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Biomedical subjects

P M Murphy

Publications and source records attributed to P M Murphy.

At least 37 records · Page 2Linked to original sources
PubMed2001

Cloning, mRNA distribution, and functional expression of an avian counterpart of the chemokine receptor/HIV coreceptor CXCR4.

The chemokine signaling system, which coordinates the basal and emergency trafficking of leukocytes, presumably coevolved with the hematopoietic system. To study its phylogenetic origins, we used the open reading frame (ORF) of the human chemokine receptor CXCR4 as a genomic probe, since in mammals it is the most highly conserved chemokine receptor known. CXCR4 cross-hybridized to genomic DNA from mouse and chicken, but not zebrafish, Drosophila, or Caenorhabditis elegans. Accordingly, we cloned the corresponding chicken cDNA. The ORF is 359 codons long versus 352 for human CXCR4, and encodes a protein 82% identical to human CXCR4. In a calcium flux assay of receptor function, CHO-K1 cells stably transfected with the chicken cDNA responded specifically to human SDF-1, the specific ligand for CXCR4, but not to a panel of other chemokines tested at 100 nM. SDF-1 activated the cells in a dose-dependent manner (EC50 approximately 5 nM), whereas parental CHO-K1 cells did not respond. The CHO-K1 cell transfectants also bound 125I-SDF-1 specifically. Leukocytes from chicken peripheral blood expressed chCXCR4 mRNA and responded to human SDF-1 in a calcium flux assay with an EC50 similar to that for chCXCR4-transfected CHO cells, suggesting that this response is mediated by native chCXCR4. Analysis of chicken genomic DNA with the chicken cDNA as probe revealed a pattern consistent with a single copy gene, and the absence of any closely related genes. mRNA was detected in brain, bursa, liver, small and large intestine, embryonal fibroblasts, and blood leukocytes, but not in stomach or pancreas. These results, which identify the first functional non-viral, non-mammalian chemokine receptor, suggest that the origins of a functional chemokine system extend at least to birds and suggest that, as in mammals, CXCR4 functions in many avian tissues.

Amino Acid Sequence↗
PubMed2001

Synthetic peptide MMK-1 is a highly specific chemotactic agonist for leukocyte FPRL1.

Human phagocytic leukocytes express the seven-transmembrane G-protein-coupled receptors formyl peptide receptor (FPR) and FPR-like 1 (FPRL1). MMK-1, a synthetic peptide derived from a random peptide library, is reported to induce calcium mobilization specifically in human FPRL1 gene-transfected cells. However, its actions on human phagocytic leukocytes remain poorly defined. We found that MMK-1 is a potent chemotactic and calcium-mobilizing agonist for human monocytes, neutrophils, and FPRL1-transfected human embryonic kidney (HEK) 293 cells but is inactive in cells transfected with FPR. MMK-1 also activated HEK 293 cells transfected with FPR2, a mouse counterpart of human FPRL1. Furthermore, MMK-1 increased pertussis toxin-sensitive production of inflammatory cytokines in human monocytes. MMK-1 signaling in human phagocytes was completely desensitized by a well-defined FPRL1 agonist, suggesting that FPRL1 is likely a receptor that mediates the action of MMK-1 in primary cells. Since MMK-1 is one of the most potent FPRL1-specific agonists identified so far, it can serve as a modulator of the host defense and a useful agent for further studying the signaling and function of FPRL1.

Animals↗
PubMed2000

Chemokine RANTES promoter polymorphism affects risk of both HIV infection and disease progression in the Multicenter AIDS Cohort Study.

OBJECTIVE: To examine whether polymorphism in the RANTES gene is associated with HIV disease outcome. DESIGN: RANTES, a ligand of the major HIV co-receptor, CCR5, is known to block HIV-CCR5 interactions. Recently, two single nucleotide polymorphisms in the RANTES gene promoter region, designated -403G/A and -28C/G, have been described. Both polymorphisms can affect in-vitro promoter activity, and the RANTES -403A, -28G haplotype has been associated with a slower CD4 cell count decline rate in a Japanese cohort. METHODS: We compared RANTES compound genotype frequencies between HIV-positive and exposed-uninfected participants of the Multicenter AIDS Cohort Study (MACS) and rates of progression to AIDS for MACS seroconverters. RESULTS: We found that the two most common RANTES promoter compound genotypes, G1 (-403G/G, -28C/C) found in 67% of Caucasians, and G4 (-403G/A, -28C/C) found in 23% of Caucasians, were associated with altered risk of HIV transmission and progression, particularly in individuals who lacked the protective CCR5 mutation, CCR5delta32. In this study, individuals with a G4 compound genotype were more likely to acquire HIV than individuals with a G1 compound genotype (OR 1.72, P = 0.016) and the risk increased when individuals possessing CCR5delta32 were omitted from consideration (OR 2.13, P = 0.005). Among seroconverters lacking CCR5delta32, those who had the G4 compound genotype progressed significantly slower to AIDS-1993 than those with the G1 compound genotype (median time to AIDS 7.6 versus 5.4 years; RH 0.65; P = 0.007). CONCLUSIONS: These data implicate the RANTES-403A allele as a risk factor for HIV transmission and as a protective factor for HIV progression.

Acquired Immunodeficiency Syndrome↗
PubMed2000

MIP-1alpha is produced but it does not control pulmonary inflammation in response to respiratory syncytial virus infection in mice.

The intent of this study was to compare the cellular and biochemical inflammatory responses of mice infected with the paramyxovirus pathogens respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM). Although RSV is not a natural pathogen of mice, it has been used extensively in mouse models of the human disease, as a limited respiratory infection can be established via intranasal inoculation of virus at high titer. In earlier work, we found that acute infection with the natural rodent pathogen, PVM, elicited a rapid and sustained pulmonary inflammatory response (peak, 1.7 x 10(6) leukocytes/ml BAL fluid) that was dependent on both local production of MIP-1alpha and signaling via its receptor, CCR1. We find here that MIP-1alpha is also produced in response to RSV, although relatively few leukocytes (<200 ml BAL fluid) are recruited to the lungs in response. Further experiments with CCR1-deficient mice confirm the finding that although MIP-1alpha is produced in response to RSV infection, leukocytes do not respond via this pathway. Among the explanations for these findings, we propose that there are other, as yet to be identified proinflammatory mediators elicited in response to PVM (but not in response to RSV) that serve to prime the leukocytes in vivo, thus enabling them to respond to MIP-1alpha signaling via CCR1. Furthermore, the differences in disease pathogenesis seen in response to each of these pneumovirus infections in mice raise questions regarding the extent to which primary RSV infection in mice can be used as a model of primary RSV infection in humans.

Acute Disease↗
PubMed2000

Selective CXCR4 antagonism by Tat: implications for in vivo expansion of coreceptor use by HIV-1.

Chemokines and chemokine receptors play important roles in HIV-1 infection and tropism. CCR5 is the major macrophage-tropic coreceptor for HIV-1 whereas CXC chemokine receptor 4 (CXCR4) serves the counterpart function for T cell-tropic viruses. An outstanding biological mystery is why only R5-HIV-1 is initially detected in new seroconvertors who are exposed to R5 and X4 viruses. Indeed, X4 virus emerges in a minority of patients and only in the late stage of disease, suggesting that early negative selection against HIV-1-CXCR4 interaction may exist. Here, we report that the HIV-1 Tat protein, which is secreted from virus-infected cells, is a CXCR4-specific antagonist. Soluble Tat selectively inhibited the entry and replication of X4, but not R5, virus in peripheral blood mononuclear cells (PBMCs). We propose that one functional consequence of secreted Tat is to select against X4 viruses, thereby influencing the early in vivo course of HIV-1 disease.

Antiviral Agents↗
PubMed2000

The chemokine macrophage-inflammatory protein-1 alpha and its receptor CCR1 control pulmonary inflammation and antiviral host defense in paramyxovirus infection.

In this work, we explore the responses of specific gene-deleted mice to infection with the paramyxovirus pneumonia virus of mice (PVM). We have shown previously that infection of wild type mice with PVM results in pulmonary neutrophilia and eosinophilia accompanied by local production of macrophage-inflammatory protein-1 alpha (MIP-1 alpha). Here we examine the role of MIP-1 alpha in the pathogenesis of this disease using mice deficient in MIP-1 alpha or its receptor, CCR1. The inflammatory response to PVM in MIP-1 alpha-deficient mice was minimal, with approximately 10-60 neutrophils/ml and no eosinophils detected in bronchoalveolar lavage fluid. Higher levels of infectious virus were recovered from lung tissue excised from MIP-1 alpha-deficient than from fully competent mice, suggesting that the inflammatory response limits the rate of virus replication in vivo. PVM infection of CCR1-deficient mice was also associated with attenuated inflammation, with enhanced recovery of infectious virus, and with accelerated mortality. These results suggest that the MIP-1 alpha/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of infection.

Animals↗
PubMed2000

The HIV-1 cell entry inhibitor T-20 potently chemoattracts neutrophils by specifically activating the N-formylpeptide receptor.

T-20, a synthetic peptide corresponding to the heptad repeat sequence of HIV-1 gp41, blocks HIV-1 entry by targeting gp41, and is currently in clinical trials as an anti-retroviral agent. We recently reported that in vitro T-20 also functions as a phagocyte chemoattractant and a chemotactic agonist at the phagocyte N-formylpeptide receptor (FPR). Here we show that T-20 is also a potent chemotactic agonist in vitro at a related human phagocyte receptor FPRL1R. To test the relative importance of FPR and FPRL1R in primary cells, we identified the corresponding mouse T-20 receptors, mFPR and FPR2, which are both expressed in neutrophils, and compared T-20 action on neutrophils from wild type and mFPR knockout mice. Surprisingly, although T-20 activates mFPR and FPR2 in transfected cells with equal potency and efficacy in both calcium flux and chemotaxis assays, neutrophils from mFPR knockout mice did not respond to T-20. These results provide genetic evidence that FPR is the major phagocyte T-20 receptor in vivo and point to the potential feasibility of studying T-20 effects on immunity in a mouse model. This may help define the cause of local inflammation after T-20 injection that has recently been reported in Phase I clinical trials.

Animals↗
PubMed2000

Activation of the chemotactic peptide receptor FPRL1 in monocytes phosphorylates the chemokine receptor CCR5 and attenuates cell responses to selected chemokines.

FPRL1 is a seven-transmembrane (STM), G-protein coupled receptor which was originally identified as a low affinity receptor for the bacterial chemotactic formyl peptide and a high affinity receptor for the lipid metabolite lipoxin A4. We recently discovered that a number of peptides, including several synthetic domains of the HIV-1 envelope proteins and the serum amyloid A, use FPRL1 to induce migration and calcium mobilization in human monocytes and neutrophils. In this study, we report that a synthetic peptide domain of the V3 region of the HIV-1 envelope gp120, activates the FPRL1 receptor in monocytes and neutrophils. Furthermore, monocytes prestimulated with V3 peptide showed reduced response to several chemokines that use multiple cell receptors. This is associated with a rapid phosphorylation of the chemokine receptor CCR5 on the serine residues. Our study suggests that FPRL1, as a classical chemoattractant receptor, may play an important role in modulating monocyte activation in the presence of multiple stimuli.

Amino Acid Sequence↗
PubMed2000

Effect of chemokine receptor gene polymorphisms on the response to potent antiretroviral therapy.

BACKGROUND: Both the natural history of HIV infection and the response to antiretroviral therapy are heterogeneous. Polymorphisms in chemokine receptor genes modulate the natural history of HIV-1 infection. In comparison with subjects with other genotypes, the prognosis for HIV-1-infected CCR5-delta32 heterozygotes is more favorable and that for CCR5 promoter allele 59029A homozygotes is less favorable. METHODS: HIV-1-infected adults with a CD4+ lymphocyte count > or = 200 cells x 10(6)/l and a plasma HIV RNA level > or = 1000 copies/ml were treated with indinavir, zidovudine and lamivudine for 6 months. HIV RNA levels were measured at 4-week intervals. Genotyping for chemokine receptor gene polymorphisms (CCR5-delta32, CCR5 59029A/G, CCR2-641) was performed. We examined whether the time to first HIV RNA < 200 copies/ml, frequency of viral suppression failure (HIV RNA > or = 200 copies/ml between weeks 16 and 28 of therapy), or reduction from the pre-treatment HIV RNA level differed by genotype. RESULTS: Time to first HIV RNA < 200 copies/ml was not predicted by genotype. Among 272 Caucasian patients, viral suppression failure was more common among patients with the CCR5 +/+ ¿ CCR2+/+ ¿ CCR5-59029 A/A genotype (28%) than among all other subjects combined (relative risk, 2.0; P = 0.06). After 24 weeks of therapy, genotype groups differed in the reduction of the HIV RNA level from baseline (P = 0.02); patients with the CCR5 +/+ ¿ CCR2+/+ ¿ CCR5-59029 A/A genotype had a mean reduction of 2.12 log10 copies/ml compared to 2.64 log10 copies/ml among all other groups combined. CONCLUSION: Polymorphisms in chemokine receptor genes may explain some of the heterogeneity in sustaining viral suppression observed among patients receiving potent antiretroviral therapy.

Adult↗
PubMed2000

Serum amyloid A is a chemotactic agonist at FPR2, a low-affinity N-formylpeptide receptor on mouse neutrophils.

Serum amyloid A (SAA) is an acute-phase plasma protein and the source of amyloid A, which accumulates in lesions of secondary amyloidosis. SAA can induce phagocyte migration in vitro and in vivo, and is a specific chemotactic agonist for the human low-affinity N-formylpeptide receptor FPRL1R, a G-protein-coupled receptor expressed on phagocytes. Here we show that FPR2, a mouse counterpart of FPRL1R, is also an SAA receptor. SAA selectively induced calcium flux and chemotaxis in mouse PMN, which express FPR2, as well as in HEK 293 cells expressing recombinant FPR2 but not in HEK 293 cells expressing FPR, a closely related high affinity N-formylpeptide receptor. Consistent with this, SAA activity on PMN from FPR+/+ and FPR-/- mice was indistinguishable. Moreover, the prototype N-formylpeptide fMLF desensitized SAA-induced calcium flux in a dose-dependent manner in both mouse neutrophils and HEK 293/FPR2 transfectants. Our results suggest that FPR2 specifically mediates mouse neutrophil migration in response to SAA.

Animals↗
PubMed2000

Rapid progression to AIDS in HIV+ individuals with a structural variant of the chemokine receptor CX3CR1.

Human immunodeficiency virus (HIV) enters cells in vitro via CD4 and a coreceptor. Which of 15 known coreceptors are important in vivo is poorly defined but may be inferred from disease-modifying mutations, as for CCR5. Here two single nucleotide polymorphisms are described in Caucasians in CX3CR1, an HIV coreceptor and leukocyte chemotactic/adhesion receptor for the chemokine fractalkine. HIV-infected patients homozygous for CX3CR1-I249 M280, a variant haplotype affecting two amino acids (isoleucine-249 and methionine-280), progressed to AIDS more rapidly than those with other haplotypes. Functional CX3CR1 analysis showed that fractalkine binding is reduced among patients homozygous for this particular haplotype. Thus, CX3CR1-I249 M280 is a recessive genetic risk factor in HIV/AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Regulator of G protein signaling 1 (RGS1) markedly impairs Gi alpha signaling responses of B lymphocytes.

Regulator of G protein signaling (RGS) proteins modulate signaling through pathways that use heterotrimeric G proteins as transducing elements. RGS1 is expressed at high levels in certain B cell lines and can be induced in normal B cells by treatment with TNF-alpha. To determine the signaling pathways that RGS1 may regulate, we examined the specificity of RGS1 for various G alpha subunits and assessed its effect on chemokine signaling. G protein binding and GTPase assays revealed that RGS1 is a Gi alpha and Gq alpha GTPase-activating protein and a potential G12 alpha effector antagonist. Functional studies demonstrated that RGS1 impairs platelet activating factor-mediated increases in intracellular Ca+2, stromal-derived factor-1-induced cell migration, and the induction of downstream signaling by a constitutively active form of G12 alpha. Furthermore, germinal center B lymphocytes, which are refractory to stromal-derived factor-1-triggered migration, express high levels of RGS1. These results indicate that RGS proteins can profoundly effect the directed migration of lymphoid cells.

Animals↗
PubMed2000

Host-related immunomodulators encoded by poxviruses and herpesviruses.

In the past year, important advances have been made in the area of host-related immunomodulatory genes encoded by the larger DNA viruses, particularly for the poxviruses and herpesviruses. Not only has the repertoire of viral immunomodulator homologs expanded as a result of sequencing the complete genome of another six, large DNA viruses, but also new concepts of how they work have been proposed and in some cases supported by in vivo evidence. Recent developments have been made in understanding a spectrum of host-related viral modulators, including complement control proteins, TNF-receptor homologs, IL-18 binding proteins, viral interleukins (vIL-6 and vIL-10), chemokine mimics and chemokine receptor homologs.

Adjuvants, Immunologic↗
PubMed2000

Use of hydrostatic pressure for inactivation of microbial contaminants in cheese.

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30 degrees C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10 degrees C and by 6-log-unit cycles at temperatures of 20 and 30 degrees C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20 degrees C and >400 MPa at 30 degrees C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20 degrees C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.

Cheese↗
PubMed2000

Identification of a gammaherpesvirus selective chemokine binding protein that inhibits chemokine action.

Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of gammaHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1alpha (MIP-1alpha), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX(3)C chemokine fractalkine with high affinity (K(d) = 1. 6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines (K(d) > 1 microM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1alpha and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.

Animals↗
PubMed2000

CCR8 on human thymocytes functions as a human immunodeficiency virus type 1 coreceptor.

To determine whether human immunodeficiency virus type 1 (HIV-1) coreceptors besides CXCR4 and CCR5 are involved in HIV-1 infection of the thymus, we focused on CCR8, a receptor for the chemokine I-309, because of its high expression in the thymus. Similar levels of CCR8 mRNA were detected in immature and mature primary human thymocytes. Consistent with this, [(125)I]I-309 was shown to bind specifically and with similar affinity to the surface of immature and mature human thymocytes. Fusion of human thymocytes with cells expressing HIV-1 X4 or X4R5 envelope glycoprotein was inhibited by I-309 in a dose-dependent manner. In addition, I-309 partially inhibited productive infection of human thymocytes by X4, R5, and X4R5 HIV-1 strains. Our data provide the first evidence that CCR8 functions as an HIV-1 coreceptor on primary human cells and suggest that CCR8 may contribute to HIV-1-induced thymic pathogenesis.

Chemokine CCL1↗