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P J Nelson

Publications and source records attributed to P J Nelson.

At least 19 recordsLinked to original sources
PubMed2000

A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection.

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.

Animals↗
PubMed2000

Interleukin-1beta induction of the chemokine RANTES promoter in the human astrocytoma line CH235 requires both constitutive and inducible transcription factors.

The chemokine RANTES is an important mediator of inflammatory processes. In this report, we describe the DNA sequence and transcription factor requirements for interleukin-1beta (IL-1beta) induction of the RANTES promoter in the human astrocytoma line CH235. RANTES promoter sequences between -278 and +55 are sufficient for IL-1beta-inducibility. In vitro DNA binding assays demonstrate constitutive binding of Sp1, HMG, Ets domain, and bZIP family members to their cognate sites in the RANTES promoter, whereas NF-kappaB and IRF-1 bind in an IL-1beta-inducible manner. IL-1beta-inducibility of the RANTES promoter requires both constitutive and inducible transcription factors. The formation of a higher order nucleoprotein complex, or 'enhanceosome', may be critical for IL-1beta induction of the RANTES promoter.

Astrocytoma↗
PubMed2000

Association of antiretroviral therapy with detection of HIV-1 RNA and DNA in the anorectal mucosa of homosexual men.

OBJECTIVE: To determine whether combination antiretroviral therapy is associated with reduced detection of HIV-1 RNA and DNA in the anorectal mucosa of men who have sex with men (MSM). DESIGN: Cross-sectional study of 233 MSM recruited from community and clinic sites in Seattle, Washington between July 1996 and December 1997. METHODS: HIV-1 RNA and HIV-1 DNA were detected in anorectal swab specimens by polymerase chain reaction amplification assays. RESULTS: HIV-1 RNA was detected significantly less often in anorectal specimens from users of combination antiretroviral therapies, whether a protease inhibitor was received (15/89; 17%) or not (16/53; 30%), than in men not receiving therapy (43/88; 49%) (P < 0.001, P = 0.03, respectively). In contrast, HIV-1 DNA was detected only slightly less frequently in anorectal specimens obtained from men receiving protease inhibitors (35/81; 43%) or reverse transcriptase inhibitors alone (22/48; 46%) than in specimens from men not receiving therapy (45/78; 58%) (P = 0.07, P = 0.20, respectively). Among men with < 50 copies HIV-1 RNA/ml plasma, detection of HIV-1 RNA in anorectal specimens was rare (1/54; 2%) but detection of HIV-1 DNA was common (14/50; 28%). CONCLUSIONS: Combination antiretroviral therapy is associated with reductions in HIV-1 RNA, but HIV-1 DNA remains detectable in the anorectal canal of almost half of MSM receiving such therapy. Condom use during anal intercourse should be encouraged, regardless of plasma viral load response to potent antiretroviral therapy.

Adult↗
PubMed2000

ATF and Jun transcription factors, acting through an Ets/CRE promoter module, mediate lipopolysaccharide inducibility of the chemokine RANTES in monocytic Mono Mac 6 cells.

The chemokine RANTES is produced by a variety of tissues, including cells of the monocyte/macrophage lineage. RANTES expression is rapidly and transiently up-regulated in primary monocytes and the monocytic cell line Mono Mac 6 in response to stimulation by the bacterial product lipopolysaccharide (LPS). Transient transfection of Mono Mac 6 cells with RANTES reporter-promoter deletion constructs, in conjunction with DNase I footprinting and heterologous reporter gene assays, allowed identification of an LPS-responsive region within the RANTES promoter. Electrophoretic mobility shift assays (EMSA), methylation interference and EMSA supershift experiments were used to characterize sequences and transcription factors responsible for this LPS inducibility. The region, termed RANTES site G [R(G)], contains consensus sites for Ets and CRE/AP-1-like elements. Site-directed mutagenesis of the Ets site resulted in a loss of only 15 % of promoter activity, while mutation of the CRE/AP-1 site led to a loss of 40 % of LPS-induced promoter activity. The Ets site constitutively binds the Ets family member PU.1. LPS stimulation leads to an induction of ATF-3 and JunD factor binding to the CRE/AP-1 site. Thus, LPS induction of RANTES transcription is mediated, in part, through the activation and selective binding of ATF and Jun nuclear factors to the R(G) promoter module.

Activating Transcription Factor 3↗
PubMed2000

Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection.

The release of microparticles from eukaryotic cells is a well-recognized phenomenon. We demonstrate here that the chemokine receptor CCR5, the principal co-receptor for macrophage-tropic human immunodeficiency virus (HIV)-1, can be released through microparticles from the surface of CCR5+ Chinese hamster ovary cells and peripheral blood mononuclear cells. Microparticles containing CCR5 can transfer the receptor to CCR5- cells and render them CCR5+. The CCR5 transfer to CCR5-deficient peripheral blood mononuclear cells homozygous for a 32-base-pair deletion in the CCR5 gene enabled infection of these cells with macrophage-tropic HIV-1. In monocytes, the transfer of CCR5 could be inhibited by cytochalasin D, and transferred CCR5 could be downmodulated by chemokines. A transfer of CCR5 from peripheral blood mononuclear cells to endothelial cells during transendothelial migration could be demonstrated. Thus, the transfer of CCR5 may lead to infection of tissues without endogenous CCR5 expression. Moreover, the intercellular transfer of membrane proteins by microparticles might have broader consequences for intercellular communication beyond the effects seen for HIV-1.

Animals↗
PubMed2000

Yersinia enterocolitica invasin protein triggers IL-8 production in epithelial cells via activation of Rel p65-p65 homodimers.

Enteropathogenic Yersinia bacteria trigger the production of the proinflammatory chemokine IL-8, an important chemokine for the recruitment of polymorphonuclear leukocytes (PMN). Yersinia is resistant to phagocytosis by PMN, and the recruitment of these cells is thought to be part of a pathogenic strategy of Yersinia to establish infection by allowing the pathogen to gain access to, and disseminate within, host tissue. We report here that Yersinia expressing the outer membrane protein invasin triggers IL-8 production in epithelial cells. The 195 carboxyl-terminal amino acids of invasin when linked to latex beads are sufficient to trigger IL-8 production. By means of IL-8 promoter reporter gene assays and electrophoretic mobility shift assay experiments, the minimal optimal region of the IL-8 promoter responsive to invasin was identified and invasin-responsive control elements were characterized. Invasin-induced activation of the IL-8 promoter was found to be mediated through a previously identified NF-kappaB element. This NF-kappaB binding site preferentially binds Rel p65-p65 homodimers as well as some p50-p65 heterodimers in response to stimulation by invasin. Invasin-induced NF-kappaB activation correlated with degradation of IkappaBalpha and the inhibition of NF-kappaB by specific inhibitors of IkappaB activation blocked invasin-induced IL-8 secretion. Invasin-triggered IL-8 production does not depend on invasin-triggered uptake of bacteria, and is independent of a functional PI3-kinase. This report is the first to demonstrate the molecular basis of IL-8 production triggered by enteropathogenic bacteria. Together, these data elucidate the possible early pathomechanisms operating in Yersinia infection and may have implications for the design of novel therapeutics directed against this enteropathogen.

Adhesins, Bacterial↗
PubMed2000

A gene encoding a novel multidomain beta-1,4-mannanase from Caldibacillus cellulovorans and action of the recombinant enzyme on kraft pulp.

Genomic walking PCR was used to obtained a 4,567-bp nucleotide sequence from Caldibacillus cellulovorans. Analysis of this sequence revealed that there were three open reading frames, designated ORF1, ORF2, and ORF3. Incomplete ORF1 encoded a putative C-terminal cellulose-binding domain (CBD) homologous to members of CBD family IIIb, while putative ORF3 encoded a protein of unknown function. The putative ManA protein encoded by complete manA ORF2 was an enzyme with a novel multidomain structure and was composed of four domains in the following order: a putative N-terminal domain (D1) of unknown function, an internal CBD (D2), a beta-mannanase catalytic domain (D3), and a C-terminal CBD (D4). All four domains were linked via proline-threonine-rich peptides. Both of the CBDs exhibited sequence similarity to family IIIb CBDs, while the mannanase catalytic domain exhibited homology to the family 5 glycosyl hydrolases. The purified recombinant enzyme ManAd3 expressed from the cloned catalytic domain (D3) exhibited optimum activity at 85 degrees C and pH 6.0 and was extremely thermostable at 70 degrees C. This enzyme exhibited high specificity with the substituted galactomannan locust bean gum, while more substituted galacto- and glucomannans were poorly hydrolyzed. Preliminary studies to determine the effect of the recombinant ManAd3 and a recombinant thermostable beta-xylanase on oxygen-delignified Pinus radiata kraft pulp revealed that there was an increase in the brightness of the bleached pulp.

Amino Acid Sequence↗
PubMed1999

Cycling of human dendritic cell effector phenotypes in response to TNF-alpha: modification of the current 'maturation' paradigm and implications for in vivo immunoregulation.

Dendritic cells (DCs) are potent antigen presenting cells reported to undergo irreversible functional 'maturation' in response to inflammatory signals such as TNF-alpha. The current paradigm holds that this DC maturation event is required for full functional capacity and represents terminal differentiation of this cell type, culminating in apoptotic cell death. This provides a possible mechanism for avoiding dysregulated immunostimulatory activity, but imposes constraints on the capacity of DCs to influence subsequent immune responses and to participate in immunological memory. We report that the cell surface and functional effects induced by TNF-alpha are reversible and reinducible. These effects are accompanied by a concordant modulation of cytokine mRNA expression that includes the induction of proinflammatory factors (IL-15, IL-12, LT-alpha, LT-beta, TNF-alpha, RANTES) which is coincident with the down-regulation of counter-regulatory cytokines (IL-10, TGF-beta1, TGF-beta2, IL-1 RA, MCP-1). The resultant net effect is a dendritic cell activation state characterized by a transient proinflammatory posture. These results demonstrate that 1) human DCs do not undergo terminal 'maturation' in response to TNF-alpha, 2) DC phenotypes are more pleiotropic than previously thought, and 3) DCs are potential immunoregulatory effector cells with implications for control of immune responses in both in vivo and in vitro systems.

Antigens, CD↗
PubMed1999

Expression of CCR2 by endothelial cells : implications for MCP-1 mediated wound injury repair and In vivo inflammatory activation of endothelium.

Endothelial cell proliferation and migration may play a central role in angiogenesis, wound healing, and atherosclerosis. Although CXC chemokines can act on endothelial cells by influencing proliferation, an involvement of CC chemokines and endothelial expression of chemokine receptors remains to be elucidated. Reverse transcription-polymerase chain reaction, RNase protection, Western blot, and flow cytometric analysis showed that human umbilical vein endothelial cells express mRNA and surface protein of the monocyte chemotactic protein-1 (MCP-1) receptor CCR2, which was upregulated by inflammatory cytokines. MCP-1 induced migration of endothelial cells in a transwell assay, which was inhibited by the 9-76 MCP-1 receptor antagonist. Increased secretion of MCP-1 or interleukin-8, but not RANTES, on endothelial injury suggested a functional role of CCR2 in wound repair as measured by ELISA. After mechanical injury to endothelial monolayers, which spontaneously closed within 24 hours, wound repair was delayed by the 9-76 antagonist and by a blocking monoclonal antibody to MCP-1, but not to interleukin-8, and was improved by exogenous MCP-1. This was confirmed by quantification of cell migration into the wound area, whereas proliferation and viability were unaltered by MCP-1 or its analogue. Notably, immunohistochemistry of inflamed tissue revealed CCR2 staining on arterial, venous, and venular endothelium affected by cellular infiltration. This is the first demonstration of endothelial CCR2 expression ex vivo, inferring its involvement in inflammatory conditions. Thus endothelial cells express functional CCR2 that may have important implications for endothelial wound repair and inflammatory reactions.

Cell Division↗
PubMed1999

Involvement of the protein kinase C substrate, SSeCKS, in the actin-based stellate morphology of mesangial cells.

Activation of protein kinase C is a key signal transduction event in mesangial cell dedifferentiation and proliferation, yet little is known about downstream substrates or their roles in normal or diseased glomeruli. SSeCKS, a novel protein kinase C substrate originally isolated as a src-suppressed negative mitogenic regulator in fibroblasts, controls actin-based cytoskeletal architecture and scaffolds key signaling kinases such as protein kinase C and protein kinase A. Based on the morphologic similarity between SSeCKS-overexpressing fibroblasts and stellate mesangial cells, we hypothesized that SSeCKS might play a role in mesangial cell morphology in a protein kinase C-dependent manner. Immunoblotting, in situ staining and northern blotting detected abundant expression of SSeCKS in human and rodent mesangial cells and glomerular parietal cells but not in renal tubular epithelia. Immunofluorescence analysis showed enrichment of SSeCKS in mesangial cell podosomes and along a cytoskeletal network distinct from F-actin. Activation of protein kinase C by phorbol ester resulted in a rapid serine phosphorylation of SSeCKS and its subsequent translocation to perinuclear sites, coincident with the retraction of stellate processes. These effects were blocked by concentrations of bis-indolylmaleimide that selectively inhibit protein kinase C. Finally, ablation of SSeCKS expression using retroviral anti-sense vectors induced (1) an elongated, fibroblastic cell morphology, (2) production of thick, longitudinal stress fibers and (3) repositioning of vinculin-associated focal complexes away from the cell edges. These data suggest a role for SSeCKS as a downstream mediator of protein kinase C-controlled, actin-based mesangial cell cytoskeletal architecture.

A Kinase Anchor Proteins↗
PubMed1999

Chemokine and chemokine receptor expression in a novel human mesangial cell line.

Chemokines are thought to play a pivotal role in mediating the selective migration of leukocytes into sites of tissue injury. The local production of chemokines by mesangial cells (MC) has been linked to inflammatory processes within the glomerulus. To study the chemokine biology of human MC, an immortalized human MC line was generated and then chemokine and chemokine receptor expression was examined in response to various proinflammatory stimuli. The results show that human MC have a specific and limited repertoire of chemokine expression. The stimulus-specific regulation of the chemokines monocyte chemoattractant protein- (MCP- 1), regulated upon activation, normal T cell expressed and secreted (RANTES), interleukin-8 (IL-8), and IP-10 was demonstrated using RNase protection assays. Transcripts for the chemokines MIP-1alpha, MIP-1beta, I-309, or lymphotactin could not be detected. The expression of CC chemokine receptors was investigated by reverse transcription-PCR and RNase protection assays. MC stimulated with interferon-gamma (IFN-gamma) expressed mRNA for the chemokine receptor CCR1. The expression could be further increased by activating the cells with a combination of tumor necrosis factor-a (TNF-alpha), IL-1beta, and IFN-gamma. Under these conditions, no mRNA for CCR2, CCR3, CCR4, CCR5, or CCR8 was detected. A comparison of the immortalized human mesangial cells with primary cells showed identical expression patterns of chemokine receptors. To demonstrate functional activity of chemokine receptors expressed by human MC, chemotaxis assays were performed. MC stimulated with a combination of TNF-alpha, IL-1beta, and IFN-gamma, but not unstimulated MC, migrated toward a RANTES gradient. Eotaxin did not enhance the migratory activity of human MC. In summary, a novel human mesangial cell line was established and the pattern of chemokine expression was examined. For the first time, the inducible expression of functionally active CCR1 by human MC was shown.

Cell Line↗
PubMed1999

Met-RANTES reduces vascular and tubular damage during acute renal transplant rejection: blocking monocyte arrest and recruitment.

Chemokines are thought to contribute to the cellular infiltrate characteristic of renal transplant rejection. We show that Met-RANTES, a chemokine receptor antagonist, suppresses recruitment of inflammatory cells into renal allografts. In a renal transplant model (Fisher RT1(lvl) rat kidney into Lewis RT1(l) rat) where no additional immune suppressant was used, Met-RANTES-treated animals showed a significant reduction in vascular injury score (16.10 +/- 5.20 vs. 62.67 +/- 18.64) and tubular damage score (15.70 +/- 5.22 vs. 33.00 +/- 6.44) relative to untreated animals. In a more severe rejection model (Brown-Norway RT1(n) rat kidney into Lewis RT1(1) rat), Met-RANTES significantly augmented low-dose cyclosporin A treatment to reduce all aspects of renal injury including interstitial inflammation (score 71.00 +/- 6.10 vs. 157.30 +/- 21.30). The majority of infiltrating cells in these models (60-70%) consisted of monocytes. Potential mechanisms of action of Met-RANTES were tested using monocyte attachment assays on microvascular endothelium under physiological flow conditions. Preexposure of microvascular endothelium to RANTES resulted in RANTES immobilization and RANTES-induced firm adhesion of monocytes only after prestimulation of the endothelium with IL-1beta. Met-RANTES completely inhibited this RANTES-mediated arrest. Thus, Met-RANTES may counter acute rejection by blocking leukocyte firm adhesion to inflamed endothelium.

Animals↗
PubMed1999

[Reduction of acute kidney transplantation rejection by the chemokine receptor antagonist Met-RANTES].

Chemokines contribute to the mononuclear cell infiltrate in vessels and interstitium which is characteristic of renal transplant rejection. By employing the chemokine receptor blocker Met-RANTES it was shown that recruitment of inflammatory cells into renal allografts could be significantly suppressed. In a renal transplant model (Fisher RT1(1v1) rat kidney into Lewis RT1(1) rat) Met-RANTES-treated animals showed a significant reduction in vascular injury score (16.10 +/- 5.20 vs. 62.67 +/- 18.64) and tubular damage score (15.70 +/- 5.22 vs. 33.00 +/- 6.44) relative to untreated animals. In a severe rejection model (Brown-Norway RT1n rat kidney into Lewis RT1(1) rat), Met-RANTES significantly augmented low-dose cyclosporin A treatment to reduce all aspects of renal injury including interstitial inflammation (score 71.00 +/- 6.10 vs. 157.30 +/- 21.30). In a monocyte attachment assay on microvascular endothelium under physiological flow conditions exposure of microvascular endothelium to RANTES resulted in RANTES immobilization and RANTES-induced firm adhesion of monocytes only after prestimulation of the endothelium with IL-1 beta. Met-RANTES completely inhibited this RANTES-mediated arrest. Thus, Met-RANTES can reduce acute rejection by impeding leukocyte arrest to inflamed endothelium.

Acute Disease↗
PubMed1998

Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity.

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.

Animals↗
PubMed1998

Chemokines, lymphocytes and viruses: what goes around, comes around.

Chemokines are believed to be the long-sought soluble mediators of selective lymphocyte recruitment. As most selectin-integrin interactions are nonselective, it is thought that the discrimination seen during lymphocyte infiltration into tissues is brought about by the actions of distinct chemokines. Developments over the past year have demonstrated the expanding roles of these factors in lymphocyte chemoattraction, normal trafficking, and viral immunity.

Animals↗