig, which selectively binds and neutralizes all three CCR5 ligands, increased the survival of tumor-bearing mice. This was associated with a reduced migration and immunosuppressive potential of tumor MDSCs. In melanoma patients, circulating CCR5(+) MDSCs were increased as compared to healthy donors. Like in melanoma-bearing mice, we observed an enrichment of these cells and CCR5 ligands in tumors as compared to the peripheral blood. Our findings define a critical role for CCR5 not only in the recruitment but also in the activation of MDSCs in tumor lesions, suggesting that novel strategies of melanoma treatment could be based on blocking CCR5/CCR5 ligand interactions.

  10. CCR5 deficiency increased susceptibility to lipopolysaccharide-induced acute renal injury.

    PubMed

    Lee, Dong Hun; Park, Mi Hee; Hwang, Chul Ju; Hwang, Jae Yeon; Yoon, Hae Suk; Yoon, Do Young; Hong, Jin Tae

    2016-05-01

    C-C chemokine receptor 5 (CCR5) regulates leukocyte chemotaxis and activation, and its deficiency exacerbates development of nephritis. Therefore, we investigated the role of CCR5 during lipopolysaccharide (LPS)-induced acute kidney injury. CCR5-deficient (CCR5-/-) and wild-type (CCR5+/+) mice, both aged about 10 months, had acute renal injury induced by intraperitoneal injection of LPS (10 mg/kg). Compared with CCR5+/+ mice, CCR5-/- mice showed increased mortality and renal injury, including elevated creatinine and blood urea nitrogen levels, following LPS challenge. Compared to CCR5+/+ mice, CCR5-/- mice also exhibited greater increases in the serum concentrations of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β following LPS challenge. Furthermore, infiltration of macrophages and neutrophils, expression of intracellular adhesion molecule (ICAM)-1, and the number of apoptotic cells were more greatly increased by LPS treatment in CCR5-/- mice than in CCR5+/+ mice. The concentrations of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β were also significantly increased in the kidney of CCR5-/- mice after LPS challenge. Moreover, primary kidney cells from CCR5-/- mice showed greater increases in TNF-α production and p38 MAP kinase activation following treatment with LPS compared with that observed in the cells from CCR5+/+ mice. LPS-induced TNF-α production and apoptosis in the primary kidney cells from CCR5-/- mice were inhibited by treatment with p38 MAP kinase inhibitor. These results suggest that CCR5 deficiency increased the production of TNF-α following LPS treatment through increased activation of the p38 pathway in the kidney, resulting in renal apoptosis and leukocyte infiltration and led to exacerbation of LPS-induced acute kidney injury.

  11. CCR5 in cancer immunotherapy: More than an "attractive" receptor for T cells.

    PubMed

    González-Martín, Alicia; Mira, Emilia; Mañes, Santos

    2012-01-01

    Despite intensive study, the role of CCR5 in cancer remains elusive. We showed that CCR5 expression by both CD4+ and CD8+ T cells is necessary to boost anti-tumor responses by optimizing helper-dependent CD8+ T cell priming. Our findings could have implications for cancer treatment in patients with defective CCR5 expression.

  12. HIV co-receptor tropism prediction remains stable over time in treatment-naïve patients.

    PubMed

    Philip, Keir Ej; Macartney, Malcolm J; Conibear, Tim Cr; Smith, Colette J; Marshall, Neal; Johnson, Margaret A; Haque, Tanzina; Webster, Daniel P

    2016-06-01

    HIV co-receptor tropism determination is essential before prescribing the CCR5 antagonist maraviroc. British HIV Association guidelines suggest tropism testing may remain valid for only 90 days in antiretroviral-naïve patients. We aimed to determine the accuracy of this figure. Tropism was assessed in 26 antiretroviral-naïve patients with ongoing viral replication, sampled yearly from first clinic visit. The V3 region of HIV-1 was sequenced in triplicate, then tropism predicted using the Geno2Pheno system. Baseline tropism prediction remained valid for a median of 52 months (range 7-81). For 19/26 individuals baseline tropism remained unchanged throughout a median of 54 months follow-up; 18 R5 tropic and 1 X4 tropic. In seven patients (27%) baseline tropism switched at least once (range 1-4 switches) during follow-up; however, their baseline tropism prediction remained valid for a median of 45 months. Co-receptor tropism in treatment-naïve patients with ongoing viral replication appears highly stable over time, suggesting that baseline genotypic tropism prediction may be valid for a longer duration in patients delaying ART initiation. In this study, baseline tropism prediction remained valid for a median of 52 months, suggesting current guidelines recommending repeat testing after 90 days may be excessively conservative in their assessment of tropism stability.

  13. Porphyromonas gingivalis induces CCR5-dependent transfer of infectious HIV-1 from oral keratinocytes to permissive cells

    PubMed Central

    Giacaman, Rodrigo A; Asrani, Anil C; Gebhard, Kristin H; Dietrich, Elizabeth A; Vacharaksa, Anjalee; Ross, Karen F; Herzberg, Mark C

    2008-01-01

    Background Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells. Results Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced. Conclusion P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1. PMID:18371227

  14. Expression of CCR5, CXCR4 and DC-SIGN in Cervix of HIV-1 Heterosexually Infected Mexican Women

    PubMed Central

    Rivera-Morales, Lydia Guadalupe; Lopez-Guillen, Paulo; Vazquez-Guillen, Jose Manuel; Palacios-Saucedo, Gerardo C; Rosas-Taraco, Adrian G; Ramirez-Pineda, Antonio; Amaya-Garcia, Patricia Irene; Rodriguez-Padilla, Cristina

    2012-01-01

    Background: A number of studies have demonstrated that receptor and co-receptor expression levels which may affect viral entry, promoting cervical HIV infection. The aim was to evaluate the expression levels of CCR5, CXCR4and DC-SIGN mRNA in a sample of heterosexually HIV infected Mexican women. Methods: We enrolled twenty-six HIV heterosexual infected women attending a local infectious diseases medical unit.RNA was isolated from the cervix and gene expression analysis was performed using real-time PCR. Results: Expression rates for mRNA of CCR5 (median 1.82; range 0.003–2934) were higher than those observed for CXCR4 (0.79; 0.0061–3312) and DC-SIGN (0.33; 0.006–532) receptors (p < 0.05). A high correlation was found between the mRNA expression levels of these three receptors (rs = 0.52 to 0.85, p < 0.01). Conclusion: Levels of expression of the tested chemokine receptors in the cervix are different from each other and alsovary from woman to woman, and seem to support the suggestion that chemokine receptor expression in genital tissues may be playing a role in the HIV transmission. PMID:23115608

  15. Impact of heterozygosity for the chemokine receptor CCR5 32-bp-deleted allele on plasma virus load and CD4 T lymphocytes in perinatally human immunodeficiency virus-infected children at 8 years of age.

    PubMed

    Buseyne, F; Janvier, G; Teglas, J P; Ivanoff, S; Burgard, M; Bui, E; Mayaux, M J; Blanche, S; Rouzioux, C; Rivière, Y

    1998-10-01

    The CCR5 gene encodes one of the major human immunodeficiency virus type 1 (HIV-1) coreceptors. A 32-bp deletion in this gene (delta ccr5) is associated with relative resistance to disease progression in heterozygous HIV-1-infected persons. The effect of this mutation on virologic and immunologic parameters was determined in a cohort of 45 perinatally HIV-1-infected children prospectively followed after 5 years of age. At a median age of 8.3 years, heterozygous children had significantly lower virus load than homozygous children (median, 3.3 vs. 4.1 log copies/mL, P < .009) and higher percentages of CD4 T cells (median, 26% vs. 17%, P < .07). However, there was no discernible influence of the CCR5 genotype on the percentages of CD8 T cells (P < .27) or on HIV-specific cytotoxic T lymphocyte activities (P < .65). There was a trend for lower rates of progression to AIDS (CDC stage C) in heterozygous children. These data confirm a major role for the CCR5 coreceptor in HIV-1 pathogenesis in children.

  16. Peptide and protein-based inhibitors of HIV-1 co-receptors

    PubMed Central

    von Recum, Horst A; Pokorski, Jonathan K

    2014-01-01

    Human immunodeficiency virus (HIV) afflicts an estimated 30 million people globally, making it a continuing pandemic. Despite major research efforts, the rate of new infections has remained relatively static over time. This article reviews an emerging strategy for the treatment of HIV, the inhibition of the co-receptors necessary for HIV entry, CCR5 and CXCR4. The aim of this article is to highlight potential therapeutics derived from peptides and proteins that show particular promise in HIV treatment. Molecules that act on CCR5, CXCR4 or on both receptors will be discussed herein. PMID:23856897

  17. Decreased HIV Type 1 Transcription in CCR5-Δ32 Heterozygotes During Suppressive Antiretroviral Therapy

    PubMed Central

    Wang, Charlene; Abdel-Mohsen, Mohamed; Strain, Matthew C.; Lada, Steven M.; Yukl, Steven; Cockerham, Leslie R.; Pilcher, Christopher D.; Hecht, Frederick M.; Sinclair, Elizabeth; Liegler, Teri; Richman, Douglas D.; Deeks, Steven G.; Pillai, Satish K.

    2014-01-01

    Individuals who are heterozygous for the CCR5-Δ32 mutation provide a natural model to examine the effects of reduced CCR5 expression on human immunodeficiency virus (HIV) persistence. We evaluated the HIV reservoir in 18 CCR5-Δ32 heterozygotes and 54 CCR5 wild-type individuals during suppressive antiretroviral therapy. Cell-associated HIV RNA levels (P = .035), RNA to DNA transcriptional ratios (P = .013), and frequency of detectable HIV 2–long terminal repeat circular DNA (P = .013) were significantly lower in CD4+ T cells from CCR5-Δ32 heterozygotes. Cell-associated HIV RNA was significantly correlated with CCR5 surface expression on CD4+ T cells (r2 = 0.136; P = .002). Our findings suggest that curative strategies should further explore manipulation of CCR5. PMID:24935955

  18. Decreased HIV type 1 transcription in CCR5-Δ32 heterozygotes during suppressive antiretroviral therapy.

    PubMed

    Wang, Charlene; Abdel-Mohsen, Mohamed; Strain, Matthew C; Lada, Steven M; Yukl, Steven; Cockerham, Leslie R; Pilcher, Christopher D; Hecht, Frederick M; Sinclair, Elizabeth; Liegler, Teri; Richman, Douglas D; Deeks, Steven G; Pillai, Satish K

    2014-12-01

    Individuals who are heterozygous for the CCR5-Δ32 mutation provide a natural model to examine the effects of reduced CCR5 expression on human immunodeficiency virus (HIV) persistence. We evaluated the HIV reservoir in 18 CCR5-Δ32 heterozygotes and 54 CCR5 wild-type individuals during suppressive antiretroviral therapy. Cell-associated HIV RNA levels (P=.035), RNA to DNA transcriptional ratios (P=.013), and frequency of detectable HIV 2-long terminal repeat circular DNA (P=.013) were significantly lower in CD4+ T cells from CCR5-Δ32 heterozygotes. Cell-associated HIV RNA was significantly correlated with CCR5 surface expression on CD4+ T cells (r2=0.136; P=.002). Our findings suggest that curative strategies should further explore manipulation of CCR5.

  19. AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor.

    PubMed

    Donzella, G A; Schols, D; Lin, S W; Esté, J A; Nagashima, K A; Maddon, P J; Allaway, G P; Sakmar, T P; Henson, G; De Clercq, E; Moore, J P

    1998-01-01

    The bicyclam AMD3100 (formula weight 830) blocks HIV-1 entry and membrane fusion via the CXCR4 co-receptor, but not via CCR5. AMD3100 prevents monoclonal antibody 12G5 from binding to CXCR4, but has no effect on binding of monoclonal antibody 2D7 to CCR5. It also inhibits binding of the CXC-chemokine, SDF-1alpha, to CXCR4 and subsequent signal transduction, but does not itself cause signaling and has no effect on RANTES signaling via CCR5. Thus, AMD3100 prevents CXCR4 functioning as both a HIV-1 co-receptor and a CXC-chemokine receptor. Development of small molecule inhibitors of HIV-1 entry is feasible.

  20. Influence of nucleotide polymorphisms in the CCR2 gene and the CCR5 promoter on the expression of cell surface CCR5 and CXCR4.

    PubMed

    Shieh, B; Liau, Y E; Hsieh, P S; Yan, Y P; Wang, S T; Li, C

    2000-09-01

    Polymorphisms in the CCR2 gene (CCR2-64I) and the CCR5 promoter (pCCR5-59029G) have been correlated with slower HIV-1 disease progression. How these polymorphisms influence the rate of AIDS progression has remained unclear. We have therefore investigated whether these nucleotide polymorphisms will reduce the expression levels of surface CCR5 and CXCR4, and thus lead to slower AIDS progression. For this, a cohort of Chinese volunteers in Taiwan was subjected to the determination of CCR2 and pCCR5 genotypes followed by analysis of the surface CCR5 and CXCR4 expression on five cell types derived from peripheral blood mononuclear cells by flow cytometry. Several significant associations were detected between genotypes and expression levels of the proteins. The most important finding was that an increased number of CD4(+) cells expressing CCR5 correlated with pCCR5-59029A homozygosity without the interference of both the CCR2-64 and the CCR5 delta 32 (deleted 32 bp) mutations (P: = 0.0453), which is consistent with the previous data on the association of the genotype to AIDS progression. Since different genetic polymorphisms co-exist in human beings, the rate of AIDS progression as well as the risk of rheumatoid arthritis may be governed by the interplay of the array of nucleotide changes and their affected proteins.

  1. Temporary CXCR3 and CCR5 Antagonism Following Vaccination Enhances Memory CD8 T Cell Immune Responses

    PubMed Central

    Li, Rui; Zhang, Nan; Tian, Miaomiao; Ran, Zihan; Zhu, Mingjun; Zhu, Haiyan; Han, Fangting; Yin, Juan; Zhong, Jiang

    2016-01-01

    Although current vaccination strategies have been successful at preventing a variety of human diseases, attempts at vaccinating against some pathogens such as AIDS and tuberculosis (TB) have been more problematic, largely because abnormally high numbers of antigen-specific CD8 + T cells are required for protection. This study assessed the effect on host immune response of temporarily dampening the chemokine receptors CXCR3 and CCR5 after vaccination by administration of TAK-779, a small-molecule CXCR3 and CCR5 antagonist commonly used to inhibit HIV infection. Our results showed that use of TAK-779 enhanced memory CD8 + T cell immune responses both qualitatively and quantitatively. Treatment with TAK-779 following vaccination of an influenza virus antigen resulted in enhanced memory generation, with more CD8 + CD127 + memory precursors and fewer terminally differentiated effector CD8 + CD69 + T cells. These memory T cells were able to become IFN-γ–secreting effector cells when re-encountering the same antigen, which can further enhance the efficacy of vaccination. The mice vaccinated in the presence of TAK-779 were better protected upon influenza virus challenge than the controls. These results show that vaccination, while temporarily inhibiting chemokine receptors CXCR3 and CCR5 by TAK-779, could be a promising strategy to generate large numbers of protective memory CD8 + T cells. PMID:27447731

  2. Effects of genetic variants of CCR5 chemokine receptors on oral squamous cell carcinoma.

    PubMed

    Tanyel, C R; Cincin, Z B; Gokcen-Rohlig, B; Bektas-Kayhan, K; Unur, M; Cakmakoglu, B

    2013-11-18

    We aimed to evaluate the effect of genetic variants of the chemokine C-C motif receptor (CCR5) in the pathogenesis of oral squamous cell carcinoma (OSCC). A total of 127 patients diagnosed with OSCC and 104 healthy individuals were included in the study. The polymorphisms CCR5 59029 and CCR5-delta32 were assessed with the polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) method from peripheral blood samples of both groups. There was a statistically significant difference between the control and patient groups for CCR5 59029 A/G genotypes (P < 0.01). Individuals carrying the CCR5 59029 G allele (GG +AG genotypes) had a 2.84-fold increased risk for OSCC (P < 0.0001), and the CCR5 59029 AA genotype frequency was higher in the control group than in the patient group (P < 0.0001). The CCR5-delta 32 genotype frequencies did not differ significantly between controls and cases (P > 0.05). CCR5 59029 GG and CCR5-delta32 DD + ID genotype frequencies were significantly increased in Grade II-III OSCC patients compared with Grade I-II OSCC patients. In conclusion, these results suggested that the G allele of the CCR5 59029 polymorphism might be a risk factor due to the loss of receptor function that might cause increased inflammation leading to the development of OSCC.

  3. Variants of CCR5, which are permissive for HIV-1 infection, show distinct functional responses to CCL3, CCL4 and CCL5

    PubMed Central

    Dong, H-F; Wigmore, K; Carrington, MN; Dean, M; Turpin, JA; Howard, OMZ

    2006-01-01

    CCR5 is one of the primary coreceptors for Env-mediated fusion between cells and human immunodeficiency virus type 1 (HIV-1). Analyses of CCR5 variants in cohorts of HIV-1 high-risk individuals led to the identification of multiple single amino-acid substitutions, which may have functional consequences. This study focused on eight naturally occurring allelic variants located between amino-acid residues 60 and 334 of CCR5. All studied allelic variants were highly expressed on the cell surface of HEK-293 cells and permissive for HIV-1 infection. Variant G301V showed 3.5-fold increase in 50% effective concentration (EC50) for CCL4 (MIP 1beta) in a competitive binding assay. There was also a significant reduction in CCL5 (RANTES) EC50 for the R223Q, A335V and Y339F variants. The most unexpected functional abnormality was exhibited by the R60S variant that exhibited a loss of ligand-induced desensitization in chemotaxis assays, but showed normal CCL4 and CCL5 binding avidity. This mutation is located in the first intracellular loop, a domain that has not previously been shown to be involved in receptor desensitization. In conclusion, our results support earlier studies showing that these naturally occurring point mutations do not limit HIV-1 infection, and indicated that single amino-acid changes can have unexpected functional consequences. PMID:16015368

  4. Use of a Small Molecule CCR5 Inhibitor in Macaques to Treat Simian Immunodeficiency Virus Infection or Prevent Simian–Human Immunodeficiency Virus Infection

    PubMed Central

    Veazey, Ronald S.; Klasse, Per Johan; Ketas, Thomas J.; Reeves, Jacqueline D.; Piatak, Michael; Kunstman, Kevin; Kuhmann, Shawn E.; Marx, Preston A.; Lifson, Jeffrey D.; Dufour, Jason; Mefford, Megan; Pandrea, Ivona; Wolinsky, Steven M.; Doms, Robert W.; DeMartino, Julie A.; Siciliano, Salvatore J.; Lyons, Kathy; Springer, Martin S.; Moore, John P.

    2003-01-01

    Human immunodeficiency virus type 1 (HIV-1) fuses with cells after sequential interactions between its envelope glycoproteins, CD4 and a coreceptor, usually CC chemokine receptor 5 (CCR5) or CXC receptor 4 (CXCR4). CMPD 167 is a CCR5-specific small molecule with potent antiviral activity in vitro. We show that CMPD 167 caused a rapid and substantial (4–200-fold) decrease in plasma viremia in six rhesus macaques chronically infected with simian immunodeficiency virus (SIV) strains SIVmac251 or SIVB670, but not in an animal infected with the X4 simian–human immunodeficiency virus (SHIV), SHIV-89.6P. In three of the SIV-infected animals, viremia reduction was sustained. In one, there was a rapid, but partial, rebound and in another, there was a rapid and complete rebound. There was a substantial delay (>21 d) between the end of therapy and the onset of full viremia rebound in two animals. We also evaluated whether vaginal administration of gel-formulated CMPD 167 could prevent vaginal transmission of the R5 virus, SHIV-162P4. Complete protection occurred in only 2 of 11 animals, but early viral replication was significantly less in the 11 CMPD 167-recipients than in 9 controls receiving carrier gel. These findings support the development of small molecule CCR5 inhibitors as antiviral therapies, and possibly as components of a topical microbicide to prevent HIV-1 sexual transmission. PMID:14623909

  5. Frequency of CCR5 delta-32 mutation in human immunodeficiency virus (HIV)-seropositive and HIV-exposed seronegative individuals and in general population of Medellin, Colombia.

    PubMed

    Díaz, F J; Vega, J A; Patiño, P J; Bedoya, G; Nagles, J; Villegas, C; Vesga, R; Rugeles, M T

    2000-01-01

    Repeated exposure to human immunodeficiency virus (HIV) does not always result in seroconversion. Modifications in coreceptors for HIV entrance to target cells are one of the factors that block the infection. We studied the frequency of Delta-32 mutation in ccr5 gene in Medellin, Colombia. Two hundred and eighteen individuals distributed in three different groups were analyzed for Delta-32 mutation in ccr5 gene by polymerase chain reaction (PCR): 29 HIV seropositive (SP), 39 exposed seronegative (ESN) and 150 individuals as a general population sample (GPS). The frequency of the Delta-32 mutant allele was 3.8% for ESN, 2.7% for GPS and 1.7% for SP. Only one homozygous mutant genotype (Delta-32/Delta-32) was found among the ESN (2.6%). The heterozygous genotype (ccr5/Delta-32) was found in eight GPS (5.3%), in one SP (3.4%) and in one ESN (2.6%). The differences in the allelic and genotypic frequencies among the three groups were not statistically significant. A comparison between the expected and the observed genotypic frequencies showed that these frequencies were significantly different for the ESN group, which indirectly suggests a protective effect of the mutant genotype (Delta-32/Delta-32). Since this mutant genotype explained the resistance of infection in only one of our ESN persons, different mechanisms of protection must be playing a more important role in this population.

  6. Different Pathogenesis of CCR5-Using Primary HIV-1 Isolates from Non-Switch and Switch Virus Patients in Human Lymphoid Tissue Ex Vivo

    NASA Technical Reports Server (NTRS)

    Iarlsson, Ingrid; Grivel, Jean-Charles; Chen. Silvia; Karlsson, Anders; Albert, Jan; Fenyol, Eva Maria; Margolis, Leonid B.

    2005-01-01

    CCR5-utilizing HIV-1 variants (R5) typically transmit infection and dominate its early stages, whereas emergence of CXCR4-using (X4 or R5X4) HIV-1 is often associated with disease progression. However, such a switch in co-receptor usage can only be detected in approximately onehalf of HIV-infected patients (switch virus patients), and progression to immunodeficiency may also occur in patients without detectable switch in co-receptor usage (non-switch virus patients). Here, we used a system of ex vivo-infected tonsillar tissue to compare the pathogenesis of sequential primary R5 HIV-1 isolates from the switch and non-switch patients. Inoculation of ex vivo tissue with these R5 isolates resulted in viral replication and CCR5(+)CD4(+) T cell depletion. The levels of such depletion by HIV-1 isolated from non-switch virus patients were significantly higher than those by R5 HIV-1 isolates from switch virus patients. T cell depletion seemed to be controlled by viral factors and did not significantly vary between tissues from different donors. In contrast, viral replication did not correlate with the switch status of the patients; in tissues fiom different donors it varied 30-fold and seemed to be controlled by a combination of viral and tissue factors. Nevertheless, replication-level hierarchy among sequential isolates remained constant in tissues from various donors. Viral load in vivo was higher in switch virus patients compared to non-switch virus patients. The high cytopathogenicity of CCR5(+)CD4(+) T cells by R5 HIV-1 isolates from non-switch virus patients may explain the steady decline of CD4(+) T cells in the absence of CXCR4 using virus; elimination of target cells by these isolates may limit their own replication in vivo.

  7. Sensitive Cell-Based Assay for Determination of Human Immunodeficiency Virus Type 1 Coreceptor Tropism

    PubMed Central

    Weber, Jan; Vazquez, Ana C.; Winner, Dane; Gibson, Richard M.; Rhea, Ariel M.; Rose, Justine D.; Wylie, Doug; Henry, Kenneth; Wright, Alison; King, Kevin; Archer, John; Poveda, Eva; Soriano, Vicente; Robertson, David L.; Olivo, Paul D.; Arts, Eric J.

    2013-01-01

    CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454

  8. CCR5-CCL Axis in PDL during Orthodontic Biophysical Force Application.

    PubMed

    Lee, S Y; Yoo, H I; Kim, S H

    2015-12-01

    Tooth movement by application of orthodontic biophysical force primarily reflects the role of soluble molecules released from the periodontal ligament (PDL). Thus far, many factors have been reported to be involved in orthodontic tooth movement (OTM), but key molecules that orchestrate responses of periodontal tissues to biophysical force are still enigmatic. In this in vivo study, in which the upper first molars in rats were moved, differential display-polymerase chain reaction revealed that CC chemokine receptor 5 (CCR5) level was differentially increased during OTM. Strong immunoreactivity for CCR5 was found in the PDL undergoing force application. Moreover, the in vitro compression or tension force application to primary cultured human PDL cells increased the expression of CCR5 and CCR5 ligands. In vitro tension force on human PDL cells did not induce RANKL, an osteoclastogenesis-inducing factor, but did induce the upregulation of IL12, an osteoclast inhibitory factor, and osteoblast differentiation factors, including Runx2, which was attenuated under tension by CCR5 gene silencing whereas augmented with CCR5 ligands. In contrast, in vitro compression force did not induce the expression of osteoprotegerin, a decoy receptor for RANKL and Runx2, but did induce the upregulation of RANKL, which was attenuated under compression by CCR5 gene silencing. These results suggest that the CCR5-CCR5 ligands axis in PDL cells may play a crucial role in the remodeling of periodontal tissues and can be a therapeutic target for achieving efficient OTM.

  9. CCR5 is a suppressor for cortical plasticity and hippocampal learning and memory.

    PubMed

    Zhou, Miou; Greenhill, Stuart; Huang, Shan; Silva, Tawnie K; Sano, Yoshitake; Wu, Shumin; Cai, Ying; Nagaoka, Yoshiko; Sehgal, Megha; Cai, Denise J; Lee, Yong-Seok; Fox, Kevin; Silva, Alcino J

    2016-12-20

    Although the role of CCR5 in immunity and in HIV infection has been studied widely, its role in neuronal plasticity, learning and memory is not understood. Here, we report that decreasing the function of CCR5 increases MAPK/CREB signaling, long-term potentiation (LTP), and hippocampus-dependent memory in mice, while neuronal CCR5 overexpression caused memory deficits. Decreasing CCR5 function in mouse barrel cortex also resulted in enhanced spike timing dependent plasticity and consequently, dramatically accelerated experience-dependent plasticity. These results suggest that CCR5 is a powerful suppressor for plasticity and memory, and CCR5 over-activation by viral proteins may contribute to HIV-associated cognitive deficits. Consistent with this hypothesis, the HIV V3 peptide caused LTP, signaling and memory deficits that were prevented by Ccr5 knockout or knockdown. Overall, our results demonstrate that CCR5 plays an important role in neuroplasticity, learning and memory, and indicate that CCR5 has a role in the cognitive deficits caused by HIV.

  10. CCR5 is a suppressor for cortical plasticity and hippocampal learning and memory

    PubMed Central

    Zhou, Miou; Greenhill, Stuart; Huang, Shan; Silva, Tawnie K; Sano, Yoshitake; Wu, Shumin; Cai, Ying; Nagaoka, Yoshiko; Sehgal, Megha; Cai, Denise J; Lee, Yong-Seok; Fox, Kevin; Silva, Alcino J

    2016-01-01

    Although the role of CCR5 in immunity and in HIV infection has been studied widely, its role in neuronal plasticity, learning and memory is not understood. Here, we report that decreasing the function of CCR5 increases MAPK/CREB signaling, long-term potentiation (LTP), and hippocampus-dependent memory in mice, while neuronal CCR5 overexpression caused memory deficits. Decreasing CCR5 function in mouse barrel cortex also resulted in enhanced spike timing dependent plasticity and consequently, dramatically accelerated experience-dependent plasticity. These results suggest that CCR5 is a powerful suppressor for plasticity and memory, and CCR5 over-activation by viral proteins may contribute to HIV-associated cognitive deficits. Consistent with this hypothesis, the HIV V3 peptide caused LTP, signaling and memory deficits that were prevented by Ccr5 knockout or knockdown. Overall, our results demonstrate that CCR5 plays an important role in neuroplasticity, learning and memory, and indicate that CCR5 has a role in the cognitive deficits caused by HIV. DOI: http://dx.doi.org/10.7554/eLife.20985.001 PMID:27996938

  11. Sequential CCR5-Tropic HIV-1 Reactivation from Distinct Cellular Reservoirs following Perturbation of Elite Control

    PubMed Central

    Watters, Sarah A.; Mlcochova, Petra; Maldarelli, Frank; Goonetilleke, Nilu; Pillay, Deenan; Gupta, Ravindra K.

    2016-01-01

    Background HIV Elite Controllers may reveal insights into virus persistence given they harbour small reservoir sizes, akin to HIV non-controllers treated early with combination antiretroviral therapy. Both groups of patients represent the most promising candidates for interventions aimed at sustained remission or ‘cure’. Analytic treatment interruption (ATI) in the latter group leads to stochastic rebound of virus, though it is unclear whether loss of elite control is also associated with similar rebound characteristics. Methods We studied three discrete periods of virus rebound during myeloma related immune disruption over 2.5 years in an elite controller who previously underwent autologous stem cell transplantation (ASCT) in the absence of any antiretroviral therapy. Single genome sequencing of the V1-V4 region of env in PBMC and plasma was performed and phylogenies reconstructed. Average pairwise distance (APD) was calculated and non-parametric methods used to assess compartmentalisation. Coreceptor usage was predicted based on genotypic algorithms. Results 122 single genome sequences were obtained (median 26 sequences per rebound). The initial rebounding plasma env sequences following ASCT represented two distinct lineages, and clustered with proviral DNA sequences isolated prior to ASCT. One of the lineages was monophyletic, possibly indicating reactivation from clonally expanded cells. The second rebound occurred 470 days after spontaneous control of the first rebound and was phylogenetically distinct from the first, confirmed by compartmentalisation analysis, with a different cellular origin rather than ongoing replication. By contrast, third rebound viruses clustered with second rebound viruses, with evidence for ongoing evolution that was associated with lymphopenia and myeloma progression. Following ASCT a shift in tropism from CXCR4-tropic viruses to a CCR5-tropic population was observed to persist through to the third rebound. Conclusions Our data

  12. Highly Accurate Structure-Based Prediction of HIV-1 Coreceptor Usage Suggests Intermolecular Interactions Driving Tropism.

    PubMed

    Kieslich, Chris A; Tamamis, Phanourios; Guzman, Yannis A; Onel, Melis; Floudas, Christodoulos A

    2016-01-01

    HIV-1 entry into host cells is mediated by interactions between the V3-loop of viral glycoprotein gp120 and chemokine receptor CCR5 or CXCR4, collectively known as HIV-1 coreceptors. Accurate genotypic prediction of coreceptor usage is of significant clinical interest and determination of the factors driving tropism has been the focus of extensive study. We have developed a method based on nonlinear support vector machines to elucidate the interacting residue pairs driving coreceptor usage and provide highly accurate coreceptor usage predictions. Our models utilize centroid-centroid interaction energies from computationally derived structures of the V3-loop:coreceptor complexes as primary features, while additional features based on established rules regarding V3-loop sequences are also investigated. We tested our method on 2455 V3-loop sequences of various lengths and subtypes, and produce a median area under the receiver operator curve of 0.977 based on 500 runs of 10-fold cross validation. Our study is the first to elucidate a small set of specific interacting residue pairs between the V3-loop and coreceptors capable of predicting coreceptor usage with high accuracy across major HIV-1 subtypes. The developed method has been implemented as a web tool named CRUSH, CoReceptor USage prediction for HIV-1, which is available at http://ares.tamu.edu/CRUSH/.

  13. CCR5 small interfering RNA ameliorated joint inflammation in rats with adjuvant-induced arthritis.

    PubMed

    Duan, Hongmei; Yang, Pingting; Fang, Fang; Ding, Shuang; Xiao, Weiguo

    2014-12-01

    Rheumatoid arthritis (RA) is a systemic inflammatory disease. C-C chemokine receptor type 5 (CCR5) is found in inflamed synovium of RA patients and is necessary for formation of RA. We aimed to check whether delivery of CCR5-specific small interfering RNA (siRNA) via electroporation suppresses local inflammation in arthritis rats. Vectors encoding siRNA that target CCR5 or negative control siRNA were constructed for gene silencing and the silencing effects of suppressing CCR5 expression in synovium examined by western blot. The vector with strongest effect was delivered into the knee joint of adjuvant-induced arthritis (AIA) rats by the in vivo electroporation method 7, 10, 13, and 16 days after immunization with Complete Freund's adjuvant. During an observation of 28 days, behavior, paw swelling, arthritis and histopathologic scoring were estimated. The expression level of CCR5 in synovium was evaluated by western blot and real-time PCR. Anti-CCR5 D1 siRNA was effectively inhibited CCR5 expression in vitro. Moreover, delivery of the siRNA into inflammatory joint also suppressed the expression of CCR5 in vivo and markedly suppressed paw swelling and inflammation. Local electroporation of anti-CCR5 siRNA into the left inflamed joints could achieve the silencing of CCR5 gene and alleviate local inflammation just in the knee joint injected with siRNA other than the opposite joint. Inhibition of CCR5 expression may provide a potential for treatment of RA.

  14. Genetic variation at the chemokine receptors CCR5/CCR2 in myocardial infarction.

    PubMed

    González, P; Alvarez, R; Batalla, A; Reguero, J R; Alvarez, V; Astudillo, A; Cubero, G I; Cortina, A; Coto, E

    2001-06-01

    Our objective was to examine the association between myocardial infarction (MI) and two DNA-polymorphisms at the proinflammatory chemokine receptors CCR2 (I64V) and CCR5 (32 bp deletion, (Delta)ccr5), defining if these polymorphisms influence the age for the onset of MI. A total of 214 patients with an age at the first MI episode <55 years, 96 patients that suffered the first MI episode when older than 60 years, and 360 population controls were polymerase chain reaction genotyped for the CCR2-V64I and CCR5-Delta32/wt polymorphisms. Patients and controls were male from the same Caucasian population (Asturias, northern Spain). The frequency of the Deltaccr5 allele was significantly higher in controls compared to patients <55 years (P = 0.004), or in patients >60 years compared to patients <55 years (P = 0.002). Taking the patients >60 years as the reference group, non-carriers of the (Delta)ccr5-allele would have a three-fold higher risk of suffering an episode of MI at <55 years of age (OR = 3.06; 95% CI = 1.46-6.42). Gene and genotype frequencies for the CCR2 polymorphism did not differ between patients <55 years and controls or patients >60 years. Our data suggest that the variation at the CCR5 gene could modulate the age at the onset of MI. Patients carrying the (Delta)ccr5-allele would be protected against an early episode of MI. CCR5 and the CCR5-ligands are expressed by cells in the arteriosclerotic plaque. Thus, the protective role of (Delta)ccr5 could be a consequence of an attenuated inflammatory response, that would determine a slower progression of the arteriosclerotic lesion among (Delta)ccr5-carriers. Our work suggests that the pharmacological blockade of CCR5 could be a valuable therapy in the treatment of MI.

  15. Amino- and Carboxyl-Terminal CCR5 Mutations in Brazilian HIV-1-Infected Women and Homology Model of p.L55Q CCR5 Mutant.

    PubMed

    Costa, Giselle Calasans de Souza; Nunes, Marcio Roberto T; Jesus, Jaqueline Goes; Novaes, Thiago; Cardoso, Jedson Ferreira; Sousa Júnior, Edivaldo Costa; Santos, Edson de Souza; Galvão-Castro, Bernardo; Zanette, Dalila Luciola; Gonçalves, Marilda de Souza; Alcantara, Luiz Carlos Junior

    2015-07-01

    Genetic factors from an HIV-1 host can affect the rate of progression to AIDS and HIV infection. To investigate the frequency of mutations in the CCR5 gene, HIV-1 samples from infected women and uninfected individuals were selected for sequencing of the CCR5 gene regions encoding the N- and C-terminal protein domains. Physicochemical CCR5 modeling and potential protein domain analysis were performed in order to evaluate the impact of the mutations found in the properties and structure of CCR5. The p.L55Q mutation in the N-terminal protein domain was observed only in uninfected individuals, with an allelic frequency of 1.8%. Physicochemical analysis revealed that the p.L55Q mutation magnified the flexibility and accessibility profiles and the modeling of CCR5 structures showed resulting in a small deviation to the right, as well as a hydrophobic to hydrophilic property alteration. The p.L55Q mutation also resulted in a slight modification of the electrostatic load of this region. Additionally, three novel silent mutations were found at the C-terminal coding region among HIV-1-infected women. The results suggest that the p.L55Q mutation might alter CCR5 conformation. Further studies should be conducted to verify the role of this mutation in HIV-1 susceptibility.

  16. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.

    PubMed

    Dorr, Patrick; Westby, Mike; Dobbs, Susan; Griffin, Paul; Irvine, Becky; Macartney, Malcolm; Mori, Julie; Rickett, Graham; Smith-Burchnell, Caroline; Napier, Carolyn; Webster, Rob; Armour, Duncan; Price, David; Stammen, Blanda; Wood, Anthony; Perros, Manos

    2005-11-01

    Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 microM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.

  17. Experimental Infection with Schistosoma mansoni in CCR5-Deficient Mice Is Associated with Increased Disease Severity, as CCR5 Plays a Role in Controlling Granulomatous Inflammation▿

    PubMed Central

    Souza, Adriano L. S.; Souza, Patrícia R. S.; Pereira, Cíntia A.; Fernandes, Adriana; Guabiraba, Rodrigo; Russo, Remo C.; Vieira, Leda Q.; Corrêa, Ary; Teixeira, Mauro M.; Negrão-Corrêa, Deborah

    2011-01-01

    The plasma level of the chemokine CCL3 is elevated in patients with chronic severe schistosomiasis mansoni. We have previously shown that CCL3−/− mice with experimental infection showed diminished pathology and worm burden compared to those of wild-type (WT) mice. To elucidate further the role of CC chemokines during schistosomiasis mansoni infection, we evaluated the course of infection in C57BL/6J mice deficient in CCR5, one of the receptors for CCL3. The CCR5 deficiency proved to be remarkably deleterious to the host, since mortality rates reached 70% at 14 weeks postinfection in CCR5−/− mice and 19% in WT mice. The increased lethality was not associated with an increased parasite burden, since similar numbers of eggs and adult worms were found in mice from both groups. Liver granulomas of chronically infected CCR5−/− mice were larger and showed greater numbers of cells and collagen deposition than liver granulomas from WT mice. This was associated with higher levels of production of intereleukin-5 (IL-5), IL-13, CCL3, and CCL5 in infected CCR5−/− mice than in infected WT mice. Moreover, at 8 weeks after infection, just before changes in pathology and mortality, the numbers of FoxP3-positive cells were lower in liver granulomas of CCR5−/− mice than in WT mice. In conclusion, the CCR5 deletion is deleterious to mice infected with Schistosoma mansoni, and this is associated with enhanced fibrosis and granulomatous inflammation. PMID:21263020

  18. Immune Subversion by Mycobacterium tuberculosis through CCR5 Mediated Signaling: Involvement of IL-10

    PubMed Central

    Das, Shibali; Banerjee, Sayantan; Majumder, Saikat; Paul Chowdhury, Bidisha; Goswami, Avranil; Halder, Kuntal; Chakraborty, Urmita; Pal, Nishith K.; Majumdar, Subrata

    2014-01-01

    Tuberculosis is characterized by severe immunosuppression of the host macrophages, resulting in the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates C-C Chemokine Receptor 5 (CCR5) to enhance IL-10 production, indicating the possible involvement of CCR5 in regulation of the host immune response. Here, we found that Mycobacterium infection significantly increased CCR5 expression in macrophages there by facilitating the activation of its downstream signaling. These events culminated in up-regulation of the immunosuppressive cytokine IL-10 production, which was further associated with the down-regulation of macrophage MHC-II expression along with the up-regulation of CCR5 expression via engagement of STAT-3 in a positive feedback loop. Treatment of macrophages with CCR5 specific siRNA abrogated the IL-10 production and restored MHCII expression. While, in vivo CCR5 silencing was also effective for the restoration of host immune responses against tuberculosis. This study demonstrated that CCR5 played a very critical role for the immune subversion mechanism employed by the pathogen. PMID:24695099

  19. Critical roles of chemokine receptor CCR5 in regulating glioblastoma proliferation and invasion.

    PubMed

    Zhao, Lanfu; Wang, Yuan; Xue, Yafei; Lv, Wenhai; Zhang, Yufu; He, Shiming

    2015-11-01

    Glioblastoma (GBM) is the most prevalent malignant primary brain tumor in adults and exhibits a spectrum of aberrantly aggressive phenotype. Tumor cell proliferation and invasion are critically regulated by chemokines and their receptors. Recent studies have shown that the chemokine CCL5 and its receptor CCR5 play important roles in tumor invasion and metastasis. Nonetheless, the roles of the CCR5 in GBM still remain unclear. The present study provides the evidence that the chemokine receptor CCR5 is highly expressed and associated with poor prognosis in human GBM. Mechanistically, CCL5-CCR5 mediates activation of Akt, and subsequently induces proliferation and invasive responses in U87 and U251 cells. Moreover, down-regulation of CCR5 significantly inhibited the growth of glioma in U87 tumor xenograft mouse model. Finally, high CCR5 expression in GBM is correlated with increased p-Akt expression in patient samples. Together, these findings suggest that the CCR5 is a critical molecular event associated with gliomagenesis.

  20. CCR5 Δ 32 mutation is not prevalent in Iranians with chronic HBV infection.

    PubMed

    Khorramdelazad, Hossein; Hakimizadeh, Elham; Hassanshahi, Gholamhossein; Rezayati, Mohammadtaghi; Sendi, Hossein; Arababadi, Mohammad Kazemi

    2013-06-01

    CCR5 is an important chemokine receptor involved in the recruitment of specific anti-viral immune cells (e.g., NK cells and T cytotoxic cells) to the liver. Previous studies indicated that the Δ 32 mutation in CCR5 gene led to inactivation of CCR5. Several conflicting studies have suggested that this mutation may be associated with either recovery or persistence of HBV infection. The main purpose of this study was to compare the frequency of the Δ 32 mutation within the CCR5 gene in a group of patients infected chronically with HBV with healthy individuals from South-East of Iran. Sixty patients with chronic HBV infection as well as 300 age-, and sex-match healthy individuals were enrolled in this study. Gap-PCR was applied to determine the frequency of CCR5 Δ 32 mutation in both groups. The results demonstrated that none of the patients infected with HBV carried the CCR5 Δ 32 mutation while, 3 (1%) of the healthy individuals were found to be heterozygotic for this mutation. The CCR5 Δ 32 mutation is not a prevalent mutation in either the patients infected chronically with HBV or their health counterparts in the South-East region of Iran. This may be attributed to either different genetic settings of the investigated population or lack of any significant correlation between this mutation and HBV pathogenicity.

  1. The CCR5 deletion mutation fails to protect against multiple sclerosis.

    PubMed

    Bennetts, B H; Teutsch, S M; Buhler, M M; Heard, R N; Stewart, G J

    1997-11-01

    Recent advances in the understanding and identification of chemokines and their receptors have provided evidence for their consideration as candidate loci with respect to genetic susceptibility/resistance to MS. Increased levels of the chemokine, macrophage inflammatory protein (MIP)-1 alpha, have been demonstrated in the cerebrospinal fluid of both patients with MS and mice with EAE, and anti-MIP-1 alpha antibodies have been shown to prevent EAE. Recently, a common deletion mutation in the gene for the major receptor for MIP-1 alpha, chemokine receptor 5 (CCR5) has been described. Homozygotes for the mutation fail to express this receptor. Moreover, homozygotes are highly protected against HIV infection this has potential implications for the cell entry of infectious agents in other multifactorial disease where a viral component may be involved. In view of these aspects, a group of 120 unrelated Australian relapsing remitting MS and 168 unrelated control subjects were screened for the CCR5 delta 32 mutation. There was no significant difference in the allele frequency of CCR5 delta 32 gene between the MS patients (0.1125) and the control population (0.0921). The presence of two CCR5 delta 32 homozygotes in the MS patients indicates that the absence of CCR5 is not protective against MS. These data suggest that CCR5 is not an essential component in MS expression, though this may be due to redundancy in the chemokine system where different chemokine receptors may substitute for CCR5 when it is absent.

  2. CCR5 (chemokine receptor-5) DNA-polymorphism influences the severity of rheumatoid arthritis.

    PubMed

    Zapico, I; Coto, E; Rodríguez, A; Alvarez, C; Torre, J C; Alvarez, V

    2000-01-01

    Chemokines are critical for the inflammatory process in autoimmune diseases such as rheumatoid arthritis (RA). The chemokine receptor-5 (CCR5) mediates chemotaxis by CC-chemokines and is expressed by lymphocytes with the Th1 phenotype and monocyte/macrophages. A 32 bp deletion in the CCR5 (CCR5-delta 32 allele) abolishes receptor expression in homozygotes, while CCR5-delta 32 carriers would express less receptor than wild-type homozygotes. This polymorphism is related to the resistance to HIV-1 infection and progression towards AIDS. We hypothesized that the CCR5-delta 32 allele may modulate the severity of disease in RA. A total of 160 RA-patients (71 and 89 with severe and non-severe phenotypes, respectively) and 500 healthy individuals from the same Caucasian population (Asturias, northern Spain) were genotyped. Carriers of the CCR5-delta 32 allele were at a significantly higher frequency (P = 0.012) in non-severe compared to severe patients (17% vs 4%). Our results suggest that the CCR5-delta 32 polymorphism is a genetic marker related to the severity of RA.

  3. CCR5 deletion protects against inflammation-associated mortality in dialysis patients.

    PubMed

    Muntinghe, Friso L H; Verduijn, Marion; Zuurman, Mike W; Grootendorst, Diana C; Carrero, Juan Jesus; Qureshi, Abdul Rashid; Luttropp, Karin; Nordfors, Louise; Lindholm, Bengt; Brandenburg, Vincent; Schalling, Martin; Stenvinkel, Peter; Boeschoten, Elisabeth W; Krediet, Raymond T; Navis, Gerjan; Dekker, Friedo W

    2009-07-01

    The CC-chemokine receptor 5 (CCR5) is a receptor for various proinflammatory chemokines, and a deletion variant of the CCR5 gene (CCR5 Delta 32) leads to deficiency of the receptor. We hypothesized that CCR5 Delta 32 modulates inflammation-driven mortality in patients with ESRD. We studied the interaction between CCR5 genotype and levels of high-sensitivity C-reactive protein (hsCRP) in 603 incident dialysis patients from the multicenter, prospective NEtherlands COoperative Study on the Adequacy of Dialysis (NECOSAD) cohort. CCR5 genotype and hsCRP levels were both available for 413 patients. During 5 yr of follow-up, 170 patients died; 87 from cardiovascular causes. Compared with the reference group of patients who had the wild-type CCR5 genotype and hsCRP 10 mg/L (n = 108) had an increased risk for mortality (HR: 1.82; 95% CI: 1.29 to 2.58). However, those carrying the deletion allele with hsCRP > 10 mg/L (n = 25) had a mortality rate similar to the reference group; this seemingly protective effect of the CCR5 deletion was even more pronounced for cardiovascular mortality. We replicated these findings in an independent Swedish cohort of 302 ESRD patients. In conclusion, the CCR5 Delta 32 polymorphism attenuates the adverse effects of inflammation on overall and cardiovascular mortality in ESRD.

  4. Distribution of the CCR5 gene 32-basepair deletion in 11 Chinese populations.

    PubMed

    Zhang, Chunyu; Fu, Songbin; Xue, Yali; Wang, Qi; Huang, Xiaoyi; Wang, Baiqiu; Liu, An; Ma, Linlin; Yu, Yang; Shi, Rongqian; Lv, Fuqu; Shi, Zhongcheng; Zhang, Yu; Cheng, Wenhong; Ai, Qionghua; Xu, Fang; Huang, Chengbin; Chen, Baibin; Kang, Xianghua; Sun, Yanyang; Zhang, Guiyin; Li, Pu

    2002-09-01

    A mutant allele of the chemokine receptor gene CCR5 bearing a 32-basepair deletion (delta 32CCR5) could increase the resistance to HIV-1 infection or delayed progression to AIDS. The frequency of this mutation is higher in Europeans than in Asians. To investigate the distribution of this polymorphism in China, 715 individuals from 11 Chinese populations were screened by PCR, including the Han and 10 other ethnic groups. The delta 32CCR5 gene was found in 16 individuals from 5 ethnic groups. All of them were heterozygous. The frequency of the mutant alleles of delta 32CCR5 is low in China and reflects (or might reflect) ancestral gene flow from Europe to Chinese ethnic groups and recent intermarriage within the ethnic groups.

  5. Delta 32 deletion of CCR5 gene and association with asthma or atopy.

    PubMed

    Mitchell, T J; Walley, A J; Pease, J E; Venables, P J; Wiltshire, S; Williams, T J; Cookson, W O

    2000-10-28

    The CCR5-delta32 deletion polymorphism (CCR5-delta32) was investigated for linkage and association to asthma and atopy using two panels of nuclear families containing 1284 individuals. No statistically significant linkage to asthma/wheeze or atopy was observed in either of the two panels of families. Multiallelic transmission disequilibrium tests (TDT) of the combined data found no significant association for atopy (52 independent alleles transmitted, 51 non-transmitted) or asthma/wheeze (39 transmitted, 44 non-transmitted). Although functional evidence might suggest that CCR5 is a good candidate gene for atopic asthma, this study provides no genetic evidence from CCR5-delta32 polymorphism to support this hypothesis.

  6. CCR5 as a potential target in cancer therapy: inhibition or stimulation?

    PubMed

    González-Martin, Alicia; Mira, Emilia; Mañes, Santos

    2012-11-01

    Extensive evidence implicates CCR5 and its ligands in the biology of tumors, although there is considerable controversy regarding the role of this chemokine receptor in cancer progression. The discrepancies between the pro- and anti-tumor effects of CCR5 might derive from its expression by cell types with opposing functions in tumor progression and the context in which tumors originate. We propose that CCR5 is necessary for optimal activation of the adaptive immune response to tumors, and for the success of certain immunotherapeutic strategies. Since efficient activation of T cell responses has broad implications in the success of some chemoand radiotherapy protocols, activation of CCR5, rather than its inhibition, might provide new therapeutic opportunities for cancer treatment.

  7. Stromal down-regulation of macrophage CD4/CCR5 expression and NF-κB activation mediates HIV-1 non-permissiveness in intestinal macrophages.

    PubMed

    Shen, Ruizhong; Meng, Gang; Ochsenbauer, Christina; Clapham, Paul R; Grams, Jayleen; Novak, Lea; Kappes, John C; Smythies, Lesley E; Smith, Phillip D

    2011-05-01

    Tissue macrophages are derived exclusively from blood monocytes, which as monocyte-derived macrophages support HIV-1 replication. However, among human tissue macrophages only intestinal macrophages are non-permissive to HIV-1, suggesting that the unique microenvironment in human intestinal mucosa renders lamina propria macrophages non-permissive to HIV-1. We investigated this hypothesis using blood monocytes and intestinal extracellular matrix (stroma)-conditioned media (S-CM) to model the exposure of newly recruited monocytes and resident macrophages to lamina propria stroma, where the cells take up residence in the intestinal mucosa. Exposure of monocytes to S-CM blocked up-regulation of CD4 and CCR5 expression during monocyte differentiation into macrophages and inhibited productive HIV-1 infection in differentiated macrophages. Importantly, exposure of monocyte-derived macrophages simultaneously to S-CM and HIV-1 also inhibited viral replication, and sorted CD4+ intestinal macrophages, a proportion of which expressed CCR5+, did not support HIV-1 replication, indicating that the non-permissiveness to HIV-1 was not due to reduced receptor expression alone. Consistent with this conclusion, S-CM also potently inhibited replication of HIV-1 pseudotyped with vesicular stomatitis virus glycoprotein, which provides CD4/CCR5-independent entry. Neutralization of TGF-β in S-CM and recombinant TGF-β studies showed that stromal TGF-β inhibited macrophage nuclear translocation of NF-κB and HIV-1 replication. Thus, the profound inability of intestinal macrophages to support productive HIV-1 infection is likely the consequence of microenvironmental down-regulation of macrophage HIV-1 receptor/coreceptor expression and NF-κB activation.

  8. Low-cost simultaneous detection of CCR5-delta32 and HLA-B*5701 alleles in human immunodeficiency virus type 1 infected patients by selective multiplex endpoint PCR.

    PubMed

    Rosi, Andrea; Meini, Genny; Materazzi, Angelo; Vicenti, Ilaria; Saladini, Francesco; Zazzi, Maurizio

    2015-11-01

    Host genetic traits impact susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, disease progression as well as antiretroviral drug pharmacokinetics and toxicity. Remarkable examples include a 32-bp deletion in the CCR5 coreceptor molecule (CCR5-delta32) impairing attachment of monocytotropic HIV-1 to the host cell membrane and the HLA-B*5701 allele, strongly associated with a potentially fatal hypersensitivity reaction triggered by abacavir, a nucleoside inhibitor of HIV reverse transcriptase. We developed a simple selective multiplex endpoint PCR method for simultaneous analysis of both genetic traits. Two primers were designed for amplification of a region surrounding the CCR5 32-bp deletion site. One common forward primer and two reverse primers with different 3' termini targeting the HLA-B*570101 and HLA-B*570102 alleles were designed for HLA-B*5701 analysis. A panel of 110 reference DNA samples typed in the HLA-B locus was used for development and blind validation of the assay. All the 45 HLA-B*5701 positive and the 55 HLA-B*5701 negative samples were correctly identified. The CCR5-delta32 allele was readily detected in 7 samples and did not interfere with detection of HLA-B*5701 while providing an internal amplification control. Multiplex PCR products were easily identified in agarose gels with no background noise. This simple and low-cost end-point selective multiplex PCR can conveniently screen HIV patients for the protective CCR5-delta32 allele and the risk of developing abacavir hypersensitivity reaction.

  9. Analysis of the CC chemokine receptor 5 (CCR5) delta-32 polymorphism in inflammatory bowel disease.

    PubMed

    Rector, A; Vermeire, S; Thoelen, I; Keyaerts, E; Struyf, F; Vlietinck, R; Rutgeerts, P; Van Ranst, M

    2001-03-01

    The inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC) are complex multifactorial traits involving both environmental and genetic factors. Recent studies have shown the important role of pro-inflammatory cytokines and chemokines, including RANTES, in IBD. RANTES is the natural ligand for the CC-chemokine receptor 5 (CCR5). The chromosomal location of the CCR5 gene on 3p21 coincides with an IBD-susceptibility locus identified by genome-wide scanning. A 32-bp deletion (A32) in the CCR5 gene results in a nonfunctional receptor and is found with high frequency in Caucasians. In this study, we investigated the presence of the CCR5delta32 allele in a large cohort of IBD patients and in a healthy control population. Blood samples were obtained from 538 unselected IBD cases (433 unrelated IBD patients: 289 CD, 142 UC, 2 indeterminate colitis; 105 affected first-degree relatives) and 135 unaffected first-degree family members. Of the IBD patients, 36% had familial IBD with at least two members being affected. There were no significant differences in the CCR5delta32 mutation frequency between IBD patients and healthy controls, nor between CD and UC patients. There was no correlation between the CCR5delta32 genotype and the age at IBD-diagnosis, the frequency of surgical intervention, or disease localization. Only the association between CCR5delta32 homozygosity and the presence of anal lesions in CD patients was statistically significant (P=0.007). Analysis by the transmission/disequilibrium test showed no significant transmission distortion to the probands or their clinically silent siblings. Based on these results, it is unlikely that the CCR5delta32 allele is an important marker for predisposition to IBD.

  10. CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape.

    PubMed

    Hütter, Gero; Bodor, Josef; Ledger, Scott; Boyd, Maureen; Millington, Michelle; Tsie, Marlene; Symonds, Geoff

    2015-07-27

    Allogeneic transplantation with CCR5-delta 32 (CCR5-d32) homozygous stem cells in an HIV infected individual in 2008, led to a sustained virus control and probably eradication of HIV. Since then there has been a high degree of interest to translate this approach to a wider population. There are two cellular ways to do this. The first one is to use a CCR5 negative cell source e.g., hematopoietic stem cells (HSC) to copy the initial finding. However, a recent case of a second allogeneic transplantation with CCR5-d32 homozygous stem cells suffered from viral escape of CXCR4 quasi-species. The second way is to knock down CCR5 expression by gene therapy. Currently, there are five promising techniques, three of which are presently being tested clinically. These techniques include zinc finger nucleases (ZFN), clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9), transcription activator-like effectors nuclease (TALEN), short hairpin RNA (shRNA), and a ribozyme. While there are multiple gene therapy strategies being tested, in this review we reflect on our current knowledge of inhibition of CCR5 specifically and whether this approach allows for consequent viral escape.

  11. Is the CCR5 Δ 32 mutation associated with immune system-related diseases?

    PubMed

    Ghorban, Khodayar; Dadmanesh, Maryam; Hassanshahi, Gholamhossein; Momeni, Mohammad; Zare-Bidaki, Mohammad; Arababadi, Mohammad Kazemi; Kennedy, Derek

    2013-06-01

    Hypersensitivity and autoimmunity are the main features of immune system-related diseases such as type 2 diabetes (T2D), multiple sclerosis (MS), and asthma. It has been established that chemokines play key roles in the activation and regulation of immune cell migration which is important in the pathogenesis of the diseases mentioned. CC chemokines receptor 5 or CCR5 is a receptor for RANTES, MIP-1α, and MIP-1β and is expressed by several immune cells including NK cells, T lymphocytes, and macrophages. It plays key roles in the regulation of migration and activation of the immune cells during immune responses against microbe and self-antigens during autoimmunity and hypersensitivity disorders. Therefore, any alteration in the sequence of CCR5 gene or in its expression could be associated with immune system-related diseases. Previous studies revealed that a 32-base pair deletion (Δ 32) in exon 1 of the CCR5 gene led to downregulation of the gene. Previous studies demonstrated that not only CCR5 expression was altered in autoimmune and hypersensitivity disorders, but also that the mutation is associated with the diseases. This review addresses the recent information regarding the association of the CCR5 Δ 32 mutation in immune-related diseases including T2D with and without nephropathy, MS, and asthma. Based on the collected data, it seems that the CCR5 Δ 32 mutation can be considered as a risk factor for MS, but not asthma and T2D with and without nephropathy.

  12. CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape

    PubMed Central

    Hütter, Gero; Bodor, Josef; Ledger, Scott; Boyd, Maureen; Millington, Michelle; Tsie, Marlene; Symonds, Geoff

    2015-01-01

    Allogeneic transplantation with CCR5-delta 32 (CCR5-d32) homozygous stem cells in an HIV infected individual in 2008, led to a sustained virus control and probably eradication of HIV. Since then there has been a high degree of interest to translate this approach to a wider population. There are two cellular ways to do this. The first one is to use a CCR5 negative cell source e.g., hematopoietic stem cells (HSC) to copy the initial finding. However, a recent case of a second allogeneic transplantation with CCR5-d32 homozygous stem cells suffered from viral escape of CXCR4 quasi-species. The second way is to knock down CCR5 expression by gene therapy. Currently, there are five promising techniques, three of which are presently being tested clinically. These techniques include zinc finger nucleases (ZFN), clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9), transcription activator-like effectors nuclease (TALEN), short hairpin RNA (shRNA), and a ribozyme. While there are multiple gene therapy strategies being tested, in this review we reflect on our current knowledge of inhibition of CCR5 specifically and whether this approach allows for consequent viral escape. PMID:26225991

  13. Distribution of the CCR5delta32 allele (gene variant CCR5) in Rondônia, Western Amazonian region, Brazil

    PubMed Central

    de Farias, Josileide Duarte; Santos, Marlene Guimarães; de França, Andonai Krauze; Delani, Daniel; Tada, Mauro Shugiro; Casseb, Almeida Andrade; Simões, Aguinaldo Luiz; Engracia, Vera

    2012-01-01

    Since around 1723, on the occasion of its initial colonization by Europeans, Rondonia has received successive waves of immigrants. This has been further swelled by individuals from northeastern Brazil, who began entering at the beginning of the twentieth century. The ethnic composition varies across the state according to the various sites of settlement of each wave of immigrants. We analyzed the frequency of the CCR5Δ32 allele of the CCR5 chemokine receptor, which is considered a Caucasian marker, in five sample sets from the population. Four were collected in Porto Velho, the state capital and the site of several waves of migration. Of these, two, from the Hospital de Base were comprised of HB Mothers and HB Newborns presenting allele frequencies of 3.5% and 3.1%, respectively, a third from the peri-urban neighborhoods of Candelária/Bate-Estaca (1.8%), whereas a fourth, from the Research Center on Tropical Medicine/CEPEM (0.6%), was composed of malaria patients under treament. The fifth sample (3.4%) came from the inland Quilombola village of Pedras Negras. Two homozygous individuals (CCR5Δ32/CCR5Δ32) were detected among the HB Mother samples. The frequency of this allele was heterogeneous and higher where the European inflow was more pronounced. The presence of the allele in Pedras Negras revealed European miscegenation in a community largely comprising Quilombolas. PMID:22481870

  14. CCR5 Polymorphism as a Protective Factor for Hepatocellular Carcinoma in Hepatitis B Virus-Infected Iranian Patients

    PubMed

    Abdolmohammadi, Reza; Shahbazi Azar, Saleh; Khosravi, Ayyoob; Shahbazi, Majid

    2016-10-01

    The CC chemokine receptor 5 (CCR5) delta 32 allele results in a nonfunctional form of the chemokine receptor and has been implicated in a variety of immune-mediated diseases. CCR5Δ32 may also predispose one to chronic liver disease or be linked with resistance to HBV infection.This study was undertaken to investigate any association between CCR5 polymorphism with resistance to hepatitis B or susceptibility to HBV infection. A total of 812 Iranian individuals were enrolled into two groups: HBV infected cases (n=357), who were HBsAg-positive, and healthy controls (n=455). We assessed polymorphisms in the CCR5 gene using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion. Genotype distributions of the HBV infected cases and healthy controls were determined and compared. The CCR5/CCR5 (WW) and CCR5/ CCR5Δ32 (W/D) genotypes were found in (98%) and (2%) of HBV infected cases, respectively. The CCR5 Δ32/ Δ32genotype was not found in HBV infected cases. Genotype distributions of CCR5 in healthy controls were W/W genotype in (87.3%), W/D genotype in (11.2%) and D/D genotype in (1.5%). Heterozygosity for CCR5/ CCR5Δ32 (W/D) in healthy controls was greater than in HBV infected cases (11.2% vs 2%, p < 0.001). W/D and D/D genotypes were more prominent in healthy controls than in HBV infected cases. This study provides evidence that the CCR5Δ32 polymorphism may have a protective effect in resistance to HBV infection at least in the Iranian population.

  15. A simple structure-based model for the prediction of HIV-1 co-receptor tropism

    PubMed Central

    2014-01-01

    Background Human Immunodeficiency Virus 1 enters host cells through interaction of its V3 loop (which is part of the gp120 protein) with the host cell receptor CD4 and one of two co-receptors, namely CCR5 or CXCR4. Entry inhibitors binding the CCR5 co-receptor can prevent viral entry. As these drugs are only available for CCR5-using viruses, accurate prediction of this so-called co-receptor tropism is important in order to ensure an effective personalized therapy. With the development of next-generation sequencing technologies, it is now possible to sequence representative subpopulations of the viral quasispecies. Results Here we present T-CUP 2.0, a model for predicting co-receptor tropism. Based on our recently published T-CUP model, we developed a more accurate and even faster solution. Similarly to its predecessor, T-CUP 2.0 models co-receptor tropism using information of the electrostatic potential and hydrophobicity of V3-loops. However, extracting this information from a simplified structural vacuum-model leads to more accurate and faster predictions. The area-under-the-ROC-curve (AUC) achieved with T-CUP 2.0 on the training set is 0.968±0.005 in a leave-one-patient-out cross-validation. When applied to an independent dataset, T-CUP 2.0 has an improved prediction accuracy of around 3% when compared to the original T-CUP. Conclusions We found that it is possible to model co-receptor tropism in HIV-1 based on a simplified structure-based model of the V3 loop. In this way, genotypic prediction of co-receptor tropism is very accurate, fast and can be applied to large datasets derived from next-generation sequencing technologies. The reduced complexity of the electrostatic modeling makes T-CUP 2.0 independent from third-party software, making it easy to install and use. PMID:25120583

  16. Establishment of mouse leukemia cell lines expressing human CD4/CCR5 using lentiviral vectors.

    PubMed

    Li, Ya-Jing; ZhuGe, Fu-Yan; Zeng, Chang-Chun; He, Jin-Yang; Tan, Ning; Liang, Juan

    2017-04-01

    A low-cost rodent model of HIV infection and which presents high application value is an effective tool to investigate HIV infection and pathogenesis. However, development of such a small animal model has been hampered by the unsuitability of rodent cells for HIV-1 replication given that the retrovirus HIV-1 has high selectivity to its host cell. Our study used the mouse leukemia cell lines L615 and L1210 that were induced by murine leukemia virus and transfected with hCD4/CCR5 loaded-lentiviral vector. Lentiviral vectors containing the genes hCD4/CCR5 under the transcriptional control of cytomegalovirus promoter were designed. Transfection efficiencies of human CD4 and CCR5 in L615 and L1210 cells were analyzed by quantitative real-time polymerase chain reaction (RT-PCR) and Western blot assay. Results showed that hCD4 and CCR5 proteins were expressed on the cell surface, demonstrating that the L615 and L1210 cells were humanized and that they possess the characteristics necessary for HIV infection of human host cells. Moreover, the sensitivity of human CD4/CCR5 transgenic mouse cells to HIV infection was confirmed by RT-PCR and ELISA. Mouse leukemia cell lines that could express hCD4 and CCR5 were thus established to facilitate normal entry of HIV-1 so that a human CD4/CCR5 transgenic mice cell model can be used to investigate the transmission and pathogenesis of HIV/AIDS and potential antiviral drugs against this disease.

  17. Polymorphism of CC chemokine receptors CCR2 and CCR5 in Crohn's disease.

    PubMed

    Herfarth, H; Pollok-Kopp, B; Göke, M; Press, A; Oppermann, M

    2001-06-01

    Crohn's disease (CD) is a chronic inflammatory disease of the intestine that is characterized by mononuclear cell infiltration and a predominant Th1 lymphocyte response. We tested the hypothesis that CC chemokine receptors CCR2 and CCR5 might be important in the regulation of the intestinal immune response in this disease, and we speculated that carriers of a defective 32 base pair deletion mutant of CCR5, CCR5Delta32, which results in a non-functional receptor, might be protected from CD. Using polymerase chain reaction (PCR) and PCR restriction fragment length polymorphism (PCR-RFLP) gene frequencies of CCR5Delta32 and of CCR2-641 (replacement of valine-64 by isoleucine in the CCR2 gene) in healthy controls (n=346) and in CD patients (n=235) were determined. In CD patients, subgroup phenotypic analyses were performed according to the Vienna classification. The overall gene frequency of CCR5Delta32 (9.8%) and CCR2-641 (7.6%) in CD patients did not deviate significantly from healthy controls (9.2 and 8.2%, respectively), nor did we observe a significant deviation from the predicted Hardy-Weinberg distribution. No significant differences in the CD phenotype classification for the different CCR5 and CCR2 alleles were observed, except for a trend to disease sparing of the upper gastrointestinal tract (carrier frequency 0 versus 19.6%, Delta=1 9.6%, P=0.079) as well as a more stricturing disease behaviour (23.5 versus 16.2%, Delta=7.3%, P=0.136) in carriers of the mutant CCR5Delta32 allele. These results indicate that the different CCR5 but not CCR2 alleles may influence disease behaviour and thereby contribute to the observed heterogeneity of CD. However, the associations observed are limited and await replication in other datasets. CCR2 and CCR5 polymorphisms are unlikely to be important determinants of overall disease susceptibility.

  18. Distribution of the HIV resistance CCR5-Delta32 allele among Egyptians and Syrians.

    PubMed

    Salem, Abdel-Halim; Batzer, Mark A

    2007-03-01

    A mutant allele of the beta-chemokine receptor gene CCR5 bearing a 32-basepair (bp) deletion that prevents cell invasion by the primary transmitting strain of HIV-1 has recently been characterized. Individuals homozygous for the mutation are resistant to infection, even after repeated high-risk exposure, but this resistance appears not absolute, as isolated cases of HIV-positive deletion homozygotes are emerging. The consequence of the heterozygous state is not clear, but it may delay the progression to AIDS in infected individuals. In order to evaluate the frequency distribution of CCR5-Delta32 polymorphism among Egyptians, a total of 200 individuals (154 from Ismailia and 46 from Sinai) were tested. Only two heterozygous individuals from Ismailia carried the CCR5-Delta32 allele (0.6%), and no homozygous (Delta32/Delta32) individuals were detected among the tested samples. The presence of the CCR5-Delta32 allele among Egyptians may be attributed to the admixture with people of European descent. Thus we conclude that the protective deletion CCR5-Delta32 is largely absent in the Egyptian population.

  19. CCR5 polymorphism and plague resistance in natural populations of the black rat in Madagascar.

    PubMed

    Tollenaere, C; Rahalison, L; Ranjalahy, M; Rahelinirina, S; Duplantier, J-M; Brouat, C

    2008-12-01

    Madagascar remains one of the world's largest plague foci. The black rat, Rattus rattus, is the main reservoir of plague in rural areas. This species is highly susceptible to plague in plague-free areas (low-altitude regions), whereas rats from the plague focus areas (central highlands) have evolved a disease-resistance polymorphism. We used the candidate gene CCR5 to investigate the genetic basis of plague resistance in R. rattus. We found a unique non-synonymous substitution (H184R) in a functionally important region of the gene. We then compared (i) CCR5 genotypes of dying and surviving plague-challenged rats and (ii) CCR5 allelic frequencies in plague focus and plague-free populations. Our results suggested a higher prevalence of the substitution in resistant animals compared to susceptible individuals, and a tendency for higher frequencies in plague focus areas compared to plague-free areas. Therefore, the CCR5 polymorphism may be involved in Malagasy black rat plague resistance. CCR5 and other undetermined plague resistance markers may provide useful biological information about host evolution and disease dynamics.

  20. CCR5 deficiency predisposes to fatal outcome in influenza virus infection.

    PubMed

    Falcon, A; Cuevas, M T; Rodriguez-Frandsen, A; Reyes, N; Pozo, F; Moreno, S; Ledesma, J; Martínez-Alarcón, J; Nieto, A; Casas, I

    2015-08-01

    Influenza epidemics affect all age groups, although children, the elderly and those with underlying medical conditions are the most severely affected. Whereas co-morbidities are present in 50% of fatal cases, 25-50% of deaths are in apparently healthy individuals. This suggests underlying genetic determinants that govern infection severity. Although some viral factors that contribute to influenza disease are known, the role of host genetic factors remains undetermined. Data for small cohorts of influenza-infected patients are contradictory regarding the potential role of chemokine receptor 5 deficiency (CCR5-Δ32 mutation, a 32 bp deletion in the CCR5 gene) in the outcome of influenza virus infection. We tested 171 respiratory samples from influenza patients (2009 pandemic) for CCR5-Δ32 and evaluated its correlation with patient mortality. CCR5-Δ32 patients (17.4%) showed a higher mortality rate than WT individuals (4.7%; P = 0.021), which indicates that CCR5-Δ32 patients are at higher risk than the normal population of a fatal outcome in influenza infection.

  1. Genotypes of CCR2 and CCR5 chemokine receptors in human myasthenia gravis.

    PubMed

    Zhao, Xiaoyan; Gharizadeh, Baback; Hjelmström, Peter; Pirskanen, Ritva; Nyrén, Pål; Lefvert, Ann-Kari; Ghaderi, Mehran

    2003-11-01

    The aim of this study was to examine the association of human autoimmune myasthenia gravis (MG) with two DNA polymorphisms of the chemokine receptors CCR5-Delta 32 and CCR2-64I. CCR2 and CCR5 interact primarily with the human CC family ligands CCL2 (formerly called monocyte chemoattractant protein; MCP-1), CCL3 and CCL4 (macrophage inflammatory protein-1 alpha and -1 beta; MIP-1 alpha/beta), and their main function is to recruit leukocytes from circulation into the tissues, thus playing an important role in human inflammatory disorders. A PCR-based genotyping method was used to determine the genetic variation at the CCR5 gene and an automated real-time Pyrosequencing technology was employed for the analysis of G right curved arrow A point mutation at the CCR2 gene. Results obtained from 158 patients and 272 healthy controls demonstrate no evidence of association between genetic variants of CCR2 and CCR5 with MG and its clinical manifestations. CCR2-64I and CCR5-Delta 32 genotypes are thus unlikely to be involved in protection or predisposition to MG.

  2. Prevalence of mutant CCR5 allele in Slovenian HIV-1-infected and non-infected individuals.

    PubMed

    Poljak, M; Tomazic, J; Seme, K; Maticic, M; Vidmar, L

    1998-02-01

    A 32 bp deletion in the CCR5 gene designated CCR5 delta 32 has been identified recently as the cellular basis for resistance to human immunodeficiency virus type 1 (HIV-1) in some individuals which remained non-infected despite a repeated exposure to this virus. The prevalence of this deletion was examined by polymerase chain reaction (PCR) on 51 HIV-1-infected and 385 non-infected individuals from all parts of Slovenia. 84.4% of the the HIV-1-infected and 83.2% of the non-infected individuals were homozygous for wild type CCR5, and 19.6% and 16.3%, respectively, were heterozygous. No homozygous mutant genotype was observed among the HIV-1-infected patients. Of the non-infected individuals, 2 women (0.5%) were found to harbour the CCR5 delta 32/CCR5 delta 32 genotype only, which is, to the best of our knowledge, the lowest prevalence of this particular genotype found among Caucasians to date.

  3. Genetic polymorphism of chemokine receptors CCR2 and CCR5 in Swedish cervical cancer patients.

    PubMed

    Zheng, Biying; Wiklund, Fredrik; Gharizadeh, Baback; Sadat, Mehdi; Gambelunghe, Giovanni; Hallmans, Göran; Dillner, Joakim; Wallin, Keng-Ling; Ghaderi, Mehran

    2006-01-01

    Chemokines are chemotactic cytokines that orchestrate leukocyte trafficking in tissues, thus, playing an important role in regulation of immunological processes. The aim of this study was to investigate the association of human papillomavirus (HPV) infection and cervical cancer with two DNA polymorphisms of the chemokine receptors CCR5-delta32 and CCR2-64I. The study material consisted of 50 cervical intraepithelial neoplasia (CIN) cases and 50 of age and sampling-date matched controls, 100 invasive cervix cancer cases and 100 of their corresponding matched disease-free controls. Pyrosequencing was employed to genotype the CCR2-64I polymorphism. CCR5-delta32 was genotyped using standard PCR fragment length analysis. The frequencies of CCR2 and CCR5 genotypes from 150 patients and 150 healthy controls were representative of the general population according to the Hardy-Weinberg equilibrium analysis. Risk association was computed with conditional logistic regression analysis. HPV-positive individuals with the rare CCR5deelta32/delta32 genotype have a risk of 4.58 (CI = 0.40-52.64, p-value = 0.045) compare to HPV negative group. The delta-32 mutation on the CCR locus is imperceptibly associated with increased risk of HPV infection. In total, cervical neoplasia was not associated with genetic polymorphism of CCR2 and CCR5.

  4. Anti-infective peptide IDR-1002 augments monocyte chemotaxis towards CCR5 chemokines.

    PubMed

    Madera, Laurence; Hancock, Robert E W

    2015-08-28

    Innate defense regulator (IDR) peptides are a class of immunomodulators which enhance and modulate host innate immune responses against microbial pathogens. While IDR-mediated protection against a range of bacterial pathogens is dependent on enhanced monocyte recruitment to the site of infection, the mechanisms through which they increase monocyte trafficking remain unclear. In this study, anti-infective peptide IDR-1002 was shown to enhance monocyte chemotaxis towards chemokines CCL3 and CCL5. This enhancement correlated with the selective upregulation of CCR5 surface expression by peptide-treated monocytes. It was found that IDR-1002 enhancement of monocyte chemotaxis was fully dependent on CCR5 function. Furthermore, IDR-1002 enhanced chemokine-induced monocyte p38 MAPK phosphorylation in a CCR5-dependent fashion. Overall, these results indicate that peptide IDR-1002 can selectively influence monocyte recruitment by host chemokines through the regulation of chemokine receptors.

  5. Frequency of the CCR5-delta 32 chemokine receptor gene mutation in the Lebanese population.

    PubMed

    Karam, W; Jurjus, R; Khoury, N; Khansa, H; Assad, C; Zalloua, P; Jurjus, A

    2004-01-01

    A direct correlation between HIV infection and mutation in the chemokine receptor (CCR5) gene has been established. However, such correlation has never been investigated in Lebanon. We report the frequency of the CCR5-delta 32 mutation in a random sample of 209 healthy, HIV-1 seronegative Lebanese aged 19-68. Overall, 4.8% were heterozygous for the mutation. Homozygosity was absent from our sample. The frequency for the CCR5-delta 32 allele was 2.5%. Distribution of the mutation was unaffected by sex, age, religion or educational level. The frequency in the Lebanese population is consistent with that in the origin of the mutation in northern Europe. This could be attributed to a gene flow into the Middle East from northern Europe.

  6. CCR5 chemokine receptor genotype frequencies among Puerto Rican HIV-1-seropositive individuals.

    PubMed

    Gonzalez, S; Tirado, G; Revuelta, G; Yamamura, Y; Lu, Y; Nerurkar, V R; Yanagihara, R

    1998-01-01

    Some individuals remain uninfected by human immunodeficiency virus type 1 (HIV-1), despite multiple sexual contacts with subjects with confirmed HIV-1 infection. Several studies have confirmed that individuals who are homozygous for a 32 base pair (bp) deletion mutation in the chemokine receptor gene CCR5, designated as delta 32/ delta 32, are protected against HIV-1 infection. Heterozygotes of the same chemokine receptor deletion mutation are, however, not protected from acquiring HIV-1 infection but seemingly have slower progression to acquired immunodeficiency syndromes (AIDS). Genotype frequencies of the delta 32 CCR5 mutation vary markedly among different ethnic groups; heterozygosity is found in approximately 15% of Caucasians, about 5-7% of Hispanics and African Americans and 1% or less of Asians. The ethnic background of Puerto Ricans is highly complex and usually includes admixture of Caucasian, Caribbean Indian and African traits to a varying extent. This study was conducted to examine the frequencies of the delta 32 CCR5 mutation among Puerto Ricans who are infected with HIV-1. Samples were received from different geographical regions of the island. Of 377 samples tested, 94.2% were wild type (non-deletion mutant) homozygotes, 5.8% were delta 32 CCR5 heterozygotes, and none were delta 32 CCR5 homozygotes. The incidence of CCR5 delta 32/w heterozygous mutation among Puerto Ricans seems to be somewhat lower than what was reported with US Hispanics. Some age and gender associated bias of the mutation frequency were observed with the study population, the reason for which is unclear at present.

  7. Association analysis of a CCR5 variant with ewe lifetime production.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A deletion in the promoter region of CCR5 associates with a 50% reduction in proviral concentration (log10 env copies/microgram DNA) of ovine progressive pneumonia virus (OPPV) in sheep blood. Because OPP provirus blood concentrations correlate with the degree of histological lesions in affected ti...

  8. Association analysis of a CCR5 variant with ewe lifetime production in 3 breeds of sheep.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A deletion in the promoter region of CCR5 associates with a 50% reduction in proviral concentration (log10 env copies/microgram DNA) of ovine progressive pneumonia virus (OPPV) in blood. Nearly half of all flocks in the U.S. have at least one sheep infected with OPPV. Because OPP provirus concentr...

  9. Enhanced CCR5+/CCR3+ T helper cell ratio in patients with active cutaneous lupus erythematosus.

    PubMed

    Freutel, S; Gaffal, E; Zahn, S; Bieber, T; Tüting, T; Wenzel, J

    2011-10-01

    Cutaneous lupus erythematosus (CLE) is characterized by enhanced interferon α (IFNα) levels in serum and in tissue. Since IFNα promotes a Th1-biased immune response, we hypothesized that a Th1-associated chemokine receptor profile should be a typical finding in patients with active CLE. Therefore, peripheral blood mononuclear cells were isolated from patients with different CLE subsets (n = 15), healthy controls (n = 13) and patients under immunotherapy with IFNα (n = 7). T helper cells were analysed by flow cytometry for the expression of the chemokines receptor CCR5, indicative for Th1 cells, and of CCR3, indicating Th2. In addition, intracellular levels of the type I IFN-inducible MxA protein were measured. Patients with widespread active CLE skin lesions had a significantly increased expression of CCR5, whereas expression of CCR3 was decreased when compared with healthy controls. MxA expression was significantly enhanced in all investigated CLE subtypes, with the highest levels in patients with widespread skin lesions. The enhanced CCR5/CCR3 ratio closely correlated with the MxA levels in peripheral lymphocytes and with disease activity. Our analyses revealed that active CLE is associated with a systemic type I IFN effect that appears to induce a shift towards a Th1-associated chemokine receptor profile. The CCR5/CCR3 T-helper cell ratio might therefore represent an indirect marker for the disease activity in CLE.

  10. Interfering with CCL5/CCR5 at the Tumor-Stroma Interface.

    PubMed

    Bronte, Vincenzo; Bria, Emilio

    2016-04-11

    In this issue of Cancer Cell, Halama et al. (2016) further advance chemokine interference as a therapeutic option for cancer by demonstrating the effect of CCR5 blockade in reshaping macrophage polarization toward an anti-tumor functional state in patient-derived tumor models and liver metastases of colorectal cancer patients.

  11. HIV-1 adaptation to low levels of CCR5 results in V3 and V2 loop changes that increase envelope pathogenicity, CCR5 affinity and decrease susceptibility to Maraviroc.

    PubMed

    Garg, Himanshu; Lee, Raphael T C; Maurer-Stroh, Sebastian; Joshi, Anjali

    2016-06-01

    Variability in CCR5 levels in the human population is suggested to affect virus evolution, fitness and the course of HIV disease. We previously demonstrated that cell surface CCR5 levels directly affect HIV Envelope mediated bystander apoptosis. In this study, we attempted to understand HIV evolution in the presence of low levels of CCR5, mimicking the limiting CCR5 levels inherent to the host. HIV-1 adaptation in a T cell line expressing low levels of CCR5 resulted in two specific mutations; N302Y and E172K. The N302Y mutation led to accelerated virus replication, increase in Maraviroc IC50 and an increase in Envelope mediated bystander apoptosis in low CCR5 expressing cells. Analysis of subtype B sequences showed that N302Y is over-represented in CXCR4 tropic viruses in comparison to CCR5 tropic isolates. Considering the variability in CCR5 levels between individuals, our findings have implications for virus evolution, MVC susceptibility as well as HIV pathogenesis.

  12. The chemokine receptor CCR5 deletion mutation is associated with MS in HLA-DR4-positive Russians.

    PubMed

    Favorova, O O; Andreewski, T V; Boiko, A N; Sudomoina, M A; Alekseenkov, A D; Kulakova, O G; Slanova, A V; Gusev, E I

    2002-11-26

    The authors studied the possible association between the presence of a 32-base pair deletion allele in CC chemokine receptor 5 gene [3p21] (CCR5 Delta 32 allele) and the occurrence of MS. The presence of CCR5 Delta 32 homozygotes among patients with MS indicates that the absence of CCR5 did not protect against MS. Moreover, the CCR5 Delta 32 mutation was associated with MS in HLA-DR4-positive Russians (p(corr) < 0.001, odds ratio [OR] = 25.0). The (CCR5 Delta 32,DR4)-positive phenotype was negatively associated with early MS onset (at ages < or = 18 years) (p = 0.0115, OR = 0.1).

  13. Do human polyoma viruses and human immunodeficiency virus share common co-receptors?

    PubMed

    Borissov, Kalin; Tsekov, Iliya; Gavazova, Rayna; Kalvatchev, Zlatko; Argirova, Radka

    2010-01-01

    Host and/or viral factors involved in human polyomavirus (HPoV) infection in persons living with HIV remain unknown. A hypothesis is outlined suggesting the importance of the co-receptors CCR5, CCR2, and CXCR4 not only for HIV, but also for HPoV. Functionally capable receptors coded by wild-type (wt) genotypes could facilitate internalization of HPoV in the cell resulting in brain and/or kidney infection/s in HIV infected individuals. Forty-nine Bulgarians with HIV, all treated by HAART, without neurological and/or kidney disorders, were tested for JCV and BKV and genotyped for CCR5 (CCR5del32), CCR2 (CCR2-64I), and CXCR4 (SDF1-3'A). In 27/49 (55.1%) individuals a co-infection with HPoV was identified-BKV in 12/49 (24.5%), JCV-in another 12/49 (24.5%), and both viruses-in 3/49 (6.1%). A high frequency of wt CCR5 was found in patients with HPoV (91.7% for BKV and JCV and in 100% with both viruses). V/V of CCR2 was presented in 75% for BKV and JCV and in 66.7% for BKV plus JCV. SDF1-3'G/G predominated in JCV infected patients (75%), while G/A and A/A genotypes were more frequent in patients with BKV (41.7%). Also, 21/22 (95.4%) persons without HPoV infection were heterozygous for SDF1 and CCR2. The number of individuals bearing wt of all co-receptors in the group of persons not infected with HPoV was lower (P = 0.03) than that with polymorphism/s in one or two genes (SDF1 and CCR2) in the same group. The results suggest a probable role of co-receptors used by HIV to facilitate infection with HPoV.

  14. Distribution of CCR5-delta 32 gene deletion across the Russian part of Eurasia.

    PubMed

    Yudin, N S; Vinogradov, S V; Potapova, T A; Naykova, T M; Sitnikova, V V; Kulikov, I V; Khasnulin, V I; Konchuk, C; Vloschinskii, P E; Ivanov, S V; Kobzev, V F; Romaschenko, A G; Voevoda, M I

    1998-06-01

    32-bp inactivating deletion in the beta-chemokine receptor 5 (CCR5) gene, common in Nothern European populations, is associated with reduced HIV-1 transmission risk and delayed disease progression. We have studied the deletion distribution in many populations in Eurasia by polymerase chain reaction analysis of 531 DNA samples representing West and East Siberian, Central Asian, and Far Eastern parts of Russia. An unusually high frequency (11.1%) of the deleted variant in natives of West Siberia, of Finno-Ugrian descent, was observed. Furthermore, the deletion was infrequent in indigenous populations of Central Asia, East Siberia, the Russian Far East, and Canada. We conclude that the delta(ccr5) distribution is limited primarily to Europeans and related western Siberian Finno-Ugrian populations, with a sharp negative gradient toward the east along the territory of Russian Asia.

  15. CCR5 Blockade Suppresses Melanoma Development Through Inhibition of IL-6-Stat3 Pathway via Upregulation of SOCS3.

    PubMed

    Tang, Qiu; Jiang, Jun; Liu, Jian

    2015-12-01

    In order to understand how tumor cells can escape immune surveillance mechanisms and thus develop antitumor therapies, it is critically important to investigate the mechanisms by which the immune system interacts with the tumor microenvironment. In our current study, we found that chemokine receptor 5 (CCR5) neutralization resulted in reduced melanoma tumor size, decreased percentage of CD11b+ Gr-1(+) myeloid-derived suppressor cells (MDSCs), and increased proportion of cluster of differentiation (CD)3+ T cells in tumor tissues. Suppressive activity of MDSCs on CD4+ T cells and CD8+ T cell proliferation is significantly inhibited by anti-CCR5 antibody. CCR5 blockade also suppresses interleukin (IL)-6 induction, which in turn deactivates signal transducer and activator of transcription 3 (Stat3) in tumors. Furthermore, the suppressed B16 tumor growth induced by CCR5 blockade is abolished with additional administration of recombinant IL-6. CCR5 blockade also induces suppressor of cytokine signaling 3 (SOCS3) upregulations, and anti-CCR5 antibody fails to suppress expression of phospho-Stat3 (p-Stat3), matrix metallopeptidase 9 (MMP9), and IL-6 in cells transfected with SOCS3 short-interfering RNA (SiRNA). All these data suggest that CCR5 blockade suppresses melanoma development through inhibition of IL-6-Stat3 pathway via upregulation of SOCS3.

  16. Lack of Correlation Between the CCR5-Δ32 Mutation and Acute Myeloid Leukemia in Iranian Patients.

    PubMed

    Khorramdelazad, Hossein; Mortazavi, Yousef; Momeni, Mohammad; Arababadi, Mohammad Kazemi; Khandany, Behjat Kalantary; Moogooei, Mozhgan; Hassanshahi, Gholamhossein

    2015-03-01

    Chemokines and their receptors are crucially important in the pathogenesis of acute myeloblastic leukemia (AML). The CC chemokine receptor 5 (CCR5) is a specific chemokine receptor for CC chemokine ligand 3 (CCL3), CCL4 and CCL5 which all play key roles in identifying cancer properties and localization of leukemia cells. It has been demonstrated that the known mutation in CCR5 gene (CCR5-Δ32) leads to mal-expression of the receptor and affect its function. The aim of this study was to determine the rate of CCR5-Δ32 mutation within Iranian AML patients. In this study, blood samples were obtained from 60 AML patients and 300 healthy controls. The CCR5-Δ32 mutation was evaluated using Gap-PCR technique. Our results showed that CCR5-Δ32 mutation was not found in the patients, while three out of the controls had hetrozygotic form of this mutation. The rest of studied samples had the wild form of the gene. According to these findings, it can probably be concluded that the CCR5-Δ32 is not associated with susceptibility to AML in Iranian patients.

  17. Elucidating a Key Anti-HIV-1 and Cancer-Associated Axis: The Structure of CCL5 (Rantes) in Complex with CCR5

    NASA Astrophysics Data System (ADS)

    Tamamis, Phanourios; Floudas, Christodoulos A.

    2014-06-01

    CCL5 (RANTES) is an inflammatory chemokine which binds to chemokine receptor CCR5 and induces signaling. The CCL5:CCR5 associated chemotactic signaling is of critical biological importance and is a potential HIV-1 therapeutic axis. Several studies provided growing evidence for the expression of CCL5 and CCR5 in non-hematological malignancies. Therefore, the delineation of the CCL5:CCR5 complex structure can pave the way for novel CCR5-targeted drugs. We employed a computational protocol which is primarily based on free energy calculations and molecular dynamics simulations, and report, what is to our knowledge, the first computationally derived CCL5:CCR5 complex structure which is in excellent agreement with experimental findings and clarifies the functional role of CCL5 and CCR5 residues which are associated with binding and signaling. A wealth of polar and non-polar interactions contributes to the tight CCL5:CCR5 binding. The structure of an HIV-1 gp120 V3 loop in complex with CCR5 has recently been derived through a similar computational protocol. A comparison between the CCL5 : CCR5 and the HIV-1 gp120 V3 loop : CCR5 complex structures depicts that both the chemokine and the virus primarily interact with the same CCR5 residues. The present work provides insights into the blocking mechanism of HIV-1 by CCL5.

  18. Peptide ligands selected with CD4-induced epitopes on native dualtropic HIV-1 envelope proteins mimic extracellular coreceptor domains and bind to HIV-1 gp120 independently of coreceptor usage.

    PubMed

    Dervillez, Xavier; Klaukien, Volker; Dürr, Ralf; Koch, Joachim; Kreutz, Alexandra; Haarmann, Thomas; Stoll, Michaela; Lee, Donghan; Carlomagno, Teresa; Schnierle, Barbara; Möbius, Kalle; Königs, Christoph; Griesinger, Christian; Dietrich, Ursula

    2010-10-01

    During HIV-1 entry, binding of the viral envelope glycoprotein gp120 to the cellular CD4 receptor triggers conformational changes resulting in exposure of new epitopes, the highly conserved CD4-induced (CD4i) epitopes that are essential for subsequent binding to chemokine receptor CCR5 or CXCR4. Due to their functional conservation, CD4i epitopes represent attractive viral targets for HIV-1 entry inhibition. The aim of the present study was to select peptide ligands for CD4i epitopes on native dualtropic (R5X4) HIV-1 envelope (Env) glycoproteins by phage display. Using CD4-activated retroviral particles carrying Env from the R5X4 HIV-1 89.6 strain as the target, we performed screenings of random peptide phage libraries under stringent selection conditions. Selected peptides showed partial identity with amino acids in the extracellular domains of CCR5/CXCR4, including motifs rich in tyrosines and aspartates at the N terminus known to be important for gp120 binding. A synthetic peptide derivative (XD3) corresponding to the most frequently selected phages was optimized for Env binding on peptide arrays. Interestingly, the optimized peptide could bind specifically to gp120 derived from HIV-1 strains with different coreceptor usage, competed with binding of CD4i-specific monoclonal antibody (MAb) 17b, and interfered with entry of both a CCR5 (R5)-tropic and a CXCR4 (X4)-tropic Env pseudotyped virus. This peptide ligand therefore points at unique properties of CD4i epitopes shared by gp120 with different coreceptor usage and could thus serve to provide new insight into the conserved structural details essential for coreceptor binding for further drug development.

  19. Folding of newly translated membrane protein CCR5 is assisted by the chaperonin GroEL-GroES

    PubMed Central

    Chi, Haixia; Wang, Xiaoqiang; Li, Jiqiang; Ren, Hao; Huang, Fang

    2015-01-01

    The in vitro folding of newly translated human CC chemokine receptor type 5 (CCR5), which belongs to the physiologically important family of G protein-coupled receptors (GPCRs), has been studied in a cell-free system supplemented with the surfactant Brij-35. The freshly synthesized CCR5 can spontaneously fold into its biologically active state but only slowly and inefficiently. However, on addition of the GroEL-GroES molecular chaperone system, the folding of the nascent CCR5 was significantly enhanced, as was the structural stability and functional expression of the soluble form of CCR5. The chaperonin GroEL was partially effective on its own, but for maximum efficiency both the GroEL and its GroES lid were necessary. These results are direct evidence for chaperone-assisted membrane protein folding and therefore demonstrate that GroEL-GroES may be implicated in the folding of membrane proteins. PMID:26585937

  20. Folding of newly translated membrane protein CCR5 is assisted by the chaperonin GroEL-GroES

    NASA Astrophysics Data System (ADS)

    Chi, Haixia; Wang, Xiaoqiang; Li, Jiqiang; Ren, Hao; Huang, Fang

    2015-11-01

    The in vitro folding of newly translated human CC chemokine receptor type 5 (CCR5), which belongs to the physiologically important family of G protein-coupled receptors (GPCRs), has been studied in a cell-free system supplemented with the surfactant Brij-35. The freshly synthesized CCR5 can spontaneously fold into its biologically active state but only slowly and inefficiently. However, on addition of the GroEL-GroES molecular chaperone system, the folding of the nascent CCR5 was significantly enhanced, as was the structural stability and functional expression of the soluble form of CCR5. The chaperonin GroEL was partially effective on its own, but for maximum efficiency both the GroEL and its GroES lid were necessary. These results are direct evidence for chaperone-assisted membrane protein folding and therefore demonstrate that GroEL-GroES may be implicated in the folding of membrane proteins.

  1. Distribution of CCR5-Delta 32 and CCR2-64I alleles in an Argentine Amerindian population.

    PubMed

    Mangano, A; Theiler, G; Sala, L; Capucchio, M; Fainboim, L; Sen, L

    2001-08-01

    In order to evaluate the frequency distributions of CCR5-Delta 32 and CCR2-64I polymorphisms in an Amerindian population, we tested a total of 42 Chiriguanos individuals that are aboriginal inhabitants of the north west of Argentina. Only one carried the CCR5-Delta 32 allele (0.0238), while 17 out of 35 carried the CCR2-64I mutation, including one homozygous for the mutated allele (0.2571). Although the cohort studied is considered highly endogamic, the HLA genotyping revealed that 8 out of 42 subjects had a gene flow from Caucasian populations. The only heterozygous CCR5+/Delta 32 found and three heterozygous CCR2+/64I belonged to the admix group. In conclusion, the protective deletion CCR5-Delta 32 is practically absent in Chiriguanos whereas the CCR2-64I allele is highly frequent.

  2. C-C chemokine receptor type five (CCR5): An emerging target for the control of HIV infection.

    PubMed

    Barmania, Fatima; Pepper, Michael S

    2013-12-01

    When HIV was initially discovered as the causative agent of AIDS, many expected to find a vaccine within a few years. This has however proven to be elusive; it has been approximately 30 years since HIV was first discovered, and a suitable vaccine is still not in effect. In 2009, a paper published by Hutter et al. reported on a bone marrow transplant performed on an HIV positive individual using stem cells that were derived from a donor who was homozygous for a mutation in the CCR5 gene known as CCR5 delta-32 (Δ32) (Hütter et al., 2009). The HIV positive individual became HIV negative and remained free of viral detection after transplantation despite having halted anti-retroviral (ARV) treatment. This review will focus on CCR5 as a key component in HIV immunity and will discuss the role of CCR5 in the control of HIV infection.

  3. Folding of newly translated membrane protein CCR5 is assisted by the chaperonin GroEL-GroES.

    PubMed

    Chi, Haixia; Wang, Xiaoqiang; Li, Jiqiang; Ren, Hao; Huang, Fang

    2015-11-20

    The in vitro folding of newly translated human CC chemokine receptor type 5 (CCR5), which belongs to the physiologically important family of G protein-coupled receptors (GPCRs), has been studied in a cell-free system supplemented with the surfactant Brij-35. The freshly synthesized CCR5 can spontaneously fold into its biologically active state but only slowly and inefficiently. However, on addition of the GroEL-GroES molecular chaperone system, the folding of the nascent CCR5 was significantly enhanced, as was the structural stability and functional expression of the soluble form of CCR5. The chaperonin GroEL was partially effective on its own, but for maximum efficiency both the GroEL and its GroES lid were necessary. These results are direct evidence for chaperone-assisted membrane protein folding and therefore demonstrate that GroEL-GroES may be implicated in the folding of membrane proteins.

  4. Virus entry via the alternative coreceptors CCR3 and FPRL1 differs by human immunodeficiency virus type 1 subtype.

    PubMed

    Nedellec, R; Coetzer, M; Shimizu, N; Hoshino, H; Polonis, V R; Morris, L; Mårtensson, U E A; Binley, J; Overbaugh, J; Mosier, D E

    2009-09-01

    Human immunodeficiency virus type 1 (HIV-1) infects target cells by binding to CD4 and a chemokine receptor, most commonly CCR5. CXCR4 is a frequent alternative coreceptor (CoR) in subtype B and D HIV-1 infection, but the importance of many other alternative CoRs remains elusive. We have analyzed HIV-1 envelope (Env) proteins from 66 individuals infected with the major subtypes of HIV-1 to determine if virus entry into highly permissive NP-2 cell lines expressing most known alternative CoRs differed by HIV-1 subtype. We also performed linear regression analysis to determine if virus entry via the major CoR CCR5 correlated with use of any alternative CoR and if this correlation differed by subtype. Virus pseudotyped with subtype B Env showed robust entry via CCR3 that was highly correlated with CCR5 entry efficiency. By contrast, viruses pseudotyped with subtype A and C Env proteins were able to use the recently described alternative CoR FPRL1 more efficiently than CCR3, and use of FPRL1 was correlated with CCR5 entry. Subtype D Env was unable to use either CCR3 or FPRL1 efficiently, a unique pattern of alternative CoR use. These results suggest that each subtype of circulating HIV-1 may be subject to somewhat different selective pressures for Env-mediated entry into target cells and suggest that CCR3 may be used as a surrogate CoR by subtype B while FPRL1 may be used as a surrogate CoR by subtypes A and C. These data may provide insight into development of resistance to CCR5-targeted entry inhibitors and alternative entry pathways for each HIV-1 subtype.

  5. Partial protective effect of CCR5-Delta 32 heterozygosity in a cohort of heterosexual Italian HIV-1 exposed uninfected individuals.

    PubMed

    Trecarichi, Enrico M; Tumbarello, Mario; de Gaetano Donati, Katleen; Tamburrini, Enrica; Cauda, Roberto; Brahe, Christina; Tiziano, Francesco D

    2006-09-25

    Despite multiple sexual exposure to HIV-1 virus, some individuals remain HIV-1 seronegative (exposed seronegative, ESN). The mechanisms underlying this resistance remain still unclear, although a multifactorial pathogenesis can be hypothesised. Although several genetic factors have been related to HIV-1 resistance, the homozigosity for a mutation in CCR5 gene (the 32 bp deletion, i.e. CCR5-Delta32 allele) is presently considered the most relevant one. In the present study we analysed the genotype at CCR5 locus of 30 Italian ESN individuals (case group) who referred multiple unprotected heterosexual intercourse with HIV-1 seropositive partner(s), for at least two years. One hundred and twenty HIV-1 infected patients and 120 individuals representative of the general population were included as control groups. Twenty percent of ESN individuals had heterozygous CCR5-Delta 32 genotype, compared to 7.5% of HIV-1 seropositive and 10% of individuals from the general population, respectively. None of the analysed individuals had CCR5-Delta 32 homozygous genotype. Sequence analysis of the entire open reading frame of CCR5 was performed in all ESN subjects and no polymorphisms or mutations were identified. Moreover, we determined the distribution of C77G variant in CD45 gene, which has been previously related to HIV-1 infection susceptibility. The frequency of the C77G variant showed no significant difference between ESN subjects and the two control groups. In conclusion, our data show a significantly higher frequency of CCR5-Delta 32 heterozygous genotype (p = 0.04) among the Italian heterosexual ESN individuals compared to HIV-1 seropositive patients, suggesting a partial protective role of CCR5-Delta 32 heterozygosity in this cohort.

  6. Maraviroc decreases CCL8-mediated migration of CCR5+ regulatory T cells and reduces metastatic tumor growth in the lungs

    PubMed Central

    Halvorsen, E. C.; Hamilton, M. J.; Young, A.; Wadsworth, B. J.; LePard, N. E.; Lee, H. N.; Firmino, N.; Collier, J. L.; Bennewith, K. L.

    2016-01-01

    ABSTRACT Regulatory T cells (Tregs) play a crucial physiological role in the regulation of immune homeostasis, although recent data suggest Tregs can contribute to primary tumor growth by suppressing antitumor immune responses. Tregs may also influence the development of tumor metastases, although there is a paucity of information regarding the phenotype and function of Tregs in metastatic target organs. Herein, we demonstrate that orthotopically implanted metastatic mammary tumors induce significant Treg accumulation in the lungs, which is a site of mammary tumor metastasis. Tregs in the primary tumor and metastatic lungs express high levels of C–C chemokine receptor type 5 (CCR5) relative to Tregs in the mammary fat pad and lungs of tumor-free mice, and Tregs in the metastatic lungs are enriched for CCR5 expression in comparison to other immune cell populations. We also identify that C–C chemokine ligand 8 (CCL8), an endogenous ligand of CCR5, is produced by F4/80+ macrophages in the lungs of mice with metastatic primary tumors. Migration of Tregs toward CCL8 ex vivo is reduced in the presence of the CCR5 inhibitor Maraviroc. Importantly, treatment of mice with Maraviroc (MVC) reduces the level of CCR5+ Tregs and metastatic tumor burden in the lungs. This work provides evidence of a CCL8/CCR5 signaling axis driving Treg recruitment to the lungs of mice bearing metastatic primary tumors, representing a potential therapeutic target to decrease Treg accumulation and metastatic tumor growth. PMID:27471618

  7. Maraviroc decreases CCL8-mediated migration of CCR5(+) regulatory T cells and reduces metastatic tumor growth in the lungs.

    PubMed

    Halvorsen, E C; Hamilton, M J; Young, A; Wadsworth, B J; LePard, N E; Lee, H N; Firmino, N; Collier, J L; Bennewith, K L

    2016-06-01

    Regulatory T cells (Tregs) play a crucial physiological role in the regulation of immune homeostasis, although recent data suggest Tregs can contribute to primary tumor growth by suppressing antitumor immune responses. Tregs may also influence the development of tumor metastases, although there is a paucity of information regarding the phenotype and function of Tregs in metastatic target organs. Herein, we demonstrate that orthotopically implanted metastatic mammary tumors induce significant Treg accumulation in the lungs, which is a site of mammary tumor metastasis. Tregs in the primary tumor and metastatic lungs express high levels of C-C chemokine receptor type 5 (CCR5) relative to Tregs in the mammary fat pad and lungs of tumor-free mice, and Tregs in the metastatic lungs are enriched for CCR5 expression in comparison to other immune cell populations. We also identify that C-C chemokine ligand 8 (CCL8), an endogenous ligand of CCR5, is produced by F4/80(+) macrophages in the lungs of mice with metastatic primary tumors. Migration of Tregs toward CCL8 ex vivo is reduced in the presence of the CCR5 inhibitor Maraviroc. Importantly, treatment of mice with Maraviroc (MVC) reduces the level of CCR5(+) Tregs and metastatic tumor burden in the lungs. This work provides evidence of a CCL8/CCR5 signaling axis driving Treg recruitment to the lungs of mice bearing metastatic primary tumors, representing a potential therapeutic target to decrease Treg accumulation and metastatic tumor growth.

  8. Is the CCR5-59029-G/G genotype a protective factor for cardiomyopathy in Chagas disease?

    PubMed

    Fernández-Mestre, M T; Montagnani, S; Layrisse, Z

    2004-07-01

    Investigated were two CCR5 gene polymorphisms, the CCR5 Delta 32 deletion and the pCCR5 59029 A-->G promoter point mutation, in 107 ethnically mixed Venezuelan patients serologically positive for Trypanosoma cruzi (34 asymptomatic, 38 arrhythmic, 35 cardiomyopathic). No difference in the distribution of CCR5 Delta 32 among asymptomatic and symptomatic patients was found. We have observed an increase of the 59029-G phenotype among asymptomatic compared with symptomatic chagasic patients (68% vs. 58%), in agreement with previously reported data (57% vs. 31%). This frequency difference, although not statistically significant, is more marked when the 59029-G allele is present in homozygous form. However, a similar distribution of the G/G genotype is present among asymptomatic patients and patients with heart failure. Because it has been reported that the 59029G/G genotype associates with lower CCR5 expression, 37% of our T. cruzi-infected patients with heart failure are genetically predisposed to express low levels of CCR5 on the surface of CD8(+) T cells, contrary to what would be expected if an inflammatory response is required for severe cardiac damage. If confirmed, the possible protection that might be conferred by the G/G genotype may be due to the existence of other genes in linkage disequilibria.

  9. CCR5 and CCR3 expression on T CD3+ lymphocytes from HIV/Leishmania co-infected subjects.

    PubMed

    Nigro, L; Rizzo, M L; Vancheri, C; La Rosa, R; Mastruzzo, C; Tomaselli, V; Ragusa, A; Manuele, R; Cacopardo, B

    2007-12-01

    CC chemokine receptor 5 (CCR5) and CC chemokine receptor 3 (CCR3) are membrane-bound proteins involved in HIV-1 entry into susceptible cells. All T lymphocyte subsets display CCR5 and CCR3 on their membrane surface. T helper 1 cells are known to express CCR5 but not CCR3, and most of T cells expressing CCR3 are T helper 2. This study aimed to assess the expression of CCR5 and CCR3 on peripheral blood CD3+ T lymphocytes of HIV-Leishmania co-infected individuals. A total of 36 subjects were enrolled; nine had HIV-Leishmania co-infection; nine were HIV-infected without Leishmania, nine had visceral leishmaniasis without HIV co-infection and nine were healthy blood donors. HIV-Leishmania co-infected subjects showed a significantly higher rate of CCR5+CD3+ T lymphocytes in comparison with the other studied groups. The higher rate of CD3+ T-cells expressing CCR5 found in HIV-Leishmania co-infected subjects may be related to the role of Leishmania as an enhancer of the progression to AIDS.

  10. CCR5-Δ32 Heterozygosity, HIV-1 Reservoir Size, and Lymphocyte Activation in Individuals Receiving Long-term Suppressive Antiretroviral Therapy.

    PubMed

    Henrich, Timothy J; Hanhauser, Emily; Harrison, Linda J; Palmer, Christine D; Romero-Tejeda, Marisol; Jost, Stephanie; Bosch, Ronald J; Kuritzkes, Daniel R

    2016-03-01

    We conducted a case-controlled study of the associations of CCR5-Δ32 heterozygosity with human immunodeficiency virus type 1 (HIV-1) reservoir size, lymphocyte activation, and CCR5 expression in 114 CCR5(Δ32/WT) and 177 wild-type CCR5 AIDS Clinical Trials Group participants receiving suppressive antiretroviral therapy. Overall, no significant differences were found between groups for any of these parameters. However, higher levels of CCR5 expression correlated with lower amounts of cell-associated HIV-1 RNA. The relationship between CCR5-Δ32 heterozygosity, CCR5 expression, and markers of HIV-1 persistence is likely to be complex and may be influenced by factors such as the duration of ART.

  11. Prevention of vaginal SHIV transmission in rhesus macaques through inhibition of CCR5.

    PubMed

    Lederman, Michael M; Veazey, Ronald S; Offord, Robin; Mosier, Donald E; Dufour, Jason; Mefford, Megan; Piatak, Michael; Lifson, Jeffrey D; Salkowitz, Janelle R; Rodriguez, Benigno; Blauvelt, Andrew; Hartley, Oliver

    2004-10-15

    Topical agents, such as microbicides, that can protect against human immunodeficiency virus (HIV) transmission are urgently needed. Using a chimeric simian/human immunodeficiency virus (SHIV SF162), which is tropic for the chemokine receptor CCR5, we report that topical application of high doses of PSC-RANTES, an amino terminus-modified analog of the chemokine RANTES, provided potent protection against vaginal challenge in rhesus macaques. These experimental findings have potentially important implications for understanding vaginal transmission of HIV and the design of strategies for its prevention.

  12. Induction of HIV-Blocking Anti-CCR5 IgA in Peyers's Patches without Histopathological Alterations

    PubMed Central

    Pastori, Claudia; Diomede, Lorenzo; Venuti, Assunta; Fisher, Gregory; Jarvik, Jonathan; Bomsel, Morgane; Sanvito, Francesca

    2014-01-01

    ABSTRACT The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathological analysis revealed mild to moderate chronic inflammation in some tissues but failed in reporting signs of autoimmune dysfunction associated with immunizations. Antisera against immunogens representing the N terminus and extracellular loops 1 and 2 (Nter1 and ECL1 and ECL2) of CCR5 were generated. All showed specific anti-HIV activity, which was stronger in the anti-ECL1 and -ECL2 sera than in the anti-Nter sera. ECL1 and ECL2 antisera induced nearly complete long-lasting CCR5 downregulation of the receptor, and especially, their IgG-depleted fractions prevented HIV infection in neutralization and transcytosis assays. In conclusion, the ECL1 and ECL2 domains could offer a promising path to achieve significant anti-HIV activity in vivo. IMPORTANCE The study was the first to adopt a systematic strategy to compare the immunogenicities of all extracellular domains of the CCR5 molecule and to set optimal conditions leading to generation of specific antibodies in the mouse model. There

  13. Long-lasting CCR5 internalization by antibodies in a subset of long-term nonprogressors: a possible protective effect against disease progression

    PubMed Central

    Pastori, Claudia; Weiser, Barbara; Barassi, Claudia; Uberti-Foppa, Caterina; Ghezzi, Silvia; Longhi, Renato; Calori, Giliola; Burger, Harold; Kemal, Kimdar; Poli, Guido; Lazzarin, Adriano; Lopalco, Lucia

    2006-01-01

    Exposure to HIV-1 does not necessarily result in infection and progression toward disease, thus suggesting that the control of viral infection may be achieved. Antibodies to CCR5 have been detected in HIV-exposed but uninfected subjects (ESNs); thus, these antibodies could be involved in HIV protection. To assess whether anti-CCR5 antibodies may also contribute to slow HIV disease progression, we searched for anti-CCR5 antibodies in 497 subjects, including 85 long-term nonprogressors (LTNPs), 70 progressors, 135 HIV+ patients treated with highly active antiretroviral therapy (HAART), and 207 seronegative donors. We found anti-CCR5 antibodies in a fraction of the LTNPs(23.5%) but not in the other populations studied (P < .001). These antibodies recognized a conformational epitope within the first extramembrane loop of CCR5, and they induced a stable and long-lasting downregulation of CCR5 on the surface of T lymphocytes, which inhibited HIV entry. In addition, CD4+ lymphocytes from LTNPs having anti-CCR5 antibodies are resistance to R5 strains of HIV-1. Follow-up studies showed that the loss of anti-CCR5 antibodies occurred in some subjects, and this loss was significantly associated with a progression toward disease, whereas subjects who retained anti-CCR5 Abs maintained their LTNP status. Induction of anti-CCR5 Abs could be relevant to vaccine design and therapeutics. PMID:16522810

  14. Lack of CCR5 modifies glial phenotypes and population of the nigral dopaminergic neurons, but not MPTP-induced dopaminergic neurodegeneration.

    PubMed

    Choi, Dong-Young; Lee, Myung Koo; Hong, Jin Tae

    2013-01-01

    Constitutive expression of C-C chemokine receptor (CCR) 5 has been detected in astrocytes, microglia and neurons, but its physiological roles in the central nervous system are obscure. The bidirectional interactions between neuron and glial cells through CCR5 and its ligands were thought to be crucial for maintaining normal neuronal activities. No study has described function of CCR5 in the dopaminergic neurodegeneration in Parkinson's disease. In order to examine effects of CCR5 on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration, we employed CCR5 wild type (WT) and knockout (KO) mice. Immunostainings for tyrosine hydroxylase (TH) exhibited that CCR5 KO mice had lower number of TH-positive neurons even in the absence of MPTP. Difference in MPTP (15mg/kg×4 times, 2hr interval)-mediated loss of TH-positive neurons was subtle between CCR5 WT and KO mice, but there was larger dopamine depletion, behavioral impairments and microglial activation in CCR5 deficient mice. Intriguingly, CCR5 KO brains contained higher immunoreactivity for monoamine oxidase (MAO) B which was mainly localized within astrocytes. In agreement with upregulation of MAO B, concentration of MPP+ was higher in the substantia nigra and striatum of CCR5 KO mice after MPTP injection. We found remarkable activation of p38 MAPK in CCR5 deficient mice, which positively regulates MAO B expression. These results indicate that CCR5 deficiency modifies the nigrostriatal dopaminergic neuronal system and bidirectional interaction between neurons and glial cells via CCR5 might be important for dopaminergic neuronal survival.

  15. Chemokine receptor CCR5 Delta 32 genetic analysis using multiple specimen types and the NucliSens Basic Kit.

    PubMed

    Tetali, S; Lee, E M; Kaplan, M H; Romano, J W; Ginocchio, C C

    2001-09-01

    Resistance to HIV-1 infection and delayed disease progression have been associated with a 32-bp deletion (Delta32) in the gene encoding the CCR5 chemokine receptor. In the present study we describe the modification of a nucleic acid sequence-based amplification (NASBA)-based CCR5 genotyping assay for a NucliSens Basic Kit (Organon Teknika, Durham, N.C.) format using a new target-specific sandwich oligonucleotide detection methodology. The new method permitted the use of generic electrochemiluminescent probes supplied in the NucliSens Basic Kit, whereas the original NASBA method required expensive target-specific ruthenium detection probes. The Basic Kit CCR5 Delta32 genotypic analysis was in 100% concordance with both the original NASBA assay and DNA PCR results. This study also evaluated the use of multiple specimen types, including peripheral blood mononuclear cells (PBMC), whole blood, dried blood spots, buccal scrapings, and plasma, for CCR5 genotype analysis. The sensitivities of the three assays were comparable when PBMC or whole blood was the specimen source. In contrast, when dried blood spots, buccal scrapings, or plasma was used as the sample source, the sensitivity of DNA PCR was 80.95, 42.8, or 0%, respectively, compared to 100% sensitivity obtained with the original NASBA and Basic Kit NASBA assays. Our study indicates that the NucliSens Basic Kit NASBA assay is very sensitive and specific for CCR5 Delta32 genotyping using multiple sample types.

  16. Chemokine Receptor CCR5 Δ32 Genetic Analysis Using Multiple Specimen Types and the NucliSens Basic Kit†

    PubMed Central

    Tetali, Suryakumari; Lee, Eun Mi; Kaplan, Mark H.; Romano, Joseph W.; Ginocchio, Christine C.

    2001-01-01

    Resistance to HIV-1 infection and delayed disease progression have been associated with a 32-bp deletion (Δ32) in the gene encoding the CCR5 chemokine receptor. In the present study we describe the modification of a nucleic acid sequence-based amplification (NASBA)-based CCR5 genotyping assay for a NucliSens Basic Kit (Organon Teknika, Durham, N.C.) format using a new target-specific sandwich oligonucleotide detection methodology. The new method permitted the use of generic electrochemiluminescent probes supplied in the NucliSens Basic Kit, whereas the original NASBA method required expensive target-specific ruthenium detection probes. The Basic Kit CCR5 Δ32 genotypic analysis was in 100% concordance with both the original NASBA assay and DNA PCR results. This study also evaluated the use of multiple specimen types, including peripheral blood mononuclear cells (PBMC), whole blood, dried blood spots, buccal scrapings, and plasma, for CCR5 genotype analysis. The sensitivities of the three assays were comparable when PBMC or whole blood was the specimen source. In contrast, when dried blood spots, buccal scrapings, or plasma was used as the sample source, the sensitivity of DNA PCR was 80.95, 42.8, or 0%, respectively, compared to 100% sensitivity obtained with the original NASBA and Basic Kit NASBA assays. Our study indicates that the NucliSens Basic Kit NASBA assay is very sensitive and specific for CCR5 Δ32 genotyping using multiple sample types. PMID:11527812

  17. Coreceptor usage of Chinese HIV-1 and impact of X4/DM transmission clusters among recently infected men who have sex with men

    PubMed Central

    Li, Xiaoshan; Zhu, Kexin; Li, Wei; Fang, Kun; Musa, Taha Hussein; Song, Yue; Du, Guoping; Gao, Rong; Guo, Yan; Yan, Wenjuan; Xuan, Yang; Zhong, Ping; Wei, Pingmin

    2016-01-01

    Abstract To characterize the current frequency of HIV-1 coreceptor usage in China and assess the candidacy of CCR5 antagonists for treatment of HIV infections. In addition, we aimed to evaluate the potential of X4/DM virus transmission in recently infected men who have sex with men (MSM) individuals. Viral tropism testing was performed on samples from 399 MSM individuals and on 2408 available Chinese HIV-1 V3 sequences downloaded from the Los Alamos database using Geno2pheno and WebPSSM in combination. The transmission clusters were evaluated using pol sequences from 291 recently infected MSM with a maximum likelihood, maximum pairwise distance, and Bayesian inference. A higher prevalence of X4/DM viruses was observed in individuals infected with CRF01_AE strains than with subtype B (27.8% vs 12.2%, P < 0.001) and CRF07_BC/CRF08_BC/C (27.8% vs 1.0%, P < 0.001). Seven clusters containing only X4/DM viruses were detected in 40 transmission clusters. No significant difference in proportions between clustered X4/DM viruses and R5 viruses was found (P = 0.683). The high proportion of CXCR4 usage for CRF01_AE strains may result in the loss of susceptibility to maraviroc since CRF01_AE has become the most prevalent strains in China. The high prevalence of X4/DM viruses among recently CRF01_AE-infected individuals may be attributed to the stochasticity of HIV transmission, which implied that the early viral tropism screening and treatment would be the key for controlling the epidemic of CRF01_AE strains in China. PMID:27684870

  18. Coreceptor usage of Chinese HIV-1 and impact of X4/DM transmission clusters among recently infected men who have sex with men.

    PubMed

    Li, Xiaoshan; Zhu, Kexin; Li, Wei; Fang, Kun; Musa, Taha Hussein; Song, Yue; Du, Guoping; Gao, Rong; Guo, Yan; Yan, Wenjuan; Xuan, Yang; Zhong, Ping; Wei, Pingmin

    2016-09-01

    To characterize the current frequency of HIV-1 coreceptor usage in China and assess the candidacy of CCR5 antagonists for treatment of HIV infections. In addition, we aimed to evaluate the potential of X4/DM virus transmission in recently infected men who have sex with men (MSM) individuals.Viral tropism testing was performed on samples from 399 MSM individuals and on 2408 available Chinese HIV-1 V3 sequences downloaded from the Los Alamos database using Geno2pheno and WebPSSM in combination. The transmission clusters were evaluated using pol sequences from 291 recently infected MSM with a maximum likelihood, maximum pairwise distance, and Bayesian inference.A higher prevalence of X4/DM viruses was observed in individuals infected with CRF01_AE strains than with subtype B (27.8% vs 12.2%, P < 0.001) and CRF07_BC/CRF08_BC/C (27.8% vs 1.0%, P < 0.001). Seven clusters containing only X4/DM viruses were detected in 40 transmission clusters. No significant difference in proportions between clustered X4/DM viruses and R5 viruses was found (P = 0.683).The high proportion of CXCR4 usage for CRF01_AE strains may result in the loss of susceptibility to maraviroc since CRF01_AE has become the most prevalent strains in China. The high prevalence of X4/DM viruses among recently CRF01_AE-infected individuals may be attributed to the stochasticity of HIV transmission, which implied that the early viral tropism screening and treatment would be the key for controlling the epidemic of CRF01_AE strains in China.

  19. CCR2+CCR5+ T Cells Produce Matrix Metalloproteinase-9 and Osteopontin in the Pathogenesis of Multiple Sclerosis

    PubMed Central

    Sato, Wakiro; Tomita, Atsuko; Ichikawa, Daijyu; Lin, Youwei; Kishida, Hitaru; Miyake, Sachiko; Ogawa, Masafumi; Okamoto, Tomoko; Murata, Miho; Kuroiwa, Yoshiyuki

    2012-01-01

    Multiple sclerosis (MS) is a demyelinating disease of the CNS that is presumably mediated by CD4+ autoimmune T cells. Although both Th1 and Th17 cells have the potential to cause inflammatory CNS pathology in rodents, the identity of pathogenic T cells remains unclear in human MS. Given that each Th cell subset preferentially expresses specific chemokine receptors, we were interested to know whether T cells defined by a particular chemokine receptor profile play an active role in the pathogenesis of MS. In this article, we report that CCR2+CCR5+ T cells constitute a unique population selectively enriched in the cerebrospinal fluid of MS patients during relapse but not in patients with other neurologic diseases. After polyclonal stimulation, the CCR2+CCR5+ T cells exhibited a distinct ability to produce matrix metalloproteinase-9 and osteopontin, which are involved in the CNS pathology of MS. Furthermore, after TCR stimulation, the CCR2+CCR5+ T cells showed a higher invasive potential across an in vitro blood–brain barrier model compared with other T cells. Of note, the CCR2+CCR5+ T cells from MS patients in relapse are reactive to myelin basic protein, as assessed by production of IFN-γ. We also demonstrated that the CCR6−, but not the CCR6+, population within CCR2+CCR5+ T cells was highly enriched in the cerebrospinal fluid during MS relapse (p < 0.0005) and expressed higher levels of IFN-γ and matrix metalloproteinase-9. Taken together, we propose that autoimmune CCR2+CCR5+CCR6− Th1 cells play a crucial role in the pathogenesis of MS. PMID:23071279

  20. Association study of CCR5 delta 32 polymorphism among the HLA-DRB1 Caucasian population in Northern Paraná, Brazil.

    PubMed

    Muxel, Sandra Marcia; Borelli, Sueli Donizete; Amarante, Marla Karine; Voltarelli, Julio Cesar; Aoki, Mateus Nobrega; de Oliveira, Carlos Eduardo Coral; Ehara Watanabe, Maria Angelica

    2008-01-01

    Chemokines are important determinants of early inflammatory response. The CC chemokine receptor 5 (CCR5) delta 32 variant results in a nonfunctional form of the chemokine receptor and has been implicated in a variety of immune-mediated diseases. In the present study, polymerase chain reaction (PCR) for genomic deoxyribonucleic acid (DNA) samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 225-basepair (bp) product from the normal CCR5 allele and a 193-bp product from the 32 bp deletion allele. Human leukocyte antigen (HLA) class II (DRB1) typing was performed by PCR-sequence-specific primer (PCR-SSP). The aim of this study was to evaluate the association of HLA-DRB1 and CCR5 genetic polymorphisms. To evaluate the frequency distributions of CCR5 delta 32 polymorphisms in a Brazilian population and their association with allelic distribution of HLA genes, DRB1; a total of 120 Caucasian individuals from northern Paraná, Brazil, were tested. The CCR5/CCR5 genotype was found in 108 individuals (90%) and only one carried the CCR5 delta 32 allele homozygous genotype (0.0238), while 12 (10%) carried the CCR5 delta 32 allele heterozygous genotype. The observed frequency for the CCR5 delta 32 allele was 0.05 in the population studied. The results revealed a CCR5 delta 32 allele occurrence with HLA-DRB1(*)01 and DRB1(*)04 (P<0.05). It is possible that HLA-DRB1(*)01 and DRB1(*)04 alleles could be associated with the delta 32-bp deletion of CCR5.

  1. CCR2 and CCR5 genes polymorphisms in benign prostatic hyperplasia and prostate cancer.

    PubMed

    Zambra, Francis Maria Báo; Biolchi, Vanderlei; Brum, Ilma Simoni; Chies, José Artur Bogo

    2013-08-01

    Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are two chronic conditions, very common in aged men, that have been associated to inflammatory process. Chemokines and their receptors are recognized as critical mediators of inflammatory responses, they regulate immune cell migration and are implicated in tumor pathogenesis. The impact of two chemokine receptor gene polymorphisms, CCR2-64I (rs1799864) and CCR5-Δ32 (rs333), was evaluated in BPH and PCa. 385 DNA samples (130 BPH, 136 PCa, 119 healthy control) were genotyped. The allele frequencies were similar among control, BPH and PCa groups. Median of serum PSA levels was different between groups: 0.79, 1.45 and 6.91 ng/mL in control, BPH and PCa groups, respectively (all p<0.001). The prostate volume median was 20.00 cm(3) in the control group, thus, lower than BPH (35.35 cm(3)) and PCa (35.80 cm(3)) (both p<0.001), nevertheless no statistical significant difference was observed between BPH and PCa patients (p=0.172). Remarkably, CCR2-64I was a protective factor to PCa when compared with BPH (OR=0.550; 95%CI=0.311-0.975), although the statistically significant difference was lost after correction for multiple comparisons. No significant associations of CCR5-Δ32 variant were observed with BPH, PCa or PCa clinicopathologic status. Our data suggest the influence of CCR2-64I variant in the development of prostate cancer.

  2. Extracellular disulfide bridges serve different purposes in two homologous chemokine receptors, CCR1 and CCR5.

    PubMed

    Rummel, Pia C; Thiele, Stefanie; Hansen, Lærke S; Petersen, Trine P; Sparre-Ulrich, Alexander H; Ulven, Trond; Rosenkilde, Mette M

    2013-09-01

    In addition to the 7 transmembrane receptor (7TM)-conserved disulfide bridge between transmembrane (TM) helix 3 and extracellular loop (ECL)-2, chemokine receptors (CCR) contain a disulfide bridge between the N terminus and what previously was believed to be ECL-3. Recent crystal and NMR structures of the CXC chemokine receptors (CXCR) CXCR4 and CXCR1, combined with structural analysis of all endogenous chemokine receptors indicate that this chemokine receptor-conserved bridge in fact connects the N terminus to the top of TM-7. By employing chemokine ligands that mainly target extracellular receptor regions and small-molecule ligands that predominantly interact with residues in the main binding crevice, we show that the 7TM-conserved bridge is essential for all types of ligand-mediated activation, whereas the chemokine-conserved bridge is dispensable for small-molecule activation in CCR1. However, in striking contrast to previous studies in other chemokine receptors, high-affinity CCL3 chemokine binding was maintained in the absence of either bridge. In the highly related CCR5, a completely different dependency was observed as neither activation nor binding of the same chemokines was retained in the absence of either bridge. In contrast, both bridges were dispensable for activation by the same small molecules. This indicates that CCR5 activity is independent of extracellular regions, whereas in CCR1 the preserved folding of ECL-2 is necessary for activation. These results indicate that conserved structural features in a receptor subgroup do not necessarily provide specific traits for the whole subgroup but rather provide unique traits to the single receptors.

  3. Frequency of the CCR5-delta32 allele in Brazilian populations: A systematic literature review and meta-analysis.

    PubMed

    Silva-Carvalho, Wlisses Henrique Veloso; de Moura, Ronald Rodrigues; Coelho, Antonio Victor Campos; Crovella, Sergio; Guimarães, Rafael Lima

    2016-09-01

    The CCR5 is a chemokine receptor widely expressed by several immune cells that are engaged in inflammatory responses. Some populations have individuals exhibiting a 32bp deletion in the CCR5 gene (CCR5-delta32) that produces a truncated non-functional protein not expressed on the cell surface. This polymorphism, known to be associated with susceptibility to infectious and inflammatory diseases, such as osteomyelitis, pre-eclampsia, systemic lupus erythematous, juvenile idiopathic arthritis, rheumatoid arthritis and HIV/AIDS, is more commonly found in European populations with average frequency of 10%. However, it is also possible to observe a significant frequency in other world populations, such as the Brazilian one. We performed a systematic review and meta-analysis of CCR5-delta32 genetic association studies in Brazilian populations throughout the country to estimate the frequency of this polymorphism. We also compared CCR5-delta32 frequencies across Brazilian regions. The systematic literature reviewed studies involving delta32 allele in Brazilian populations published from 1995 to 2015. Among the reviewed literature, 25 studies including 30 Brazilian populations distributed between the North, Northeast, South and Southeast regions were included in our meta-analysis. We observed an overall allelic frequency of 4% (95%-CI, 0.03-0.05), that was considered moderate and, notably, higher than some European populations, such as Cyprus (2.8%), Italy (3%) and Greece (2.4%). Regarding the regional frequency comparisons between North-Northeast (N-NE) and South-Southeast (S-SE) regions, we observed an allelic frequency of 3% (95%-CI, 0.02-0.04) and 4% (95%-CI, 0.03-0.05), respectively. The populations from S-SE regions had a slightly higher CCR5-delta32 frequency than N-NE regions (OR=1.41, p=0.002). Although there are several studies about the CCR5-delta32 polymorphism and its effect on the immune response of some infectious diseases, this report is the first meta

  4. Evaluating plague and smallpox as historical selective pressures for the CCR5-Delta 32 HIV-resistance allele.

    PubMed

    Galvani, Alison P; Slatkin, Montgomery

    2003-12-09

    The high frequency, recent origin, and geographic distribution of the CCR5-Delta 32 deletion allele together indicate that it has been intensely selected in Europe. Although the allele confers resistance against HIV-1, HIV has not existed in the human population long enough to account for this selective pressure. The prevailing hypothesis is that the selective rise of CCR5-Delta 32 to its current frequency can be attributed to bubonic plague. By using a population genetic framework that takes into account the temporal pattern and age-dependent nature of specific diseases, we find that smallpox is more consistent with this historical role.

  5. The role of the alternative coreceptor GPR15 in SIV tropism for human cells.

    PubMed

    Kiene, Miriam; Marzi, Andrea; Urbanczyk, Andreas; Bertram, Stephanie; Fisch, Tanja; Nehlmeier, Inga; Gnirss, Kerstin; Karsten, Christina B; Palesch, David; Münch, Jan; Chiodi, Francesca; Pöhlmann, Stefan; Steffen, Imke

    2012-11-10

    Many SIV isolates can employ the orphan receptor GPR15 as coreceptor for efficient entry into transfected cell lines, but the role of endogenously expressed GPR15 in SIV cell tropism is largely unclear. Here, we show that several human B and T cell lines express GPR15 on the cell surface, including the T/B cell hybrid cell line CEMx174, and that GPR15 expression is essential for SIV infection of CEMx174 cells. In addition, GPR15 expression was detected on subsets of primary human CD4(+), CD8(+) and CD19(+) peripheral blood mononuclear cells (PBMCs), respectively. However, GPR15(+) PBMCs were not efficiently infected by HIV and SIV, including cells from individuals homozygous for the defective Δ32 ccr5 allele. These results suggest that GPR15 is coexpressed with CD4 on PBMCs but that infection of CD4(+), GPR15(+) cells is not responsible for the well documented ability of SIV to infect CCR5(-) blood cells.

  6. X4-tropic human immunodeficiency virus IIIB utilizes CXCR4 as coreceptor, as distinct from R5X4-tropic viruses.

    PubMed

    Islam, Salequl; Hoque, Sheikh Ariful; Adnan, Nihad; Tanaka, Atsushi; Jinno-Oue, Atsushi; Hoshino, Hiroo

    2013-06-01

    Human immunodeficiency viruses initiate infections via CCR5 coreceptors and then change their tropism to C-X-C chemokine receptor type 4 (CXCR4), this change being associated with rapid disease progression. HIV-1IIIB, a widely described pure X4-tropic strain, is distinct from R5X4-tropic viruses. In this study, the requirement for amino terminal regions (NTRs) of CXCR4 for entry of HIV-1IIIB virus into host cells was examined and compared to that of R5X4-tropic viruses. CXCR4 and its deletion mutant (CXCR4ΔNTR23; first 23 amino acids removed from NTR) were amplified to examine their coreceptor activities. NP-2/CD4/CXCR4 and NP-2/CD4/CXCR4ΔNTR23 cell lines were prepared accordingly. Indirect immune fluorescence assay (IFA), PCR, and reverse transcriptase (RT) activity were used to compare the process of infection of host cells by HIV-1IIIB virus, one R5-tropic and five other R5X4-tropic viruses. All the R5X4-tropic HIVs were found to utilize both CCR5 and CXCR4 but unable to use CXCR4ΔNTR23 as coreceptors. In contrast, X4-tropic HIV-1IIIB was found to preferentially infect through CXCR4ΔNTR23. Viral antigens in infected NP-2/CD4/CXCR4ΔNTR23 cells were detected by IFA and confirmed by detection of proviral DNA and by performing RT assays on the spent cell-supernatants. In dual tropic viruses, deletion of 23 amino acids from NTR abrogates the coreceptor activity of CXCR4. This observation demonstrates that NTR of CXCR4 have an obligatory coreceptor role for dual tropic viruses. However, HIV-1IIIB may have different requirements for NTR than R5X4 viruses or may infect host cells independent of NTR of CXCR4.

  7. Determinants for Sensitivity of Human Immunodeficiency Virus Coreceptor CXCR4 to the Bicyclam AMD3100

    PubMed Central

    Labrosse, Béatrice; Brelot, Anne; Heveker, Nikolaus; Sol, Nathalie; Schols, Dominique; De Clercq, Erik; Alizon, Marc

    1998-01-01

    The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4. PMID:9658078

  8. HIV-1 envelope, integrins and co-receptor use in mucosal transmission of HIV.

    PubMed

    Cicala, Claudia; Arthos, James; Fauci, Anthony S

    2011-01-27

    It is well established that HIV-1 infection typically involves an interaction between the viral envelope protein gp120/41 and the CD4 molecule followed by a second interaction with a chemokine receptor, usually CCR5 or CXCR4. In the early stages of an HIV-1 infection CCR5 using viruses (R5 viruses) predominate. In some viral subtypes there is a propensity to switch to CXCR4 usage (X4 viruses). The receptor switch occurs in ~ 40% of the infected individuals and is associated with faster disease progression. This holds for subtypes B and D, but occurs less frequently in subtypes A and C. There are several hypotheses to explain the preferential transmission of R5 viruses and the mechanisms that lead to switching of co-receptor usage; however, there is no definitive explanation for either. One important consideration regarding transmission is that signaling by R5 gp120 may facilitate transmission of R5 viruses by inducing a permissive environment for HIV replication. In the case of sexual transmission, infection by HIV requires the virus to breach the mucosal barrier to gain access to the immune cell targets that it infects; however, the immediate events that follow HIV exposure at genital mucosal sites are not well understood. Upon transmission, the HIV quasispecies that is replicating in an infected donor contracts through a "genetic bottleneck", and often infection results from a single infectious event. Many details surrounding this initial infection remain unresolved. In mucosal tissues, CD4(+) T cells express high levels of CCR5, and a subset of these CD4(+)/CCR5(high) cells express the integrin α₄β₇, the gut homing receptor. CD4(+)/CCR5(high)/ α4β7(high) T cells are highly susceptible to infection by HIV-1 and are ideal targets for an efficient productive infection at the point of transmission. In this context we have demonstrated that the HIV-1 envelope protein gp120 binds to α₄β₇ on CD4(+) T cells. On CD4(+)/CCR5(high)/ α4β7(high) T cells,

  9. Human immunodeficiency virus receptor and coreceptor expression on human uterine epithelial cells: regulation of expression during the menstrual cycle and implications for human immunodeficiency virus infection

    PubMed Central

    Yeaman, Grant R; Howell, Alexandra L; Weldon, Sally; Demian, Douglas J; Collins, Jane E; O'Connell, Denise M; Asin, Susana N; Wira, Charles R; Fanger, Michael W

    2003-01-01

    Human immunodeficiency virus-1 (HIV-1) is primarily a sexually transmitted disease. Identification of cell populations within the female reproductive tract that are initially infected, and the events involved in transmission of infection to other cells, remain to be established. In this report, we evaluated expression of HIV receptors and coreceptors on epithelial cells in the uterus and found they express several receptors critical for HIV infection including CD4, CXCR4, CCR5 and galactosylceramide (GalC). Moreover, expression of these receptors varied during the menstrual cycle. Expression of CD4 and CCR5 on uterine epithelial cells is high throughout the proliferative phase of the menstrual cycle when blood levels of oestradiol are high. In contrast, CXCR4 expression increased gradually throughout the proliferative phase. During the secretory phase of the cycle when both oestradiol and progesterone are elevated, CD4 and CCR5 expression decreased whereas CXCR4 expression remained elevated. Expression of GalC on endometrial glands is higher during the secretory phase than during the proliferative phase of the menstrual cycle. Because epithelial cells line the female reproductive tract and express HIV receptors and coreceptors, it is likely that they are one of the first cell types to become infected. The hormonal regulation of HIV receptor expression may affect a woman's susceptibility to HIV infection during her menstrual cycle. Moreover, selective coreceptor expression could account for the preferential transmission of R5-HIV-1 strains to women. In addition, these studies provide evidence that the uterus, and potentially the entire upper reproductive tract, are important sites for the initial events involved in HIV infection. PMID:12709027

  10. Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

    SciTech Connect

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Sanders, Rogier W.; Johan Klasse, Per; Moore, John P.

    2012-07-05

    A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

  11. R5 HIV productively infects Langerhans cells, and infection levels are regulated by compound CCR5 polymorphisms.

    PubMed

    Kawamura, Tatsuyoshi; Gulden, Forrest O; Sugaya, Makoto; McNamara, David T; Borris, Debra L; Lederman, Michael M; Orenstein, Jan M; Zimmerman, Peter A; Blauvelt, Andrew

    2003-07-08

    Langerhans cells (LCs) are suspected to be initial targets for HIV after sexual exposure (by becoming infected or by capturing virus). Here, productive R5 HIV infection of LC ex vivo and LC-mediated transmission of virus to CD4+ T cells were both found to depend on CCR5. By contrast, infection of monocyte-derived dendritic cells and transfer of infection from monocyte-derived dendritic cells to CD4+ T cells were mediated by CCR5-dependent as well as DC-specific ICAM-3-grabbing nonintegrin-dependent pathways. Furthermore, in 62 healthy individuals, R5 HIV infection levels in LCs ex vivo were associated with CCR5 genotype. Specifically, genotyping for ORF Delta 32 revealed that LCs isolated from ORF Delta 32/wt individuals were significantly less susceptible to HIV when compared with LCs isolated from ORFwt/wt individuals (P = 0.016). Strikingly, further genetic analyses of the A-2459G CCR5 promoter polymorphism in ORF Delta 32/wt heterozygous individuals revealed that LCs isolated from -2459A/G + ORF Delta 32/wt individuals were markedly less susceptible to HIV than were LCs from -2459A/A + ORF Delta 32/wt individuals (P = 0.012). Interestingly, these genetic susceptibility data in LCs parallel those of genetic susceptibility studies performed in cohorts of HIV-infected individuals. Thus, we suggest that CCR5-mediated infection of LCs, and not capture of virus by LCs, provides a biologic basis for understanding certain aspects of host genetic susceptibility to initial HIV infection.

  12. Maximal T cell-mediated antitumor responses rely upon CCR5 expression in both CD4(+) and CD8(+) T cells.

    PubMed

    González-Martín, Alicia; Gómez, Lucio; Lustgarten, Joseph; Mira, Emilia; Mañes, Santos

    2011-08-15

    Immune responses against cancer rely upon leukocyte trafficking patterns that are coordinated by chemokines. CCR5, the receptor for chemotactic chemokines MIP1alpha, MIP1beta, and RANTES (CCL3, CCL4, CCL5), exerts major regulatory effects on CD4(+)- and CD8(+) T cell-mediated immunity. Although CCR5 and its ligands participate in the response to various pathogens, its relevance to tumoral immune control has been debated. Here, we report that CCR5 has a specific, ligand-dependent role in optimizing antitumor responses. In adoptive transfer studies, efficient tumor rejection required CCR5 expression by both CD4(+) and CD8(+) T cells. CCR5 activation in CD4(+) cells resulted in CD40L upregulation, leading to full maturation of antigen-presenting cells and enhanced CD8(+) T-cell crosspriming and tumor infiltration. CCR5 reduced chemical-induced fibrosarcoma incidence and growth, but did not affect the onset or progression of spontaneous breast cancers in tolerogenic Tg(MMTV-neu) mice. However, CCR5 was required for TLR9-mediated reactivation of antineu responses in these mice. Our results indicate that CCR5 boosts T-cell responses to tumors by modulating helper-dependent CD8(+) T-cell activation.

  13. Coreceptor gene imprinting governs thymocyte lineage fate

    PubMed Central

    Adoro, Stanley; McCaughtry, Thomas; Erman, Batu; Alag, Amala; Van Laethem, François; Park, Jung-Hyun; Tai, Xuguang; Kimura, Motoko; Wang, Lie; Grinberg, Alex; Kubo, Masato; Bosselut, Remy; Love, Paul; Singer, Alfred

    2012-01-01

    Immature thymocytes are bipotential cells that are signalled during positive selection to become either helper- or cytotoxic-lineage T cells. By tracking expression of lineage determining transcription factors during positive selection, we now report that the Cd8 coreceptor gene locus co-opts any coreceptor protein encoded within it to induce thymocytes to express the cytotoxic-lineage factor Runx3 and to adopt the cytotoxic-lineage fate, findings we refer to as ‘coreceptor gene imprinting'. Specifically, encoding CD4 proteins in the endogenous Cd8 gene locus caused major histocompatibility complex class II-specific thymocytes to express Runx3 during positive selection and to differentiate into CD4+ cytotoxic-lineage T cells. Our findings further indicate that coreceptor gene imprinting derives from the dynamic regulation of specific cis Cd8 gene enhancer elements by positive selection signals in the thymus. Thus, for coreceptor-dependent thymocytes, lineage fate is determined by Cd4 and Cd8 coreceptor gene loci and not by the specificity of T-cell antigen receptor/coreceptor signalling. This study identifies coreceptor gene imprinting as a critical determinant of lineage fate determination in the thymus. PMID:22036949

  14. Downregulation of CCR5 inhibits the proliferation and invasion of cervical cancer cells and is regulated by microRNA-107.

    PubMed

    Che, Li-Fan; Shao, Su-Fang; Wang, Li-Xin

    2016-02-01

    Cervical cancer is among the most prevalent forms of cancer worldwide. C-C chemokine receptor type 5 (CCR5) is hypothesized to be a key functional protein involved in tumorigenesis. However, the role of CCR5 in cervical cancer remains unclear. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to evaluate the mRNA and protein expression levels of CCR5 in human cervical carcinoma tissues. Furthermore, a small interfering RNA was employed to knockdown CCR5 in HeLa and C33A cells. MTT, colony formation and Transwell assays were performed to determine the effects of this knockdown on cell viability, proliferation and invasion. In addition, micro RNA (miR)-107 was identified as a potential candidate regulator of CCR5 using miR prediction algorithms, and the effects of miR-107 and its antisense miR on CCR5 mRNA expression were determined. The results of the present study indicated that CCR5 is overexpressed in human cervical cancer tissues compared with adjacent normal tissues, and its downregulation inhibits cervical cancer cell growth and proliferation. Furthermore, the downregulation of CCR5 appears to suppress cervical cancer cell invasion. Finally, the tumor suppressor miR-107 was able to directly target CCR5 and inhibit its expression. These results suggest that the upregulation of CCR5, which is inhibited by miR-107, may play a carcinogenic role in cervical cancer and could provide a novel therapeutic target in the future.

  15. Assessment of the pharmacokinetics, safety and tolerability of maraviroc, a novel CCR5 antagonist, in healthy volunteers

    PubMed Central

    Abel, Samantha; van der Ryst, Elna; Rosario, Maria C; Ridgway, Caroline E; Medhurst, Christine G; Taylor-Worth, Richard J; Muirhead, Gary J

    2008-01-01

    Aims To evaluate the pharmacokinetics, safety and tolerability of single and multiple oral doses of maraviroc in healthy volunteers. Methods Three double-blind, placebo-controlled, dose-escalation studies with either single or multiple doses of maraviroc were conducted in healthy volunteers. Plasma and urine samples were collected to investigate the pharmacokinetics of maraviroc and evaluate any changes with respect to dose and duration/frequency of dosing. Safety and toleration of maraviroc were also assessed. Results Maraviroc is rapidly absorbed following oral administration, and plasma Tmax is achieved within 0.5–4.0 h postdose. Steady-state plasma concentrations are achieved after 7 consecutive days of dosing. Although the pharmacokinetics of maraviroc is nonproportional over the dose range studied (3–1200 mg), the degree of nonproportionality is small at clinically relevant doses. Renal clearance is approximately 10–12 l h−1 and appears unaffected by increasing maraviroc doses. Maraviroc does not significantly modulate the activity of CYP2D6 or CYP3A4 at clinically relevant doses. There were no serious adverse events in any of these studies, and doses up to 900 mg were generally well tolerated, with postural hypotension being the dose-limiting event. There was no pattern or dose relationship observed with maraviroc with regard to laboratory abnormalities, including hepatic transaminases. No clinically significant increases in QTc were noted at clinically relevant doses. Conclusions Maraviroc is absorbed into the systemic circulation and reaches steady state by day 7 of multiple dosing. It does not significantly influence the activity of major drug-metabolizing enzymes and is well tolerated at clinically relevant doses, with most adverse events being mild or moderate. PMID:18333861

  16. [The HIV-infection outbreak in the town Lys'va (Perm region): homozygote genotype CCR5 delta32/CCR5 delta32 provides the high level of the persistence in the parenteral transmission of the virus].

    PubMed

    Riabov, G S; Kazennova, E V; Korepanova, L B; Mal'tseva, E A; Zhalnin, V V; Krasnikova, L A; Zverev, S Ia; Pokrovskiĭ, V V; Bobkov, A F; Weber, J N

    2002-01-01

    Specific features of human immunodeficiency virus type 1 (HIV-1) transmission among injecting drug users were studied on HIV infection outbreak in Lysva, the Perm region. During the period from November 1998 to March 2000, 32 injecting drug users infected with the subtype A HIV-1 variant originating from the same source, were found in this town. To understand the role of the CCR5 delta 32 mutation in parenteral transmission of HIV-1 the distribution of the mutant CCR5 delta 32 allele in HIV-infected and in non-infected but HIV-exposed drug users (n = 74) was analysed. The percentage of the homozygous CCR5 delta 32 genotype among HIV-exposed individuals (4/74, 5.4%) was significantly (p < 0.05) higher than the analogous rate for healthy blood donors in Russia (1/163, 0.6%). Thus, the homozygosity for this mutant allele confers a high resistance level to HIV even in parenteral transmission.

  17. Genetic Susceptibility to Cardiac and Digestive Clinical Forms of Chronic Chagas Disease: Involvement of the CCR5 59029 A/G Polymorphism

    PubMed Central

    de Oliveira, Amanda Priscila; Bernardo, Cássia Rubia; Camargo, Ana Vitória da Silveira; Ronchi, Luiz Sérgio; Borim, Aldenis Albaneze; Brandão de Mattos, Cinara Cássia; de Campos Júnior, Eumildo; Castiglioni, Lílian; Netinho, João Gomes; Cavasini, Carlos Eugênio; Bestetti, Reinaldo Bulgarelli; de Mattos, Luiz Carlos

    2015-01-01

    The clinical manifestations of chronic Chagas disease include the cardiac form of the disease and the digestive form. Not all the factors that act in the variable clinical course of this disease are known. This study investigated whether the CCR5Δ32 (rs333) and CCR5 59029 A/G (promoter region—rs1799987) polymorphisms of the CCR5 gene are associated with different clinical forms of chronic Chagas disease and with the severity of left ventricular systolic dysfunction in patients with chronic Chagas heart disease (CCHD). The antibodies anti-T. cruzi were identified by ELISA. PCR and PCR-RFLP were used to identify the CCR5Δ32 and CCR5 59029 A/G polymorphisms. The chi-square test was used to compare variables between groups. There was a higher frequency of the AA genotype in patients with CCHD compared with patients with the digestive form of the disease and the control group. The results also showed a high frequency of the AG genotype in patients with the digestive form of the disease compared to the other groups. The results of this study show that the CCR5Δ32 polymorphism does not seem to influence the different clinical manifestations of Chagas disease but there is involvement of the CCR5 59029 A/G polymorphism in susceptibility to the different forms of chronic Chagas disease. Besides, these polymorphisms do not influence left ventricular systolic dysfunction in patients with CCHD. PMID:26599761

  18. Genetic variations in the receptor-ligand pair CCR5 and CCL3L1 are important determinants of susceptibility to Kawasaki disease.

    PubMed

    Burns, Jane C; Shimizu, Chisato; Gonzalez, Enrique; Kulkarni, Hemant; Patel, Sukeshi; Shike, Hiroko; Sundel, Robert S; Newburger, Jane W; Ahuja, Sunil K

    2005-07-15

    Kawasaki disease (KD) is an enigmatic, self-limited vasculitis of childhood that is complicated by development of coronary-artery aneurysms. The high incidence of KD in Asian versus European populations prompted a search for genetic polymorphisms that are differentially distributed among these populations and that influence KD susceptibility. Here, we demonstrate a striking, inverse relationship between the worldwide distribution of CCR5- Delta 32 allele and the incidence of KD. In 164 KD patient-parent trios, 4 CCR5 haplotypes including the CCR5- Delta 32 allele were differentially transmitted from heterozygous parents to affected children. However, the magnitude of the reduced risk of KD associated with the CCR5- Delta 32 allele and certain CCR5 haplotypes was significantly greater in individuals who also possessed a high copy number of the gene encoding CCL3L1, the most potent CCR5 ligand. These findings, derived from the largest genetic study of any systemic vasculitis, suggest a central role of CCR5-CCL3L1 gene-gene interactions in KD susceptibility and the importance of gene modifiers in infectious diseases.

  19. CCR5-delta 32 allele is associated with the risk of developing multiple sclerosis in the Iranian population.

    PubMed

    Shahbazi, Majid; Ebadi, Hamid; Fathi, Davood; Roshandel, Danial; Mahamadhoseeni, Mana; Rashidbaghan, Azam; Mahammadi, Narges; Mahammadi, Mahammad Reza; Zamani, Mahdi

    2009-12-01

    The 32-base pair deletion on the C-C chemokine receptor 5 gene (CCR5-delta 32) is known as a protective allele against immune system disorders. We have studied this variation in Iranian multiple sclerosis (MS) patients and healthy controls. DNA samples were prepared from the whole blood of 254 patients with MS and 380 healthy controls. We amplified the fragment including the CCR5-delta 32 polymorphism and visualized the products in a documentation system after agarose gel electrophoresis. Data were analysed using one-way ANOVA and Fisher's exact tests with SPSS-v13 and STATA-v8 software. The delta 32 allele was more frequent in MS patients when compared with controls (OR = 2.3, P < 0.0001). Also, we found a significant difference in the frequency of the delta 32/delta 32 genotype among patients and controls (OR = 7.4, P < 0.001). The mean age at onset and progression index was not significantly different between patients with various genotypes. According to our study, the delta 32 allele of the CCR5 gene might be a predisposing factor for MS development in the Iranian population. However, there were no associations between this polymorphism and the clinical course of the disease in this study.

  20. Catalytic antibody light chain capable of cleaving a chemokine receptor CCR-5 peptide with a high reaction rate constant.

    PubMed

    Mitsuda, Yukie; Hifumi, Emi; Tsuruhata, Kumi; Fujinami, Hiroko; Yamamoto, Naoki; Uda, Taizo

    2004-04-20

    A monoclonal antibody (MAb), ECL2B-2, was obtained by immunizing a peptide possessing a part of a sequence of a chemokine receptor, CCR-5, which is present as a membrane protein on the macrophage surface, and which plays an important role in human immunodeficiency virus (HIV) infection. From the DNA and the deduced amino acid sequences of the light and heavy chains of ECL2B-2 MAb, molecular modeling was conducted to calculate the steric conformation of the antibody. Modeling suggested that the structure of ECL2B-2 could possess one or two catalytic triad(s), composed of Asp(1), Ser(27a) (or Ser(27e)), and His(93) (or His(27d)), in the light chain of ECL2B-2. The three amino acid residues, Asp(1), Ser(27a), and His(93), are identical to those of catalytic antibody light chains such as VIPase and i41SL1-2. The light chain of ECL2B-2 MAb degraded the antigenic peptide CCR-5 within about 100 h. Surprisingly, the light chain had a very high catalytic reaction rate constant (k(cat)) of 2.23 min(-1), which is greater by factors of tens to hundreds than those of natural catalytic antibodies obtained previously. The heavy chain of ECL2B-2 MAb, which has no catalytic triad because of a lack of His residue, did not degrade the CCR-5 peptide.

  1. Splenic differentiation and emergence of CCR5+CXCL9+CXCL10+ monocyte-derived dendritic cells in the brain during cerebral malaria

    PubMed Central

    Hirako, Isabella C.; Ataide, Marco A.; Faustino, Lucas; Assis, Patricia A.; Sorensen, Elizabeth W.; Ueta, Hisashi; Araújo, Natalia M.; Menezes, Gustavo B.; Luster, Andrew D.; Gazzinelli, Ricardo T.

    2016-01-01

    Dendritic cells have an important role in immune surveillance. After being exposed to microbial components, they migrate to secondary lymphoid organs and activate T lymphocytes. Here we show that during mouse malaria, splenic inflammatory monocytes differentiate into monocyte-derived dendritic cells (MO-DCs), which are CD11b+F4/80+CD11c+MHCIIhighDC-SIGNhighLy6c+ and express high levels of CCR5, CXCL9 and CXCL10 (CCR5+CXCL9/10+ MO-DCs). We propose that malaria-induced splenic MO-DCs take a reverse migratory route. After differentiation in the spleen, CCR5+CXCL9/10+ MO-DCs traffic to the brain in a CCR2-independent, CCR5-dependent manner, where they amplify the influx of CD8+ T lymphocytes, leading to a lethal neuropathological syndrome. PMID:27808089

  2. Mucosal transmissibility, disease induction and coreceptor switching of R5 SHIVSF162P3N molecular clones in rhesus macaques

    PubMed Central

    2013-01-01

    Background Mucosally transmissible and pathogenic CCR5 (R5)-tropic simian-human immunodeficiency virus (SHIV) molecular clones are useful reagents to identity neutralization escape in HIV-1 vaccine experiments and to study the envelope evolutionary process and mechanistic basis for coreceptor switch during the course of natural infection. Results We observed progression to AIDS in rhesus macaques infected intrarectally with molecular clones of the pathogenic R5 SHIVSF162P3N isolate. Expansion to CXCR4 usage was documented in one diseased macaque that mounted a neutralizing antibody response and in another that failed to do so, with the latter displaying a rapid progressor phenotype. V3 loop envelop glycoprotein gp120 sequence changes that are predictive of a CXCR4 (X4)-using phenotype in HIV-1 subtype B primary isolates, specifically basic amino acid substations at positions 11 (S11R), 24 (G24R) and 25 (D25K) of the loop were detected in the two infected macaques. Functional assays showed that envelopes with V3 S11R or D25K mutation were dual-tropic, infecting CD4+ target cells that expressed either the CCR5 or CXCR4 coreceptor. And, consistent with findings of coreceptor switching in macaques infected with the pathogenic isolate, CXCR4-using variant was first detected in the lymph node of the chronically infected rhesus monkey several weeks prior to its presence in peripheral blood. Moreover, X4 emergence in this macaque coincided with persistent peripheral CD4+ T cell loss and a decline in neutralizing antibody titer that are suggestive of immune deterioration, with macrophages as the major virus-producing cells at the end-stage of disease. Conclusions The data showed that molecular clones derived from the R5 SHIVSF162P3N isolate are mucosally transmissible and induced disease in a manner similar to that observed in HIV-1 infected individuals, providing a relevant and useful animal infection model for in-depth analyses of host selection pressures and the env

  3. Evidence for recent selection of the CCR5-delta 32 deletion from differences in its frequency between Ashkenazi and Sephardi Jews.

    PubMed

    Maayan, S; Zhang, L; Shinar, E; Ho, J; He, T; Manni, N; Kostrikis, L G; Neumann, A U

    2000-08-01

    Recent studies have shown higher frequencies of the CCR5-delta 32 allele and the CCR5-delta 32/delta 32 genotype, which confers protection against HIV infection, in northern Europe as compared to Mediterranean countries. Here, we analyse the prevalence of CCR5-delta 32 in 922 HIV seronegative blood donors in Israel to verify its frequency in Jews of Ashkenazi and Sephardi origin. A significant difference (P < 0.001) was found between the CCR5-delta 32 allele frequency in Ashkenazi (13.8%) vs (4.9%) Jews. In contrast, no significant difference was observed in the frequency of the CCR2-641 mutation between Ashkenazi (9.2%) and Sephardi (13.4%) Jews. Using the Island model we calculate that a minimal genetic migration rate of 3% per generation would have been necessary if the higher CCR5-delta 32 prevalence in Ashkenazi is to be fully explained by mixing with the indigenous north-European populations. This putative migration rate is 20-fold higher than that currently estimated from other genes, and would correspond to a non-realistic minimal current admixture of 80%. Thus, our results suggest that a positive selection process for CCR5-delta 32 should have occurred in northern Europe at most a 1000 years ago, after the Ashkenazi Jews separated from their Sephardi kin and moved to north Europe.

  4. Allo-immunization elicits CCR5 antibodies, SDF-1 chemokines, and CD8-suppressor factors that inhibit transmission of R5 and X4 HIV-1 in women.

    PubMed

    Wang, Y; Underwood, J; Vaughan, R; Harmer, A; Doyle, C; Lehner, T

    2002-09-01

    Studies in humans suggest that allo-immunization induces CC-chemokines, CD8-suppressor factors (SF) and anti-HIV immunity. Here we report that allo-immunization with unmatched leucocytes from partners of women with recurrent spontaneous abortion elicits specific antibodies to the CCR5 receptor. Such antibodies inhibit replication of M-tropic HIV-1 (R5) and MIP-1beta-mediated chemotaxis. These CCR5 antibodies were also found in the sera of multiparous women that were naturally immunized by semi-allogeneic fetal antigens. The specificity of these antibodies was demonstrated by adsorption with CCR5 transfected HEK-293 cells, a baculovirus CCR5 preparation and a peptide of the 2nd extra-cellular loop of CCR5. Allo-immunization also stimulated increased concentrations of the CXC chemokine, SDF-1alpha and CD8-SF that inhibit T-tropic HIV-1 (X4) replication. We suggest that allo- immunization may elicit (a) CC chemokines, CCR5 antibodies and CD8-SF that inhibit M-tropic HIV-1 infection and (b) the CXC chemokine SDF-1alpha and CD8-SF that inhibit T-tropic HIV-1 infection.

  5. Prevalence of CCR5-Δ32 and CCR2-V64I polymorphisms in a mixed population from northeastern Brazil.

    PubMed

    Ferreira-Fernandes, H; Santos, A C C; Motta, F J N; Canalle, R; Yoshioka, F K N; Burbano, R R; Rey, J A; da Silva, B B; Pinto, G R

    2015-10-02

    Chemokines are low-molecular weight proteins that play a key role in inflammatory processes. Genomic variations in chemokine receptors are associated with the susceptibility to various diseases. Polymorphisms in chemokine receptor type 5 (CCR5)-Δ32 and CCR2-V64I are related to human immunodeficiency virus infection resistance, which has led to genetic association studies for several other diseases. Given the heterogeneous distribution of these polymorphisms in different global populations and within Brazilian populations, we analyzed the prevalence of CCR5-Δ32 and CCR2-V64I polymorphisms in a mixed population from northeastern Brazil. The study included 223 individuals from the general population of the city of Parnaíba, Piauí, who had a mean age of 73 years. Of these individuals, 37.2% were men and 62.8% were women. Polymorphisms were analyzed using DNA extracted from peripheral blood leukocytes by using polymerase chain reaction alone (CCR5-Δ32) or accompanied by restriction endonuclease digestion (CCR2-V64I). In both cases, the genotypes were determined using 8% polyacrylamide gel electrophoresis and silver nitrate staining. The population conformed to Hardy-Weinberg equilibrium for both the loci studied. No individuals were homozygous for allele-Δ32, which was present in 1.8% of the population, whereas allele-64I was present in 13.9% of the participants studied; 74.9% were homozygous for the wild-type allele, while 22.4 and 2.7% were heterozygous and homozygous for the mutant allele, respectively. Additional studies are needed to investigate the relationship between these polymorphisms and disease etiopathogenesis in reference populations.

  6. Influence of CCR5 and CCR2 Genetic Variants in the Resistance/Susceptibility to HIV in Serodiscordant Couples from Colombia

    PubMed Central

    Zapata, Wildeman; Aguilar-Jiménez, Wbeimar; Pineda-Trujillo, Nicolás; Rojas, Winston; Estrada, Hernando

    2013-01-01

    Abstract The main genetic factor related to HIV-1 resistance is the CCR5-Δ32 mutation; however, the homozygous genotype is uncommon. The CCR5-Δ32 mutation along with single nucleotide polymorphisms (SNPs) in the CCR5 promoter and the CCR2-V64I mutation have been included in seven human haplogroups (HH) previously associated with resistance/susceptibility to HIV-1 infection and different rates of AIDS progression. Here, we determined the association of the CCR5 promoter SNPs, the CCR5-Δ32 mutation, CCR2-V64I SNP, and HH frequencies with resistance/susceptibility to HIV-1 infection in a cohort of HIV-1-serodiscordant couples from Colombia. Seventy HIV-1-exposed, but seronegative (HESN) individuals, 57 seropositives (SP), and 112 healthy controls (HC) were included. The CCR5-Δ32 mutation and CCR2-V64I SNP were identified by PCR, and the CCR5 promoter SNPs were evaluated by sequencing. None of the individuals exhibited a homozygous Δ32 genotype; the CCR2-I allele was more frequent in HESN (34%) than HC (23%) (p=0.039, OR=1.672). The frequency of the 29G allele was higher in SP than HC (p=0.003, OR=3). HHF2 showed a higher frequency in HC (19%) than SP (9%) (p=0.027), while HHG1 was more frequent in SP (11.1%) than in HC (4.2%) (p=0.019). The AGACCAC-CCR2-I-CCR5 wild-type haplotype showed a higher frequency in SP (14.2%) than in HC (3.7%) (p=0.001). In conclusion, the CCR5-Δ32 allele is not responsible for HIV-1 resistance in this HESN group; however, the CCR2-I allele could be protective, while the 29G allele might increase the likelihood of acquiring HIV-1 infection. HHG1 and the AGACCAC-CCR2-I-CCR5 wild-type haplotype might promote HIV-1 infection while HHF2 might be related to resistance. However, additional studies are required to evaluate the implications of these findings. PMID:24098976

  7. Astrocyte elevated gene-1 regulates CCL3/CCR5-induced epithelial-to-mesenchymal transition via Erk1/2 and Akt signaling in cardiac myxoma.

    PubMed

    Shi, Ping; Fang, Changcun; Pang, Xinyan

    2015-09-01

    In recent years, astrocyte elevated gene-1 (AEG-1) has been reported as a key mediator that is involved in the epithelial-to-mesenchymal transition (EMT) process. However, the mechanisms underlying CCL3/CCR5-AEG-1 pathway-mediated EMT in cardiac myxoma (CM) has not been well featured till now. We used immnohistochemistry and immunoblotting to assess the expression of CCR5 and AEG-1 in 30 cases of CM tissues and cells. Subsequently, cultured CM cells were treated with si-AEG-1 or si-CCR5 and then subjected to in vitro assays. We observed that CCR5 and AEG-1 proteins were highly expressed in CM tissues (73.3 and 76.7%, respectively) and closely correlated with tumor size (>5 cm). Importantly, we validated the expression of AEG-1, p-Erk1/2, p-Akt, vimentin, N-cadherin and MMP2 increased in the CM cell with CCL3 treatment in a time- and concentration-dependent manner. When CM cells were treated with si-CCR5, the expression of AEG-1, p-Erk1/2, p-Akt, vimentin, N-cadherin and MMP2 was downregulated. In addition, when CM cells were treated with si-AEG-1, the expression of p-Erk1/2, p-Akt, vimentin, N-cadherin and MMP2 was also downregulated. Using the cell cycle and proliferation assay, the knockdown of AEG-1 inhibited the entry of G1 into S phase and the proliferation capacity of CM cells. In conclusion, AEG-1 mediates CCL3/CCR5-induced EMT development via both Erk1/2 and Akt signaling pathway in CM patients, which indicates CCL3/CCR5-AEG-1-EMT pathway could be suggested as a useful target to affect the progression of CM.

  8. A role for CCR5(+)CD4 T cells in cutaneous psoriasis and for CD103(+) CCR4(+) CD8 Teff cells in the associated systemic inflammation.

    PubMed

    Sgambelluri, Francesco; Diani, Marco; Altomare, Andrea; Frigerio, Elena; Drago, Lorenzo; Granucci, Francesca; Banfi, Giuseppe; Altomare, Gianfranco; Reali, Eva

    2016-06-01

    Recent results have identified critical components of the T cell response involved in the initiation and amplification phases of psoriasis. However the link between T cell responses arising in the skin and the systemic inflammation associated with severe psoriasis is largely unknown. We hypothesized that specific subsets of memory T cells recirculating from the skin could play a role. We therefore dissected the circulating memory T cell compartment in patients by analyzing the TCM, TEM and Teff phenotype, the pattern of CCR4 and CCR5 chemokine receptor expression and the expression of the tissue homing molecule CD103. For each subset we calculated the correlation with the Psoriasis Area and Severity Index (PASI) and with the extent of systemic inflammation measured as serum level of the prototypic short pentraxin, C reactive protein (CRP). Validation was performed by comparison with gene expression data in psoriatic plaques. We found that circulating CD103(+)CCR4(+)CCR5(+) and CCR4(+)CCR6(-) CD8(+) Teff cells, were highly correlated with CRP levels as well as with the validated index PASI, reflecting a link between skin involvement and systemic inflammation in patients with severe psoriasis. In addition we observed a contraction of circulating CCR5(+) T cells in psoriasis patients, with a highly significant inverse correlation between CCR5(+)CD4 T cells and the PASI score. Increased expression of CCR5 and CCL5 genes in psoriatic skin lesions was consistent with an accumulation of CCR5(+) cells in psoriatic plaques indicating a role for CCR5/CCL5 axis in disease pathogenesis.

  9. 27-Hydroxycholesterol and 7alpha-hydroxycholesterol trigger a sequence of events leading to migration of CCR5-expressing Th1 lymphocytes

    SciTech Connect

    Kim, Sun-Mi; Kim, Bo-Young; Lee, Sae-A; Eo, Seong-Kug; Yun, Yungdae; Kim, Chi-Dae; Kim, Koanhoi

    2014-02-01

    Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. A high cholesterol diet resulted in enhanced expression of CCR5 ligands, including CCL3 and CCL4, but not of proatherogenic CXCR3 ligands, in atherosclerotic arteries of ApoE{sup −/−} mice. 27-Hydroxycholesterol and 7α-hydroxycholesterol, cholesterol oxides (oxysterols) detected in abundance in atherosclerotic lesions, greatly induced the transcription of CCL3 and CCL4 genes in addition to enhancing secretion of corresponding proteins by THP-1 monocytic cells. However, an identical or even higher concentration of cholesterol, 7β-hydroxycholesterol, and 7-ketocholsterol did not influence expression of these chemokines. Conditioned media containing the CCR5 ligands secreted from THP-1 cells induced migration of Jurkat T cells expressing CCR5, a characteristic chemokine receptor of Th1 cells, but not of Jurkat T cells that do not express CCR5. The migration of CCR5-expressing Jurkat T cells was abrogated in the presence of a CCR5-neutralizing antibody. 27-Hydroxycholesterol and 7α-hydroxycholesterol enhanced phosphorylation of Akt. Pharmacological inhibitors of phosphoinositide-3-kinase/Akt pathways blocked transcription as well as secretion of CCL3 and CCL4 in conjunction with attenuated migration of CCR5-expressing Jurkat T cells. This is the first report on the involvement of cholesterol oxides in migration of distinct subtype of T cells. We propose that 27-hydroxycholesterol and 7α-hydroxycholesterol can trigger a sequence of events that leads to recruitment of Th1 lymphocytes and phosphoinositide-3-kinase/Akt pathways play a major role in the process. - Graphical abstract: Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of

  10. Impact of maraviroc-resistant and low-CCR5-adapted mutations induced by in vitro passage on sensitivity to anti-envelope neutralizing antibodies.

    PubMed

    Yoshimura, Kazuhisa; Harada, Shigeyoshi; Boonchawalit, Samatchaya; Kawanami, Yoko; Matsushita, Shuzo

    2014-08-01

    The aim of this study was to generate maraviroc (MVC)-resistant viruses in vitro using a human immunodeficiency virus type 1 subtype B clinical isolate (HIV-1KP-5) to understand the mechanism(s) of resistance to MVC. To select HIV-1 variants resistant to MVC in vitro, we exposed high-chemokine (C-C motif) receptor 5 (CCR5)-expressing PM1/CCR5 cells to HIV-1KP-5 followed by serial passage in the presence of MVC. We also passaged HIV-1KP-5 in PM1 cells, which were low CCR5 expressing to determine low-CCR5-adapted substitutions and compared the Env sequences of the MVC-selected variants. Following 48 passages with MVC (10 µM), HIV-1KP-5 acquired a resistant phenotype [maximal per cent inhibition (MPI) 24%], whilst the low-CCR5-adapted variant had low sensitivity to MVC (IC50 ~200 nM), but not reduction of the MPI. The common substitutions observed in both the MVC-selected and low-CCR5-adapted variants were selected from the quasi-species, in V1, V3 and V5. After 14 passages, the MVC-selected variants harboured substitutions around the CCR5 N-terminal-binding site and V3 (V200I, T297I, K305R and M434I). The low-CCR5-adapted infectious clone became sensitive to anti-CD4bs and CD4i mAbs, but not to anti-V3 mAb and autologous plasma IgGs. Conversely, the MVC-selected clone became highly sensitive to the anti-envelope (Env) mAbs tested and the autologous plasma IgGs. These findings suggest that the four MVC-resistant mutations required for entry using MVC-bound CCR5 result in a conformational change of Env that is associated with a phenotype sensitive to anti-Env neutralizing antibodies.

  11. CCL3L1-CCR5 genotype influences durability of immune recovery during antiretroviral therapy of HIV-1–infected individuals

    PubMed Central

    Ahuja, Sunil K; Kulkarni, Hemant; Catano, Gabriel; Agan, Brian K; Camargo, Jose F; He, Weijing; O'Connell, Robert J; Marconi, Vincent C; Delmar, Judith; Eron, Joseph; Clark, Robert A; Frost, Simon; Martin, Jeffrey; Ahuja, Seema S; Deeks, Steven G; Little, Susan; Richman, Douglas; Hecht, Frederick M; Dolan, Matthew J

    2008-01-01

    The basis for the extensive variability seen in the reconstitution of CD4+ T cell counts in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) is not fully known. Here, we show that variations in CCL3L1 gene dose and CCR5 genotype, but not major histocompatibility complex HLA alleles, influence immune reconstitution, especially when HAART is initiated at <350 CD4+ T cells/mm3. The CCL3L1-CCR5 genotypes favoring CD4+ T cell recovery are similar to those that blunted CD4+ T cell depletion during the time before HAART became available (pre-HAART era), suggesting that a common CCL3L1-CCR5 genetic pathway regulates the balance between pathogenic and reparative processes from early in the disease course. Hence, CCL3L1-CCR5 variations influence HIV pathogenesis even in the presence of HAART and, therefore, may prospectively identify subjects in whom earlier initiation of therapy is more likely to mitigate immunologic failure despite viral suppression by HAART. Furthermore, as reconstitution of CD4+ cells during HAART is more sensitive to CCL3L1 dose than to CCR5 genotypes, CCL3L1 analogs might be efficacious in supporting immunological reconstitution. PMID:18376407

  12. Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce β-chemokines

    PubMed Central

    Liao, Hua-Xin; Alam, S. Munir; Scearce, Richard M.; Plonk, M. Kelly; Kozink, Daniel M.; Drinker, Mark S.; Zhang, Ruijun; Xia, Shi-Mao; Sutherland, Laura L.; Tomaras, Georgia D.; Giles, Ian P.; Kappes, John C.; Ochsenbauer-Jambor, Christina; Edmonds, Tara G.; Soares, Melina; Barbero, Gustavo; Forthal, Donald N.; Landucci, Gary; Chang, Connie; King, Steven W.; Kavlie, Anita; Denny, Thomas N.; Hwang, Kwan-Ki; Chen, Pojen P.; Thorpe, Philip E.; Montefiori, David C.

    2010-01-01

    Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes. PMID:20368576

  13. Differences in T cell distribution and CCR5 expression in HIV-positive and HIV-exposed seronegative persons who inject drugs.

    PubMed

    Kallas, Eveli; Huik, Kristi; Türk, Silver; Pauskar, Merit; Jõgeda, Ene-Ly; Šunina, Marina; Karki, Tõnis; Des Jarlais, Don; Uusküla, Anneli; Avi, Radko; Lutsar, Irja

    2016-06-01

    Some individuals remain uninfected despite repeated exposure to HIV. This protection against HIV has been partly associated with altered T cell subset distributions and CCR5 expression levels. However, the majority of studies have been conducted in sexually exposed subjects. We aimed to assess whether HIV infection and intravenous drug use were associated with differences in CCR5 expression, immune activation on the CD4+ and CD8+ T cells and T cell distribution among Caucasian persons who inject drugs (PWIDs). Analyses of the data from 41 HIV-positive PWIDs, 47 HIV-exposed seronegative PWIDs (ESNs) and 47 age- and gender-matched HIV-negative non-drug users are presented. Of all of the study subjects, 111 (82 %) were male, and the median age was 29 years. T cell phenotyping was performed in peripheral blood mononuclear cells with multicolour flow cytometry using anti-CD3, CD4, CD8, CD45RA, CD45RO, HLA-DR and CCR5 antibodies. The ESNs exhibited greater levels of immune activation and higher percentages of CD4+ CD45RA+RO+ and CD8+ CD45RA+RO+ cells compared to the controls but not the HIV-positive people. The CCR5 expression on the CD4+ T cell subsets in the ESNs was lower than that in the controls but similar to that the HIV positives. The percentages of CCR5+ T cells were similar in all study groups and in most of the studied cell populations. Intravenous drug use was similarly associated with differences in T cell subset distributions and CCR5 expression among both the HIV-positive and HIV-negative PWIDs compared with the controls.

  14. Characterization of cellular infiltrate, detection of chemokine receptor CCR5 and interleukin-8 and RANTES chemokines in adult periodontitis.

    PubMed

    Gamonal, J; Acevedo, A; Bascones, A; Jorge, O; Silva, A

    2001-06-01

    Leukocyte migration is essential for immune surveillance of tissues by focusing immune cells to sites of antigenic challenge. The control of leukocyte migration depends on the combined actions of adhesion molecules and a vast array of chemokines and their receptors. The purpose of the present study was to investigate the involvement of Interleukin-8 (IL-8), RANTES, the associated infiltrating cells and expression of CCR5 chemokine receptors in periodontitis; furthermore, the effect of periodontal therapy on these parameters was evaluated. Patients included in the study had moderate to advanced periodontal disease with at least 5-6 teeth with probing depth > 6 mm, attachment loss > or =3 mm and extensive radiographic bone loss. The inflammatory infiltrate was analyzed by immunohistochemistry in gingival biopsies obtained from subjects at the beginning of the study and 2 months after periodontal treatment. Gingival crevicular fluid (GCF) was collected for 30 seconds using periopaper strips, and chemokines were quantified by ELISA. The cellular components of the inflammatory infiltrate included B (CD19) and T (CD3, CD4+ and CD8+) lymphocytes and monocytes/macrophages (CD11c). CCR5 chemokine receptor expressing cells were exclusively found in periodontitis gingiva. IL-8 and RANTES were detected in the periodontitis group, obtaining a total amount of 212.5 pg and 42.0 pg, respectively. However, IL-8 was also detectable in the GCF of the healthy group (total amount of 44.8 pg). Periodontal therapy reduced the cell number in the infiltrate and the levels of IL-8 and RANTES, suggesting a relationship between these chemokines and periodontal status. We propose that the presence of these chemokines and the expression of chemokine receptors may represent a marker of lymphocyte subsets with the ability to migrate to inflammatory sites.

  15. Enhanced monocyte migration to CXCR3 and CCR5 chemokines in COPD.

    PubMed

    Costa, Claudia; Traves, Suzanne L; Tudhope, Susan J; Fenwick, Peter S; Belchamber, Kylie B R; Russell, Richard E K; Barnes, Peter J; Donnelly, Louise E

    2016-04-01

    Chronic obstructive pulmonary disease (COPD) patients exhibit chronic inflammation, both in the lung parenchyma and the airways, which is characterised by an increased infiltration of macrophages and T-lymphocytes, particularly CD8+ cells. Both cell types can express chemokine (C-X-C motif) receptor (CXCR)3 and C-C chemokine receptor 5 and the relevant chemokines for these receptors are elevated in COPD. The aim of this study was to compare chemotactic responses of lymphocytes and monocytes of nonsmokers, smokers and COPD patients towards CXCR3 ligands and chemokine (C-C motif) ligand (CCL)5. Migration of peripheral blood mononuclear cells, monocytes and lymphocytes from nonsmokers, smokers and COPD patients toward CXCR3 chemokines and CCL5 was analysed using chemotaxis assays. There was increased migration of peripheral blood mononuclear cells from COPD patients towards all chemokines studied when compared with nonsmokers and smokers. Both lymphocytes and monocytes contributed to this enhanced response, which was not explained by increased receptor expression. However, isolated lymphocytes failed to migrate and isolated monocytes from COPD patients lost their enhanced migratory capacity. Both monocytes and lymphocytes cooperate to enhance migration towards CXCR3 chemokines and CCL5. This may contribute to increased numbers of macrophages and T-cells in the lungs of COPD patients, and inhibition of recruitment using selective antagonists might be a treatment to reduce the inflammatory response in COPD.

  16. Improving HIV coreceptor usage prediction in the clinic using hints from next-generation sequencing data

    PubMed Central

    Pfeifer, Nico; Lengauer, Thomas

    2012-01-01

    Motivation: Due to the high mutation rate of human immunodeficiency virus (HIV), drug-resistant-variants emerge frequently. Therefore, researchers are constantly searching for new ways to attack the virus. One new class of anti-HIV drugs is the class of coreceptor antagonists that block cell entry by occupying a coreceptor on CD4 cells. This type of drug just has an effect on the subset of HIVs that use the inhibited coreceptor. A good prediction of whether the viral population inside a patient is susceptible to the treatment is hence very important for therapy decisions and pre-requisite to administering the respective drug. The first prediction models were based on data from Sanger sequencing of the V3 loop of HIV. Recently, a method based on next-generation sequencing (NGS) data was introduced that predicts labels for each read separately and decides on the patient label through a percentage threshold for the resistant viral minority. Results: We model the prediction problem on the patient level taking the information of all reads from NGS data jointly into account. This enables us to improve prediction performance for NGS data, but we can also use the trained model to improve predictions based on Sanger sequencing data. Therefore, also laboratories without NGS capabilities can benefit from the improvements. Furthermore, we show which amino acids at which position are important for prediction success, giving clues on how the interaction mechanism between the V3 loop and the particular coreceptors might be influenced. Availability: A webserver is available at http://coreceptor.bioinf.mpi-inf.mpg.de. Contact: nico.pfeifer@mpi-inf.mpg.de PMID:22962486

  17. Cytomegalovirus upregulates expression of CCR5 in central memory cord blood mononuclear cells, which may facilitate in utero HIV type 1 transmission.

    PubMed

    Johnson, Erica L; Howard, Chanie L; Thurman, Joy; Pontiff, Kyle; Johnson, Elan S; Chakraborty, Rana

    2015-01-15

    Administration of combination antiretroviral therapy to human immunodeficiency virus type 1 (HIV-1)-infected pregnant women significantly reduces vertical transmission. In contrast, maternal co-opportunistic infection with primary or reactivated cytomegalovirus (CMV) or other pathogens may facilitate in utero transmission of HIV-1 by activation of cord blood mononuclear cells (CBMCs). Here we examine the targets and mechanisms that affect fetal susceptibility to HIV-1 in utero. Using flow cytometry, we demonstrate that the fraction of CD4(+)CD45RO(+) and CD4(+)CCR5(+) CBMCs is minimal, which may account for the low level of in utero HIV-1 transmission. Unstimulated CD4(+) CBMCs that lack CCR5/CD45RO showed reduced levels of HIV-1 infection. However, upon in vitro stimulation with CMV, CBMCs undergo increased proliferation to upregulate the fraction of T central memory cells and expression of CCR5, which enhances susceptibility to HIV-1 infection in vitro. These data suggest that activation induced by CMV in vivo may alter CCR5 expression in CD4(+) T central memory cells to promote in utero transmission of HIV-1.

  18. Marsdenia tenacissima extract suppresses A549 cell migration through regulation of CCR5-CCL5 axis, Rho C, and phosphorylated FAK.

    PubMed

    Lin, Sen-Sen; Li, Fang-Fang; Sun, Li; Fan, Wei; Gu, Ming; Zhang, Lu-Yong; Qin, Song; Yuan, Sheng-Tao

    2016-03-01

    Marsdenia tenacissima, a traditional Chinese medicine, is long been used to treat various diseases including asthma, cancer, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia. Although Marsdenia tenacissima has been demonstrated to have strong anti-tumor effects against primary tumors, its effect on cancer metastasis remains to be defined, and the molecular mechanism underlying the anti-metastatic effect is unknown. In the present study, we investigated the effects of XAP (an extract of Marsdenia tenacissima) on A549 lung cancer cell migration and explored the role of CCR5-CCL5 axis in the anti-metastatic effects of XAP. Our resutls showed that XAP inhibited A549 lung cancer cell migration and invasion in a dose-dependent manner. The protein levels of CCR5, but not CCR9 and CXCR4, were decreased by XAP. The secretion of CCL5, the ligand of CCR5, was reduced by XAP. XAP down-regulated Rho C expression and FAK phosphorylation. In conclusion, XAP inhibited A549 cell migration and invasion through down-regulation of CCR5-CCL5 axis, Rho C, and FAK.

  19. Potent in vitro inactivation of both free and cell-associated CCR5- and CXCR4-tropic HIV-1 by common commercial soap bars from South Africa.

    PubMed

    Jenabian, Mohammad-Ali; Auvert, Bertran; Saïdi, Héla; Lissouba, Pascale; Matta, Mathieu; Bélec, Laurent

    2010-08-01

    We showed herein the potent virucidal effect of soap and water solutions against both CCR5-tropic and CXCR4-tropic cell-free HIV-1 strains, and cytotoxicity for HIV-1-infected lymphocytes during short incubation durations, ranging from 30 seconds to 2 minutes. These observations indicate a rapid inhibitory effect of soap and water on viral infectivity.

  20. Association of TGFβ1, TNFα, CCR2 and CCR5 gene polymorphisms in type-2 diabetes and renal insufficiency among Asian Indians

    PubMed Central

    Prasad, Pushplata; Tiwari, Arun K; Kumar, KM Prasanna; Ammini, AC; Gupta, Arvind; Gupta, Rajeev; Thelma, BK

    2007-01-01

    Background Cytokines play an important role in the development of diabetic chronic renal insufficiency (CRI). Transforming growth factor β1 (TGF β1) induces renal hypertrophy and fibrosis, and cytokines like tumor necrosis factor-alpha (TNFα), chemoattractant protein-1 (MCP-1), and regulated upon activation and normal T cell expressed and secreted (RANTES) mediate macrophage infiltration into kidney. Over expression of these chemokines leads to glomerulosclerosis and interstitial fibrosis. The effect of MCP-1 and RANTES on kidney is conferred by their receptors i.e., chemokine receptor (CCR)-2 and CCR-5 respectively. We tested association of nine single nucleotide polymorphisms (SNPs) from TGFβ1, TNFα, CCR2 and CCR5 genes among individuals with type-2 diabetes with and without renal insufficiency. Methods Type-2 diabetes subjects with chronic renal insufficiency (serum creatinine ≥ 3.0 mg/dl) constituted the cases, and matched individuals with diabetes of duration ≥ 10 years and normoalbuminuria were evaluated as controls from four centres in India. Allelic and genotypic contributions of nine SNPs from TGFβ1, TNFα, CCR2 and CCR5 genes to diabetic CRI were tested by computing odds ratio (OR) and 95% confidence intervals (CI). Sub-analysis of CRI cases diabetic retinopathy status as dependent variable and SNP genotypes as independent variable in a univariate logistic regression was also performed. Results SNPs Tyr81His and Thr263Ile in TGF β1 gene were monomorphic, and Arg25Pro in TGF β1 gene and Δ32 polymorphism in CCR5 gene were minor variants (minor allele frequency <0.05) and therefore were not considered for case-control analysis. A significant allelic association of 59029G>A SNP of CCR5 gene has been observed and the allele 59029A seems to confer predisposition to development of diabetic CRI (OR 1.39; CI 1.04–1.84). In CRI subjects a compound group of genotypes "GA and AA" of SNP G>A -800 was found to confer predisposition for proliferative

  1. Allelic variants of human beta-chemokine receptor 5 (CCR5) promoter: evolutionary relationships and predictable associations with HIV-1 disease progression.

    PubMed

    Tang, J; Rivers, C; Karita, E; Costello, C; Allen, S; Fultz, P N; Schoenbaum, E E; Kaslow, R A

    1999-09-01

    Variability in the natural history of HIV-1 infection has been repeatedly associated with genetic variants in the beta-chemokine receptor 5 (CCR5) locus. While CCR5 coding sequences have demonstrated relatively limited variation, sequences of its promoter appear polymorphic in all major populations. Our studies revealed five major CCR5 promoter alleles with distributions that differed widely among the four distinct ethnic groups from Kigali, Rwanda and Bronx, New York. In particular, promoter allele P*0103 (G59029-T59353-T59356-A59402-C59653) was largely restricted to black subjects. The promoter allele P*0202 (A59029-C59353-C59356-A59402-T59653) was tightly linked to the slightly less frequent CCR2b-641, a variant of the CCR2b gene, which is about 12.7 kbp upstream from the promoter region. Another closely related promoter allele P*0201 (A59029-C59353-C59356-A59402-C59653) exclusively carried the far less common CCR5-delta 32, a 32-bp deletion in the CCR5 coding sequence 2 kbp downstream from the promoter. The homozygous P*0201/*0201 genotype can be predicted as a risk factor for more rapid disease progression. Among human, chimpanzee, pig-tailed macaque, and sooty mangabey promoter allelic sequences, the apparent ancestral lineage of the promoter sequence (G59029-T59353-C59356-A59402-C59653 = human P*0102) was highly conserved across the primate species analyzed here while P*0201 and P*0202 arose more recently than the other three major alleles. Further effort to establish the mechanism by which CCR chemokine receptor polymorphisms govern the initiation and pathogenesis of primate lentivirus infection apparently requires fully detailed genotypic characterization of the affected populations.

  2. Acute response of peripheral CCr5 chemoreceptor and NK cells in individuals submitted to a single session of low-intensity strength exercise with blood flow restriction.

    PubMed

    Dorneles, Gilson Pires; Colato, Alana Schraiber; Galvão, Simone Lunelli; Ramis, Thiago Rozales; Ribeiro, Jerri Luiz; Romão, Pedro Roosevelt; Peres, Alessandra

    2016-07-01

    The purpose of this study was to compare the peripheral expression of natural killers and CCR5 in a session of low-intensity strength training with vascular occlusion and in high-intensity training. Young males were randomized into session groups of a high-intensity strength training (HI) and a session group of low-intensity strength training with vascular occlusion (LI-BFR). The exercise session consisted in knee extension and bicep curl in 80% 1RM (HI) and 30% 1RM (LI-BFR) with equalized volumes. Blood collection was made before, immediately after and 24 h after each training session. Immunophenotyping was carried out through CD195+ (CCR5) e CD3-CD16+CD56+ (NK) in peripheral blood and analysed by flow cytometry and presented in frequency (%). Peripheral frequency of NK cells showed no significant difference in LI-BFR group in time effect, while a gradual reduction of NK cells was identified in HI group in before-24 h postexercise and after-24 h postexercise comparison. However, significant differences have been found in relative change of NK cells immediately after exercise between sessions. In addition, HI and LI-BFR groups showed a significant reduction in the cells expressed CCR5 during 24 h postsession compared to the postsession, but CCR5 also differed when comparing before-24 h after session in the HI group. No differences were observed amongst the groups. LIO induced CCR5 response similar to the HI session, while the NK cells remained in similar frequency during the studied moments in LI-BFR, but not in HI group, suggesting that local hypoxia created by the blood flow restriction was able to prevent a change in the frequency of peripheral cells and a possible immunosuppression.

  3. High levels of CC-chemokine expression and downregulated levels of CCR5 during HIV-1/HTLV-1 and HIV-1/HTLV-2 coinfections.

    PubMed

    Oo, Z; Barrios, C S; Castillo, L; Beilke, M A

    2015-05-01

    The human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 are common copathogens among Human Immunodeficiency Virus (HIV)-infected individuals. HTLV-2 may confer a survival benefit among patients with HIV-1/HTLV-2 coinfections, along with lower plasma HIV-1 levels and delayed rates of CD4(+) T-cell decline. These effects have been attributed to the ability of the HTLV-2 viral transactivating Tax2 protein to induce the production of high levels of antiviral CC-chemokines and to downregulate expression of the CCR5 receptor, resulting in impaired entry of HIV-1 into CD4(+) T-cells. This study investigated the innate immunity of coinfected HIV/HTLV individuals by testing the ability of patient PBMCs to produce CC-chemokines in association CCR5 receptor modulation. The cellular proliferative responses of HIV/HTLV coinfected versus HIV monoinfected individuals were also evaluated. Higher levels of MIP-1α, MIP-1β, and RANTES (P < 0.05) were found in HIV-1/HTLV-2 coinfected group compared to HIV-1 monoinfected population. Upregulated levels of RANTES were shown in HIV-1/HTLV-1 after 1 and 3 days of culture (P < 0.05). Lymphocytes from HIV-1/HTLV-2 coinfected individuals showed significant CCR5 downregulation after 1 and 3 days of culture compared to lymphocytes from HIV-1 and uninfected groups (P < 0.05). Lower percentages of CCR5-positive cells were found in HIV-1/HTLV-1 coinfected after 3 days of incubation (P < 0.05). Levels of proliferation were significantly higher in the HIV-1/HTLV-1 group compared to HIV-1 alone (P < 0.05). HTLV-2 and HTLV-1 infections may induce the involvement of innate immunity against HIV-1 via stimulation of CC-chemokines and receptors, potentially modifying CCR5/HIV-1 binding and HIV-1 progression in coinfected individuals.

  4. CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

    SciTech Connect

    Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael; Ellett, Anne; Farrugia, William; Wesselingh, Steven L.; Cunningham, Anthony L.; Ramsland, Paul A.; Gorry, Paul R.

    2011-02-20

    CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.

  5. Variants of the chemokine receptor CCR5 are associated with severe bronchiolitis caused by respiratory syncytial virus.

    PubMed

    Hull, Jeremy; Rowlands, Kate; Lockhart, Elizabeth; Moore, Catrin; Sharland, Mike; Kwiatkowski, Dominic

    2003-09-15

    Respiratory syncytial virus (RSV) bronchiolitis is characterized by intense inflammation of the airways, and high levels of proinflammatory cytokines and chemokines can be found in respiratory secretions of affected infants. Important among these chemokines are RANTES (regulated on activation, normal T cell-expressed and -secreted) and macrophage inflammatory-protein alpha, MIP-1alpha, both of which show correlation with severe RSV bronchiolitis. It is not clear whether high levels of these chemokines are important in disease pathogenesis, and this study addresses this question by studying genetic variants of their major receptor, CC chemokine receptor 5. Results from both a case-control and family-based genetic-association analysis show that the -2459G and -2554T variants are associated with severe RSV bronchiolitis (P=.01). It is proposed that these CCR5 variants influence the inflammatory response, and these data provide further evidence of the important role that host genetic variability plays in the determination of disease severity in RSV bronchiolitis.

  6. Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes

    PubMed Central

    Fine, Eli J.; Appleton, Caleb M.; White, Douglas E.; Brown, Matthew T.; Deshmukh, Harshavardhan; Kemp, Melissa L.; Bao, Gang

    2015-01-01

    CRISPR/Cas9 systems have been used in a wide variety of biological studies; however, the large size of CRISPR/Cas9 presents challenges in packaging it within adeno-associated viruses (AAVs) for clinical applications. We identified a two-cassette system expressing pieces of the S. pyogenes Cas9 (SpCas9) protein which splice together in cellula to form a functional protein capable of site-specific DNA cleavage. With specific CRISPR guide strands, we demonstrated the efficacy of this system in cleaving the HBB and CCR5 genes in human HEK-293T cells as a single Cas9 and as a pair of Cas9 nickases. The trans-spliced SpCas9 (tsSpCas9) displayed ~35% of the nuclease activity compared with the wild-type SpCas9 (wtSpCas9) at standard transfection doses, but had substantially decreased activity at lower dosing levels. The greatly reduced open reading frame length of the tsSpCas9 relative to wtSpCas9 potentially allows for more complex and longer genetic elements to be packaged into an AAV vector including tissue-specific promoters, multiplexed guide RNA expression, and effector domain fusions to SpCas9. For unknown reasons, the tsSpCas9 system did not work in all cell types tested. The use of protein trans-splicing may help facilitate exciting new avenues of research and therapeutic applications through AAV-based delivery of CRISPR/Cas9 systems. PMID:26126518

  7. Lack of association of CCR2-64I and CCR5-Delta 32 with type 1 diabetes and latent autoimmune diabetes in adults.

    PubMed

    Gambelunghe, Giovanni; Ghaderi, Mehran; Brozzetti, Annalisa; Del Sindaco, Paola; Gharizadeh, Babeck; Nyren, Paul; Hjelmström, Peter; Nikitina-Zake, Liene; Sanjeevi, Carani B; Falorni, Alberto

    2003-06-01

    It is well known that type 1 diabetes mellitus (T1DM) is a complex genetic disease resulting from the autoimmune destruction of pancreatic beta cells. Several genes have been associated with susceptibility and/or protection for T1DM, but the disease risk is mostly influenced by genes located in the class II region of the major histocompatibility complex. The attraction of leukocytes to tissues is essential for inflammation and the beginning of autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytolines. Some studies have shown that CCR2-64I and CCR5-Delta 32 might be important for protection of susceptibility to some immunologically-mediated disorders. In the present study, we demonstrate the lack of association between CCR2-64I and CCR5-Delta 32 gene polymorphism and TIDM and we describe a new method for a simple and more precise genotyping of the CCR2 gene.

  8. Elucidation of the CCR1- and CCR5-binding modes of MIP-1α by application of an NMR spectra reconstruction method to the transferred cross-saturation experiments.

    PubMed

    Yoshiura, Chie; Ueda, Takumi; Kofuku, Yutaka; Matsumoto, Masahiko; Okude, Junya; Kondo, Keita; Shiraishi, Yutaro; Shimada, Ichio

    2015-12-01

    C-C chemokine receptor 1 (CCR1) and CCR5 are involved in various inflammation and immune responses, and regulate the progression of the autoimmune diseases differently. However, the number of residues identified at the binding interface was not sufficient to clarify the differences in the CCR1- and CCR5-binding modes to MIP-1α, because the NMR measurement time for CCR1 and CCR5 samples was limited to 24 h, due to their low stability. Here we applied a recently developed NMR spectra reconstruction method, Conservation of experimental data in ANAlysis of FOuRier, to the amide-directed transferred cross-saturation experiments of chemokine receptors, CCR1 and CCR5, embedded in lipid bilayers of the reconstituted high density lipoprotein, and MIP-1α. Our experiments revealed that the residues on the N-loop and β-sheets of MIP-1α are close to both CCR1 and CCR5, and those in the C-terminal helix region are close to CCR5. These results suggest that the genetic influence of the single nucleotide polymorphisms of MIP-1α that accompany substitution of residues in the C-terminal helix region, E57 and V63, would provide clues toward elucidating how the CCR5-MIP-1α interaction affects the progress of autoimmune diseases.

  9. Detection of intermolecular transferred-NOE interactions in small and medium size protein complexes: RANTES complexed with a CCR5 N-terminal peptide.

    PubMed

    Abayev, Meital; Srivastava, Gautam; Arshava, Boris; Naider, Fred; Anglister, Jacob

    2017-02-01

    NMR is a powerful tool for studying structural details of protein/peptide complexes exhibiting weak to medium binding (KD > 10 μm). However, it has been assumed that intermolecular nuclear Overhauser effect (NOE) interactions are difficult to observe in such complexes. We demonstrate that intermolecular NOEs can be revealed by combining the (13) C-edited/(13) C-filtered experiment with the transferred NOE effect (TRNOE). Due to the TRNOE phenomenon, intermolecular NOE cross peaks are characterized by both the chemical shifts (CSs) of the protein protons and the average CSs of the peptide protons, which are dominated by the CSs of the protons of the free peptide. Previously, the TRNOE phenomenon was used almost exclusively to investigate the conformation of small ligands bound to large biomolecules. Here, we demonstrate that TRNOE can be extended to enable the study of intermolecular interactions in small- and medium-sized protein complexes. We used the (13) C-edited/(13) C-filtered TRNOE experiment to study the interactions of the chemokine regulated upon activation, normal T cell, expressed and secreted (RANTES) with a 27-residue peptide, containing two sulfotyrosine residues, representing the N-terminal segment of the CCR5 receptor ((Nt-CCR5(1-27). The TRNOE phenomenon led to more than doubling of the signal-to-noise ratios (SNRs) for the intermolecular NOEs observed in the (13) C-edited/(13) C-filtered experiment for the 11.5-kDa monomeric RANTES/Nt-CCR5(1-27) complex. An even better improvement in the SNR was achieved with dimeric Nt-CCR5(1-27)/RANTES (23 kDa), especially in comparison with the spectra measured with a 1 : 1 protein to peptide ratio. In principle, the isotope-edited/isotope-filtered TRNOE spectrum can discern all intermolecular interactions involving nonexchangeable protons in the complex.

  10. CCR2 and CCR5 genes polymorphisms in women with cervical lesions from Pernambuco, Northeast Region of Brazil: a case-control study

    PubMed Central

    dos Santos, Erinaldo Ubirajara Damasceno; de Lima, Géssica Dayane Cordeiro; Oliveira, Micheline de Lucena; Heráclio, Sandra de Andrade; da Silva, Hildson Dornelas Angelo; Crovella, Sergio; Maia, Maria de Mascena Diniz; de Souza/, Paulo Roberto Eleutério

    2016-01-01

    Polymorphisms in chemokine receptors play an important role in the progression of cervical intraepithelial neoplasia (CIN) to cervical cancer (CC). Our study examined the association of CCR2-64I (rs1799864) andCCR5-Δ32 (rs333) polymorphisms with susceptibility to develop cervical lesion (CIN and CC) in a Brazilian population. The genotyping of 139 women with cervical lesions and 151 women without cervical lesions for the CCR2-64I and CCR5-Δ32 polymorphisms were performed using polymerase chain reaction-restriction fragment length polymorphism. The individuals carrying heterozygous or homozygous genotypes (GA+AA) for CCR2-64I polymorphisms seem to be at lower risk for cervical lesion [odds ratio (OR) = 0.37, p = 0.0008)]. The same was observed for the A allele (OR = 0.39, p = 0.0002), while no association was detected (p > 0.05) with CCR5-Δ32 polymorphism. Regarding the human papillomavirus (HPV) type, patients carrying the CCR2-64Ipolymorphism were protected against infection by HPV type 16 (OR = 0.35, p = 0.0184). In summary, our study showed a protective effect ofCCR2-64I rs1799864 polymorphism against the development of cervical lesions (CIN and CC) and in the susceptibility of HPV 16 infection. PMID:26982176

  11. Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

    SciTech Connect

    Anastassopoulou, Cleo G. Ketas, Thomas J.; Depetris, Rafael S.; Thomas, Antonia M.; Klasse, Per Johan; Moore, John P.

    2011-04-25

    Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.

  12. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection.

    PubMed

    Ye, Lin; Wang, Jiaming; Beyer, Ashley I; Teque, Fernando; Cradick, Thomas J; Qi, Zhongxia; Chang, Judy C; Bao, Gang; Muench, Marcus O; Yu, Jingwei; Levy, Jay A; Kan, Yuet Wai

    2014-07-01

    Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.

  13. Selected amino acid changes in HIV-1 subtype-C gp41 are associated with specific gp120(V3) signatures in the regulation of co-receptor usage.

    PubMed

    Dimonte, Salvatore; Babakir-Mina, Muhammed; Mercurio, Fabio; Di Pinto, Domenico; Ceccherini-Silberstein, Francesca; Svicher, Valentina; Perno, Carlo-Federico

    2012-09-01

    The majority of studies have characterized the tropism of HIV-1 subtype-B isolates, but little is known about the determinants of tropism in other subtypes. So, the goal of the present study was to genetically characterize the envelope of viral proteins in terms of co-receptor usage by analyzing 356 full-length env sequences derived from HIV-1 subtype-C infected individuals. The co-receptor usage of V3 sequences was inferred by using the Geno2Pheno and PSSM algorithms, and also analyzed to the "11/25 rule". All reported env sequences were also analyzed with regard to N-linked glycosylation sites, net charge and hydrophilicity, as well as the binomial correlation phi coefficient to assess covariation among gp120(V3) and gp41 signatures and the average linkage hierarchical agglomerative clustering were also performed. Among env sequences present in Los Alamos Database, 255 and 101 sequences predicted as CCR5 and CXCR4 were selected, respectively. The classical V3 signatures at positions 11 and 25, and other specific V3 and gp41 amino acid changes were found statistically associated with different co-receptor usage. Furthermore, several statistically significant associations between V3 and gp41 signatures were also observed. The dendrogram topology showed a cluster associated with CCR5-usage composed by five gp41 mutated positions, A22V, R133M, E136G, N140L, and N166Q that clustered with T2V(V3) and G24T(V3) (bootstrap=1). Conversely, a heterogeneous cluster with CXCR4-usage, involving S11GR(V3), 13-14insIG/LG(V3), P16RQ(V3), Q18KR(V3), F20ILV(V3), D25KRQ(V3), Q32KR(V3) along with A30T(gp41), S107N(gp41), D148E(gp41), A189S(gp41) was identified (bootstrap=0.86). Our results show that as observed for HIV-1 subtype-B, also in subtype-C specific and different gp41 and gp120V3 amino acid changes are associated individually or together with CXCR4 and/or CCR5 usage. These findings strengthen previous observations that determinants of tropism may also reside in the gp41

  14. Intensification of Antiretroviral Therapy with a CCR5 Antagonist in Patients with Chronic HIV-1 Infection: Effect on T Cells Latently Infected

    PubMed Central

    Vallejo, Alejandro; Hernández-Novoa, Beatriz; Abad, María; Madrid, Nadia; Dahl, Viktor; Rubio, Rafael; Moreno, Ana M.; Dronda, Fernando; Casado, José Luis; Navas, Enrique; Pérez-Elías, María Jesús; Zamora, Javier; Palmer, Sarah; Muñoz, Eduardo; Muñoz-Fernández, María Ángeles; Moreno, Santiago

    2011-01-01

    Objective The primary objective was to assess the effect of MVC intensification on latently infected CD4+ T cells in chronically HIV-1-infected patients receiving antiretroviral therapy. Methods We performed an open-label pilot phase II clinical trial involving chronically HIV-1-infected patients receiving stable antiretroviral therapy whose regimen was intensified with 48 weeks of maraviroc therapy. We analyzed the latent reservoir, the residual viremia and episomal 2LTR DNA to examine the relationship between these measures and the HIV-1 latent reservoir, immune activation, lymphocyte subsets (including effector and central memory T cells), and markers associated with bacterial translocation. Results Overall a non significant reduction in the size of the latent reservoir was found (p = 0.068). A mean reduction of 1.82 IUPM was observed in 4 patients with detectable latent reservoir at baseline after 48 weeks of intensification. No effect on plasma residual viremia was observed. Unexpectedly, all the patients had detectable 2LTR DNA circles at week 24, while none of them showed those circles at the end of the study. No changes were detected in CD4+ or CD8+ counts, although a significant decrease was found in the proportion of HLA-DR+/CD38+ CD4+ and CD8+ T-cells. LPS and sCD14 levels increased. Conclusions Intensification with MVC was associated with a trend to a decrease in the size of the latent HIV-1 reservoir in memory T cells. No impact on residual viremia was detected. Additional studies with larger samples are needed to confirm the results. Trial Registration ClinicalTrials.gov NCT00795444 PMID:22174752

  15. The inner foreskin of healthy males at risk of HIV infection harbors epithelial CD4+ CCR5+ cells and has features of an inflamed epidermal barrier.

    PubMed

    Lemos, Maria P; Lama, Javier R; Karuna, Shelly T; Fong, Youyi; Montano, Silvia M; Ganoza, Carmela; Gottardo, Raphael; Sanchez, Jorge; McElrath, M Juliana

    2014-01-01

    Male circumcision provides partial protection against multiple sexually transmitted infections (STIs), including HIV, but the mechanisms are not fully understood. To examine potential vulnerabilities in foreskin epithelial structure, we used Wilcoxon paired tests adjusted using the false discovery rate method to compare inner and outer foreskin samples from 20 healthy, sexually active Peruvian males who have sex with males or transgender females, ages 21-29, at elevated risk of HIV infection. No evidence of epithelial microtrauma was identified, as assessed by keratinocyte activation, fibronectin deposition, or parakeratosis. However, multiple suprabasal tight junction differences were identified: 1) inner foreskin stratum corneum was thinner than outer (p = 0.035); 2) claudin 1 had extended membrane-bound localization throughout inner epidermis stratum spinosum (p = 0.035); 3) membrane-bound claudin 4 was absent from inner foreskin stratum granulosum (p = 0.035); and 4) occludin had increased membrane deposition in inner foreskin stratum granulosum (p = 0.042) versus outer. Together, this suggests subclinical inflammation and paracellular transport modifications to the inner foreskin. A setting of inflammation was further supported by inner foreskin epithelial explant cultures secreting higher levels of GM-CSF (p = 0.029), IP-10 (p = 0.035) and RANTES (p = 0.022) than outer foreskin, and also containing an increased density of CCR5+ and CD4+ CCR5+ cells (p = 0.022). Inner foreskin dermis also secreted more RANTES than outer (p = 0.036), and had increased density of CCR5+ cells (p = 0.022). In conclusion, subclinical changes to the inner foreskin of sexually active males may support an inflammatory state, with availability of target cells for HIV infection and modifications to epidermal barriers, potentially explaining the benefits of circumcision for STI prevention.

  16. Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

    SciTech Connect

    Mefford, Megan E.; Kunstman, Kevin; Wolinsky, Steven M.; Gabuzda, Dana

    2015-07-15

    Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions.

  17. Investigation of substituent effect of 1-(3,3-diphenylpropyl)-piperidinyl phenylacetamides on CCR5 binding affinity using QSAR and virtual screening techniques

    NASA Astrophysics Data System (ADS)

    Afantitis, Antreas; Melagraki, Georgia; Sarimveis, Haralambos; Koutentis, Panayiotis A.; Markopoulos, John; Igglessi-Markopoulou, Olga

    2006-02-01

    A linear quantitative-structure activity relationship model is developed in this work using Multiple Linear Regression Analysis as applied to a series of 51 1-(3,3-diphenylpropyl)-piperidinyl phenylacetamides derivatives with CCR5 binding affinity. For the selection of the best variables the Elimination Selection-Stepwise Regression Method (ES-SWR) is utilized. The predictive ability of the model is evaluated against a set of 13 compounds. Based on the produced QSAR model and an analysis on the domain of its applicability, the effects of various structural modifications on biological activity are investigated. The study leads to a number of guanidine derivatives with significantly improved predicted activities.

  18. High-level production, solubilization and purification of synthetic human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1.

    PubMed

    Ren, Hui; Yu, Daoyong; Ge, Baosheng; Cook, Brian; Xu, Zhinan; Zhang, Shuguang

    2009-01-01

    Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two

  19. Possible Impact of 190G > A CCR2 and Δ32 CCR5 Mutations on Decrease of the HBV Vaccine Immunogenicity—A Preliminary Report

    PubMed Central

    Ganczak, Maria; Skonieczna-Żydecka, Karolina; Drozd-Dąbrowska, Marzena; Adler, Grażyna

    2017-01-01

    Background: Chemokine genetic variations are involved in infectious diseases such as hepatitis B virus (HBV). Several allelic variants might, in theory, affect the outcome of vaccination. Objectives: This study was carried out to examine the associations of Δ32 CCR5 and 190G > A CCR2 polymorphisms with a response to a primary course of three HBV vaccinations. Methods: Between December 2014 and December 2016, patients from three randomly selected primary care clinics in the West Pomeranian region (Poland), 1 month after receiving the third dose of HBV vaccine, were enrolled. Enzyme-linked immunosorbent assay (ELISA) system version 3.0 was used to detect anti-HBs and anti-HBc totals. The identification of polymorphisms were performed by a polymerase chain reaction technique using a single primer extension assay. Genotype distributions of responders versus non-responders to HBV vaccination were compared on the basis of anti-HBs level. Results: In 149 patients (mean age 60 years) the mean anti-HBs level was 652.2 ± 425.9 mIU/mL (range: 0–1111.0 mIU/mL). There were 14.1% (n = 21) non-responders to the HBV vaccine (anti-HBs < 10.0 mIU/mL). The wild type/Δ32 genotype of CCR5 gene was found in 18.1% participants, and 1.3% were Δ32/Δ32 homozygotes. The frequency of allele A of the CCR2 gene was 11.1%. Lower anti-HBs levels in Δ32/Δ32 homozygotes were observed (Me = 61 mIU/mL vs. Me = 660.2 mIU/mL; p = 0.048). As age was found to be a correlate to the anti-HBs titer (r = −0.218, p = 0.0075; 95% CI: −0.366–−0.059)—an analysis of a co-variance was performed which found a statistically significant (p = 0.04) difference in anti-HBs titres between Δ32/Δ32 homozygotes and other CCR5 genotypes. The association between anti-HBs titres and CCR2 genotypes was not statistically significant. Conclusions: Our study—which is a preliminary report that suggest this topic deserves further observation with larger sample sizes, different ethnicities, and other single

  20. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

    PubMed Central

    Myburgh, Renier; Ivic, Sandra; Pepper, Michael S.; Gers-Huber, Gustavo; Li, Duo; Audigé, Annette; Rochat, Mary-Aude; Jaquet, Vincent; Regenass, Stephan; Manz, Markus G.; Salmon, Patrick; Krause, Karl-Heinz

    2015-01-01

    ABSTRACT Gene-engineered CD34+ hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+ cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4+ T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34+ cells that produce a constant supply of HIV-resistant progeny. IMPORTANCE Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4+ T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a

  1. Enhanced CD4+ cellular apoptosis by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with progressive HIV-1 infection

    SciTech Connect

    Wade, Jessica; Sterjovski, Jasminka; Gray, Lachlan; Roche, Michael; Chiavaroli, Lisa; Ellett, Anne; Jakobsen, Martin R.; Cowley, Daniel; Fonseca Pereira, Candida da; Saksena, Nitin; Wang, Bin; Purcell, Damian F.J.; Karlsson, Ingrid; Fenyoe, Eva-Maria; Churchill, Melissa; Gorry, Paul R.

    2010-01-20

    CCR5-using (R5) human immunodeficiency virus type 1 (HIV-1) strains cause CD4+ T-cell loss in most infected individuals, but mechanisms underlying cytopathicity of R5 viruses are poorly understood. We investigated mechanisms contributing to R5 envelope glycoprotein (Env)-mediated cellular apoptosis by constructing a panel of retroviral vectors engineered to co-express GFP and R5 Envs derived from two HIV-1-infected subjects spanning asymptomatic (Early, E-R5 Envs) to late stages of infection (Late, L-R5 Envs). The L-R5 Envs induced significantly more cellular apoptosis than E-R5 Envs, but only in Env-expressing (GFP-positive) cells, and only in cells where CD4 and CCR5 levels were limiting. Studies with fusion-defective Env mutants showed induction of apoptosis required membrane-fusing events. Our results provide evidence for an intracellular mechanism of R5 Env-induced apoptosis of CD4+ cells that requires membrane fusion. Furthermore, they contribute to a better understanding of mechanisms involved in CD4+ T-cell loss in subjects experiencing progressive R5 HIV-1 infection.

  2. CXCR3, CCR5, and CRTH2 Chemokine Receptor Expression in Lymphocytes Infiltrating Thyroid Nodules with Coincident Hashimoto's Thyroiditis Obtained by Fine Needle Aspiration Biopsy

    PubMed Central

    Antošová, Marie; Krátký, Jan; Vítková, Hana; Límanová, Zdeňka; Marečková, Helena; Potluková, Eliška

    2016-01-01

    Objective. To determine the expression of chemokine receptors in lymphocytes from thyroid nodules and peripheral blood in patients with and without Hashimoto's thyroiditis (HT). Patients and Methods. The study included 46 women with thyroid nodules and HT and 60 women with thyroid nodules without HT (controls) who underwent a fine needle aspiration biopsy (FNAB). Expression of chemokine receptors CXCR3, CCR5, and CRTH2 was assessed by flow cytometry in lymphocytes from FNAB samples and from peripheral blood. Results. The percentage of CRTH2+ lymphocytes was higher in nodules with HT in comparison with controls, both in FNAB samples (13.95 versus 6.7%, p = 0.008) and in peripheral blood (6.7 versus 5.13%, p = 0.047), and positively correlated with serum antibodies to thyroid peroxidase (r = 0.243; p = 0.026) and negatively correlated with thyroid volume (r = −0.346; p = 0.008). Lymphocytes from neoplastic nodules showed a higher expression of both CXCR3 and CCR5 than those from hyperplastic ones. Conclusion. Flow cytometry performed in FNAB samples may serve as a good tool in investigation of intrathyroidal expression of immunological parameters. In our study, the CRTH2 expression on thyroid-infiltrating lymphocytes as well as on lymphocytes from peripheral blood was increased in HT as compared to controls. PMID:27872865

  3. Humanized mice dually challenged with R5 and X4 HIV-1 show preferential R5 viremia and restricted X4 infection of CCR5(+)CD4(+) T cells.

    PubMed

    Terahara, Kazutaka; Ishige, Masayuki; Ikeno, Shota; Okada, Seiji; Kobayashi-Ishihara, Mie; Ato, Manabu; Tsunetsugu-Yokota, Yasuko

    2015-05-01

    CCR5-tropic (R5) immunodeficiency virus type 1 (HIV-1) strains are highly transmissible during the early stage of infection in humans, whereas CXCR4-tropic (X4) strains are less transmissible. This study aimed to explore the basis for early phase R5 and X4 HIV-1 infection in vivo by using humanized mice dually challenged with R5 HIV-1NLAD8-D harboring DsRed and X4 HIV-1(NL-E) harboring EGFP. Whereas R5 HIV-1 replicated well, X4 HIV-1 caused only transient viremia with variable kinetics; however, this was distinct from the low level but persistent viremia observed in mice challenged with X4 HIV-1 alone. Flow cytometric analysis of HIV-1-infected cells revealed that X4 HIV-1 infection of CCR5(+)CD4(+) T cells was significantly suppressed in the presence of R5 HIV-1. X4 HIV-1 was more cytopathic than R5 HIV-1; however, this was not the cause of restricted X4 HIV-1 infection because there were no significant differences in the mortality rates of CCR5(+) and CCR5(-) cells within the X4 HIV-1-infected cell populations. Taken together, these results suggest that restricted infection of CCR5(+)CD4(+) T cells by X4 HIV-1 (occurring via a still-to-be-identified mechanism) might contribute to the preferential transmission of R5 HIV-1 during the early phase of infection.

  4. Astragaloside IV Alleviates Lipopolysaccharide-Induced Acute Kidney Injury Through Down-Regulating Cytokines, CCR5 and p-ERK, and Elevating Anti-Oxidative Ability

    PubMed Central

    Zhou, Wei; Chen, Yi; Zhang, Xingyu

    2017-01-01

    Background Astragaloside IV (AS-IV) has been shown to prevent ischemia-induced acute kidney injury (AKI) in rat models of ischemia and reperfusion. However, the effects of AS-IV on AKI during sepsis and endotoxinemia is unclear. The current study aimed to investigate the effects and molecular mechanisms of AS-IV on lipopolysaccharide (LPS)-induced AKI. Material/Methods Adult male CD-1 mice were randomly assigned into 6 groups (n=8/group): control group: mice were intraperitoneally (i.p.) injected with normal saline; LPS group (10 mg/kg, i.p.); low-dose AS-IV (25 mg/kg; gavage for 7 days) + LPS (i.p., 1 hour after last gavage) group; medial-dose AS-IV (50 mg/kg) + LPS group; high-dose AS-IV (100 mg/kg) + LPS group; high-dose AS-IV alone (100 mg/kg; gavage for 7 days) group. Blood samples were collected at 24 hours after LPS injection, and plasma uric acid and BUN were measured with colorimetric detection kits. The concentration of plasma tumor necrosis factor (TNF)-α and interleukin 1β, renal p-extracellular signal-regulated kinases, and urinary albumin were evaluated by ELISA. The expression of CCR5 in renal tissue was evaluated by PCR and Western blotting. Concentrations of glutathione (GSH) and reactive oxygen species (ROS) in renal tissue were also measured. Results AS-IV decreased LPS-stimulated production of blood TNF-α and IL-6, LPS-induced the expression of CCR5, and activation of ERK in the kidneys in a rodent model of endotoxinemia. AS-IV attenuated LPS-caused decreased GSH and increased ROS. It also attenuated LPS-induced increases in plasma uric acid, BUN, and urinary albumin. Conclusions AS-IV protects against AKI during bacterial endotoxinemia by attenuating expression of cytokines, CCR5, and p-ERK, and elevating anti-oxidative ability. PMID:28328867

  5. Effect of TNF-α genetic variants and CCR5Δ32 on the vulnerability to HIV-1 infection and disease progression in Caucasian Spaniards

    PubMed Central

    2010-01-01

    Background Tumor necrosis factor alpha (TNF-α) is thought to be involved in the various immunogenetic events that influence HIV-1 infection. Methods We aimed to determine whether carriage of the TNF-α-238G>A, -308G>A and -863 C>A gene promoter single nucleotide polymorphisms (SNP) and the CCR5Δ32 variant allele influence the risk of HIV-1 infection and disease progression in Caucasian Spaniards. The study group consisted of 423 individuals. Of these, 239 were uninfected (36 heavily exposed but uninfected [EU] and 203 healthy controls [HC]) and 184 were HIV-1-infected (109 typical progressors [TP] and 75 long-term nonprogressors [LTNP] of over 16 years' duration). TNF-α SNP and the CCR5Δ32 allele were assessed using PCR-RFLP and automatic sequencing analysis methods on white blood cell DNA. Genotype and allele frequencies were compared using the χ 2 test and the Fisher exact test. Haplotypes were compared by logistic regression analysis. Results The distribution of TNF-α-238G>A, -308G>A and -863 C>A genetic variants was non-significantly different in HIV-1-infected patients compared with uninfected individuals: -238G>A, p = 0.7 and p = 0.3; -308G>A, p = 0.05 and p = 0.07; -863 C>A, p = 0.7 and p = 0.4, for genotype and allele comparisons, respectively. Haplotype analyses, however, indicated that carriers of the haplotype H3 were significantly more common among uninfected subjects (p = 0.04). Among the infected patients, the distribution of the three TNF-α genetic variants assessed was non-significantly different between TP and LTNP: -238G>A, p = 0.35 and p = 0.7; -308G>A, p = 0.7 and p = 0.6: -863 C>A, p = 0.2 and p = 0.2, for genotype and allele comparisons, respectively. Haplotype analyses also indicated non-significant associations. Subanalyses in the LTNP subset indicated that the TNF-α-238A variant allele was significantly overrepresented in patients who spontaneously controlled plasma viremia compared with those who had a detectable plasma viral load

  6. Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit

    PubMed Central

    Chen, Sisi; Luetje, Charles W.

    2014-01-01

    Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control.  Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry.  Synthetic Orco directed agonists and antagonists have recently been identified.  Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants.  The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants.  To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists.  Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system.  Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants.  Tyramine had effects similar to those of tryptamine, but was less potent.  Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae).  Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants. PMID:25075297

  7. Is long-term virological response related to CCR5 Δ32 deletion in HIV-1-infected patients started on highly active antiretroviral therapy?

    PubMed Central

    Laurichesse, Jean-Jacques; Taieb, Audrey; Capoulade-Metay, Corinne; Katlama, Christine; Villes, Virginie; Drobacheff-Thiebaud, Marie-Christine; Raffi, François; Chêne, Genevieve; Theodorou, Ioannis; Leport, Catherine

    2010-01-01

    Objective To examine whether CCR5 Δ32 deletion is associated with long-term response to combination antiretroviral treatment (cART) in HIV-1 infected patients. Methods The genetic sub-study of ANRS CO8 APROCO-COPILOTE cohort included 609 patients who started a protease inhibitor-containing cART in 1997–99. Patients were considered to have a sustained virological response if all plasma HIV-RNA measurements between month 4 and years 3–5 were <500 copies/ml, allowing for a single blip. Virological response was compared between patients heterozygous for CCR5 Δ32 (Δ32/wt) and wild-type patients (wt/wt) from month 4 to year 3 and month 4 to year 5. Logistic regression analysis was used to adjust for baseline demographical data, HIV-RNA, CD4 cell counts, antiretroviral naive status, time spent on antiretroviral therapy at year 3 and 5 and adherence to treatment (month 4 to year 3 and 5). Results Sustained virological response was better in Δ32/wt than in wt/wt patients: 66% versus 52% up to year 3 (p=0.02), nearly significant after adjustment to potential cofounders (p=0.07). Δ32/wt patients had a better virological response, up to year 5, 48% versus 35% (p=0.01), and remained significantly better, after adjustment, associated with a better virological response up to 5 years post initiation of cART (p=0.04). There was no association with CD4 response. Conclusion Δ32/wt deletion is associated with a beneficial virological response to cART on the long-term. Whether this association can be a direct effect of Δ32/wt deletion remains questionable and needs confirmation in other observational studies. PMID:20050936

  8. Genetic analysis of HIV-1 Circulating Recombinant Form 02_AG, B and C subtype-specific envelope sequences from Northern India and their predicted co-receptor usage

    PubMed Central

    2009-01-01

    HIV-1 epidemic in India is largely driven by subtype C but other subtypes or recombinants have also been reported from several states of India. This is mainly due to the co-circulation of other genetic subtypes that potentially can recombine to generate recombinant/mosaic genomes. In this study, we report detail genetic characterization of HIV-1 envelope sequences from North India (Delhi and neighboring regions). Six of 13 were related to subtype C, one B and the rest six showed relatedness with CRF02_AG strain. The subtype C possessed the highly conserved GPGQ motif but subtype B possessed the GPGR motif in the V3 loop as observed earlier. While most of the sequences suggested CCR5 co-receptor usage, one subtype C sample clearly indicated CXCR4 usage. A successful mother to child transmission was established in two pairs. Thus, co-circulation of multiple subtypes (B and C) and the recombinant CRF02_AG strains in North India suggests a rapidly evolving scenario of HIV-1 epidemic in this region with impact on vaccine formulation. Since this is the first report of CRF02_AG envelope from India, it will be important to monitor the spread of this strain and its impact on HIV-1 transmission in India. PMID:19954551

  9. CCR5 delta32 deletion and reduced risk of toxoplasmosis in persons infected with human immunodeficiency virus type 1. The SEROCO-HEMOCO-SEROGEST Study Groups.

    PubMed

    Meyer, L; Magierowska, M; Hubert, J B; Mayaux, M J; Misrahi, M; Le Chenadec, J; Debre, P; Rouzioux, C; Delfraissy, J F; Theodorou, I

    1999-09-01

    This study attempted to determine whether the CCR5 Delta32 deletion affected progression to certain first AIDS-defining illnesses in human immunodeficiency virus type 1-infected patients enrolled in the French SEROCO/HEMOCO/SEROGEST cohorts. Toxoplasmosis onset as a first AIDS-defining illness was significantly delayed in 253 heterozygous patients, compared with 1404 wild type patients. The relative risk of toxoplasmosis associated with heterozygosity was 0. 39 (95% confidence interval, 0.16-0.96) after adjustment for age, CD4 cell count, and primary specific prophylaxis. A nonsignificant protective trend was observed with regard to the onset of mycobacterial, cytomegalovirus, and herpesvirus diseases, but these events were less frequent than toxoplasmosis. Progression to other conditions (e.g., wasting, non-Hodgkin's lymphoma, Kaposi's sarcoma) was similar in the 2 groups as was the frequency of toxoplasmosis as a subsequent AIDS-defining illness. As chemokines are involved in numerous infectious processes, the Delta32 deletion could delay progression to certain opportunistic infections such as toxoplasmosis.

  10. Stable Delivery of CCR5-Directed shRNA into Human Primary Peripheral Blood Mononuclear Cells and Hematopoietic Stem/Progenitor Cells via a Lentiviral Vector

    PubMed Central

    Shimizu, Saki; Yadav, Swati Seth; An, Dong Sung

    2016-01-01

    RNAi is a powerful tool to achieve suppression of a specific gene expression and therefore it has tremendous potential for gene therapy applications. A number of vector systems have been developed to express short-hairpin RNAs (shRNAs) to produce siRNAs within mammalian T-cells, primary hematopoietic stem/progenitor cells (HSPC), human peripheral blood mononuclear cells, and in animal model systems. Among these, vectors based on lentivirus backbones have significantly transformed our ability to transfer shRNAs into nondividing cells, such as HSPC, resulting in high transduction efficiencies. However, delivery and long-term expression of shRNAs should be carefully optimized for efficient knock down of target gene without causing cytotoxicity in mammalian cells. Here, we describe our protocols for the development of shRNA against a major HIV co-receptor/chemokine receptor CCR5 and the use of lentiviral vectors for stable shRNA delivery and expression in primary human PBMC and HSPC. PMID:26472455

  11. Generation of lineage-related, mucosally transmissible subtype C R5 simian-human immunodeficiency viruses capable of AIDS development, induction of neurological disease, and coreceptor switching in rhesus macaques.

    PubMed

    Ren, Wuze; Mumbauer, Alexandra; Gettie, Agegnehu; Seaman, Michael S; Russell-Lodrigue, Kasi; Blanchard, James; Westmoreland, Susan; Cheng-Mayer, Cecilia

    2013-06-01

    Most human immunodeficiency virus (HIV) transmissions are initiated with CCR5 (R5)-using viruses across mucosal surfaces, with the majority in regions where HIV type 1 (HIV-1) clade C predominates. Mucosally transmissible, highly replication competent, pathogenic R5 simian-human immunodeficiency viruses (SHIVs) encoding biologically relevant clade C envelopes are therefore needed as challenge viruses in vaccine efficacy studies with nonhuman primates. Here we describe the generation of three lineage-related subtype C SHIVs through four successive rapid transfers in rhesus macaques of SHIVC109F.PB4, a molecular clone expressing the soluble-CD4 (sCD4)-sensitive CCR5-tropic clade C envelope of a recently infected subject in Zambia. The viruses differed in their monkey passage histories and neutralization sensitivities but remained R5 tropic. SHIVC109P3 and SHIVC109P3N were recovered from a passage-3 rapid-progressor animal during chronic infection (24 weeks postinfection [wpi]) and at end-stage disease (34 wpi), respectively, and are classified as tier 1B strains, whereas SHIVC109P4 was recovered from a passage-4 normal-progressor macaque at 22 wpi and is a tier 2 virus, more difficult to neutralize. All three viruses were transmitted efficiently via intrarectal inoculation, reaching peak viral loads of 10(7) to 10(9) RNA copies/ml plasma and establishing viremia at various set points. Notably, one of seven (GC98) and two of six (CL31, FI08) SHIVC109P3- and SHIVC109P3N-infected macaques, respectively, progressed to AIDS, with neuropathologies observed in GC98 and FI08, as well as coreceptor switching in the latter. These findings support the use of these new SHIVC109F.PB4-derived viruses to study the immunopathology of HIV-1 clade C infection and to evaluate envelope-based AIDS vaccines in nonhuman primates.

  12. Tick saliva inhibits the chemotactic function of MIP-1alpha and selectively impairs chemotaxis of immature dendritic cells by down-regulating cell-surface CCR5.

    PubMed

    Oliveira, Carlo José F; Cavassani, Karen A; Moré, Daniela D; Garlet, Gustavo P; Aliberti, Julio C; Silva, João S; Ferreira, Beatriz R

    2008-05-01

    Ticks are blood-feeding arthropods that secrete immunomodulatory molecules through their saliva to antagonize host inflammatory and immune responses. As dendritic cells (DCs) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC migration and function. Bone marrow-derived immature DCs pre-exposed to tick saliva showed reduced migration towards macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and regulated upon activation, normal T cell expressed and secreted (RANTES) chemokines in a Boyden microchamber assay. This inhibition was mediated by saliva which significantly reduced the percentage and the average cell-surface expression of CC chemokine receptor CCR5. In contrast, saliva did not alter migration of DCs towards MIP-3beta, not even if the cells were induced for maturation. Next, we evaluated the effect of tick saliva on the activity of chemokines related to DC migration and showed that tick saliva per se inhibits the chemotactic function of MIP-1alpha, while it did not affect RANTES, MIP-1beta and MIP-3beta. These data suggest that saliva possibly reduces immature DC migration, while mature DC chemotaxis remains unaffected. In support of this, we have analyzed the percentage of DCs on mice 48h after intradermal inoculation with saliva and found that the DC turnover in the skin was reduced compared with controls. Finally, to test the biological activity of the saliva-exposed DCs, we transferred DCs pre-cultured with saliva and loaded with the keyhole limpet haemocyanin (KLH) antigen to mice and measured their capacity to induce specific T cell cytokines. Data showed that saliva reduced the synthesis of both T helper (Th)1 and Th2 cytokines, suggesting the induction of a non-polarised T cell response. These findings propose that the inhibition of DCs migratory ability and function may be a relevant mechanism used by ticks to subvert the immune response of the host.

  13. Generation of a neutralization-resistant CCR5 tropic simian/human immunodeficiency virus (SHIV-MK38) molecular clone, a derivative of SHIV-89.6.

    PubMed

    Ishida, Yuki; Yoneda, Mai; Otsuki, Hiroyuki; Watanabe, Yuji; Kato, Fumihiro; Matsuura, Kanako; Kikukawa, Minako; Matsushita, Shuzo; Hishiki, Takayuki; Igarashi, Tatsuhiko; Miura, Tomoyuki

    2016-05-01

    Previously, we reported that a new genetically diverse CCR5 (R5) tropic simian/human immunodeficiency virus (SHIV-MK38) adapted to rhesus monkeys became more neutralization resistant to SHIV-infected plasma than did the parental SHIV-KS661 clone. Here, to clarify the significance of the neutralization-resistant phenotype of SHIV in a macaque model, we initially investigated the precise neutralization phenotype of the SHIVs, including SHIV-MK38 molecular clones, using SHIV-MK38-infected plasma, a pooled plasma of human immunodeficiency virus (HIV)-infected individuals, soluble CD4 and anti-HIV-1 neutralizing mAbs, the epitopes of which were known. The results show that SHIV-KS661 had tier 1 neutralization sensitivity, but monkey-adapted R5 tropic SHIV-MK38 acquired neutralization resistance similar to that of tier 2 or 3 as a clone virus. Sequence analysis of the env gene suggested that the neutralization-resistant phenotype of SHIV-MK38 was acquired by conformational changes in Env associated with the net charge and potential N-linked glycosylation sites. To examine the relationship between neutralization phenotype and stably persistent infection in monkeys, we performed in vivo rectal inoculation experiments using a SHIV-MK38 molecular clone. The results showed that one of three rhesus monkeys exhibited durable infection with a plasma viral load of 105 copies ml- 1 despite the high antibody responses that occurred in the host. Whilst further improvements are required in the development of a challenge virus, it will be useful to generate a neutralization-resistant R5 tropic molecular clone of the SHIV-89.6 lineage commonly used for vaccine development - a result that can be used to explore the foundation of AIDS pathogenesis.

  14. Microparticles derived from obese adipose tissue elicit a pro-inflammatory phenotype of CD16(+), CCR5(+) and TLR8(+) monocytes.

    PubMed

    Renovato-Martins, Mariana; Matheus, Maria Eline; de Andrade, Isadora Ramos; Moraes, João Alfredo; da Silva, Simone Vargas; Citelli Dos Reis, Marta; de Souza, Antônio Augusto Peixoto; da Silva, César Cláudio; Bouskela, Eliete; Barja-Fidalgo, Christina

    2017-01-01

    Macrophage infiltration into adipose tissue (AT) is a hallmark of the chronic inflammatory response in obesity and is supported by an intense monocyte migration towards AT. Although it has been detected an increased proportion of circulating CD16(+) monocyte subsets in obese subjects, the mechanisms underlying this effect and the contribution of these cells to the inflamed profile of obese AT are still poorly understood. We investigated whether factors secreted by human obese omental AT could polarize monocytes to CD16(+) enriched phenotype, and how these changes could modify their migratory capacity towards adipose tissue itself. We show that explants of human obese omental AT, obtained during bariatric surgery, released higher levels of MIP1-α, TNFα, leptin and also VEGF, together with increasing amounts of microparticles (MP), when compared to explants of lean subcutaneous AT. A higher content of circulating MP derived from preadipocytes and leukocytes was also detected in plasma of obese subjects. Conditioned media or MP released from obese omental AT increased CD16 and CCR5 expression on CD14(+)CD16(-) monocytes and augmented their migratory capacity towards the conditioned media from obese omental AT, itself. This effect was inhibited when MIP1-α was neutralized. Additionally, we demonstrate that MP derived from obese omental AT carry and transfer TLR8 to monocytes, thus triggering an increase in CD16 expression in those cells. Our data shows a positive feedback loop between blood monocytes and obese omental AT, which releases chemotactic mediators and TLR8-enriched MP, thus inducing an up-regulation of CD16(+) monocytes, favoring leukocyte infiltration in the obese omental AT.

  15. Progesterone Levels Associate with a Novel Population of CCR5+CD38+ CD4 T Cells Resident in the Genital Mucosa with Lymphoid Trafficking Potential.

    PubMed

    Swaims-Kohlmeier, Alison; Haaland, Richard E; Haddad, Lisa B; Sheth, Anandi N; Evans-Strickfaden, Tammy; Lupo, L Davis; Cordes, Sarah; Aguirre, Alfredo J; Lupoli, Kathryn A; Chen, Cheng-Yen; Ofotukun, Igho; Hart, Clyde E; Kohlmeier, Jacob E

    2016-07-01

    The female genital tract (FGT) provides a means of entry to pathogens, including HIV, yet immune cell populations at this barrier between host and environment are not well defined. We initiated a study of healthy women to characterize resident T cell populations in the lower FGT from lavage and patient-matched peripheral blood to investigate potential mechanisms of HIV sexual transmission. Surprisingly, we observed FGT CD4 T cell populations were primarily CCR7(hi), consistent with a central memory or recirculating memory T cell phenotype. In addition, roughly half of these CCR7(hi) CD4 T cells expressed CD69, consistent with resident memory T cells, whereas the remaining CCR7(hi) CD4 T cells lacked CD69 expression, consistent with recirculating memory CD4 T cells that traffic between peripheral tissues and lymphoid sites. HIV susceptibility markers CCR5 and CD38 were increased on FGT CCR7(hi) CD4 T cells compared with blood, yet migration to the lymphoid homing chemokines CCL19 and CCL21 was maintained. Infection with GFP-HIV showed that FGT CCR7(hi) memory CD4 T cells are susceptible HIV targets, and productive infection of CCR7(hi) memory T cells did not alter chemotaxis to CCL19 and CCL21. Variations of resident CCR7(hi) FGT CD4 T cell populations were detected during the luteal phase of the menstrual cycle, and longitudinal analysis showed the frequency of this population positively correlated to progesterone levels. These data provide evidence women may acquire HIV through local infection of migratory CCR7(hi) CD4 T cells, and progesterone levels predict opportunities for HIV to access these novel target cells.

  16. Rubratoxin-B-induced secretion of chemokine ligands of cysteine-cysteine motif chemokine receptor 5 (CCR5) and its dependence on heat shock protein 90 in HL60 cells.

    PubMed

    Nagashima, Hitoshi

    2015-11-01

    To elucidate the mechanism underlying rubratoxin B toxicity, the effects of rubratoxin B on the secretion of CCR5 chemokines, CCL3, CCL4, and CCL5, in a human promyelocytic leukemia cell line, HL60, were investigated. In addition, to examine whether the molecular chaperone 90-kDa heat shock protein (Hsp90) contributes to rubratoxin B toxicity, the effects of Hsp90-specific inhibitors, radicicol and geldanamycin, were investigated. Exposure to rubratoxin B for 24h induced secretion of each CCR5 chemokine, although the effect on CCL5 secretion was modest, and it enhanced secretion of proinflammatory cytokines tumor necrosis factor-α, CXCL8, and CCL2. Concomitant treatment with radicicol abolished the rubratoxin-induced secretion of all cytokines investigated. Geldanamycin antagonized the rubratoxin B-induced effects on CCL3 and CCL5, but not CCL4; the effects of geldanamycin were less than that of radicicol. Taken together, the results suggest that rubratoxin B, with the contribution of Hsp90, induces secretion of CCR5 chemokines.

  17. Analysis of CCR5-Delta 32 and CCR2-V64I polymorphisms in a cohort of Spanish HCV patients using real-time polymerase chain reaction and fluorescence resonance energy transfer technologies.

    PubMed

    Ruiz-Ferrer, M; Barroso, N; Antiñolo, G; Aguilar-Reina, J

    2004-07-01

    Nowadays it is clear that chemokine-chemokine receptor interactions are important in chronic hepatitis C virus (HCV) infection. The objective of the present study was to elucidate the involvement of the CCR5-Delta 32 and CCR2-V64I polymorphisms in the response to the HCV infection, as well as in the histological damage and the outcome of the infection. A cohort of 139 patients with hepatitis C and 100 healthy blood donors were analysed for both polymorphisms using real-time polymerase chain reaction (PCR) and LightCycler technology. We have detected the CCR5-Delta 32 allele in 15 of 278 HCV chromosomes (5.4%) and 15 of 200 control chromosomes (7.5%). The CCR2-V64I allele was present in 24 of 278 HCV chromosomes (8.6%) and 19 of 200 control chromosomes (9.5%). Analysis of the histological parameters showed no statistical significance when comparing the patients carrying the variants vs the cases with the wild-type allele. Our results seem to indicate that the CCR5-Delta 32 and CCR2-V64I polymorphisms are not related to the response to HCV infection, histological damage and outcome of infection in our cohort of Spanish HCV patients.

  18. Microinjection of Cre recombinase protein into zygotes enables specific deletion of two eukaryotic selection cassettes and enhances the expression of a DsRed2 reporter gene in Ccr2/Ccr5 double-deficient mice.

    PubMed

    Luckow, Bruno; Hänggli, Amy; Maier, Holger; Chilla, Silvia; Loewe, Robert P; Dehmel, Stefan; Schlöndorff, Detlef; Loetscher, Pius; Zerwes, Hans-Günter; Müller, Matthias

    2009-08-01

    The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double-deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double-deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose-binding protein and Cre (MBP-Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks.

  19. Virologic failure in first-line human immunodeficiency virus therapy with a CCR5 entry inhibitor, aplaviroc, plus a fixed-dose combination of lamivudine-zidovudine: nucleoside reverse transcriptase inhibitor resistance regardless of envelope tropism.

    PubMed

    Demarest, James F; Amrine-Madsen, Heather; Irlbeck, David M; Kitrinos, Kathryn M

    2009-03-01

    The CCR102881 (ASCENT) study evaluated the antiviral activity of the novel CCR5 entry inhibitor aplaviroc plus a fixed-dose combination of lamivudine-zidovudine (Combivir) in drug-naïve human immunodeficiency virus type 1-infected subjects with only CCR5-tropic virus detected in plasma. Although the trial was stopped prematurely due to idiosyncratic hepatotoxicity, eight subjects met protocol-defined virologic failure criteria. Clonal analyses of the viral envelope tropism, aplaviroc susceptibility, and env sequencing were performed on plasma at baseline and at the time of virologic failure. Molecular evolutionary analyses were also performed. The majority of the subjects with virologic failure (six of eight) acquired the lamivudine resistance-associated mutation M184V, and none had evidence of reduced susceptibility to aplaviroc at the time of virologic failure, even at the clonal level. Six subjects with virologic failure maintained CCR5 tropism, while two exhibited a change in population tropism readout to dual/mixed-tropic with R5X4-tropic clones detected prior to therapy. Two evolutionary patterns were observed: five subjects had no evidence of population turnover, while three subjects had multiple lines of evidence for env population turnover. The acquisition of the M184V mutation is the primary characteristic of virologic failure in first-line therapy with aplaviroc plus lamivudine-zidovudine, regardless of the envelope tropism.

  20. MIF allele-dependent regulation of the MIF coreceptor CD44 and role in rheumatoid arthritis.

    PubMed

    Yoo, Seung-Ah; Leng, Lin; Kim, Bum-Joon; Du, Xin; Tilstam, Pathricia V; Kim, Kyung Hee; Kong, Jin-Sun; Yoon, Hyung-Ju; Liu, Aihua; Wang, Tian; Song, Yan; Sauler, Maor; Bernhagen, Jurgen; Ritchlin, Christopher T; Lee, Patty; Cho, Chul-Soo; Kim, Wan-Uk; Bucala, Richard

    2016-12-06

    Fibroblast-like synoviocytes mediate joint destruction in rheumatoid arthritis and exhibit sustained proinflammatory and invasive properties. CD44 is a polymorphic transmembrane protein with defined roles in matrix interaction and tumor invasion that is also a signaling coreceptor for macrophage migration inhibitory factor (MIF), which engages cell surface CD74. High-expression MIF alleles (rs5844572) are associated with rheumatoid joint erosion, but whether MIF signaling through the CD74/CD44 receptor complex promotes upstream autoimmune responses or contributes directly to synovial joint destruction is unknown. We report here the functional regulation of CD44 by an autocrine pathway in synovial fibroblasts that is driven by high-expression MIF alleles to up-regulate an inflammatory and invasive phenotype. MIF increases CD44 expression, promotes its recruitment into a functional signal transduction complex, and stimulates alternative exon splicing, leading to expression of the CD44v3-v6 isoforms associated with oncogenic invasion. CD44 recruitment into the MIF receptor complex, downstream MAPK and RhoA signaling, and invasive phenotype require MIF and CD74 and are reduced by MIF pathway antagonists. These data support a functional role for high-MIF expression alleles and the two-component CD74/CD44 MIF receptor in rheumatoid arthritis and suggest that pharmacologic inhibition of this pathway may offer a specific means to interfere with progressive joint destruction.

  1. Kisspeptin antagonists.

    PubMed

    Roseweir, Antonia Kathryn; Millar, Robert P

    2013-01-01

    Kisspeptin is now known to be an important regulator of the hypothalamic--pituitary-gonadal axis and is the target of a range of regulators, such as steroid hormone feedback, nutritional and metabolic regulation. Kisspeptin binds to its cognate receptor, KISS1R (also called GPR54), on GnRH neurons and stimulates their activity, which in turn provides an obligatory signal for GnRH secretion-thus gating down-stream events supporting reproduction. The development of peripherally active kisspeptin antagonists could offer a unique therapeutic agent for treating hormone-dependent disorders of reproduction, including precocious puberty, endometriosis, and metastatic prostate cancer. The following chapter discusses the advances made in the search for both peptide and small molecule kisspeptin antagonists and their use in delineating the role of kisspeptin within the reproductive system. To date, four peptide antagonists and one small molecule antagonist have been designed.

  2. [Coreceptors of HIV infection and the development of HIV entry inhibitors: overview and targets].

    PubMed

    Hoshino, Hiroo

    2002-01-01

    In 1996 CXCR4 was identified as a coreceptor for HIV-1. This finding has lead to further identification of more than ten G-protein-coupled receptors (GPCRs) as coreceptors for HIV/SIV. Cell tropisms and coreceptor uses of HIV during the course of HIV infection are summarized. Promiscuous properties of correlations between chemokines and their chemokine receptor uses and also between variable amino acid sequences in the V3 region of HIV gp120 Env and HIV coreceptor uses are discussed. This promiscuous property of HIV-1 is claimed to be a possible cause of a difficulty in developing highly effective entry inhibitors and in addition to allow rapid appearance of immune escape HIV mutants. Representative agents that inhibit HIV entry with a special reference to inhibitors of coreceptor use and gp41 function are summarized. gp41 is discussed as a promising target for the development of effective entry inhibitors.

  3. Structural analysis of a novel rabbit monoclonal antibody R53 targeting an epitope in HIV-1 gp120 C4 region critical for receptor and co-receptor binding

    DOE PAGES

    Pan, Ruimin; Chen, Yuxin; Vaine, Michael; ...

    2015-07-15

    The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitopemore » peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence 433AMYAPPI439, it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.« less

  4. Structural analysis of a novel rabbit monoclonal antibody R53 targeting an epitope in HIV-1 gp120 C4 region critical for receptor and co-receptor binding

    SciTech Connect

    Pan, Ruimin; Chen, Yuxin; Vaine, Michael; Hu, Guangnan; Wang, Shixia; Lu, Shan; Kong, Xiang -Peng

    2015-07-15

    The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitope peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence 433AMYAPPI439, it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.

  5. The odorant receptor co-receptor from the bed bug, Cimex lectularius L.

    PubMed

    Hansen, Immo A; Rodriguez, Stacy D; Drake, Lisa L; Price, David P; Blakely, Brittny N; Hammond, John I; Tsujimoto, Hitoshi; Monroy, Erika Y; Maio, William A; Romero, Alvaro

    2014-01-01

    Recently, the bed bug, Cimex lectularius L. has re-emerged as a serious and growing problem in many parts of the world. Presence of resistant bed bugs and the difficulty to eliminate them has renewed interest in alternative control tactics. Similar to other haematophagous arthropods, bed bugs rely on their olfactory system to detect semiochemicals in the environment. Previous studies have morphologically characterized olfactory organs of bed bugs' antenna and have physiologically evaluated the responses of olfactory receptor neurons (ORNs) to host-derived chemicals. To date, odorant binding proteins (OBPs) and odorant receptors (ORs) associated with these olfaction processes have not been studied in bed bugs. Chemoreception in insects requires formation of heteromeric complexes of ORs and a universal OR coreceptor (Orco). Orco is the constant chain of every odorant receptor in insects and is critical for insect olfaction but does not directly bind to odorants. Orco agonists and antagonists have been suggested as high-value targets for the development of novel insect repellents. In this study, we have performed RNAseq of bed bug sensory organs and identified several odorant receptors as well as Orco. We characterized Orco expression and investigated the effect of chemicals targeting Orco on bed bug behavior and reproduction. We have identified partial cDNAs of six C. lectularius OBPs and 16 ORs. Full length bed bug Orco was cloned and sequenced. Orco is widely expressed in different parts of the bed bug including OR neurons and spermatozoa. Treatment of bed bugs with the agonist VUAA1 changed bed bug pheromone-induced aggregation behavior and inactivated spermatozoa. We have described and characterized for the first time OBPs, ORs and Orco in bed bugs. Given the importance of these molecules in chemoreception of this insect they are interesting targets for the development of novel insect behavior modifiers.

  6. The Odorant Receptor Co-Receptor from the Bed Bug, Cimex lectularius L

    PubMed Central

    Hansen, Immo A.; Rodriguez, Stacy D.; Drake, Lisa L.; Price, David P.; Blakely, Brittny N.; Hammond, John I.; Tsujimoto, Hitoshi; Monroy, Erika Y.; Maio, William A.; Romero, Alvaro

    2014-01-01

    Recently, the bed bug, Cimex lectularius L. has re-emerged as a serious and growing problem in many parts of the world. Presence of resistant bed bugs and the difficulty to eliminate them has renewed interest in alternative control tactics. Similar to other haematophagous arthropods, bed bugs rely on their olfactory system to detect semiochemicals in the environment. Previous studies have morphologically characterized olfactory organs of bed bugs’ antenna and have physiologically evaluated the responses of olfactory receptor neurons (ORNs) to host-derived chemicals. To date, odorant binding proteins (OBPs) and odorant receptors (ORs) associated with these olfaction processes have not been studied in bed bugs. Chemoreception in insects requires formation of heteromeric complexes of ORs and a universal OR coreceptor (Orco). Orco is the constant chain of every odorant receptor in insects and is critical for insect olfaction but does not directly bind to odorants. Orco agonists and antagonists have been suggested as high-value targets for the development of novel insect repellents. In this study, we have performed RNAseq of bed bug sensory organs and identified several odorant receptors as well as Orco. We characterized Orco expression and investigated the effect of chemicals targeting Orco on bed bug behavior and reproduction. We have identified partial cDNAs of six C. lectularius OBPs and 16 ORs. Full length bed bug Orco was cloned and sequenced. Orco is widely expressed in different parts of the bed bug including OR neurons and spermatozoa. Treatment of bed bugs with the agonist VUAA1 changed bed bug pheromone-induced aggregation behavior and inactivated spermatozoa. We have described and characterized for the first time OBPs, ORs and Orco in bed bugs. Given the importance of these molecules in chemoreception of this insect they are interesting targets for the development of novel insect behavior modifiers. PMID:25411789

  7. Analysis of genetic polymorphisms in CCR5, CCR2, stromal cell-derived factor-1, RANTES, and dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin in seronegative individuals repeatedly exposed to HIV-1.

    PubMed

    Liu, Huanliang; Hwangbo, Yon; Holte, Sarah; Lee, Jean; Wang, Chunhui; Kaupp, Nicole; Zhu, Haiying; Celum, Connie; Corey, Lawrence; McElrath, M Juliana; Zhu, Tuofu

    2004-09-15

    To determine the influence of host genetics on human immunodeficiency virus (HIV) type 1 infection, we examined 94 repeatedly exposed seronegative (ES) individuals for polymorphisms in multiple genes and compared the results with those for 316 HIV-1-seropositive and 425 HIV-1-seronegative individuals. The frequency of homozygous C-C chemokine receptor (CCR) 5- Delta 32 was higher in ES (3.2%) than in HIV-1-seropositive individuals (0.0%; P=.012). However, the CCR5-59029A, CCR2-64I, stromal cell-derived factor (SDF)-1-3'A, RANTES (regulated on activation, normally T cell-expressed and -secreted)-403A, and RANTES-28G polymorphisms were not associated with resistance to HIV-1 infection. Furthermore, we identified novel variants in the DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) repeat region and observed that heterozygous DC-SIGN reduced the risk of HIV-1 infection (3.2% in ES individuals vs. 0.0% in HIV-1-seropositive individuals; P=.011).

  8. Natural OX40L expressed on human T cell leukemia virus type-I-immortalized T cell lines interferes with infection of activated peripheral blood mononuclear cells by CCR5-utilizing human immunodeficiency virus

    PubMed Central

    2013-01-01

    Background OX40 ligand (OX40L) co-stimulates and differentiates T cells via ligation of OX40 that is transiently induced on T cells upon activation, resulting in prolonged T cell survival and enhanced cytokine production by T cells. This view has led to the targeting of OX40 as a strategy to boost antigen specific T cells in the context of vaccination. In addition, the ligation of OX40 has also been shown to inhibit infection by CCR5-utilizing (R5) but not CXCR4-utilizing (X4) human immunodeficiency virus type-1 (HIV-1) via enhancement of production of CCR5-binding β-chemokines. It was reasoned that human T cell leukemia virus type-I (HTLV-1) immortalized T cell lines that express high levels of OX40L could serve as an unique source of physiologically functional OX40L. The fact that HTLV-1+ T cell lines simultaneously also express high levels of OX40 suggested a potential limitation. Results Results of our studies showed that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40. Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L. Functionality of the OX40L was confirmed by the fact that a paraformaldehyde (PFA)-fixed HTLV-1+ T cell lines inhibited the infection of autologous activated peripheral blood mononuclear cells (PBMCs) with R5 HIV-1 which was reversed by either anti-OX40L blocking mAb or a mixture of neutralizing mAbs against CCR5-binding β-chemokines. Conclusions Altogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L. PMID:24238037

  9. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases

    PubMed Central

    Nerys-Junior, Arildo; Costa, Lendel C.; Braga-Dias, Luciene P.; Oliveira, Márcia; Rossi, Átila D.; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S.; Tanuri, Amilcar

    2014-01-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity. PMID:24688299

  10. Use of the heteroduplex mobility assay and cell sorting to select genome sequences of the CCR5 gene in HEK 293T cells edited by transcription activator-like effector nucleases.

    PubMed

    Nerys-Junior, Arildo; Costa, Lendel C; Braga-Dias, Luciene P; Oliveira, Márcia; Rossi, Atila D; da Cunha, Rodrigo Delvecchio; Gonçalves, Gabriel S; Tanuri, Amilcar

    2014-03-01

    Engineered nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are one of the most promising tools for modifying genomes. These site-specific enzymes cause double-strand breaks that allow gene disruption or gene insertion, thereby facilitating genetic manipulation. The major problem associated with this approach is the labor-intensive procedures required to screen and confirm the cellular modification by nucleases. In this work, we produced a TALEN that targets the human CCR5 gene and developed a heteroduplex mobility assay for HEK 293T cells to select positive colonies for sequencing. This approach provides a useful tool for the quick detection and easy assessment of nuclease activity.

  11. ACTH Antagonists

    PubMed Central

    Clark, Adrian John; Forfar, Rachel; Hussain, Mashal; Jerman, Jeff; McIver, Ed; Taylor, Debra; Chan, Li

    2016-01-01

    Adrenocorticotropin (ACTH) acts via a highly selective receptor that is a member of the melanocortin receptor subfamily of type 1 G protein-coupled receptors. The ACTH receptor, also known as the melanocortin 2 receptor (MC2R), is unusual in that it is absolutely dependent on a small accessory protein, melanocortin receptor accessory protein (MRAP) for cell surface expression and function. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and high degree of ligand specificity suggests that antagonism of this receptor could provide a useful therapeutic aid and a potential investigational tool. Clinical situations in which this could be useful include (1) Cushing’s disease and ectopic ACTH syndrome – especially while preparing for definitive treatment of a causative tumor, or in refractory cases, or (2) congenital adrenal hyperplasia – as an adjunct to glucocorticoid replacement. A case for antagonism in other clinical situations in which there is ACTH excess can also be made. In this article, we will explore the scientific and clinical case for an ACTH antagonist, and will review the evidence for existing and recently described peptides and modified peptides in this role. PMID:27547198

  12. A Cinnamon-Derived Procyanidin Compound Displays Anti-HIV-1 Activity by Blocking Heparan Sulfate- and Co-Receptor- Binding Sites on gp120 and Reverses T Cell Exhaustion via Impeding Tim-3 and PD-1 Upregulation

    PubMed Central

    Connell, Bridgette Janine; Chang, Sui-Yuan; Prakash, Ekambaranellore; Yousfi, Rahima; Mohan, Viswaraman; Posch, Wilfried; Wilflingseder, Doris; Moog, Christiane; Kodama, Eiichi N.; Clayette, Pascal; Lortat-Jacob, Hugues

    2016-01-01

    Amongst the many strategies aiming at inhibiting HIV-1 infection, blocking viral entry has been recently recognized as a very promising approach. Using diverse in vitro models and a broad range of HIV-1 primary patient isolates, we report here that IND02, a type A procyanidin polyphenol extracted from cinnamon, that features trimeric and pentameric forms displays an anti-HIV-1 activity against CXCR4 and CCR5 viruses with 1–7 μM ED50 for the trimer. Competition experiments, using a surface plasmon resonance-based binding assay, revealed that IND02 inhibited envelope binding to CD4 and heparan sulphate (HS) as well as to an antibody (mAb 17b) directed against the gp120 co-receptor binding site with an IC50 in the low μM range. IND02 has thus the remarkable property of simultaneously blocking gp120 binding to its major host cell surface counterparts. Additionally, the IND02-trimer impeded up-regulation of the inhibitory receptors Tim-3 and PD-1 on CD4+ and CD8+ cells, thereby demonstrating its beneficial effect by limiting T cell exhaustion. Among naturally derived products significantly inhibiting HIV-1, the IND02-trimer is the first component demonstrating an entry inhibition property through binding to the viral envelope glycoprotein. These data suggest that cinnamon, a widely consumed spice, could represent a novel and promising candidate for a cost-effective, natural entry inhibitor for HIV-1 which can also down-modulate T cell exhaustion markers Tim-3 and PD-1. PMID:27788205

  13. Hematopoietic stem cell fate decisions are regulated by Wnt antagonists: comparisons and current controversies.

    PubMed

    Cain, Corey J; Manilay, Jennifer O

    2013-01-01

    Wingless and int (Wnt) proteins are secreted proteins that are important for regulating hematopoietic stem cell self-renewal and differentiation in the bone marrow microenvironment in mice. The mechanisms by which Wnt signaling regulates these hematopoietic cell fate decisions are not fully understood. Secreted Wnt antagonists, which are expressed in bone and bone marrow stromal cells, either bind to Wnt ligands directly or block Wnt receptors and co-receptors to halt Wnt-mediated signal transduction in both osteolineage and hematopoietic cell types. Secreted frizzled related proteins-1 and -2, Wnt inhibitory factor-1, Dickkopf-1, and Sclerostin are Wnt antagonists that influence hematopoietic cell fate decisions in the bone marrow niche. In this review, we compare and contrast the roles of these Wnt antagonists and their effects on hematopoietic development in mice, and also discuss the clinical significance of targeting Wnt antagonists within the context of hematopoietic disease.

  14. Effects of Combined CCR5/Integrase Inhibitors-Based Regimen on Mucosal Immunity in HIV-Infected Patients Naïve to Antiretroviral Therapy: A Pilot Randomized Trial

    PubMed Central

    Ma, Zhong-Min; Utay, Netanya S.; Wook-Chun, Tae; Mann, Surinder; Kashuba, Angela D.; Siewe, Basile; Albanese, Anthony; Troia-Cancio, Paolo; Sinclair, Elizabeth; Somasunderam, Anoma; Yotter, Tammy; Deeks, Steven G.; Landay, Alan; Pollard, Richard B.; Miller, Christopher J.; Moreno, Santiago; Asmuth, David M.

    2016-01-01

    Whether initiation of antiretroviral therapy (ART) regimens aimed at achieving greater concentrations within gut associated lymphoid tissue (GALT) impacts the level of mucosal immune reconstitution, inflammatory markers and the viral reservoir remains unknown. We included 12 HIV- controls and 32 ART-naïve HIV patients who were randomized to efavirenz, maraviroc or maraviroc+raltegravir, each with fixed-dose tenofovir disoproxil fumarate/emtricitabine. Rectal and duodenal biopsies were obtained at baseline and at 9 months of ART. We performed a comprehensive assay of T-cell subsets by flow cytometry, T-cell density in intestinal biopsies, plasma and tissue concentrations of antiretroviral drugs by high-performance liquid chromatography/mass spectroscopy, and plasma interleukin-6 (IL-6), lipoteichoic acid (LTA), soluble CD14 (sCD14) and zonulin-1 each measured by ELISA. Total cell-associated HIV DNA was measured in PBMC and rectal and duodenal mononuclear cells. Twenty-six HIV-infected patients completed the follow-up. In the duodenum, the quadruple regimen resulted in greater CD8+ T-cell density decline, greater normalization of mucosal CCR5+CD4+ T-cells and increase of the naïve/memory CD8+ T-cell ratio, and a greater decline of sCD14 levels and duodenal HIV DNA levels (P = 0.004 and P = 0.067, respectively), with no changes in HIV RNA in plasma or tissue. Maraviroc showed the highest drug distribution to the gut tissue, and duodenal concentrations correlated well with other T-cell markers in duodenum, i.e., the CD4/CD8 ratio, %CD4+ and %CD8+ HLA-DR+CD38+ T-cells. Maraviroc use elicited greater activation of the mucosal naïve CD8+ T-cell subset, ameliorated the distribution of the CD8+ T-cell maturational subsets and induced higher improvement of zonulin-1 levels. These data suggest that combined CCR5 and integrase inhibitor based combination therapy in ART treatment naïve patients might more effectively reconstitute duodenal immunity, decrease inflammatory

  15. Stalk region of beta-chain enhances the coreceptor function of CD8.

    PubMed

    Wong, Jenny S; Wang, Xiaosong; Witte, Torsten; Nie, Linghu; Carvou, Nicolas; Kern, Petra; Chang, Hsiu-Ching

    2003-07-15

    CD8 glycoproteins are expressed as either alphaalpha homodimers or alphabeta heterodimers on the surface of T cells. CD8alphabeta is a more efficient coreceptor than the CD8alphaalpha for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain. In an attempt to understand why CD8alphabeta is a better coreceptor than CD8alphaalpha, we engineered, expressed, and functionally tested a chimeric CD8alpha protein whose stalk region is replaced with that of CD8beta. We found that the beta stalk region enhances the coreceptor function of chimeric CD8alphaalpha to a level similar to that of CD8alphabeta. Surprisingly, the beta stalk region also restored functional activity to an inactive CD8alpha variant, carrying an Ala mutation at Arg(8) (R8A), to a level similar to that of wild-type CD8alphabeta. Using the R8A variant of CD8alpha, a panel of anti-CD8alpha Abs, and three MHC class I (MHCI) variants differing in key residues known to be involved in CD8alpha interaction, we show that the introduction of the CD8beta stalk leads to a different topology of the CD8alpha-MHCI complex without altering the overall structure of the Ig-like domain of CD8alpha or causing the MHCI to employ different residues to interact with the CD8alpha Ig domain. Our results show that the stalk region of CD8beta is capable of fine-tuning the coreceptor function of CD8 proteins as a coreceptor, possibly due to its distinct protein structure, smaller physical size and the unique glycan adducts associated with this region.

  16. The tight junction protein JAM-A functions as coreceptor for rotavirus entry into MA104 cells.

    PubMed

    Torres-Flores, Jesús M; Silva-Ayala, Daniela; Espinoza, Marco A; López, Susana; Arias, Carlos F

    2015-01-15

    Several molecules have been identified as receptors or coreceptors for rotavirus infection, including glycans, integrins, and hsc70. In this work we report that the tight junction proteins JAM-A, occludin, and ZO-1 play an important role during rotavirus entry into MA104 cells. JAM-A was found to function as coreceptor for rotavirus strains RRV, Wa, and UK, but not for rotavirus YM. Reassortant viruses derived from rotaviruses RRV and YM showed that the virus spike protein VP4 determines the use of JAM-A as coreceptor.

  17. Identification of RL-TGR, a coreceptor involved in aversive chemical signaling

    PubMed Central

    Cohen, Staci P.; Haack, Karla K. V.; Halstead-Nussloch, Gwyneth E.; Bernard, Karen F.; Hatt, Hanns; Kubanek, Julia; McCarty, Nael A.

    2010-01-01

    Chemical signaling plays an important role in predator–prey interactions and feeding dynamics. Like other organisms that are sessile or slow moving, some marine sponges contain aversive compounds that defend these organisms from predation. We sought to identify and characterize a fish chemoreceptor that detects one of these compounds. Using expression cloning in Xenopus oocytes coexpressing the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, the beta-2 adrenergic receptor (β2AR), and fractions of a zebrafish cDNA library, we isolated a cDNA clone encoding receptor activity–modifying protein (RAMP)-like triterpene glycoside receptor (RL-TGR), a novel coreceptor involved in signaling in response to triterpene glycosides. This coreceptor appears to be structurally and functionally related to RAMPs, a family of coreceptors that physically associate with and modify the activity of G protein–coupled receptors (GPCRs). In membranes from formoside-responsive oocytes, RL-TGR was immunoprecipitated in an apparent complex with β2AR. In HEK293 cells, coexpression of β2AR induced the trafficking of RL-TGR from the cytoplasm to the plasma membrane. These results suggest that RL-TGR in the predatory fish physically associates with the β2AR or another, more physiologically relevant GPCR and modifies its pharmacology to respond to triterpene glycosides found in sponges that serve as a potential food source for the fish. RL-TGR forms a coreceptor that responds to a chemical defense compound in the marine environment, and its discovery might lead the way to the identification of other receptors that mediate chemical defense signaling. PMID:20566865

  18. Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2

    PubMed Central

    Chen, Chia-Lin; Huang, Jeffrey Y.; Wang, Chun-Hsiang; Tahara, Stanley M; Zhou, Lin; Kondo, Yasuteru; Schechter, Joel; Su, Lishan; Lai, Michael M C.; Wakita, Takaji; Cosset, François-Loïc; Jung, Jae U; Machida, Keigo

    2017-01-01

    B-cell infection by hepatitis C virus (HCV) has been a controversial topic. To examine whether HCV has a genetically determined lymphotropism through a co-receptor specific for the infection by lymphotropic HCV, we established an infectious clone and chimeric virus of hepatotropic and lymphotropic HCV strains derived from an HCV-positive B-cell lymphoma. The viral envelope and 5′-UTR sequences of the lymphotropic HCV strain were responsible for the lymphotropism. Silencing of the virus sensor, RIGI, or overexpression of microRNA-122 promoted persistent viral replication in B cells. By cDNA library screening, we identified an immune cell-specific, co-stimulatory receptor B7.2 (CD86) as a co-receptor of lymphotropic HCV. Infection of B cells by HCV inhibited the recall reaction to antigen stimulation. Together, a co-receptor B7.2 enabled lymphotropic HCV to infect memory B cells, leading to inhibition of memory B-cell function and persistent HCV infection in HCV-infected hosts. PMID:28067225

  19. Anti-coreceptor therapy drives selective T cell egress by suppressing inflammation-dependent chemotactic cues

    PubMed Central

    Martin, Aaron J.; Clark, Matthew; Gojanovich, Gregory; Manzoor, Fatima; Miller, Keith; Kline, Douglas E.; Morillon, Y. Maurice; Wang, Bo

    2016-01-01

    There continues to be a need for immunotherapies to treat type 1 diabetes in the clinic. We previously reported that nondepleting anti-CD4 and -CD8 Ab treatment effectively reverses diabetes in new-onset NOD mice. A key feature of the induction of remission is the egress of the majority of islet-resident T cells. How this occurs is undefined. Herein, the effects of coreceptor therapy on islet T cell retention were investigated. Bivalent Ab binding to CD4 and CD8 blocked TCR signaling and T cell cytokine production, while indirectly downregulating islet chemokine expression. These processes were required for T cell retention, as ectopic IFN-γ or CXCL10 inhibited Ab-mediated T cell purging. Importantly, treatment of humanized mice with nondepleting anti–human CD4 and CD8 Ab similarly reduced tissue-infiltrating human CD4+ and CD8+ T cells. These findings demonstrate that Ab binding of CD4 and CD8 interrupts a feed-forward circuit by suppressing T cell–produced cytokines needed for expression of chemotactic cues, leading to rapid T cell egress from the islets. Coreceptor therapy therefore offers a robust approach to suppress T cell–mediated pathology by purging T cells in an inflammation-dependent manner. PMID:27777971

  20. Co-receptor and co-stimulation blockade for mixed chimerism and tolerance without myelosuppressive conditioning

    PubMed Central

    Graca, Luis; Daley, Stephen; Fairchild, Paul J; Cobbold, Stephen P; Waldmann, Herman

    2006-01-01

    Background A major challenge in the application of marrow transplantation as a route to immunological tolerance of a transplanted organ is to achieve hematopoietic stem cell (HSC) engraftment with minimal myelosuppressive treatments. Results We here describe a combined antibody protocol which can achieve long-term engraftment with clinically relevant doses of MHC-mismatched bone marrow, without the need for myelosuppressive drugs. Although not universally applicable in all strains, we achieved reliable engraftment in permissive strains with a two-stage strategy: involving first, treatment with anti-CD8 and anti-CD4 in advance of transplantation; and second, treatment with antibodies targeting CD4, CD8 and CD40L (CD154) at the time of marrow transplantation. Long-term mixed chimerism through co-receptor and co-stimulation blockade facilitated tolerance to donor-type skin grafts, without any evidence of donor-antigen driven regulatory T cells. Conclusion We conclude that antibodies targeting co-receptor and co-stimulatory molecules synergise to enable mixed hematopoietic chimerism and central tolerance, showing that neither cytoreductive conditioning nor 'megadoses' of donor bone marrow are required for donor HSC to engraft in permissive strains. PMID:16638128

  1. CD36 is a co-receptor for hepatitis C virus E1 protein attachment

    PubMed Central

    Cheng, Jun-Jun; Li, Jian-Rui; Huang, Meng-Hao; Ma, Lin-Lin; Wu, Zhou-Yi; Jiang, Chen-Chen; Li, Wen-Jing; Li, Yu-Huan; Han, Yan-Xing; Li, Hu; Chen, Jin-Hua; Wang, Yan-Xiang; Song, Dan-Qing; Peng, Zong-Gen; Jiang, Jian-Dong

    2016-01-01

    The cluster of differentiation 36 (CD36) is a membrane protein related to lipid metabolism. We show that HCV infection in vitro increased CD36 expression in either surface or soluble form. HCV attachment was facilitated through a direct interaction between CD36 and HCV E1 protein, causing enhanced entry and replication. The HCV co-receptor effect of CD36 was independent of that of SR-BI. CD36 monoclonal antibodies neutralized the effect of CD36 and reduced HCV replication. CD36 inhibitor sulfo-N-succinimidyl oleate (SSO), which directly bound CD36 but not SR-BI, significantly interrupted HCV entry, and therefore inhibited HCV replication. SSO’s antiviral effect was seen only in HCV but not in other viruses. SSO in combination with known anti-HCV drugs showed additional inhibition against HCV. SSO was considerably safe in mice. Conclusively, CD36 interacts with HCV E1 and might be a co-receptor specific for HCV entry; thus, CD36 could be a potential drug target against HCV. PMID:26898231

  2. Muscarinic Receptor Antagonists.

    PubMed

    Matera, Maria Gabriella; Cazzola, Mario

    2017-01-01

    Parasympathetic activity is increased in patients with chronic obstructive pulmonary disease (COPD) and asthma and appears to be the major reversible component of airway obstruction. Therefore, treatment with muscarinic receptor antagonists is an effective bronchodilator therapy in COPD and also in asthmatic patients. In recent years, the accumulating evidence that the cholinergic system controls not only contraction by airway smooth muscle but also the functions of inflammatory cells and airway epithelial cells has suggested that muscarinic receptor antagonists could exert other effects that may be of clinical relevance when we must treat a patient suffering from COPD or asthma. There are currently six muscarinic receptor antagonists licenced for use in the treatment of COPD, the short-acting muscarinic receptor antagonists (SAMAs) ipratropium bromide and oxitropium bromide and the long-acting muscarinic receptor antagonists (LAMAs) aclidinium bromide, tiotropium bromide, glycopyrronium bromide and umeclidinium bromide. Concerns have been raised about possible associations of muscarinic receptor antagonists with cardiovascular safety, but the most advanced compounds seem to have an improved safety profile. Further beneficial effects of SAMAs and LAMAs are seen when added to existing treatments, including LABAs, inhaled corticosteroids and phosphodiesterase 4 inhibitors. The importance of tiotropium bromide in the maintenance treatment of COPD, and likely in asthma, has spurred further research to identify new LAMAs. There are a number of molecules that are being identified, but only few have reached the clinical development.

  3. Functional HIV CXCR4 coreceptor on human epithelial Langerhans cells and infection by HIV strain X4.

    PubMed

    Tchou, I; Misery, L; Sabido, O; Dezutter-Dambuyant, C; Bourlet, T; Moja, P; Hamzeh, H; Peguet-Navarro, J; Schmitt, D; Genin, C

    2001-08-01

    HIV can cross the intact epithelium of genital mucosae via Langerhans cells. Fresh Langerhans cells are known to express CD4 and CCR5. The presence of CXCR4 on the surface of cultured but not freshly isolated Langerhans cells has been described. In the present study, we demonstrate that CXCR4 was expressed by fresh Langerhans cells isolated and purified from epidermis. However, the percentage of Langerhans cells expressing CXCR4 or CCR5 increased during maturation of the cells in culture, especially in the presence of exogenous granulocyte-macrophage colony-stimulating factor. To determine whether CXCR4 was functional, freshly isolated Langerhans cells were infected with HIV LAI, a T-cell-tropic strain, and p24 protein production was measured in culture supernatants. p24 production was observed when infected Langerhans cells were cocultured with SupT1 cells. However, the presence of HIV provirus DNA was evidenced within the infected Langerhans cells by nested PCR. Ultrastructural studies confirmed the formation of syncytia when Langerhans cells were cocultured with SupT1 cells. Preincubation of Langerhans cells with azidothymidine or SDF-1-alpha, a natural ligand for CXCR4, prevented infection. These data demonstrated that CXCR4 is present on the surface of Langerhans cells freshly isolated from human skin epidermis and that this expression is functional.

  4. CDO, an Hh-coreceptor, mediates lung cancer cell proliferation and tumorigenicity through Hedgehog signaling.

    PubMed

    Leem, Young-Eun; Ha, Hye-Lim; Bae, Ju-Hyeon; Baek, Kwan-Hyuck; Kang, Jong-Sun

    2014-01-01

    Hedgehog (Hh) signaling plays essential roles in various developmental processes, and its aberrant regulation results in genetic disorders or malignancies in various tissues. Hyperactivation of Hh signaling is associated with lung cancer development, and there have been extensive efforts to investigate how to control Hh signaling pathway and regulate cancer cell proliferation. In this study we investigated a role of CDO, an Hh co-receptor, in non-small cell lung cancer (NSCLC). Inhibition of Hh signaling by SANT-1 or siCDO in lung cancer cells reduced proliferation and tumorigenicity, along with the decrease in the expression of the Hh components. Histological analysis with NSCLC mouse tissue demonstrated that CDO was expressed in advanced grade of the cancer, and precisely co-localized with GLI1. These data suggest that CDO is required for proliferation and survival of lung cancer cells via Hh signaling.

  5. CDO, an Hh-Coreceptor, Mediates Lung Cancer Cell Proliferation and Tumorigenicity through Hedgehog Signaling

    PubMed Central

    Leem, Young-Eun; Ha, Hye-Lim; Bae, Ju-Hyeon; Baek, Kwan-Hyuck; Kang, Jong-Sun

    2014-01-01

    Hedgehog (Hh) signaling plays essential roles in various developmental processes, and its aberrant regulation results in genetic disorders or malignancies in various tissues. Hyperactivation of Hh signaling is associated with lung cancer development, and there have been extensive efforts to investigate how to control Hh signaling pathway and regulate cancer cell proliferation. In this study we investigated a role of CDO, an Hh co-receptor, in non-small cell lung cancer (NSCLC). Inhibition of Hh signaling by SANT-1 or siCDO in lung cancer cells reduced proliferation and tumorigenicity, along with the decrease in the expression of the Hh components. Histological analysis with NSCLC mouse tissue demonstrated that CDO was expressed in advanced grade of the cancer, and precisely co-localized with GLI1. These data suggest that CDO is required for proliferation and survival of lung cancer cells via Hh signaling. PMID:25369201

  6. Growth factor and co-receptor release by structural regulation of substrate metalloprotease accessibility

    PubMed Central

    Parra, Liseth M.; Hartmann, Monika; Schubach, Salome; Ma, Junzhi; Herrlich, Peter; Herrlich, Andreas

    2016-01-01

    Release of cytokines, growth factors and other life-essential molecules from precursors by a-disintegrin-and-metalloproteases (ADAMs) is regulated with high substrate-specificity. We hypothesized that this is achieved by cleavage-regulatory intracellular-domain (ICD)-modifications of the precursors. We show here that cleavage-stimuli-induced specific ICD-modifications cause structural substrate changes that enhance ectodomain sensitivity of neuregulin-1 (NRG1; epidermal-growth-factor) or CD44 (receptor-tyrosine-kinase (RTK) co-receptor) to chymotrypsin/trypsin or soluble ADAM. This inside-out signal transfer required substrate homodimerization and was prevented by cleavage-inhibitory ICD-mutations. In chimeras, regulation could be conferred to a foreign ectodomain, suggesting a common higher-order structure. We predict that substrate-specific protease-accessibility-regulation controls release of numerous ADAM substrates. PMID:27876763

  7. C-terminal tail of FGF19 determines its specificity toward Klotho co-receptors.

    PubMed

    Wu, Xinle; Lemon, Bryan; Li, XiaoFan; Gupte, Jamila; Weiszmann, Jennifer; Stevens, Jennitte; Hawkins, Nessa; Shen, Wenyan; Lindberg, Richard; Chen, Jin-Long; Tian, Hui; Li, Yang

    2008-11-28

    FGF19 subfamily proteins (FGF19, FGF21, and FGF23) are unique members of fibroblast growth factors (FGFs) that regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis in an endocrine fashion. Their activities require the presence of alpha or betaKlotho, two related single-pass transmembrane proteins, as co-receptors in relevant target tissues. We previously showed that FGF19 can bind to both alpha and betaKlotho, whereas FGF21 and FGF23 can bind only to either betaKlotho or alphaKlotho, respectively in vitro. To determine the mechanism regulating the binding and specificity among FGF19 subfamily members to Klotho family proteins, chimeric proteins between FGF19 subfamily members or chimeric proteins between Klotho family members were constructed to probe the interaction between those two families. Our results showed that a chimera of FGF19 with the FGF21 C-terminal tail interacts only with betaKlotho and a chimera with the FGF23 C-terminal tail interacts only with alphaKlotho. FGF signaling assays also reflected the change of specificity we observed for the chimeras. These results identified the C-terminal tail of FGF19 as a region necessary for its recognition of Klotho family proteins. In addition, chimeras between alpha and betaKlotho were also generated to probe the regions in Klotho proteins that are important for signaling by this FGF subfamily. Both FGF23 and FGF21 require intact alpha or betaKlotho for signaling, respectively, whereas FGF19 can signal through a Klotho chimera consisting of the N terminus of alphaKlotho and the C terminus of betaKlotho. Our results provide the first glimpse of the regions that regulate the binding specificity between this unique family of FGFs and their co-receptors.

  8. Immune Receptors and Co-receptors in Antiviral Innate Immunity in Plants

    PubMed Central

    Gouveia, Bianca C.; Calil, Iara P.; Machado, João Paulo B.; Santos, Anésia A.; Fontes, Elizabeth P. B.

    2017-01-01

    Plants respond to pathogens using an innate immune system that is broadly divided into PTI (pathogen-associated molecular pattern- or PAMP-triggered immunity) and ETI (effector-triggered immunity). PTI is activated upon perception of PAMPs, conserved motifs derived from pathogens, by surface membrane-anchored pattern recognition receptors (PRRs). To overcome this first line of defense, pathogens release into plant cells effectors that inhibit PTI and activate effector-triggered susceptibility (ETS). Counteracting this virulence strategy, plant cells synthesize intracellular resistance (R) proteins, which specifically recognize pathogen effectors or avirulence (Avr) factors and activate ETI. These coevolving pathogen virulence strategies and plant resistance mechanisms illustrate evolutionary arms race between pathogen and host, which is integrated into the zigzag model of plant innate immunity. Although antiviral immune concepts have been initially excluded from the zigzag model, recent studies have provided several lines of evidence substantiating the notion that plants deploy the innate immune system to fight viruses in a manner similar to that used for non-viral pathogens. First, most R proteins against viruses so far characterized share structural similarity with antibacterial and antifungal R gene products and elicit typical ETI-based immune responses. Second, virus-derived PAMPs may activate PTI-like responses through immune co-receptors of plant PTI. Finally, and even more compelling, a viral Avr factor that triggers ETI in resistant genotypes has recently been shown to act as a suppressor of PTI, integrating plant viruses into the co-evolutionary model of host-pathogen interactions, the zigzag model. In this review, we summarize these important progresses, focusing on the potential significance of antiviral immune receptors and co-receptors in plant antiviral innate immunity. In light of the innate immune system, we also discuss a newly uncovered layer of

  9. Abscisic acid sensor RCAR7/PYL13, specific regulator of protein phosphatase coreceptors.

    PubMed

    Fuchs, Stefan; Tischer, Stefanie V; Wunschel, Christian; Christmann, Alexander; Grill, Erwin

    2014-04-15

    The plant hormone abscisic acid (ABA) acts both as a developmental signal and as an integrator of environmental cues such as drought and cold. ABA perception recruits an ABA-binding regulatory component [regulatory component of ABA receptor (RCAR)/PYR1/PYL] and an associated protein phosphatase 2C (PP2C). Phytohormone binding inactivates the phosphatase activity of the coreceptor, permitting phosphorelay of the ABA signal via downstream protein kinases. RCARs and PP2C coreceptors are represented by small protein families comprising 14 and 9 members in Arabidopsis, respectively. The specificity of the RCAR-PP2C interaction and the constraints contributing to specific combinations are poorly understood. In this contribution, we analyzed RCAR7/PYL13, which is characterized by three variant amino acid residues in the conserved ABA-binding pocket. RCAR7 regulated the phosphatase activity of the PP2Cs ABI1, ABI2, and PP2CA in vitro at nanomolar ABA levels; however, it was unable to regulate the structurally related hypersensitive to ABA 1 (HAB1). Site-directed mutagenesis of HAB1 established ABA-dependent regulation by RCAR7. Conversion of the noncanonical amino acid residues of RCAR7 into the consensus ABA-binding pocket did not perceptibly change receptor function. Ectopic expression of RCAR7 in Arabidopsis resulted in ABA hypersensitivity affecting gene regulation, seed germination, and stomatal closure. The RCAR7 loss-of-function mutant revealed no changes in ABA responses, similar to the RCAR9 knockout line, whereas the combined deficiency of RCAR7 and RCAR9 resulted in ABA-insensitive seed germination. The study shows a role of RCAR7 in early plant development, proves its ABA receptor function, and identifies structural constraints of RCAR7-PP2C interaction.

  10. Neuropilins are multifunctional coreceptors involved in tumor initiation, growth, metastasis and immunity

    PubMed Central

    Prud'homme, Gérald J.; Glinka, Yelena

    2012-01-01

    The neuropilins (Nrps) are multifunctional proteins involved in development, immunity and cancer. Neuropilin-1 (Nrp1), or its homologue neuropilin-2 (Nrp2), are coreceptors that enhance responses to several growth factors (GFs) and other mediators. Nrps are coreceptors for the class 3 semaphorins (SEMA3), involved in axonal guidance, and several members of the vascular endothelial growth factor (VEGF) family. However, recent findings reveal they have a much broader spectrum of activity. They bind transforming growth factor β1 (TGF-β1) and its receptors, hepatocyte growth factor (HGF) and its receptor (cMet), platelet derived growth factor (PDGF) and its receptors, fibroblast growth factors (FGFs), and integrins. Nrps also promote Hedgehog signaling. These ligands and pathways are all relevant to angiogenesis and wound healing. In the immune system, the Nrps are expressed primarily by dendritic cells (DCs) and regulatory T cells (Tregs), and exert mainly inhibitory effects. In cancer, Nrps have been linked to a poor prognosis, which is consistent with their numerous interactions with ligands and receptors that promote tumor progression. We hypothesize that Nrps boost responses by capturing ligands, regulating GF receptor expression, endocytosis and recycling, and possibly also by signaling independently. Importantly, they promote epithelial-mesenchymal transition (EMT), and the survival of cancer stem cells. The recent finding that Nrps bind and internalize cell-penetrating peptides (CPPs) with arginine/lysine-rich C-terminal motifs (C-end rule; e.g., RXXR) is of interest. These CPPs can be coupled to large drugs for cancer therapy. Almost all studies have been preclinical, but findings suggest Nrps are excellent targets for anti-cancer drug development. PMID:22948112

  11. Opioid Antagonist Impedes Exposure.

    ERIC Educational Resources Information Center

    Merluzzi, Thomas V.; And Others

    1991-01-01

    Thirty spider-phobic adults underwent exposure to 17 phobic-related, graded performance tests. Fifteen subjects were assigned to naltrexone, an opioid antagonist, and 15 were assigned to placebo. Naltrexone had a significant effect on exposure, with naltrexone subjects taking significantly longer to complete first 10 steps of exposure and with…

  12. Maraviroc: a review of its use in HIV infection and beyond.

    PubMed

    Woollard, Shawna M; Kanmogne, Georgette D

    2015-01-01

    The human immunodeficiency virus-1 (HIV-1) enters target cells by binding its envelope glycoprotein gp120 to the CD4 receptor and/or coreceptors such as C-C chemokine receptor type 5 (CCR5; R5) and C-X-C chemokine receptor type 4 (CXCR4; X4), and R5-tropic viruses predominate during the early stages of infection. CCR5 antagonists bind to CCR5 to prevent viral entry. Maraviroc (MVC) is the only CCR5 antagonist currently approved by the United States Food and Drug Administration, the European Commission, Health Canada, and several other countries for the treatment of patients infected with R5-tropic HIV-1. MVC has been shown to be effective at inhibiting HIV-1 entry into cells and is well tolerated. With expanding MVC use by HIV-1-infected humans, different clinical outcomes post-approval have been observed with MVC monotherapy or combination therapy with other antiretroviral drugs, with MVC use in humans infected with dual-R5- and X4-tropic HIV-1, infected with different HIV-1 genotype or infected with HIV-2. This review discuss the role of CCR5 in HIV-1 infection, the development of the CCR5 antagonist MVC, its pharmacokinetics, pharmacodynamics, drug-drug interactions, and the implications of these interactions on treatment outcomes, including viral mutations and drug resistance, and the mechanisms associated with the development of resistance to MVC. This review also discusses available studies investigating the use of MVC in the treatment of other diseases such as cancer, graft-versus-host disease, and inflammatory diseases.

  13. Expression of betaglycan, an inhibin coreceptor, in normal human ovaries and ovarian sex cord-stromal tumors and its regulation in cultured human granulosa-luteal cells.

    PubMed

    Liu, Jianqi; Kuulasmaa, Tiina; Kosma, Veli-Matti; Bützow, Ralf; Vänttinen, Teemu; Hydén-Granskog, Christel; Voutilainen, Raimo

    2003-10-01

    Activins and inhibins are often antagonistic in the regulation of ovarian function. TGFbeta type III receptor, betaglycan, has been identified as a coreceptor to enhance the binding of inhibins to activin type II receptor and thus to prevent the binding of activins to their receptor. In this study we characterized the expression and regulation pattern of betaglycan gene in normal ovaries and sex cord-stromal tumors and in cultured human granulosa-luteal cells from women undergoing in vitro fertilization. Expression of betaglycan mRNA was detected by RT-PCR or Northern blotting in normal ovarian granulosa, thecal, and stroma cells as well as in granulosa-luteal cells. Immunohistochemical analysis revealed positive staining for betaglycan in antral and preovulatory follicular granulosa and thecal cells and in corpora lutea of normal ovaries. Furthermore, betaglycan expression was detected in the vast majority of granulosa cell tumors, thecomas, and fibromas, with weaker staining in granulosa cell tumors compared with fibrothecomas. In cultured granulosa-luteal cells, FSH and LH treatment increased dose-dependently the accumulation of betaglycan mRNA, as did the protein kinase A activator dibutyryl cAMP and the protein kinase C inhibitor staurosporine. In contrast, the protein kinase C activator 12-O-tetradecanoyl phorbol 13-acetate had no significant effect on betaglycan mRNA levels. Treatment with prostaglandin E(2) and with its receptor EP2 subtype agonist butaprost increased betaglycan mRNA accumulation and progesterone secretion dose- and time-dependently. In summary, betaglycan gene is expressed in normal human ovarian steroidogenic cells and sex cord-stromal ovarian tumors. The accumulation of its mRNA in cultured granulosa-luteal cells is up-regulated by gonadotropins and prostaglandin E(2), probably via the protein kinase A pathway. The specific expression and regulation pattern of betaglycan gene may be related to the functional antagonism of inhibins to

  14. Lack of prophylactic efficacy of oral maraviroc in macaques despite high drug concentrations in rectal tissues.

    PubMed

    Massud, Ivana; Aung, Wutyi; Martin, Amy; Bachman, Shanon; Mitchell, James; Aubert, Rachael; Solomon Tsegaye, Theodros; Kersh, Ellen; Pau, Chou-Pong; Heneine, Walid; García-Lerma, J Gerardo

    2013-08-01

    Maraviroc (MVC) is a potent CCR5 coreceptor antagonist that is in clinical testing for daily oral pre-exposure prophylaxis (PrEP) for HIV prevention. We used a macaque model consisting of weekly SHIV162p3 exposures to evaluate the efficacy of oral MVC in preventing rectal SHIV transmission. MVC dosing was informed by the pharmacokinetic profile seen in blood and rectal tissues and consisted of a human-equivalent dose given 24 h before virus exposure, followed by a booster postexposure dose. In rectal secretions, MVC peaked at 24 h (10,242 ng/ml) with concentrations at 48 h that were about 40 times those required to block SHIV infection of peripheral blood mononuclear cells (PBMCs) in vitro. Median MVC concentrations in rectal tissues at 24 h (1,404 ng/g) were 30 and 10 times those achieved in vaginal or lymphoid tissues, respectively. MVC significantly reduced macrophage inflammatory protein 1β-induced CCR5 internalization in rectal mononuclear cells, an indication of efficient binding to CCR5 in rectal lymphocytes. The half-life of CCR5-bound MVC in PBMCs was 2.6 days. Despite this favorable profile, 5/6 treated macaques were infected during five rectal SHIV exposures as were 3/4 controls. MVC treatment was associated with a significant increase in the percentage of CD3(+)/CCR5(+) cells in blood. We show that high and durable MVC concentrations in rectal tissues are not sufficient to prevent SHIV infection in macaques. The increases in CD3(+)/CCR5(+) cells seen during MVC treatment point to unique immunological effects of CCR5 inhibition by MVC. The implications of these immunological effects on PrEP with MVC require further evaluation.

  15. Advantages of an antagonist: bicuculline and other GABA antagonists

    PubMed Central

    Johnston, Graham AR

    2013-01-01

    The convulsant alkaloid bicuculline continues to be investigated more than 40 years after the first publication of its action as an antagonist of receptors for the inhibitory neurotransmitter GABA. This historical perspective highlights key aspects of the discovery of bicuculline as a GABA antagonist and the sustained interest in this and other GABA antagonists. The exciting advances in the molecular biology, pharmacology and physiology of GABA receptors provide a continuing stimulus for the discovery of new antagonists with increasing selectivity for the myriad of GABA receptor subclasses. Interesting GABA antagonists not structurally related to bicuculline include gabazine, salicylidene salicylhydrazide, RU5135 and 4-(3-biphenyl-5-(4-piperidyl)-3-isoxazole. Bicuculline became the benchmark antagonist for what became known as GABAA receptors, but not all ionotropic GABA receptors are susceptible to bicuculline. In addition, not all GABAA receptor antagonists are convulsants. Thus there are still surprises in store as the study of GABA receptors evolves. PMID:23425285

  16. alpha2-Adrenoreceptor antagonists.

    PubMed

    Mayer, P; Imbert, T

    2001-06-01

    A review of the literature relating to the therapeutic potential of alpha2-adrenoceptor antagonists published between 1990 and 2000 is presented. Although extensively studied since the early 1970s in a wide spectrum of therapeutic applications, the distinction of alpha2-adrenoceptor subtypes and some emerging evidence concerning new applications in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, obesity and schizophrenia, have refreshed an interest in this class of agents.

  17. Heparan Sulfate Proteoglycans Containing a Glypican 5 Core and 2-O-Sulfo-iduronic Acid Function as Sonic Hedgehog Co-receptors to Promote Proliferation*♦

    PubMed Central

    Witt, Rochelle M.; Hecht, Marie-Lyn; Pazyra-Murphy, Maria F.; Cohen, Samuel M.; Noti, Christian; van Kuppevelt, Toin H.; Fuller, Maria; Chan, Jennifer A.; Hopwood, John J.; Seeberger, Peter H.; Segal, Rosalind A.

    2013-01-01

    Sonic Hedgehog (Shh) signaling is crucial for growth, cell fate determination, and axonal guidance in the developing nervous system. Although the receptors Patched (Ptch1) and Smoothened (Smo) are required for Shh signaling, a number of distinct co-receptors contribute to these critical responses to Shh. Several membrane-embedded proteins such as Boc, Cdo, and Gas1 bind Shh and promote signaling. In addition, heparan sulfate proteoglycans (HSPGs) have also been implicated in the initiation of Shh responses. However, the attributes of HSPGs that function as co-receptors for Shh have not yet been defined. Here, we identify HSPGs containing a glypican 5 core protein and 2-O-sulfo-iduronic acid residues at the nonreducing ends of the glycans as co-receptors for Shh. These HSPG co-receptors are expressed by cerebellar granule cell precursors and promote Shh binding and signaling. At the subcellular level, these HSPG co-receptors are located adjacent to the primary cilia that act as Shh signaling organelles. Thus, Shh binds to HSPG co-receptors containing a glypican 5 core and 2-O-sulfo-iduronic acid to promote neural precursor proliferation. PMID:23867465

  18. Heparan sulfate proteoglycans containing a glypican 5 core and 2-O-sulfo-iduronic acid function as Sonic Hedgehog co-receptors to promote proliferation.

    PubMed

    Witt, Rochelle M; Hecht, Marie-Lyn; Pazyra-Murphy, Maria F; Cohen, Samuel M; Noti, Christian; van Kuppevelt, Toin H; Fuller, Maria; Chan, Jennifer A; Hopwood, John J; Seeberger, Peter H; Segal, Rosalind A

    2013-09-06

    Sonic Hedgehog (Shh) signaling is crucial for growth, cell fate determination, and axonal guidance in the developing nervous system. Although the receptors Patched (Ptch1) and Smoothened (Smo) are required for Shh signaling, a number of distinct co-receptors contribute to these critical responses to Shh. Several membrane-embedded proteins such as Boc, Cdo, and Gas1 bind Shh and promote signaling. In addition, heparan sulfate proteoglycans (HSPGs) have also been implicated in the initiation of Shh responses. However, the attributes of HSPGs that function as co-receptors for Shh have not yet been defined. Here, we identify HSPGs containing a glypican 5 core protein and 2-O-sulfo-iduronic acid residues at the nonreducing ends of the glycans as co-receptors for Shh. These HSPG co-receptors are expressed by cerebellar granule cell precursors and promote Shh binding and signaling. At the subcellular level, these HSPG co-receptors are located adjacent to the primary cilia that act as Shh signaling organelles. Thus, Shh binds to HSPG co-receptors containing a glypican 5 core and 2-O-sulfo-iduronic acid to promote neural precursor proliferation.

  19. Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor

    SciTech Connect

    Sheard, Laura B; Tan, Xu; Mao, Haibin; Withers, John; Ben-Nissan, Gili; Hinds, Thomas R; Kobayashi, Yuichi; Hsu, Fong-Fu; Sharon, Michal; Browse, John; He, Sheng Yang; Rizo, Josep; Howe, Gregg A; Zheng, Ning

    2011-11-07

    Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved {alpha}-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.

  20. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells.

    PubMed

    Kawalekar, Omkar U; O'Connor, Roddy S; Fraietta, Joseph A; Guo, Lili; McGettigan, Shannon E; Posey, Avery D; Patel, Prachi R; Guedan, Sonia; Scholler, John; Keith, Brian; Snyder, Nathaniel W; Snyder, Nathaniel; Blair, Ian A; Blair, Ian; Milone, Michael C; June, Carl H

    2016-02-16

    Chimeric antigen receptors (CARs) redirect T cell cytotoxicity against cancer cells, providing a promising approach to cancer immunotherapy. Despite extensive clinical use, the attributes of CAR co-stimulatory domains that impact persistence and resistance to exhaustion of CAR-T cells remain largely undefined. Here, we report the influence of signaling domains of coreceptors CD28 and 4-1BB on the metabolic characteristics of human CAR T cells. Inclusion of 4-1BB in the CAR architecture promoted the outgrowth of CD8(+) central memory T cells that had significantly enhanced respiratory capacity, increased fatty acid oxidation and enhanced mitochondrial biogenesis. In contrast, CAR T cells with CD28 domains yielded effector memory cells with a genetic signature consistent with enhanced glycolysis. These results provide, at least in part, a mechanistic insight into the differential persistence of CAR-T cells expressing 4-1BB or CD28 signaling domains in clinical trials and inform the design of future CAR T cell therapies.

  1. Ubiquitin-dependent folding of the Wnt signaling coreceptor LRP6

    PubMed Central

    Perrody, Elsa; Abrami, Laurence; Feldman, Michal; Kunz, Beatrice; Urbé, Sylvie; van der Goot, F Gisou

    2016-01-01

    Many membrane proteins fold inefficiently and require the help of enzymes and chaperones. Here we reveal a novel folding assistance system that operates on membrane proteins from the cytosolic side of the endoplasmic reticulum (ER). We show that folding of the Wnt signaling coreceptor LRP6 is promoted by ubiquitination of a specific lysine, retaining it in the ER while avoiding degradation. Subsequent ER exit requires removal of ubiquitin from this lysine by the deubiquitinating enzyme USP19. This ubiquitination-deubiquitination is conceptually reminiscent of the glucosylation-deglucosylation occurring in the ER lumen during the calnexin/calreticulin folding cycle. To avoid infinite futile cycles, folded LRP6 molecules undergo palmitoylation and ER export, while unsuccessfully folded proteins are, with time, polyubiquitinated on other lysines and targeted to degradation. This ubiquitin-dependent folding system also controls the proteostasis of other membrane proteins as CFTR and anthrax toxin receptor 2, two poor folders involved in severe human diseases. DOI: http://dx.doi.org/10.7554/eLife.19083.001 PMID:27751231

  2. Utilization of sialic acid as a coreceptor is required for reovirus-induced biliary disease

    PubMed Central

    Barton, Erik S.; Youree, Bryan E.; Ebert, Daniel H.; Forrest, J. Craig; Connolly, Jodi L.; Valyi-Nagy, Tibor; Washington, Kay; Wetzel, J. Denise; Dermody, Terence S.

    2003-01-01

    Infection of neonatal mice with some reovirus strains produces a disease similar to infantile biliary atresia, but previous attempts to correlate reovirus infection with this disease have yielded conflicting results. We used isogenic reovirus strains T3SA– and T3SA+, which differ solely in the capacity to bind sialic acid as a coreceptor, to define the role of sialic acid in reovirus encephalitis and biliary tract infection in mice. Growth in the intestine was equivalent for both strains following peroral inoculation. However, T3SA+ spread more rapidly from the intestine to distant sites and replicated to higher titers in spleen, liver, and brain. Strikingly, mice infected with T3SA+ but not T3SA– developed steatorrhea and bilirubinemia. Liver tissue from mice infected with T3SA+ demonstrated intense inflammation focused at intrahepatic bile ducts, pathology analogous to that found in biliary atresia in humans, and high levels of T3SA+ antigen in bile duct epithelial cells. T3SA+ bound 100-fold more efficiently than T3SA– to human cholangiocarcinoma cells. These observations suggest that the carbohydrate-binding specificity of a virus can dramatically alter disease in the host and highlight the need for epidemiologic studies focusing on infection by sialic acid–binding reovirus strains as a possible contributor to the pathogenesis of neonatal biliary atresia. PMID:12813018

  3. Neuronal apoptotic signaling pathways probed and intervened by synthetically and modularly modified (SMM) chemokines.

    PubMed

    Choi, Won-Tak; Kaul, Marcus; Kumar, Santosh; Wang, Jun; Kumar, I M Krishna; Dong, Chang-Zhi; An, Jing; Lipton, Stuart A; Huang, Ziwei

    2007-03-09

    As the main coreceptors for human immunodeficiency virus type 1 (HIV-1) entry, CXCR4 and CCR5 play important roles in HIV-associated dementia (HAD). HIV-1 glycoprotein gp120 contributes to HAD by causing neuronal damage and death, either directly by triggering apoptotic pathways or indirectly by stimulating glial cells to release neurotoxins. Here, to understand the mechanism of CXCR4 or CCR5 signaling in neuronal apoptosis associated with HAD, we have applied synthetically and modularly modified (SMM)-chemokine analogs derived from natural stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II as chemical probes of the mechanism(s) whereby these SMM-chemokines prevent or promote neuronal apoptosis. We show that inherently neurotoxic natural ligands of CXCR4, such as stromal cell-derived factor-1alpha or viral macrophage inflammatory protein-II, can be modified to protect neurons from apoptosis induced by CXCR4-preferring gp120(IIIB), and that the inhibition of CCR5 by antagonist SMM-chemokines, unlike neuroprotective CCR5 natural ligands, leads to neurotoxicity by activating a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Furthermore, we discover distinct signaling pathways activated by different chemokine ligands that are either natural agonists or synthetic antagonists, thus demonstrating a chemical biology strategy of using chemically engineered inhibitors of chemokine receptors to study the signaling mechanism of neuronal apoptosis and survival.

  4. Identification of a Putative Coreceptor on Vero Cells That Participates in Dengue 4 Virus Infection

    PubMed Central

    Martínez-Barragán, José de Jesús; del Angel, Rosa M.

    2001-01-01

    Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (α-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells. PMID:11483725

  5. A case-based reasoning system for genotypic prediction of HIV-1 co-receptor tropism.

    PubMed

    Evans, Mark C; Paquet, Agnes C; Huang, Wei; Napolitano, Laura; Frantzell, Arne; Toma, Jonathan; Stawiski, Eric W; Goetz, Matthew Bidwell; Petropoulos, Christos J; Whitcomb, Jeannette; Coakley, Eoin; Haddad, Mojgan

    2013-08-01

    Accurate co-receptor tropism (CRT) determination is critical for making treatment decisions in HIV management. We created a genotypic tropism prediction tool by utilizing the case-based reasoning (CBR) technique that attempts to solve new problems through applying the solution from similar past problems. V3 loop sequences from 732 clinical samples with diverse characteristics were used to build a case library. Additional sequence and molecular properties of the V3 loop were examined and used for similarity assessment. A similarity metric was defined based on each attribute's frequency in the CXCR4-using viruses. We implemented three other genotype-based tropism predictors, support vector machines (SVM), position specific scoring matrices (PSSM), and the 11/25 rule, and evaluated their performance as the ability to predict CRT compared to Monogram's enhanced sensitivity Trofile(®) assay (ESTA). Overall concordance of the CBR based tropism prediction algorithm was 81%, as compared to ESTA. Sensitivity to detect CXCR4 usage was 90% and specificity was at 73%. In comparison, sensitivity of the SVM, PSSM, and the 11/25 rule were 85%, 81%, and 36% respectively while achieving a specificity of 90% by SVM, 75% by PSSM, and 97% by the 11/25 rule. When we evaluated these predictors in an unseen dataset, higher sensitivity was achieved by the CBR algorithm (87%), compared to SVM (82%), PSSM (76%), and the 11/25 rule (33%), while maintaining similar level of specificity. Overall this study suggests that CBR can be utilized as a genotypic tropism prediction tool, and can achieve improved performance in independent datasets compared to model or rule based methods.

  6. Dual-mixed HIV-1 Coreceptor Tropism and HIV-Associated Neurocognitive Deficits

    PubMed Central

    Morris, Sheldon R.; Woods, Steven Paul; Deutsch, Reena; Little, Susan J.; Wagner, Gabriel; Morgan, Erin E.; Heaton, Robert K.; Letendre, Scott L.; Grant, Igor; Smith, Davey M.

    2014-01-01

    Background HIV coreceptor usage of CXCR4 (X4) is associated with decreased CD4+ T-cell counts and accelerated disease progression, but the role of X4 tropism in HIV-associated neurocognitive disorders (HAND) has not previously been described. Methods This longitudinal study evaluated data on 197 visits from 72 recently HIV-infected persons who had undergone up to 4 sequential neurocognitive assessments over a median of 160 days (IQR 138–192). Phenotypic tropism testing (Trofile ES, Monogram, Biosciences) was performed on stored blood samples. Multivariable mixed model repeated measures regression was used to determine the association between HAND and dual-mixed (DM) viral tropism, estimated duration of infection (EDI), HIV RNA, CD4 count and problematic methamphetamine use. Results Six subjects (8.3%) had dual mixed tropism (DM) at their first neurocognitive assessment and four converted to DM in subsequent sampling (for total of 10 DM) at a median EDI of 10.1 months (IQR 7.2–12.2). There were 44 (61.1%) subjects who demonstrated HAND on at least one study visit. HAND was associated with DM tropism (odds ratio 4.4, 95% CI 0.9–20.5) and shorter EDI (odds ratio 1.1 per month earlier, 95% CI 1.0–1.2). Conclusion This study found that recency of HIV-1 infection and the development of DM tropism may be associated with HAND in the relatively early stage of infection. Together these data suggest that viral interaction with cellular receptors may play an important role in the early manifestation of HAND. PMID:24078557

  7. Roles of mitogen-activated protein kinase and phosphoinositide 3'-kinase in ErbB2/ErbB3 coreceptor-mediated heregulin signaling.

    PubMed

    Vijapurkar, Ulka; Kim, Myong-Soo; Koland, John G

    2003-04-01

    ErbB2/HER2 and ErbB3/HER3, two members of the ErbB/HER family, together constitute a heregulin coreceptor complex that elicits a potent mitogenic and transforming signal. Among known intracellular effectors of the ErbB2/ErbB3 heregulin coreceptor are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. Activation of the distinct MAPK and PI 3-kinase signaling pathways by the ErbB2/ErbB3 coreceptor in response to heregulin and their relative contributions to the mitogenic and transformation potentials of the activated coreceptor were investigated here. To this end, cDNAs encoding the wild-type ErbB3 protein (ErbB3-WT) and ErbB3 proteins with amino acid substitutions in either the Shc-binding site (ErbB3-Y1325F), the six putative PI 3-kinase-binding sites (ErbB3-6F), or both (ErbB3-7F) were generated and expressed in NIH-3T3 cells to form functional ErbB2/ErbB3 heregulin coreceptors. While the coreceptor incorporating ErbB3-WT activated both the MAPK and the PI 3-kinase signaling pathways, those incorporating ErbB3-Y1325F or ErbB3-6F activated either PI 3-kinase or MAPK, respectively. The ErbB2/ErbB3-7F coreceptor activated neither. Elimination of either signaling pathway lowered basal and eliminated heregulin-dependent expression of cyclin D1, which was in each case accompanied by an attenuated mitogenic response. Selective elimination of the PI 3-kinase pathway severely impaired the ability of heregulin to transform cells expressing the coreceptor, whereas attenuation of the MAPK pathway had a lesser effect. Thus, while both pathways contributed in a roughly additive manner to the mitogenic response elicited by the activated ErbB2/ErbB3 coreceptor, the PI 3-kinase pathway predominated in the induction of cellular transformation.

  8. Maraviroc, a chemokine receptor-5 antagonist, fails to demonstrate efficacy in the treatment of patients with rheumatoid arthritis in a randomized, double-blind placebo-controlled trial

    PubMed Central

    2012-01-01

    Introduction The purpose of this study was to determine whether maraviroc, a human CC chemokine receptor 5 (CCR5) antagonist, is safe and effective in the treatment of active rheumatoid arthritis (RA) in patients on background methotrexate (MTX). Methods This phase IIa study comprised two distinct components: an open-label safety study of the pharmacokinetics (PK) of MTX in the presence of maraviroc, and a randomized, double-blind, placebo-controlled, proof-of-concept (POC) component. In the PK component, patients were randomized 1:1 to receive maraviroc 150 or 300 mg twice daily (BID) for four weeks. In the POC component, patients were randomized 2:1 to receive maraviroc 300 mg BID or placebo for 12 weeks. Patients were not eligible for inclusion in both components. Results Sixteen patients were treated in the safety/PK component. Maraviroc was well tolerated and there was no evidence of drug-drug interaction with MTX. One hundred ten patients were treated in the POC component. The study was terminated after the planned interim futility analysis due to lack of efficacy, at which time 59 patients (38 maraviroc; 21 placebo) had completed their week 12 visit. There was no significant difference in the number of ACR20 responders between the maraviroc (23.7%) and placebo (23.8%) groups (treatment difference -0.13%; 90% CI -20.45, 17.70; P = 0.504). The most common all-causality treatment-emergent adverse events in the maraviroc group were constipation (7.8%), nausea (5.2%), and fatigue (3.9%). Conclusions Maraviroc was generally well tolerated over 12 weeks; however, selective antagonism of CCR5 with maraviroc 300 mg BID failed to improve signs and symptoms in patients with active RA on background MTX. Trial Registration ClinicalTrials.gov: NCT00427934 PMID:22251436

  9. Treatment intensification with maraviroc (CCR5 antagonist) leads to declines in CD16-expressing monocytes in cART-suppressed chronic HIV-infected subjects and is associated with improvements in neurocognitive test performance: implications for HIV-associated neurocognitive disease (HAND).

    PubMed

    Ndhlovu, Lishomwa C; Umaki, Tracie; Chew, Glen M; Chow, Dominic C; Agsalda, Melissa; Kallianpur, Kalpana J; Paul, Robert; Zhang, Guangxiang; Ho, Erika; Hanks, Nancy; Nakamoto, Beau; Shiramizu, Bruce T; Shikuma, Cecilia M

    2014-12-01

    HIV-associated neurocognitive disorders (HAND) continues to be prevalent (30-50%) despite plasma HIV-RNA suppression with combination antiretroviral therapy (cART). There is no proven therapy for individuals on suppressive cART with HAND. We have shown that the degree of HIV reservoir burden (HIV DNA) in monocytes appear to be linked to cognitive outcomes. HIV infection of monocytes may therefore be critical in the pathogenesis of HAND. A single arm, open-labeled trial was conducted to examine the effect of maraviroc (MVC) intensification on monocyte inflammation and neuropsychological (NP) performance in 15 HIV subjects on stable 6-month cART with undetectable plasma HIV RNA (<48 copies/ml) and detectable monocyte HIV DNA (>10 copies/10(6) cells). MVC was added to their existing cART regimen for 24 weeks. Post-intensification change in monocytes was assessed using multiparametric flow cytometry, monocyte HIV DNA content by PCR, soluble CD163 (sCD163) by an ELISA, and NP performance over 24 weeks. In 12 evaluable subjects, MVC intensification resulted in a decreased proportion of circulating intermediate (median; 3.06% (1.93, 6.45) to 1.05% (0.77, 2.26)) and nonclassical (5.2% (3.8, 7.9) to 3.2% (1.8, 4.8)) CD16-expressing monocytes, a reduction in monocyte HIV DNA content to zero log10 copies/10(6) cells and in levels of sCD163 of 43% by 24 weeks. This was associated with significant improvement in NP performance among six subjects who entered the study with evidence of mild to moderate cognitive impairment. The results of this study suggest that antiretroviral therapy with potency against monocytes may have efficacy against HAND.

  10. Small Molecule CXCR3 Antagonists.

    PubMed

    Andrews, Stephen P; Cox, Rhona J

    2016-04-14

    Chemokines and their receptors are known to play important roles in disease. More than 40 chemokine ligands and 20 chemokine receptors have been identified, but, to date, only two small molecule chemokine receptor antagonists have been approved by the FDA. The chemokine receptor CXCR3 was identified in 1996, and nearly 20 years later, new areas of CXCR3 disease biology continue to emerge. Several classes of small molecule CXCR3 antagonists have been developed, and two have shown efficacy in preclinical models of inflammatory disease. However, only one CXCR3 antagonist has been evaluated in clinical trials, and there remain many opportunities to further investigate known classes of CXCR3 antagonists and to identify new chemotypes. This Perspective reviews the known CXCR3 antagonists and considers future opportunities for the development of small molecules for clinical evaluation.

  11. Bicyclams, a class of potent anti-HIV agents, are targeted at the HIV coreceptor fusin/CXCR-4.

    PubMed

    Schols, D; Esté, J A; Henson, G; De Clercq, E

    1997-08-01

    Bicyclams are a novel class of antiviral compounds which are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. The prototype compound, AMD3100, has an IC50 of 1-10 ng/ml, which is a least 100,000 fold lower than the cytotoxic concentration. AMD3100 does not inhibit virus binding to the CD4 receptor and based on time-of-addition experiments, has been assumed to interact with the HIV fusion-uncoating process. Resistance of HIV-1 strains to AMD3100 is associated with the accumulation of several mutations in the viral envelope glycoprotein gp120. Here, we demonstrate that AMD3100 interacts with fusin (CXCR-4), the coreceptor used by T-tropic viruses to infect the target cells. The replication of NL4-3 wild type virus and NL4-3 dextran sulfate-resistant virus was inhibited by the CXC-chemokine, stromal cell-derived factor 1 (SDF-1), the natural ligand for CXCR-4. In contrast, the replication of the HIV-1 NL4-3 AMD3100-resistant virus was no longer inhibited by SDF-1. The bicyclams are the first low-molecular-weight anti-HIV agents shown to interact with the coreceptor for T-tropic viruses.

  12. Genetic interactions between the hedgehog co-receptors Gas1 and Boc regulate cell proliferation during murine palatogenesis

    PubMed Central

    Xavier, Guilherme M.; Seppala, Maisa; Papageorgiou, Spyridon N.; Fan, Chen-Ming; Cobourne, Martyn T.

    2016-01-01

    Abnormal regulation of Sonic hedgehog (Shh) signaling has been described in a variety of human cancers and developmental anomalies, which highlights the essential role of this signaling molecule in cell cycle regulation and embryonic development. Gas1 and Boc are membrane co-receptors for Shh, which demonstrate overlapping domains of expression in the early face. This study aims to investigate potential interactions between these co-receptors during formation of the secondary palate. Mice with targeted mutation in Gas1 and Boc were used to generate Gas1; Boc compound mutants. The expression of key Hedgehog signaling family members was examined in detail during palatogenesis via radioactive in situ hybridization. Morphometric analysis involved computational quantification of BrdU-labeling and cell packing; whilst TUNEL staining was used to assay cell death. Ablation of Boc in a Gas1 mutant background leads to reduced Shh activity in the palatal shelves and an increase in the penetrance and severity of cleft palate, associated with failed elevation, increased proliferation and reduced cell death. Our findings suggest a dual requirement for Boc and Gas1 during early development of the palate, mediating cell cycle regulation during growth and subsequent fusion of the palatal shelves. PMID:27811357

  13. Coreceptor signal strength regulates positive selection but does not determine CD4/CD8 lineage choice in a physiologic in vivo model.

    PubMed

    Erman, Batu; Alag, Amala S; Dahle, Oyvind; van Laethem, François; Sarafova, Sophia D; Guinter, Terry I; Sharrow, Susan O; Grinberg, Alexander; Love, Paul E; Singer, Alfred

    2006-11-15

    TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.

  14. Control of Antagonistic Components of the Hedgehog Signaling Pathway by microRNAs in Drosophila

    PubMed Central

    Friggi-Grelin, Florence; Lavenant-Staccini, Laurence; Therond, Pascal

    2008-01-01

    Hedgehog (Hh) signaling is critical for many developmental processes and for the genesis of diverse cancers. Hh signaling comprises a series of negative regulatory steps, from Hh reception to gene transcription output. We previously showed that stability of antagonistic regulatory proteins, including the coreceptor Smoothened (Smo), the kinesin-like Costal-2 (Cos2), and the kinase Fused (Fu), is affected by Hh signaling activation. Here, we show that the level of these three proteins is also regulated by a microRNA cluster. Indeed, the overexpression of this cluster and resulting microRNA regulation of the 3′-UTRs of smo, cos2, and fu mRNA decreases the levels of the three proteins and activates the pathway. Further, the loss of the microRNA cluster or of Dicer function modifies the 3′-UTR regulation of smo and cos2 mRNA, confirming that the mRNAs encoding the different Hh components are physiological targets of microRNAs. Nevertheless, an absence of neither the microRNA cluster nor of Dicer activity creates an hh-like phenotype, possibly due to dose compensation between the different antagonistic targets. This study reveals that a single signaling pathway can be targeted at multiple levels by the same microRNAs. PMID:18493062

  15. The endocytosis and signaling of the γδ T cell coreceptor WC1 are regulated by a dileucine motif.

    PubMed

    Hsu, Haoting; Baldwin, Cynthia L; Telfer, Janice C

    2015-03-01

    WC1 proteins, which are specifically expressed by bovine γδ T cells from a gene array containing 13 members, are part of the scavenger receptor cysteine-rich family. WC1 cytoplasmic domains contains multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WC1 is endocytosed in response to PMA. Because WC1 endocytosis may play a role in the activation of γδ T cells, we examined WC1 endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WC1. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WC1 cytoplasmic domain significantly reduced PMA-induced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WC1 caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WC1 endocytosis in Jurkat cells, the pan-protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WC1 inhibited IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WC1 gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens.

  16. Bi-Directional Heterologous Desensitization Between the Major HIV-1 Co-Receptor CXCR4 and the κ-Opioid Receptor1

    PubMed Central

    Finley, Matthew J.; Chen, Xiaohong; Bardi, Guiseppe; Davey, Penny; Geller, Ellen B.; Zhang, Lily; Adler, Martin W.; Rogers, Thomas J.

    2008-01-01

    We previously characterized multiple interactions between chemokine and opioid G-protein coupled receptors (GPCR), and we found both µ and δ-opioid receptors cross-desensitize CCR1, CCR2, CCR5, but not CXCR4. Here we report that the κ-opioid receptor (KOR) is able to cross-desensitize CXCR4, and this phenomenon is bi-directional. Chemotactic responses by KOR activation are diminished with prior activation of CXCR4. Additionally, calcium mobilization assays show these cross-desensitization processes occur within seconds of receptor activation, and target receptor internalization is not responsible for desensitization between these receptors. These results have implications for several essential processes including neuronal and lymphocyte development, inflammatory responses, and pain/sensitivity. PMID:18533278

  17. CD8 Co-receptor promotes susceptibility of CD8+ T cells to transforming growth factor-β (TGF-β)-mediated suppression

    PubMed Central

    Zloza, Andrew; Jagoda, Michael C.; Lyons, Gretchen E.; Graves, Michael C.; Kohlhapp, Frederick J.; O’Sullivan, Jeremy A.; Lacek, Andrew T.; Nishimura, Michael I.

    2015-01-01

    CD8+ T cell function depends on a finely orchestrated balance of activation/suppression signals. While the stimulatory role of the CD8 co-receptor and pleiotropic capabilities of TGF-β have been studied individually, the influence of CD8 co-receptor on TGF-β function in CD8+ T cells is unknown. Here, we show that while CD8 enhances T cell activation, it also enhances susceptibility to TGF-β-mediated immune suppression. Using Jurkat cells expressing a full-length, truncated or no αβCD8 molecule, we demonstrate that cells expressing full-length αβCD8 were highly susceptible, αβCD8-truncated cells were partially susceptible, and CD8-deficient cells were completely resistant to suppression by TGF-β. Additionally, we determined that inhibition of Lck rendered mouse CD8+ T cells highly resistant to TGF-β suppression. Resistance was not associated with TGF-β receptor expression but did correlate with decreased Smad3 and increased Smad7 levels. These findings highlight a previously unrecognized third role for CD8 co-receptor which appears to prepare activated CD8+ T cells for response to TGF-β. Based on the important role which TGF-β-mediated suppression plays in tumor immunology, these findings unveil necessary considerations in formulation of CD8+ T cell-related cancer immunotherapy strategies. PMID:21193909

  18. Crystal Structure of Botulinum Neurotoxin Type a in Complex With the Cell Surface Co-Receptor GT1b-Insight Into the Toxin-Neuron Interaction

    SciTech Connect

    Stenmark, P.; Dupuy, J.; Inamura, A.; Kiso, M.; Stevens, R.C.

    2009-05-26

    Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in the toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.

  19. Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human papilloma virus co-receptor expression.

    PubMed

    Ortiz-Sánchez, Elizabeth; Santiago-López, Luz; Cruz-Domínguez, Verónica B; Toledo-Guzmán, Mariel E; Hernández-Cueto, Daniel; Muñiz-Hernández, Saé; Garrido, Efraín; Cantú De León, David; García-Carrancá, Alejandro

    2016-05-31

    Cancer stem cells (CSC) exhibit high tumorigenic capacity in several tumor models. We have now determined an extended phenotype for cervical cancer stem cells. Our results showed increased CK-17, p63+, AII+, CD49f+ expression in these cells, together with higher Aldehyde dehydrogenase (ALDHbright)activity in Cervical CSC (CCSC) enriched in cervospheres. An increase in stem cell markers, represented by OCT-4, Nanog, and β-catenin proteins, was also observed, indicating that under our culture conditions, CCSC are enriched in cervospheres, as compared to monolayer cultures. In addition, we were able to show that an increased ALDHbright activity correlated with higher tumorigenic activity. Flow cytometry and immunflorescence assays demonstrated that CCSC in cervosphere cultures contain a sub-population of cells that contain Annexin II, a Human papillomavirus (HPV) co-receptor. Taken together, under our conditions there is an increase in the number of CCSC in cervosphere cultures which exhibit the following phenotype: CK-17, p63+, AII+, CD49f+ and high ALDH activity, which in turn correlates with higher tumorigenicity. The presence of Annexin II and CD49f in CCSC opens the possibility that normal cervical stem cells could be the initial target of infection by high risk HPV.

  20. Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions

    PubMed Central

    Zeng, Xin; Huang, He; Tamai, Keiko; Zhang, Xinjun; Harada, Yuko; Yokota, Chika; Almeida, Karla; Wang, Jianbo; Doble, Brad; Woodgett, Jim; Wynshaw-Boris, Anthony; Hsieh, Jen-Chieh; He, Xi

    2016-01-01

    Canonical Wnt/β-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/β-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/β-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate β-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/β-catenin signaling. PMID:18077588

  1. Growth and turning properties of adult glial cell-derived neurotrophic factor coreceptor α1 nonpeptidergic sensory neurons.

    PubMed

    Guo, GuiFang; Singh, Vandana; Zochodne, Douglas W

    2014-09-01

    An overlapping population of adult primary sensory neurons that innervate the skin express the glial cell-derived neurotrophic factor coreceptor α1 (GFRα1), the lectin IB4, and the "regenerative brake" phosphatase and tensin homolog deleted on chromosome 10. Using an adapted turning and growth assay, we analyzed the growth cone behavior of adult immunoselected GFRα1 sensory neurons. These neurons had less robust baseline growth and reluctant responsiveness to individual growth factors but responded to synergistic types of input from glial cell-derived neurotrophic factor, hepatocyte growth factor, a phosphatase and tensin homolog deleted on chromosome 10 inhibitor, or a downstream Rho kinase inhibitor. Hepatocyte growth factor and the phosphatase and tensin homolog deleted on chromosome 10 inhibitor were associated with growth cone turning. A gradient of protein extracted from skin samples, a primary target of GFRα1 axons, replicated the impact of synergistic support. Within the skin, glial cell-derived neurotrophic factor was expressed within epidermal axons, indicating an autocrine role accompanying local hepatocyte growth factor synthesis. Taken together, our findings identify unique growth properties and plasticity of a distinct population of epidermal axons that are relevant to neurologic repair and skin reinnervation.

  2. Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human papilloma virus co-receptor expression

    PubMed Central

    Ortiz-Sánchez, Elizabeth; Santiago-López, Luz; Cruz-Domínguez, Verónica B.; Toledo-Guzmán, Mariel E.; Hernández-Cueto, Daniel; Muñiz-Hernández, Saé; Garrido, Efraín; De León, David Cantú; García-Carrancá, Alejandro

    2016-01-01

    Cancer stem cells (CSC) exhibit high tumorigenic capacity in several tumor models. We have now determined an extended phenotype for cervical cancer stem cells. Our results showed increased CK-17, p63+, AII+, CD49f+ expression in these cells, together with higher Aldehyde dehydrogenase (ALDHbright)activity in Cervical CSC (CCSC) enriched in cervospheres. An increase in stem cell markers, represented by OCT-4, Nanog, and β-catenin proteins, was also observed, indicating that under our culture conditions, CCSC are enriched in cervospheres, as compared to monolayer cultures. In addition, we were able to show that an increased ALDHbright activity correlated with higher tumorigenic activity. Flow cytometry and immunflorescence assays demonstrated that CCSC in cervosphere cultures contain a sub-population of cells that contain Annexin II, a Human papillomavirus (HPV) co-receptor. Taken together, under our conditions there is an increase in the number of CCSC in cervosphere cultures which exhibit the following phenotype: CK-17, p63+, AII+, CD49f+ and high ALDH activity, which in turn correlates with higher tumorigenicity. The presence of Annexin II and CD49f in CCSC opens the possibility that normal cervical stem cells could be the initial target of infection by high risk HPV. PMID:27008711

  3. Role of Wnt Co-receptor LRP6 in Triple Negative Breast Cancer Cell Migration and Invasion.

    PubMed

    Ma, Jinlu; Lu, Wenyan; Chen, Dongquan; Xu, Bo; Li, Yonghe

    2017-03-01

    The low-density lipoprotein receptor-related protein 6 (LRP6) is an essential Wnt co-receptor of the Wnt/β-catenin signaling pathway. Although studies have shown an increased expression of LRP6 in several types of cancer, its function in tumor development and progression remains to be elucidated. We herein demonstrated that LRP6 expression is up-regulated in human triple negative breast cancer (TNBC) patients and human TNBC cell lines, and that knockdown of LRP6 expression and treatment of recombinant Mesd protein (a specific inhibitor of LRP6) significantly decreased cell migration and invasion of TNBC MDA-MB-231 and BT549 cells. Interestingly, the effects of LRP6 knockdown and Mesd treatment on TNBC cell migration and invasion were more prominent than on TNBC cell proliferation/viability. Mechanistically, LRP6 knockdown and Mesd treatment inhibited Wnt/β-catenin signaling and decreased the expression of S100A4, a mediator of cancer metastasis and a specific target of Wnt/β-catenin signaling, in TNBC cells. Together, our data suggest that LRP6 promotes TNBC cell migration and invasion by regulating the expression and function of S100A4 via the Wnt/β-catenin signaling pathway. This article is protected by copyright. All rights reserved.

  4. Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

    PubMed

    Gelfand, Maria V; Hagan, Nellwyn; Tata, Aleksandra; Oh, Won-Jong; Lacoste, Baptiste; Kang, Kyu-Tae; Kopycinska, Justyna; Bischoff, Joyce; Wang, Jia-Huai; Gu, Chenghua

    2014-09-22

    During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

  5. Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis.

    PubMed

    Li, Chao; Yeh, Fang-Ling; Cheung, Alice Y; Duan, Qiaohong; Kita, Daniel; Liu, Ming-Che; Maman, Jacob; Luu, Emily J; Wu, Brendan W; Gates, Laura; Jalal, Methun; Kwong, Amy; Carpenter, Hunter; Wu, Hen-Ming

    2015-06-08

    The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor.

  6. The Wnt Co-Receptor Lrp5 Is Required for Cranial Neural Crest Cell Migration in Zebrafish

    PubMed Central

    Willems, Bernd; Tao, Shijie; Yu, Tingsheng; Huysseune, Ann; Witten, Paul Eckhard; Winkler, Christoph

    2015-01-01

    During vertebrate neurulation, cranial neural crest cells (CNCCs) undergo epithelial to mesenchymal transition (EMT), delaminate from the neural plate border, and migrate as separate streams into different cranial regions. There, they differentiate into distinct parts of the craniofacial skeleton. Canonical Wnt signaling has been shown to be essential for this process at different levels but the involved receptors remained unclear. Here we show that the frizzled co-receptor low-density-lipoprotein (LDL) receptor-related protein 5 (Lrp5) plays a crucial role in CNCC migration and morphogenesis of the cranial skeleton. Early during induction and migration of CNCCs, lrp5 is expressed ubiquitously but later gets restricted to CNCC derivatives in the ventral head region besides different regions in the CNS. A knock-down of lrp5 does not interfere with induction of CNCCs but leads to reduced proliferation of premigratory CNCCs. In addition, cell migration is disrupted as CNCCs are found in clusters at ectopic positions in the dorsomedial neuroepithelium after lrp5 knock-down and transient CRISPR/Cas9 gene editing. These migratory defects consequently result in malformations of the craniofacial skeleton. To date, Lrp5 has mainly been associated with bone homeostasis in mammals. Here we show that in zebrafish, lrp5 also controls cell migration during early morphogenetic processes and contributes to shaping the craniofacial skeleton. PMID:26121341

  7. Opioid antagonists for smoking cessation

    PubMed Central

    David, Sean P; Lancaster, Tim; Stead, Lindsay F; Evins, A. Eden; Prochaska, Judith J

    2014-01-01

    Background The reinforcing properties of nicotine may be mediated through release of various neurotransmitters both centrally and systemically. People who smoke report positive effects such as pleasure, arousal, and relaxation as well as relief of negative affect, tension, and anxiety. Opioid (narcotic) antagonists are of particular interest to investigators as potential agents to attenuate the rewarding effects of cigarette smoking. Objectives To evaluate the efficacy of opioid antagonists in promoting long-term smoking cessation. The drugs include naloxone and the longer-acting opioid antagonist naltrexone. Search methods We searched the Cochrane Tobacco Addiction Group Specialised Register for trials of naloxone, naltrexone and other opioid antagonists and conducted an additional search of MEDLINE using ’Narcotic antagonists’ and smoking terms in April 2013. We also contacted investigators, when possible, for information on unpublished studies. Selection criteria We considered randomised controlled trials comparing opioid antagonists to placebo or an alternative therapeutic control for smoking cessation. We included in the meta-analysis only those trials which reported data on abstinence for a minimum of six months. We also reviewed, for descriptive purposes, results from short-term laboratory-based studies of opioid antagonists designed to evaluate psycho-biological mediating variables associated with nicotine dependence. Data collection and analysis We extracted data in duplicate on the study population, the nature of the drug therapy, the outcome measures, method of randomisation, and completeness of follow-up. The main outcome measure was abstinence from smoking after at least six months follow-up in patients smoking at baseline. Abstinence at end of treatment was a secondary outcome. We extracted cotinine- or carbon monoxide-verified abstinence where available. Where appropriate, we performed meta-analysis, pooling risk ratios using a Mantel

  8. Mineralcorticoid antagonists in heart failure.

    PubMed

    D'Elia, Emilia; Krum, Henry

    2014-10-01

    Mineralocorticoid receptor antagonists (MRAs) have become mandated therapy in patients with reduced ejection fraction (systolic) heart failure (HF) across all symptom classes. These agents should also be prescribed in the early post-myocardial infarction setting in those with reduced ejection fraction and either HF symptoms or diabetes. This article explores the pathophysiological role of aldosterone, an endogenous ligand for the mineralcorticoid receptor (MR), and summarizes the clinical data supporting guideline recommendations for these agents in systolic HF. The use of MRAs in novel areas beyond systolic HF ejection is also explored. Finally, the current status of newer agents will be examined.

  9. NK-1 Antagonists and Itch.

    PubMed

    Ständer, Sonja; Luger, Thomas A

    2015-01-01

    Substance P (SP) is an important mediator of pro-inflammatory mechanisms in the skin. It targets multiple cells such as keratinocytes, mast cells, and fibroblasts which are involved in the cutaneous generation of pruritus. This suggests that SP is an interesting target for therapy. In fact, in recent case reports and case series, SP antagonists demonstrated a significant antipruritic effect in acute and chronic pruritus such as drug-induced pruritus, paraneoplastic pruritus, prurigo nodularis, cutaneous T-cell lymphoma, and brachioradial pruritus.

  10. Function-oriented development of CXCR4 antagonists as selective human immunodeficiency virus (HIV)-1 entry inhibitors.

    PubMed

    Wu, Chien-Huang; Wang, Chuan-Jen; Chang, Chun-Ping; Cheng, Yung-Chi; Song, Jen-Shin; Jan, Jiing-Jyh; Chou, Ming-Chen; Ke, Yi-Yu; Ma, Jing; Wong, Ying-Chieh; Hsieh, Tsung-Chih; Tien, Yun-Chen; Gullen, Elizabeth A; Lo, Chen-Fu; Cheng, Chia-Yi; Liu, Yu-Wei; Sadani, Amit A; Tsai, Chia-Hua; Hsieh, Hsin-Pang; Tsou, Lun K; Shia, Kak-Shan

    2015-02-12

    Motivated by the pivotal role of CXCR4 as an HIV entry co-receptor, we herein report a de novo hit-to-lead effort on the identification of subnanomolar purine-based CXCR4 antagonists against HIV-1 infection. Compound 24, with an EC50 of 0.5 nM against HIV-1 entry into host cells and an IC50 of 16.4 nM for inhibition of radioligand stromal-derived factor-1α (SDF-1α) binding to CXCR4, was also found to be highly selective against closely related chemokine receptors. We rationalized that compound 24 complementarily interacted with the critical CXCR4 residues that are essential for binding to HIV-1 gp120 V3 loop and subsequent viral entry. Compound 24 showed a 130-fold increase in anti-HIV activity compared to that of the marketed CXCR4 antagonist, AMD3100 (Plerixafor), whereas both compounds exhibited similar potency in mobilization of CXCR4(+)/CD34(+) stem cells at a high dose. Our study offers insight into the design of anti-HIV therapeutics devoid of major interference with SDF-1α function.

  11. Identification of ghrelin receptor blocker, D-[Lys3] GHRP-6 as a CXCR4 receptor antagonist.

    PubMed

    Patel, Kalpesh; Dixit, Vishwa Deep; Lee, Jun Ho; Kim, Jie Wan; Schaffer, Eric M; Nguyen, Dzung; Taub, Dennis D

    2012-01-01

    [D-Lys3]-Growth Hormone Releasing Peptide-6 (DLS) is widely utilized in vivo and in vitro as a selective ghrelin receptor (GHS-R) antagonist. Unexpectedly, we identified that DLS also has the ability to block CXCL12 binding and activity through CXCR4 on T cells and peripheral blood mononuclear cells (PBMCs). Moreover, as CXCR4 has been shown to act as a major co-receptor for HIV-1 entry into CD4 positive host cells, we have also found that DLS partially blocks CXCR4-mediated HIV-1 entry and propagation in activated human PBMCs. These data demonstrate that DLS is not the specific and selective antagonist as thought for GHS-R1a and appears to have additional effects on the CXCR4 chemokine receptor. Our findings also suggest that structural analogues that mimic DLS binding properties may also have properties of blocking HIV infectivity, CXCR4 dependent cancer cell migration and attenuating chemokine-mediated immune cell trafficking in inflammatory disorders.

  12. Bidirectional effect of Wnt signaling antagonist DKK1 on the modulation of anthrax toxin uptake.

    PubMed

    Qian, LiLi; Cai, ChangZu; Yuan, PengFei; Jeong, Sun-Young; Yang, XiaoZhou; Dealmeida, Venita; Ernst, James; Costa, Michael; Cohen, Stanley N; Wei, WenSheng

    2014-05-01

    LRP6, a co-receptor for the morphogen Wnt, aids endocytosis of anthrax complexes. Here we report that Dickkopf1 (DKK1) protein, a secreted LRP6 ligand and antagonist, is also a modulator of anthrax toxin sensitivity. shRNA-mediated gene silencing or TALEN-mediated gene knockout of DKK1 reduced sensitivity of cells to PA-dependent hybrid toxins. However, unlike the solely inhibitory effect on Wnt signaling, the effects of DKK1 overexpression on anthrax toxicity were bidirectional, depending on its endogenous expression and cell context. Fluorescence microscopy and biochemical analyses showed that DKK1 facilitates internalization of anthrax toxins and their receptors, an event mediated by DKK1-LRP6-Kremen2 complex. Monoclonal antibodies against DKK1 provided dose-dependent protection to macrophages from killing by anthrax lethal toxin (LT). Our discovery that DKK1 forms ternary structure with LRP6 and Kremen2 in promoting PA-mediated toxin internalization provides a paradigm for bacterial exploitation of mechanisms that host cells use to internalize signaling proteins.

  13. MuSK is a BMP co-receptor that shapes BMP responses and calcium signaling in muscle cells

    PubMed Central

    Yilmaz, Atilgan; Kattamuri, Chandramohan; Ozdeslik, Rana N.; Schmiedel, Carolyn; Mentzer, Sarah; Schorl, Christoph; Oancea, Elena; Thompson, Thomas B.; Fallon, Justin R.

    2017-01-01

    Bone morphogenetic proteins (BMPs) function in most tissues but have cell type-specific effects. Given the relatively small number of BMP receptors, this exquisite signaling specificity requires additional molecules to regulate this pathway’s output. The receptor tyrosine kinase MuSK (muscle-specific kinase) is critical for neuromuscular junction formation and maintenance. Here, we show that MuSK also promotes BMP signaling in muscle cells. MuSK bound to BMP4 and related BMPs with low nanomolar affinity in vitro and to the type I BMP receptors ALK3 and ALK6 in a ligand-independent manner both in vitro and in cultured myotubes. High-affinity binding to BMPs required the third, alternatively spliced MuSK immunoglobulin-like domain. In myoblasts, endogenous MuSK promoted BMP4-dependent phosphorylation of SMADs and transcription of Id1, which encodes a transcription factor involved in muscle differentiation. Gene expression profiling showed that MuSK was required for the BMP4-induced expression of a subset of genes in myoblasts, including regulator of G protein signaling 4 (Rgs4). In myotubes, MuSK enhanced the BMP4-induced expression of a distinct set of genes, including transcripts characteristic of slow muscle. MuSK-mediated stimulation of BMP signaling required type I BMP receptor activity but was independent of MuSK tyrosine kinase activity. MuSK-dependent expression of Rgs4 resulted in the inhibition of Ca2+ signaling induced by the muscarinic acetylcholine receptor in myoblasts. These findings establish that MuSK has dual roles in muscle cells, acting both as a tyrosine kinase-dependent synaptic organizing molecule and as a BMP co-receptor that shapes BMP transcriptional output and cholinergic signaling. PMID:27601729

  14. Defining Binding Efficiency and Specificity of Auxins for SCFTIR1/AFB-Aux/IAA Co-receptor Complex Formation

    PubMed Central

    2013-01-01

    Structure–activity profiles for the phytohormone auxin have been collected for over 70 years, and a number of synthetic auxins are used in agriculture. Auxin classification schemes and binding models followed from understanding auxin structures. However, all of the data came from whole plant bioassays, meaning the output was the integral of many different processes. The discovery of Transport Inhibitor-Response 1 (TIR1) and the Auxin F-Box (AFB) proteins as sites of auxin perception and the role of auxin as molecular glue in the assembly of co-receptor complexes has allowed the development of a definitive quantitative structure–activity relationship for TIR1 and AFB5. Factorial analysis of binding activities offered two uncorrelated factors associated with binding efficiency and binding selectivity. The six maximum-likelihood estimators of Efficiency are changes in the overlap matrixes, inferring that Efficiency is related to the volume of the electronic system. Using the subset of compounds that bound strongly, chemometric analyses based on quantum chemical calculations and similarity and self-similarity indices yielded three classes of Specificity that relate to differential binding. Specificity may not be defined by any one specific atom or position and is influenced by coulomb matrixes, suggesting that it is driven by electrostatic forces. These analyses give the first receptor-specific classification of auxins and indicate that AFB5 is the preferred site for a number of auxinic herbicides by allowing interactions with analogues having van der Waals surfaces larger than that of indole-3-acetic acid. The quality factors are also examined in terms of long-standing models for the mechanism of auxin binding. PMID:24313839

  15. Localization and action of Dragon (repulsive guidance molecule b), a novel bone morphogenetic protein coreceptor, throughout the reproductive axis.

    PubMed

    Xia, Yin; Sidis, Yisrael; Mukherjee, Abir; Samad, Tarek A; Brenner, Gary; Woolf, Clifford J; Lin, Herbert Y; Schneyer, Alan

    2005-08-01

    Bone morphogenetic proteins (BMPs) play important roles in reproduction including primordial germ cell formation, follicular development, spermatogenesis, and FSH secretion. Dragon, a recently identified glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, is also a BMP coreceptor. In the present study, we determined the tissue and cellular localization of Dragon in reproductive organs using immunohistochemistry and in situ hybridization. Among reproductive organs, Dragon was expressed in testis, epididymis, ovary, uterus, and pituitary. In the testis of early postnatal mice, Dragon was found in gonocytes and spermatogonia, whereas in immature testes, Dragon was only weakly expressed in spermatogonia. Interestingly, pregnant mare serum gonadotropin treatment of immature mice robustly induced Dragon production in spermatocytes. In adult testis, Dragon was found in spermatocytes and round spermatids. In the ovary, Dragon was detected exclusively within oocytes and primarily those within secondary follicles. In the pituitary, Dragon-expressing cells overlapped FSH-expressing cells. Dragon was also expressed in a number of cell lines originating from reproductive tissues including Ishikawa, Hela, LbetaT2, MCF-7, and JEG3 cells. Immunocytochemistry and gradient sucrose ultracentrifugation studies showed Dragon was localized in lipid rafts within the plasma membrane. In reproductive cell lines, Dragon expression enhanced signaling of exogenous BMP2 or BMP4. The present studies demonstrate that Dragon expression is dynamically regulated throughout the reproductive tract and that Dragon protein modulates BMP signaling in cells from reproductive tissues. The overlap between Dragon expression and the functional BMP signaling system suggests that Dragon may play a role in mammalian reproduction.

  16. Genetic predisposition of variants in TLR2 and its co-receptors to severe malaria in Odisha, India.

    PubMed

    Panigrahi, Subhendu; Kar, Avishek; Tripathy, Sagnika; Mohapatra, Manoj K; Dhangadamajhi, Gunanidhi

    2016-02-01

    Although the role of TLRs signalling in malaria pathogenesis is well established, contribution of individual TLR to clinical outcome of malaria still remains inconclusive. Given the importance of TLR2 and its co-receptors in recognising distinct structural forms of key malaria toxins and mediating innate immune response, it is essential to delineate their genetic contribution. Variants in TLR1 (I602S) and TLR6 (P249S) were genotyped by PCR-RFLP methods, and TLR2 (I/D) was genotyped by PCR in 200 samples each from uncomplicated malaria (UM) and severe malaria (SM). Further, SM was categorised into its sub-clinical groups (CM and NCSM or SOD and MODS) and analysed. The results showed the PP genotype of TLR6 (P249S) to be significantly more common in UM (P < 0.0001), whereas the 'SS' genotype was the risk factor for SM including its sub-clinical categories. The TLR1 (602S) and TLR2 (D) variants were significantly high in patients with CM; however, negative LD was observed between TLR2 and TLR6 in NCSM and MODS. Haplotype analysis showed significantly high frequency of I-I-S haplotype in all forms of subclinical SM and was associated with low parasite load in SM (P = 0.013). The haplotypes I-D-S and S-I-P were significantly high in SOD and CM, respectively. The TLR6 '249S' variant appeared to be the dominant determinant for genetic predisposition to SM and that its association with either TLR2 'D' or TLR1 '602S' modulates for CM development. The present study opens up several new avenues for their exploration and validation in future studies in different global settings for malaria.

  17. Vitamin K antagonists: beyond bleeding.

    PubMed

    Krüger, Thilo; Floege, Jürgen

    2014-01-01

    Warfarin is the most widely used oral anticoagulant in clinical use today. Indications range from prosthetic valve replacement to recurrent thromboembolic events due to antiphospholipid syndrome. In hemodialysis (HD) patients, warfarin use is even more frequent than in the nonrenal population due to increased cardiovascular comorbidities. The use of warfarin in dialysis patients with atrial fibrillation requires particular caution because side effects may outweigh the assumed benefit of reduced stroke rates. Besides increased bleeding risk, coumarins exert side effects which are not in the focus of clinical routine, yet they deserve special consideration in dialysis patients and should influence the decision of whether or not to prescribe vitamin K antagonists in cases lacking clear guidelines. Issues to be taken into consideration in HD patients are the induction or acceleration of cardiovascular calcifications, a 10-fold increased risk of calciphylaxis and problems related to maintaining a target INR range. New anticoagulants like direct thrombin inhibitors are promising but have not yet been approved for ESRD patients. Here, we summarize the nontraditional side effects of coumarins and give recommendations about the use of vitamin K antagonists in ESRD patients.

  18. Construction, purification, and immunogenicity of recombinant cystein-cystein type chemokine receptor 5 vaccine.

    PubMed

    Wu, Kongtian; Xue, Xiaochang; Wang, Zenglu; Yan, Zhen; Shi, Jihong; Han, Wei; Zhang, Yingqi

    2006-09-01

    Cystein-Cystein type chemokine receptor 5 (CCR5) is a seven-transmembrane, G-protein coupled receptor. It is a major coreceptor with CD4 glycoprotein mediating cellular entry of