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Human Cytomegalovirus Chemokine Receptor Gene US28 Is Transcribed in Latently Infected THP-1 Monocytes
Patrick S. Beisser, Lysiane Laurent, Jean-Louis Virelizier, Susan Michelson
Patrick S. Beisser
Lysiane Laurent
Jean-Louis Virelizier
Susan Michelson
DOI: 10.1128/JVI.75.13.5949-5957.2001
The human cytomegalovirus (HCMV) US28 gene product, pUS28, is a G protein-coupled receptor that interacts with both CC and CX3C chemokines. To date, the role of pUS28 in immune evasion and cell migration has been studied only in cell types that can establish productive HCMV infection. We show that HCMV can latently infect THP-1 monocytes and that during latency US28 is transcribed. We also show that the transcription is sustained during differentiation of the THP-1 monocytes. Since cells expressing pUS28 were previously shown to adhere to immobilized CX3C chemokines (C. A. Haskell, M. D. Cleary, and I. F. Charo, J. Biol. Chem. 275:34183–34189, 2000), we hypothesize that latently infected circulating monocytes express pUS28, thereby enabling adhesion of these cells to CX3C-exposing endothelium. Consequently, the US28-encoded chemokine receptor may play an important role in dissemination of latent HCMV.
The human cytomegalovirus (HCMV) genome contains four G-protein-coupled receptor-like genes, UL33, UL78, US27, and US28 (8, 11). The US28-encoded G-protein-coupled receptor pUS28 appears to be functional in many different respects. (i) It was identified as a chemokine receptor capable of binding the CC chemokines RANTES, MCP-1, MCP-3, MIP1-α, and MIP1-β (2, 10,21, 26), as well as soluble forms of the CX3C chemokine fractalkine (18). Upon interaction with the CC chemokines, pUS28 induces Ca2+ mobilization and extracellular signal-related kinase 2 activation (2, 10, 21,26). In addition, transient expression of pUS28 leads to constitutive activation of both phospholipase C and NF-κB signaling (6). In this system, fractalkine acts as an inverse agonist for pUS28 (6). (ii) Transient, high-level expression of pUS28 in smooth muscle cells induces chemokinesis in the presence of MCP-1 and chemotaxis within a RANTES gradient (38). (iii) Expression of pUS28 leads to internalization of RANTES and MCP-1 in both HCMV-infected fibroblasts and endothelial cells (3, 4). Thus, this receptor acts as a CC-chemokine sink, possibly enabling HCMV-infected cells to evade immune surveillance. (iv) The chemokine receptor pUS28 is a coreceptor for several human immunodeficiency virus strains (27, 32) and can elicit cell-to-cell fusion upon interaction with several types of viral envelope proteins (33). (v) The expression of pUS28 on the cell surface of murine pre-B-cell line 300-19 can establish cell rolling and adhesion to a fixed fractalkine surface (15). If pUS28 is expressed on the surface of HCMV-infected leukocytes, then it could play an important role in virus trafficking from the circulation to inflammatory sites. Particularly, the receptor could mediate adhesion of circulating HCMV-infected monocytes to endothelial cells and subsequent transmission of HCMV from infected monocytes to endothelial cells or, alternatively, transendothelial migration of infected monocytes toward subendothelial tissues. To date, only a few reports on US28 expression exist. US28-specific transcripts were detected in peripheral blood mononuclear cells of some naturally infected individuals (31). In addition, US28-specific transcripts were detected from 2 h to at least 24 h post-HCMV infection (p.i.) of human foreskin fibroblasts (HFF) in vitro (4,42, 44). Finally, US28-specific transcripts were detected in HCMV-infected myeloid cell cultures—at 4 h p.i. in infected U373 MG astrocytoma cells and at 4 and 24 h p.i. in infected THP-1 monocytic cells (44). Interestingly, in many cell types of myeloid origin, HCMV establishes latent infection, i.e., persistence of nonreplicating viral genomic DNA in infected cells. Latent infection occurs in progenitors of granulocytes, macrophages, and dendritic cells (13, 20), as well as in peripheral blood monocytes (5) and immature macrophages (37). Recently, HCMV major immediate early (MIE) gene-derived transcripts were identified in latently infected monocyte or granulocyte progenitor cells (20), as well as in bone marrow (BM)-derived CD33+ CD14+ and CD33+CD15+ cells (13). These cytomegalovirus latency-associated transcripts (CLTs) have either a sense or an antisense orientation relative to the conventional MIE coding region. The sense CLTs have two specific transcription start sites (LSS1 and LSS2) within the MIE promoter-enhancer region, upstream of the productive-infection transcription start site (PSS) (Fig.1). The function of neither CLTs nor their corresponding gene products is known. To date, CLTs are the only transcripts identified in latently infected cells. Here we report a system for studying HCMV gene expression in latently infected monocytic THP-1 cells. Using this system, we demonstrate that US28, like the MIE gene, is transcribed during both latent and productive HCMV infection.
RT-PCR primers and their target transcripts. Schematic representation of the HCMV Toledo genome and the relative positions of the MIE gene and US28. Black boxes represent repeat regions of the Toledo genome. The US28-specific transcripts and CLTs are indicated below the genome at a smaller scale by open arrows. The positions and polarity of the US28-, as well as the CLT-specific RT-PCR primers (Table 1) are indicated by black arrows. Transcription starts of the sense CLTs (LSS1 and LSS2) and the PSS are indicated by arrowheads.
Cells and virus.Primary HFF, the human fibroblast cell line MRC5, and the myeloid cell lines K562, KG1a, HL-60, U937, and THP-1 cells were cultured as described previously (4, 14, 44). Stocks of wild-type (Toledo) and a US27-US28 double deletion mutant virus (RV101) (4) were generated by propagation in HFF and MRC5 (4). For reverse transcription (RT)-PCR analysis, virions from HFF culture medium samples, each containing 3 × 106 PFU of either Toledo or RV101 HCMV per ml, were separated from cellular debris by low-speed centrifugation (10 min at 10,000 × g , 4°C) and pelleted by ultracentrifugation (30 min at 100,000 × g , 4°C). A sample of Toledo virus was inactivated by irradiation with 4.7 J of UV using a Stratalinker UV Crosslinker 1800 (Stratagene Cloning Systems, Amsterdam, The Netherlands).
RT-PCR.Primers specific for either the HCMV US28 gene (US28F and US28R), both sense (CLTF1, CLTF2, CLTR1, and CLTR2) and anti-sense CLTs (ANTICLTF and ANTICLTR); the HCMV DNA polymerase gene UL54 (DNAPOLF* and DNAPOLR*, and a supplemental set, DNAPOLF and DNAPOLR); or the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (GAPDHF and GAPDHR) were obtained from Eurogentec (Eurogentec, Seraing, Belgium). The nucleotide sequences of these primers are shown in Table 1. The positions of the CLT-specific and US28-specific primers relative to their corresponding transcripts are shown in Fig. 1. Primers CLTR1 and CLTR2 were derived from nucleotide sequences within CLT exon 3 and exon 2, respectively (Fig. 1). Primers CLTF1 and CLTF2 were derived from the sequence between the latency-specific transcription start site 2 (LSS2) and the PSS (Fig. 1). The sequences of primer ANTICLTF and ANTICLTR are collinear with MIE intron sequences and can therefore not detect mature sense CLTs (Fig. 1). However, it may be possible that these primers amplify either sense-polarized MIE-specific transcripts that have not been identified before, or immature CLTs containing intron sequences. Both possibilities should be taken into consideration for every reference to antisense CLTs in this report. Each target sample for RT-PCR was obtained by isolation of poly(A)+ RNA from 5 × 106 cells using a QuickPrep Micro mRNA purification kit (Amersham Pharmacia Biotech, Saclay, France). Subsequently, poly(A)+ RNA samples were treated with DNase I (Amersham Pharmacia Biotech) according to the manufacturer's protocol. They were then reverse transcribed using an Advantage RT-for-PCR Kit (Clontech-Ozyme, Montigny-Le-Bretonneux, France). RT was primed with oligo(dT) primers included in the kit. Aliquots of the resulting cDNA that corresponded to 2 × 105 cells were added to PCR mixtures. PCR mixtures were prepared using AdvanTaq Plus DNA Polymerase kit (Clontech-Ozyme) according to the manufacturer's protocol. Thermal cycling conditions were similar for PCRs with all primer sets mentioned (2 min of denaturation at 94°C, followed by 50 cycles of 5 s at 94°C and 30 s at 70°C), except for those for the GAPDH- and HCMV DNA polymerase-specific (DNAPOLF and DNAPOLR) primer sets, which were 2 min of denaturation at 94°C, followed by 50 cycles of 5 s at 94°C and 30 s at 68°C. Thermal cycling was done with a GeneAmp PCR System 9600 thermal cycler (Perkin-Elmer, Courtaboeuf, France). PCR products were visualized by agarose gel electrophoresis and ethidium bromide staining.
PCR primers used in this study
Indirect immunocytometry.The following monoclonal antibodies (MAb) were used for HCMV antigen detection in both MRC5 and THP-1 cells: (i) MAb E13 (a kind gift from M. C. Mazeron, Hôpital Lariboisière, Paris, France), which detects both HCMV immediate early 1 and 2 antigens; (ii) MAb F6a, which detects the HCMV early antigen ppUL83 (pp65) (Michelson et al., unpublished data); and (iii) an anti-pp150 MAb (a kind gift from H. P. Hartus, Universität Erlangen-Nürnberg, Erlangen, Germany), which detects the HCMV late antigen ppUL32 (pp150) (17). The secondary antibodies used were fluorescein-conjugated sheep anti-mouse immunoglobulin G (Amersham Pharmacia Biotech) for the detection of MAb F6a and fluorescein-conjugated goat anti-mouse immunoglobulin G (CALTAG Laboratories, Burlingame, Calif.) for the detection of MAb E13 and MAb anti-pp150. Samples containing 106 uninfected or HCMV-infected cells of either MRC5 or THP-1 origin were permeabilized in permeabilization buffer (phosphate-buffered saline with 0.2% bovine serum albumin [Sigma-Aldrich Chemie, Steinheim, Germany] and 0.05% saponin [Sigma-Aldrich Chemie]) for 20 min at room temperature. All subsequent incubations were done in permeabilization buffer at 4°C. Cells were incubated for 30 min with 5 μg of primary MAb per ml, washed two times, and stained with secondary MAb according to the manufacturer's protocol. Finally, the cells were fixed in phosphate-buffered saline with 4% formaldehyde and subjected to flow cytometry using a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, La Pond-de Claix, France). Data were analyzed using the CELL Quest flow cytometry analysis program (version 3.3; Becton Dickinson Immunocytometry Systems).
Detection of HCMV-specific transcripts in various infected myeloid cell lines.Previously, both HCMV-specific DNA and RNA in latently infected individuals were detected using PCR techniques. Genomic HCMV DNA can be detected in approximately 0.01% of mononuclear cells in blood and BM samples from naturally infected individuals (19, 36,39), whereas between 0.01 and 0.001% of BM-derived CD33+ CD14+ and CD33+CD15+ cells contain CLTs (13). Similar to the techniques used for the detection of genomic HCMV DNA in these cells, we developed an RT-PCR system to enable detection of HCMV-specific transcripts during latent infection in vitro. In order to determine the type of cell that would be suitable for establishing latent HCMV infection in vitro, we screened several infected myeloid cells lines for the presence of US28-specific transcripts and antisense CLTs. Each of these lines represents a specific differentiation stage in hematopoiesis toward monocytes: K562 cells (very early pluripotent hematopoietic stem cells), KG1a (pluripotent CD34+hematopoietic stem cell-like), HL-60 (monocyte or granulocyte progenitor-like), U937 (promonocytic), and THP-1 (monocytic) (reviewed by Harris 14). Cells (5 × 107 per sample) were infected with HCMV strain Toledo at a multiplicity of infection (MOI) of 4. On day 4 p.i., culture medium was refreshed. Finally, on day 8 p.i., cells were harvested and subjected to RT-PCR. Whereas all cDNA samples were positive for GAPDH transcripts with the expected size of 235 bp (Fig.2A, lanes 2 to 11, upper panel), only the sample containing cDNA from the infected THP-1 monocytic cell line was PCR positive for both US28 transcripts (Fig. 2A, lane 10, middle panel; expected size = 298 bp) and antisense CLT (Fig. 2A, lane 10, lower panel; expected size = 469 bp). Fresh samples were subjected to a new round of PCR in which various combinations of sense CLT-specific primer pairs were included (Fig. 1). As shown in Fig. 2B, PCR mixtures containing either primer pairs CLTF1 and CLTR1, CLTF2 and CLTR1, or CLTF1 and CLTR2 each produced products that matched the expected sizes of 436, 368, and 297 bp, respectively (Fig. 2B, lanes 2 to 4). In contrast, PCR samples containing the primer combination of CLTF2 and CLTR2 (expected PCR product size = 229 bp) remained negative, possibly due to incompatibility of the PCR primer pair (Fig. 2B, lane 5). Nonetheless, these results indicate that HCMV-infected THP-1 cells harbor US28-specific transcripts, as well as sense and anti-sense CLTs at day 8 p.i. We were also able to detect these transcripts at a later time point. Both US28- and antisense CLT-specific RT-PCR signals were obtained from samples of infected THP-1 cells at day 15 p.i. (Fig. 2C, lanes 7 and 10). We therefore focused on HCMV gene expression in THP-1 cells in subsequent experiments.
The US28 and MIE genes are transcribed in HCMV-infected THP-1 cells. The figure shows ethidium bromide-stained 2% agarose gels in which RT-PCR samples were separated. All agarose gel images shown in this report were digitized and contrast-inverted for clarity using a video scanner (Virbert Lourmat, Marne la Vallée, France). Molecular weight (MW) marker sizes are indicated on the left of each panel, and the primer sets corresponding to each panel are indicated on the right. Black arrowheads denote the relevant PCR products. (A) Detection of GAPDH-specific transcripts, antisense CLT-specific, and US28-specific transcripts in a panel of HCMV-infected myeloid cell types at day 8 p.i. The cell types used are indicated above the panel using the following abbreviations: H, HL-60; K5, K562; KG, KG1a; T, THP-1; U, U937. −RT, sample not treated with reverse transcriptase. (B) Detection of sense CLTs from HCMV-infected THP-1 cells at day 8 p.i. The primer combinations that were used in this experiment (see also Table 1 and Fig. 1) are indicated above each lane. (C) Detection of GAPDH-specific transcripts, anti-sense CLT-specific, and US28-specific transcripts in HCMV-infected THP-1 cells at day 15 p.i. M, mock infected; I, HCMV Toledo infected.
US28 is transcribed de novo in THP-1 cells at late time points p.i.Although we were able to detect viral transcripts in THP-1 cells by RT-PCR, we did not exclude the possibility that these transcripts could be deposited by virions into the target cells upon inoculation. To examine whether US28-specific mRNA can be deposited in THP-1 cells, we prepared virions from both HCMV Toledo and a (negative control) US27-US28 deletion mutant strain, RV101 (4), and subjected these to RT-PCR analysis. Poly(A)+ RNA was isolated from the virion preparations and subsequently reverse transcribed and included in PCR mixtures containing either US28- or GAPDH-specific primers. Surprisingly, both US28- and GAPDH-specific transcripts were detected in mixtures from the Toledo samples (Fig.3A), indicating that the viral inoculum contains both viral and host cell-specific poly(A)+ RNA. Consequently, it is possible that both types of RNA can be deposited either at the cell surface of inside THP-1 cells upon inoculation. To examine transfer of mRNA from the inoculum to the target cells, we infected THP-1 cells that were treated with actinomycin D (10 μg/ml, 1 h before, during, and after infection) to block de novo RNA synthesis. In addition, untreated THP-1 cells were inoculated with either UV-inactivated or intact HCMV. Samples of these THP-1 cultures were analyzed by RT-PCR at 1 h and 1 day p.i. Both US28- and GAPDH-specific transcripts could be detected in actinomycin-D-treated cells at 1 h p.i. (Fig. 3B, lane 1), whereas the actinomycin-D-treated cells at day 1 p.i. were PCR negative (Fig.3B, lane 4). This indicates that although both US28- and GAPDH-specific mRNA are deposited either at the cell surface or inside THP-1 cells upon infection, the transcripts cannot persist for 1 day in these cells. Similar results were obtained by infection with UV-inactivated virus. US28-specific transcripts could be detected in THP-1 cells treated with inactivated virus at 1 h p.i. but not at day 1 p.i. (Fig. 3B, lanes 2 and 5, respectively). In contrast, transcripts were detected at each of these time points in cells infected with untreated virus (Fig. 3B, lanes 3 and 6). Taken together, we conclude that US28 is transcribed de novo in THP-1 cells at very late time points (8 to at least 15 days) p.i.
US28-specific transcripts are deposited either inside or at the surface of THP-1 cells immediately after infection but transcribed de novo at later times p.i. RT-PCR samples are visualized on agarose gels as described in Fig. 2. (A) Detection of viral and cellular poly(A)+ RNA in virus inoculum from HCMV-infected fibroblasts. The primer sets that were included in the RT-PCR samples are indicated on top of the panel. (B) Detection of US28-specific transcripts either in actinomycin-D-treated HCMV-infected THP-1 cells or in THP-1 cells infected with UV-inactivated HCMV. The primer sets that were included in the RT-PCR samples are indicated at the right of each panel. Samples from which reverse transcriptase enzyme was omitted remained PCR negative (data not shown). Abbreviations: −RT, sample not treated with reverse transcriptase; +RT, reverse transcriptase treated; Δ, HCMV RV101 (US27-US28 deletion mutant) virions; WT, wild-type HCMV; h, hours p.i.; d, days p.i.; A, actinomycin D treated; UV, UV-treated; NT, not treated.
Detection of lytic HCMV antigens in infected THP-1 cells.HCMV infection in unstimulated THP-1 cells was previously defined as abortive. This was based on experiments indicating the absence of HCMV antigen expression (16, 22, 41, 44), probably due to repression of the MIE promoter-enhancer (35). However, subtle differences in culture conditions may result in the presence of small numbers of differentiated cells harboring replicating HCMV in an infected THP-1 population. Consequently, US28-specific transcripts and CLTs could be synthesized in a small fraction of productively rather than latently infected cells. To determine whether in our system a fraction of the HCMV-treated THP-1 culture contains reproductively infected cells, we subjected a large quantity (5 × 105 per sample) of HCMV-treated THP-1 cells to immunocytometric analysis. An assay was set up to enable detection of antigens representing all phases of the lytic HCMV infection program, including pUL122 and pUL123 (the MIE 1 and 2 transactivator antigens), ppUL83 (a dominant early-phase tegument phospoprotein pp65), and ppUL32 (a dominant late-phase tegument phosphoprotein pp150). By using this assay, each of these antigens was detected at day 2 p.i. in MRC5 fibroblasts that were infected at an MOI of 0.1 but not at day 7 p.i. in THP-1 monocytes that were infected at an MOI of 4 (Fig.4). These results indicate that the absence of lytic HCMV antigens in THP-1 cells is inadequate to define abortive infection, considering that both US28-specific transcripts and CLTs were detected in these cells. Instead, the detection of gene transcription in the absence of lytic HCMV antigens may indicate latent infection. Nevertheless, productively infected THP-1 cells may be present at levels below the level of antigen detection by immunocytometry. Therefore, as described in the section below, an alternative standard is needed to determine whether the US28-specific transcripts occur in productively or latently infected cells.
Expression of lytic-phase antigens in HCMV-infected THP-1 cells at day 7 p.i. The top panels show immunocytometric histograms of HCMV-infected MRC5 fibroblasts (total cell count = 10,000) at day 2 p.i. The lower panels show histograms of infected THP-1 cells (total cell count = 500,000) at day 7 p.i. FL1-H, relative intensity of fluorescence. Dotted lines represent uninfected cells.
Quantitative assessment of HCMV transcripts in infected THP-1 cells.Latent HCMV infection, as opposed to productive infection, implicates the absence of factors enabling viral replication, possibly including those required for viral genomic DNA replication. Here, we set out to determine whether US28-transcribing HCMV-infected THP-1 cells contain such factors. For this purpose, we designed an RT-PCR assay to compare the transcription levels of US28 with those of the UL54 HCMV DNA polymerase gene in infected THP-1 cells. Initially, the sensitivity of both US28- and UL54-specific RT-PCR assays (each utilizing either US28F-US28R or DNAPOLF*-DNAPOLR*, respectively [Table 1]) were determined. This was done by applying RT-PCR to target cDNA serially diluted in a cDNA background corresponding to 2 × 105 of uninfected THP-1 cells. Positive RT-PCR signals were obtained in samples initially containing a minimum of 10 copies of either US28specific (Fig. 5A, lanes 1 to 5, upper panel) or UL54-specific (Fig. 5A, lanes 1 to 5, lower panel; expected size = 245 bp) cDNA. These results indicate that the US28- and UL54-specific RT-PCR assays have similar sensitivities. Subsequently, we determined the amount of HCMV-infected THP-1 cells containing either US28- or UL54-specific transcripts at day 7 p.i.: cDNA samples were obtained from a panel of HCMV-treated THP-1 cells serially diluted with untreated cells corresponding to a total of 2 × 105 cells. These samples were subjected to RT-PCR analysis. Consequently, we found that the HCMV-treated THP-1 cell culture could be diluted at least 10-fold before the corresponding US28-specific RT-PCR signal was lost (Fig. 5, lanes 1 to 6, upper panel). This indicates that US28 is transcribed in at least 1 out of 2 × 104 HCMV-treated THP-1 cells. Surprisingly, we were also able to detect UL54-specific transcripts in HCMV-infected THP-1 cells. However, these transcripts could only be detected in the undiluted culture sample (Fig. 5, lanes 1 to 6, middle panel). Since both assays were shown to have similar sensitivities, we conclude that US28-transcribing cells are in 10-fold excess of UL54-transcribing cells within an HCMV-infected THP-1 culture at day 7 p.i. Next, we set out to compare the levels of US28 and UL54 transcription in the total cDNA fraction corresponding to 2 × 105 THP-1 cells. For this purpose, a series of diluted cDNA samples from THP-1 cells at day 7 p.i. was subjected to RT-PCR analysis. In effect, US28-specific transcripts could be detected in cDNA samples that were diluted up to 1,000-fold (Fig. 5C, lanes 1 to 6, upper panel). In contrast, UL54-specific transcripts could only be detected in undiluted samples (Fig. 5C, lanes 1 to 6, middle panel), indicating that the level of US28 transcription is 1,000-fold that of UL54.
Quantitative comparison of US28 and HCMV DNA polymerase gene transcription in HCMV-infected THP-1 cells at day 7 p.i. RT-PCR samples are visualized on agarose gels as described in Fig. 2. The primer sets that correspond to each of the panels are indicated on the right of each panel. Black arrowheads denote the relevant PCR products. (A) Sensitivity of RT-PCR for the detection of US28 and HCMV DNA polymerase cDNA. The amount of target cDNA molecules that was included in each RT-PCR sample is indicated at the top. (B) Quantification of HCMV-infected THP-1 cells transcribing either US28 or the HCMV DNA polymerase gene at day 7 p.i. The quantities on top correspond to the amount of cells that were taken from the original HCMV-infected THP-1 culture and mixed with uninfected THP-1 cells to a total of 2 × 105 prior to cDNA preparation and RT-PCR. (C) Quantification of US28- and HCMV DNA polymerase-specific transcripts in untreated and PAA-treated HCMV-infected THP-1 cells at day 7 p.i. The dilution factor of each cDNA sample relative to an initial sample representing 2 × 105 cells is indicated on the top. Abbreviations: M, mock-infected; I, HCMV-infected; I/PAA, infected and PAA-treated; −RT, sample not treated with reverse transcriptase.
Since the amount of cells transcribing US28 was estimated to be 10-fold that of UL54-transcribing cells, it is possible that the HCMV-treated THP-1 culture contains both a latently and a productively infected fraction. Alternatively, the US28-positive–UL54-negative fraction could represent THP-1 cells that absorbed HCMV particles that were passed on by neighboring UL54-positive cells at a late time point in culture. The US28-positive–UL54-negative fraction might therefore be productively infected, albeit at an early stage of infection in which UL54 transcription is not yet manifest. To determine whether US28 transcription is the result of such a productive infection phenomenon, we assessed US28 transcription in HCMV-treated THP-1 cells treated with a viral DNA replication inhibitor. THP-1 cells were infected as described above. At 4 h p.i., phosphonoacetic acid (PAA) was added to the culture medium at a final concentration of 200 μg/ml. PAA treatment was maintained until the cells were harvested at day 7 p.i. The inhibitory potential of PAA was confirmed by assessing inhibition of pp150 synthesis in HCMV-infected MRC5 fibroblasts using immunocytometry (data not shown). RT-PCR was performed on serially diluted cDNA samples derived from HCMV-infected PAA-treated THP-1 cells. Interestingly, US28-specific signals were obtained in samples diluted as much as 1,000-fold (Fig. 5C, lanes 7 to 12, upper panel), similar to what was found for infected cells that were not treated with PAA (Fig. 5C, lanes 1 to 6, upper panel). To check whether PAA treatment had no effect on the natural cellular transcription level, the dilution series from both untreated and PAA-treated cells were subjected to RT-PCR using GAPDH-specific primers. Levels of GAPDH transcription were found to be similar for both samples (Fig. 5C, lanes 1 to 12, lower panel), indicating that US28 transcript levels are also similar. Surprisingly, RT-PCR signals for UL54 could not be observed in cDNA samples from PAA-treated cells, whereas the undiluted sample of untreated HCMV-infected cells was RT-PCR positive (Fig. 5, lanes 1 to 12, middle panel). Although PAA treatment affects HCMV DNA polymerase activity, we did not expect UL54 transcript levels to be lower in PAA-treated cells than in untreated cells. This might indicate that UL54 transcription is propelled by a late-phase positive feedback mechanism. We did not investigate this possibility further. Nevertheless, we have shown that (i) US28 is transcribed in HCMV-infected THP-1 cells at least until day 15 p.i., (ii) the amount of US28-transcribing THP-1 cells is approximately 10-fold that of UL54-transcribing cells at day 7 p.i., and (iii) detection of US28 transcription is irrespective of viral replication in HCMV-infected THP-1 cells. These results indicate that US28 is transcribed in latently infected THP-1 monocytes.
US28 transcription in differentiated THP-1 cells.In addition to demonstrating that the HCMV US28 gene is transcribed during latent infection in undifferentiated THP-1 monocytes, we set out to determine whether US28 transcription also occurs in differentiated cells. Previously, HCMV-infected monocyte-derived macrophages were shown to support viral replication (22, 37, 40). Therefore, we also assessed the replication status of the HCMV-infected cells by determining whether the HCMV DNA polymerase gene UL54 would be upregulated upon differentiation. First, THP-1 cells were infected with HCMV as described above, and then a culture sample was treated with phorbol-12-myristate-13-acetate (PMA) (50 ng/ml) from 0 to 72 h p.i. In contrast to untreated THP-1 cells, PMA-treated THP-1 cells became adherent after several hours of stimulation (data not shown). Such an adhesion phenotype is typical for differentiation of monocytic cells (14). At days 1, 5, and 7 p.i., both US28 and UL54 transcription were assessed in cDNA samples derived from untreated and PMA-treated THP-1 cultures. Alternative primers for UL54 transcript detection, DNAPOLF and DNAPOLR (Table 1), were used for RT-PCR, resulting in an assay less sensitive than RT-PCR with the aforementioned DNAPOLF* and DNAPOLR* primers (Table 1). As a result, UL54-transcripts could not be detected in undifferentiated HCMV-infected THP-1 cells (Fig. 6, lanes 1 to 3, middle panel). In contrast, by using these primers, UL54 transcripts were detected in differentiated THP-1 cells at all time points (Fig. 6, lanes 4 to 6, middle panel). These results confirm upregulation of UL54 transcription and possibly HCMV replication upon differentiation of THP-1 cells. Finally, we were able to detect US28 transcript in both untreated and PMA-treated HCMV-infected cells at all time points mentioned (Fig. 6, lanes 1 to 6, upper panel). We therefore conclude that, in addition to transcription in latently infected THP-1 monocytes, US28 transcription is sustained in infected THP-1 cells throughout differentiation into adherent, macrophage-like cells.
Transcription of US28 and the HCMV DNA polymerase gene in undifferentiated (untreated) and differentiated (PMA-treated) THP-1 cells. RT-PCR samples are visualized on agarose gels as described in Fig. 2. The primer sets that correspond to each of the panels are indicated on the right. The time points (p.i.) at which samples were taken for RT-PCR are shown at the top. d, days p.i.
To date, both US28 transcripts and CLTs are the only transcripts identified in myeloid cells that are latently infected with HCMV. Yet, they are also present in productively infected cells (4, 24, 42,43, 44) and are therefore not explicit markers of latency. To determine whether cells are latently infected with HCMV, the presence of either viral DNA or RNA from candidate latency-associated genes should be demonstrated in parallel to the absence of virus production. In this report, we considered the presence of HCMV DNA polymerase UL54 transcripts to be a determinant of productive infection. However, this determinant could prove to be too stringent; in addition to being a marker for productive infection, it could indicate viral DNA replication during latency, such as in a self-renewing HCMV-infected myeloid cell population. Nevertheless, we showed that the majority of US28-transcribing THP-1 cells does not contain UL54-specific transcripts. Moreover, US28 transcription levels were not affected in the presence of an HCMV DNA replication inhibitor. These findings indicate that this majority of US28-transcribing THP-1 cells are latently infected. Similar conditions could be used as a standard for identifying other latency-associated transcripts. Thus, a strict definition of latency could comprise (i) the absence of UL54-specific transcripts and (ii) candidate HCMV gene transcription irrespective of viral DNA replication. Nonetheless, UL54 transcription does not necessarily exclude latency. Its role in viral genome replication during latent infection will have to be addressed in future studies.
We demonstrated that the lytic phase antigens IE1-IE2, pp65, and pp150 were present at levels below the detection level in undifferentiated THP-1 cells and that HCMV DNA polymerase UL54 transcription levels were higher in differentiated cells than in undifferentiated THP-1 cells. Similarly, it was previously shown that MIE protein levels are higher in differentiated THP-1 cells that were infected with HCMV than in less differentiated cells (23). In addition, the generation of HCMV particles could not be demonstrated in monocytes, whereas replication was demonstrated in monocyte-derived macrophages and dendritic cells (34, 37). Thus, the relative degree of monocyte differentiation may be an important factor for regulation of HCMV gene expression. This explains why in our study US28 transcripts were not detected in K562, KG1a, HL-60, and U937 cells, which are less differentiated than the monocyte-type THP-1 cells. In addition to containing US28 and the MIE gene, the HCMV genome may contain many other genes that could concert latency and replication in myeloid cells. However, the low amount of infected (relative to unifected) myeloid precursors and the low-level transcription of HCMV genes during latent infection currently limit the study of HCMV gene expression in vivo to RT-PCR analysis. In the future, a sensitive immunological assay will have to be developed to confirm expression of viral proteins during latent HCMV infection in monocytes.
In this report we demonstrated that HCMV inoculum contains both viral and host cell RNA. We did not determine further whether the poly(A)+ RNA originated from virions or cellular debris associated with the virions. However, Greijer et al. (12) recently reported that both viral and host cell RNA molecules can be detected in sucrose density step-gradient-purified HCMV virions. Furthermore, they showed that virion-associated RNA can be deposited either at the surface of or inside inoculated fibroblasts upon inoculation. RNA could be detected after 1 h, but not at 4 h or at later time points p.i.—similar to our observation that virus-associated transcripts can be deposited in or on THP-1 cells upon infection but that these transcripts do not persist in or on these cells for more than 1 day p.i.
Kledal et al. (18) reported that pUS28 is capable of binding soluble forms of the CX3C chemokine fractalkine (or neurotactin) with subnanomolar affinity. This chemokine exists both in a soluble and a cell membrane-bound form. The membrane-bound form consists of a chemokine domain, a mucin stalk, a transmembrane and an intracellular domain (1, 28). Interestingly, it appears to combine the properties of both chemoattractants and adhesion molecules. Fractalkine is expressed at the surface of activated endothelium, thereby enabling leukocyte capture, firm integrin-independent adhesion, and attachment of circulating monocytes under flow conditions (7,9). Additionally, it was shown that pUS28-expressing 300-19 murine pre-B cells could be captured by a fractalkine- or stalk-coated surface under physiological flow conditions (15). Although many CC chemokines can bind pUS28 (4, 10, 21, 26), fractalkine has the highest affinity, as well as a slow off-rate for this receptor (15, 18). In this report it was shown that US28 is transcribed in monocytic cells that harbor latent HCMV. Thus, expression of the US28 gene could play a role in the tethering of latently infected circulating monocytes to endothelial tissue expressing membrane-bound fractalkine. Alternatively, pUS28 could guide latently infected monocytes to other tissues expressing fractalkine or CC chemokines by chemotaxis. Fractalkine is shed from intestinal epithelial cells upon stimulation with interleukin 1β (25). Moreover, membrane-bound fractalkine is expressed by skin-derived dendritic and Langerhans cells (30), as well as by skin endothelial cells and dermal dendrocytes (29). These cell types are permissive for productive HCMV infection and therefore potential targets for monocyte-mediated HCMV dissemination. Finally, monocytes mature into either macrophages or dendritic cells upon migration into target tissues (14). We showed that transcription of US28 persists during differentiation of THP-1 cells by stimulation with PMA. It is therefore possible that pUS28 continues to guide infected monocytes during their differentiation in the target tissues. Further studies require the development of suitable antibodies against the US28 gene product. By using these antibodies, pUS28 surface expression on latently infected monocytes from healthy individuals could be confirmed, as well as pUS28-dependent interaction of these cells with the endothelium and other potential target tissues. Hence, an intriguing function of pUS28 in the dissemination of latent HCMV may be established.
We thank Maria-Paola Landini for the kind gift of Toledo virus and Tom Jones for providing the deletion mutant RV101. We also thank Françoise Bachelerie and Agustin Valenzuela-Fernandez for critically reading the manuscript.
P.S.B. is a beneficiary of a research fellowship from the European Molecular Biology Organization, Heidelberg, Germany. This work was supported by the Agence National de la Recherche contre le SIDA.
Received 29 November 2000.
Accepted 29 March 2001.
↵* Corresponding author. Present address: Department of Medical Microbiology, University Hospital of Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. Phone: 31 43 3876644. Fax: 31 43 3876643. E-mail: pbe{at}lmib.azm.nl.
Bazan J. F.,
Bacon K. B.,
Hardiman G.,
Wang W.,
Soo K.,
Rossi D.,
Greaves D. R.,
Zlotnik A.,
Schall T. J.
(1997) A new class of membrane-bound chemokine with a CX3C motif. Nature 385:640–644.
Billstrom M. A.,
Johnson G. L.,
Avdi N. J.,
Worthen G. S.
(1998) Intracellular signaling by the chemokine receptor US28 during human cytomegalovirus infection. J. Virol. 72:5535–5544.
Lehman L. A.,
(1999) Depletion of extracellular RANTES during human cytomegalovirus infection of endothelial cells. Am. J. Respir. Cell Mol. Biol. 21:163–167.
Bodaghi B.,
Jones T. R.,
Zipeto D.,
Vita C.,
Sun L.,
Laurent L.,
Arenzana-Seisdedos F.,
Virelizier J. L.,
Michelson S.
(1998) Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells. J. Exp. Med. 188:855–866.
Bolovan-Fritts C. A.,
Mocarski E. S.,
Wiedeman J. A.
(1999) Peripheral blood CD14+ cells from healthy subjects carry a circular conformation of latent cytomegalovirus genome. Blood 93:394–398.
Casarosa P.,
Bakker R. A.,
Verzijl D.,
Navis M.,
Timmerman H.,
Leurs R.,
Smit M. J.
(2001) Constitutive signalling of the human cytomegalovirus-encoded chemokine receptor US28. J. Biol. Chem. 276:1133–1137.
Chapman G. A.,
Moores K. E.,
Gohil J.,
Berkhout T. A.,
Patel L.,
Green P.,
Macphee C. H.,
Stewart B. R.
(2000) The role of fractalkine in the recruitment of monocytes to the endothelium. Eur. J. Pharmacol. 392:189–195.
Chee M. S.,
Satchwell S. C.,
Preddie E.,
Weston K. M.,
Barrell B. G.
(1990) Human cytomegalovirus encodes three G protein-coupled receptor homologues. Nature 344:774–777.
Fong A. M.,
Robinson L. A.,
Steeber D. A.,
Tedder T. F.,
Yoshie O.,
Imai T.,
Patel D. D.
(1998) Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow. J. Exp. Med. 188:1413–1419.
Gao J. L.,
Murphy P. M.
(1994) Human cytomegalovirus open reading frame US28 encodes a functional beta chemokine receptor. J. Biol. Chem. 269:28539–28542.
Gompels U. A.,
Nicholas J.,
Lawrence G.,
Jones M.,
Thomson B. J.,
Martin M. E.,
Efstathiou S.,
Craxton M.,
Macaulay H. A.
(1995) The DNA sequence of human herpesvirus-6: structure, coding content, and genome evolution. Virology 209:29–51.
Greijer A. E.,
Dekkers C. A. J.,
Middeldorp J. M.
(2000) Human cytomegalovirus virions differentially incorporate viral and host cell RNA during the assembly process. J. Virol. 74:9078–9082.
Hahn G.,
Jores R.,
Mocarski E. S.
(1998) Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc. Natl. Acad. Sci. USA 95:3937–3942.
Herzenberg L.,
Blackwell C.
Harris P.
(1996) Human myeloid cell lines. in The integrated immune system, eds Herzenberg L., Blackwell C. (Blackwell Science, Cambridge, Mass), 5th ed. IV:173.1–173.16.
Haskell C. A.,
Cleary M. D.,
Charo I. F.
(2000) Unique role of the chemokine domain of fractalkine in cell capture: kinetics of receptor dissociation correlate with cell adhesion. J. Biol. Chem. 275:34183–34189.
Huang T. H.,
Oka T.,
Asai T.,
Okada T.,
Merrills B. W.,
Gertson P. N.,
Whitson R. H.,
Itakura K.
(1996) Repression by a differentiation-specific factor of the human cytomegalovirus enhancer. Nucleic Acids Res. 24:1695–1701.
Jahn G.,
Harthus H. P.,
Broker M.,
Borisch B.,
Platzer B.,
Plachter B.
(1990) Generation and application of a monoclonal antibody raised against a recombinant cytomegalovirus-specific polypeptide. Klin. Wochenschr. 68:1003–1007.
Kledal T. N.,
Rosenkilde M. M.,
Schwartz T. W.
(1998) Selective recognition of the membrane-bound CX3C chemokine, fractalkine, by the human cytomegalovirus-encoded broad-spectrum receptor US28. FEBS Lett. 441:209–214.
Kondo K.,
Kaneshima H.,
(1994) Human cytomegalovirus latent infection of granulocyte-macrophage progenitors. Proc. Natl. Acad. Sci. USA 91:11879–11883.
Xu J.,
(1996) Human cytomegalovirus latent gene expression in granulocyte-macrophage progenitors in culture and in seropositive individuals. Proc. Natl. Acad. Sci. USA 93:11137–11142.
Kuhn D. E.,
Beall C. J.,
Kolattukudy P. E.
(1995) The cytomegalovirus US28 protein binds multiple CC chemokines with high affinity. Biochem. Biophys. Res. Commun. 211:325–330.
Lashmit P. E.,
Stinski M. F.,
Murphy E. A.,
Bullock G. C.
(1998) A cis repression sequence adjacent to the transcription start site of the human cytomegalovirus US3 gene is required to down regulate gene expression at early and late times after infection. J. Virol. 72:9575–9584.
Lee C. H.,
Lee G. C.,
Chan Y. J.,
Chiou C. J.,
Ahn J. H.,
Hayward G. S.
(1999) Factors affecting human cytomegalovirus gene expression in human monocyte cell lines. Mol. Cells 9:37–44.
Lunetta J. M.,
(2000) Latency-associated sense transcripts are expressed during in vitro human cytomegalovirus productive infection. Virology 278:467–476.
Muehlhoefer A.,
Saubermann L. J.,
Gu X.,
Luedtke-Heckenkamp K.,
Xavier R.,
Blumberg R. S.,
Podolsky D. K.,
MacDermott R. P.,
Reinecker H. C.
(2000) Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa. J. Immunol. 164:3368–3376.
Neote K.,
DiGregorio D.,
Mak J. Y.,
Horuk R.,
(1993) Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor. Cell 72:415–425.
Ohagen A.,
Li L.,
Rosenzweig A.,
Gabuzda D.
(2000) Cell-dependent mechanisms restrict the HIV type 1 coreceptor activity of US28, a chemokine receptor homolog encoded by human cytomegalovirus. AIDS Res. Hum. Retrovirus. 16:27–35.
Pan Y.,
Lloyd C.,
Zhou H.,
Dolich S.,
Deeds J.,
Gonzalo J. A.,
Vath J.,
Gosselin M.,
Dussault B.,
Woolf E.,
Alperin G.,
Culpepper J.,
Gutierrez-Ramos J. C.,
Gearing D.
(1997) Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation. Nature 387:611–617.
Papadopoulos E. J.,
Fitzhugh D. J.,
Tkaczyk C.,
Gilfillan A. M.,
Sassetti C.,
Metcalfe D. D.,
Hwang S. T.
(2000) Mast cells migrate, but do not degranulate, in response to fractalkine, a membrane-bound chemokine expressed constitutively in diverse cells of the skin. Eur. J. Immunol. 30:2355–2361.
Saeki H.,
Yamada N.,
Kawamura T.,
Saraf M. A.,
Schall T.,
Blauvelt A.,
Rosen S. D.,
(1999) Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is up-regulated upon dendritic cell maturation. Eur. J. Immunol. 29:2551–2559.
Patterson B. K.,
Landay A.,
Andersson J.,
Brown C.,
Behbahani H.,
Jiyamapa D.,
Burki Z.,
Stanislawski D.,
Czerniewski M. A.,
Garcia P.
(1998) Repertoire of chemokine receptor expression in the female genital tract: implications for human immunodeficiency virus transmission. Am. J. Pathol. 153:481–490.
Pleskoff O.,
Treboute C.,
Brelot A.,
Heveker N.,
Seman M.,
Alizon M.
(1997) Identification of a chemokine receptor encoded by human cytomegalovirus as a cofactor for HIV-1 entry. Science 276:1874–1878.
(1998) The cytomegalovirus-encoded chemokine receptor US28 can enhance cell-cell fusion mediated by different viral proteins. J. Virol. 72:6389–6397.
Riegler S.,
Hebart H.,
Einsele H.,
Brossart P.,
Sinzger C.
(2000) Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus. J. Gen. Virol. 81:393–399.
Sinclair J. H.,
Baillie J.,
Bryant L. A.,
Taylor-Wiedeman J. A.,
Sissons J. G.
(1992) Repression of human cytomegalovirus major immediate early gene expression in a monocytic cell line. J. Gen. Virol. 73:433–435.
Slobedman B.,
(1999) Quantitative analysis of latent human cytomegalovirus. J. Virol. 73:4806–4812.
Söderberg-Naucler C.,
Fish K. N.,
Nelson J. A.
(1998) Growth of human cytomegalovirus in primary macrophages. Methods 16:126–138.
Streblow D. N.,
Vieira J.,
Smith P.,
Wakabayashi E.,
Ruchti F.,
Mattison K.,
Altschuler Y.,
(1999) The human cytomegalovirus chemokine receptor US28 mediates vascular smooth muscle cell migration. Cell 99:511–520.
Tanaka N.,
Kimura H.,
Iida K.,
Saito Y.,
Tsuge I.,
Yoshimi A.,
Matsuyama T.,
Morishima T.
(2000) Quantitative analysis of cytomegalovirus load using a real-time PCR assay. J. Med. Virol. 60:455–462.
Turtinen L. W.,
Seufzer B. J.
(1994) Selective permissiveness of TPA differentiated THP-1 myelomonocytic cells for human cytomegalovirus strains AD169 and Towne. Microb. Pathog. 16:373–378.
Weinshenker B. G.,
Wilton S.,
Rice G. P.
(1988) Phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line. J. Immunol. 140:1625–1631.
Welch A. R.,
McGregor L. M.,
Gibson W.
(1991) Cytomegalovirus homologs of cellular G protein-coupled receptor genes are transcribed. J. Virol. 65:3915–3918.
White K. L.,
(2000) Human cytomegalovirus latency-associated protein pORF94 is dispensable for productive and latent infection. J. Virol. 74:9333–9337.
(1999) Kinetics of transcription of human cytomegalovirus chemokine receptor US28 in different cell types. J. Gen. Virol. 80:543–547.
Journal of Virology Jul 2001, 75 (13) 5949-5957; DOI: 10.1128/JVI.75.13.5949-5957.2001
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Avoid rushing to catch a flight at the airport and looking for airport parking. Select from a variety of park stay and fly packages in the Vancouver, BC airport area hotels. For one low price enjoy one night at a nearby hotel, shuttle service to and from the airport and car parking at the hotel while you travel. Park stay and fly packages allow you to catch your flight relaxed and rested and without the stress of rushing to the airport, getting stuck in traffic and looking for airport parking that is more expensive than a good park stay and fly package.
Excellent for this purpose. Pool & Spa not necessary for our purpose so great value.


This category includes all silicone watch straps. They come in different models, widths and colours. All watch straps come with spring bars. More details about each silicone watch strap are available on individual product page. To narrow search result to a specific width please use the navigation menu.


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Pope Francis has chosen the youth at Casal del Marmo Detention Centre for the traditional washing of the feet on Holy Thursday. This was announced in the Holy See Press Office this morning, Thursday 21 March. "As Archbishop of Buenos Aires — the communiqué reads — Cardinal Bergoglio used to celebrate" The Mass In Cena Domini "in a prison, hospital or hospice for the poor or marginalized". With the choice to go to a prison for juvenile delinquency, Pope Francis has thus decided to continue this course and to maintain the simplicity whereby he has always lived.
Therefore on 28 March the Holy Father will celebrate the Chrism Mass in the morning in St Peter's Basilica and in the afternoon at 5:30 p.m. he will go to the Correctional Facility for minors, Casal del Marmo, for the "Mass of the Lord's Supper", a celebration — continued the Press Office — characterized "by the message of the Commandment of love and by the washing of the feet". The statement also confirmed that "the other ceremonies of Holy Week will take place according to the normal custom, as will be made clear by a Notification from the Office for the Liturgical Celebrations of the Supreme Pontiff".
In the vast complex the incarcerated males are housed in two buildings with 24 rooms each; a third building houses the female section, with another 24 rooms. Scholastic activities take place there, along with exercises for the illiterate. There is also work formation with shops for carpentry, upholstery and pizzerias. For young women there is also a design studio.
Benedict XVI and John Paul II, too, visited the detention centre. Pope Benedict celebrated Mass there on 18 March 2007, the Fourth Sunday of Lent, in the chapel of the "Merciful Father". Pope John Paul went there for a visit during the afternoon of 6 January 1980, the Solemnity of the Epiphany, accompanied by his Secretary of State, Cardinal Casaroli, who was particularly involved in the pastoral care of the youth who were held in the Casal del Marmo, where he was known simply as Fr Agostino.


First Baptist Tulsa offers you the opportunity to contribute to God's work by making financial donations on-line or mobile texting.
You may contribute to FBC's General Church Budget or to a number of designated funds.
What is supported through the general budget?
The General Budget represents the annual budget approved by the church. In addition to operating and personnel costs, the General Budget supports the church's ministries to all ages, Sunday worship, evangelism, and mission efforts at the local, state, national and international level. Please contact the financial office for more information.
What are the advantages of recurring donation request?
Recurring donation requests allow you to have a stated amount automatically drawn from your bank account or charged to your debit card weekly, biweekly, or monthly. It provides not only a secure, paper-less and convenient option, but allows you to establish a disciplined giving pattern.
When will my donation be drawn from my account or charged to my debit card?
Online Giving will process your transactions on a daily basis and submit them for final processing within 24 hours. We understand that it is important to know what the status of your transaction is. Your transaction will be immediately registered in the Online Giving system and made available to you from your Online Giving pages.
Without a cancelled check or receipt, how do I document my contribution?
The First Baptist Church Finance Office mails quarterly and year-end contribution statements which serve as sufficient documentation for IRS tax purposes.
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Click on the "Online Giving" and login to your account. You can edit your profile from the 'View History' tab.
We accept stocks, bonds, property or other in-kind gifts. Call (918) 594-5079 if you have further questions or would like to donate.
We also accept clothing and other items throughout the year at both First Baptist Tulsa Caring Centers. Call (918) 594-5073 if you have further questions or to make a donation.


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Manavgat Green Canyon jeep safari tour is adrenaline and fun safari tour organized by open landrover jeeps in the Oymapınar dam (built on Manavgat river water) area where is called Green Canyon.You will be amazed by Green Canyon's beautiful view from the Taurus mountain and get action on off-road.You will have a visit to a small zoo having some local animals on our way.You will have time for swimming,canoing in the Green Lake.You can also catch fishes in the restaurant we have lunch.We recommend Green Canyon jeep safari tour having a different route than other safari tours to all safari lovers.
*Green canyon region is about 60 km from Alanya city center and takes 1-2 hours longer than normal jeep safari tour.
*If you are interested in safari toud,We recommend you firstly our normal jeep safari tour program.However,if you have already made normal Jeep safari and want to see Green canyon region,you should go this tour.
*You can pay the tour fee on the jeep to our guide.


As I come across research concerning the effects of cultivating a regular Taijiquan and/or Qigong practice, I will post summaries and links to this page. In addition to what I've listed here, you can additional resources on my Books for the Journey and Health Benefits pages.
A regular tai chi exercise regimen enlarges the brain and enhances the cognitive abilities of the elderly.
The full study, "Changes in Brain Volume and Cognition in a Randomized Trial of Exercise and Social Interaction in a Community-Based Sample of Non-Demented Chinese Elders," is published in the Journal of Alzheimer's Disease.
Qigong Reduces Stress in Computer Operators.
Chinese research indicates that the Qigong method reduces psychosomatic and physical symptoms through an effect on the sympathetic nervous system.
The aim was to investigate the effects of Qigong on stress among computer operators.
Qigong reduced noradrenaline excretion in urine (p<0.05), and influenced the heart rate and temperature, indicating reduced activity of the sympathetic nervous system. Moreover, Qigong reduced low-back symptoms (p<0.05). In conclusion, Qigong exercise may reduce stress at computerised work.
A study published in 1997 found that seniors who took 15 tai chi lessons and practiced for 15 minutes twice daily were able to significantly reduce their risk of falls. Since then, several more studies have pointed to the physical benefits of tai chi for the elderly.
One six-month study, a group of elderly people who took part in tai chi were about twice as likely to report that they were not limited in their ability to perform moderate-to-vigorous daily activities – things like walking, climbing, bending, lifting. The seniors in that study also reported better overall quality of life – in terms of bodily pain, mental health, and perceptions of health and independence.
Another study of seniors with arthritis showed that those who took a 12-week tai chi course got around better and had less pain in their legs. Yet another study found that people with arthritis who took a 12-week tai chi class had stronger abdominal muscles and better balance afterward.
Aug. 18, 2010 — Just 12 weeks of tai chi — the slow-motion Chinese martial art — relieved longstanding fibromyalgia symptoms and improved quality of life in a clinical trial.
Compared with patients who received wellness education and stretching exercises, those who practiced tai chi saw their fibromyalgia become much less severe. They also slept better, felt better, had less pain, had more energy, and had better physical and mental health, says study researcher Chenchen Wang, MD, of Tufts University School of Medicine.
It wasn't for everyone. Wang says 10% to 20% of patients randomly assigned to tai chi did not feel it helped them. But he says 50% to 60% of the patients were "really engaged," and after about eight weeks began to feel better.
Improvement was gradual but steady. Patients who benefited from tai chi asked the researchers to continue the program when the 24-week study ended.


"Our next fundraising event will be an evening with local authors Mike (writing as Alex) and Helen Walters. Titled 'Clues, Crimes and Conversations: putting Culbokie on the fiction map', it will be held on Saturday February 23rd, 7 for 7.30 at Findon Hall. In advance I've just been reading the three books by Mike in the DI Alec McKay series set on the Black Isle – and now I am completely spooked! Several locations have now assumed new identities and my imagination is in overdrive! Come and hear more – tickets (£5) are available from the Spar shop or can be reserved at event@culbokiect.org. Wine and beer will be available for purchase."


text:
Destroyed at the end of the First Age
On the upper course of the river Aros at the eastern end of Nan Dungortheb, on the border between Dor Dínen and Himlad
Sindar
Aros flowed down to the crossing from two separate sources in Ered Gorgoroth to the north
Aros ran on along the borders of Doriath to meet Sirion far to the southwest
Aros is pronounced 'a'ros'
Aros means 'bloody', referring to the river's reddish colour
Arossiach
Alphabetical: A
Updated 28 August 2020
Fords of Aros
The crossings on the upper River Aros
The River Aros had its sources in the hills of Dorthonion. Where it was met by an inflowing stream out of the same hills, an ancient east-west road crossed the river at a ford. This was the place known as the Fords of Aros, or the Arossiach in Elvish. The ford marked the boundary between two regions. To the east was Himlad, and the other lands held by the sons of Fëanor. To the west was the Silent Land of Dor Dínen, and beyond it the perilous valley of Nan Dungortheb.
The Fords of Aros seem to date back to the days before the beginning of the First Age, when the roads north of Doriath were safe to travel. After the coming of Ungoliant, and the raising of the Girdle of Melian, the lands west of the Arossiach became a haunted and dangerous place, and in later years the Fords were only used by the bravest or most desperate travellers.
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text:
2011 / Mountain - Climbing
From the director of PURE and CORE, get ready to infiltrate four of the biggest scenes in the world of climbing.
What makes a climbing mecca? The Scene takes a comprehensive look into four major epicenters of hard climbing and training, and the characters who define them by their relentless commitment to the sport. Featuring groundbreaking first ascents in bouldering, trad, and sport climbing, including Chris Sharma's Fight or Flight (5.15b) in Oliana, Spain. Filmed in stunning 4K Ultra High Definition, join an international cast of characters as they give us a glimpse into the impossible.


text:
Book Review: Our Life Is Love. The Quaker Spiritual Journey.
Anyone who has sought but not found, will be encouraged by this book. Thus Thomas Kelly:
"For God can be found. There is a last rock for your souls, a resting place of absolute peace and joy and power and radiance and security." (A Testimony of Devotion. p.17)
Marcelle Martin analyses the Quaker Spiritual Journey into ten stages, the main divisions of which are Awakening, Convincement, and Faithfulness. The ten stages fit into those; for example, the stages in Faithfulness are Leadings, The Cross, Abiding and Perfection. One half of the book consists of these stages as illustrated in the writings of early Friends, and the other half is given to illustrations from our own time. Concentration on the latter will be given here.
Martin emphasizes that people experience the Journey differently; all the same, it is easy to illustrate the ten stages from the lives of Friends. About the Awakening, Thomas Kelly wrote:
We have hints that there is a way of life vastly richer and deeper than all this harried existence, a life of unhurried serenity and power. (Our Life is Love. p170)
These hints can be reinforced by many small incidents, the effects of which may be cumulative: a time of quiet, a book, an inspiring film, comments from friends. More dramatic forms of awakening, such as illness and war, are common. At these times, people are desperate for help, wherever they may find it.
Why is it that so many seek and so few seem to find? Perhaps the answer was given by Jeremiah 29: 13 "You will seek me and find me when you seek me with all your heart." Not many put their hearts into it, but there are enough to show it works. A contemporary British Friend, Ben Pink Dandelion, writes
"I felt lifted up and cradled by what I have called God, held and reassured…It was a powerful experience for me and, delightfully, has never left me." (op. cit. p49)
Similarly, Thomas Kelly, just like St. Teresa 400 years ago:
Those who learn to abide in God`s love sometimes have the experience that they are living in two levels of reality simultaneously, interacting in daily events and at the same time abiding with God in this centre (op.cit. p. 150)
Some people are suspicious of religious experience, but that is one way in which we are given a share in the divine nature. See 2 Peter.1:4.
Exceptional among the testimonies are those of Marcelle Martin, given in twelve pages near the end of the book. She resembles the Hebrew prophets and the Desert Fathers who went out into the desert to find God. She subordinated everything to her quest. Although a contemporary American, until the age of fifty-five when she married, she never owned a house or car. For most of her life she lived without health insurance, which is perilous in the U.S. She had part-time work and was dependant on the benefactions of others. Although she felt vulnerable, she was free to do the learning, teaching, spiritual nurture, organising and writing, to which she felt led. She joined Quakers when she was 34. She wrote
A divine Light flows through all things. I felt this Light flowing through me down my arms, and out of my finger tips into the world. (op.cit. p.181)
The book sometimes seems rather dire. That may be due to Marcelle`s conviction that the cross is necessary for spiritual development, which is worth debate.
Our Life Is Love. The Quaker Spiritual Journey by Marcelle Martin is published by Inner Light Books. San Francisco. California. 2016. ISBN 978-0-9970604-1-6[paperback] pp.228. $20.
Reg Naulty, Canberra Regional Meeting


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Next month, celebrate the positive impact migration has on Sheffield as the bigger and better Migration Matters Festival returns to the city for Refugee Week. From 19 to 23 June, you'll find venues across the city hosting over 40 events in celebration of Sheffield's international community.
The five-day festival aims to unite people through the enjoyment of an eclectic mix of international and UK artists, spanning music, film, theatre, dance, spoken word, fashion and of course, food.
Now in its 20th year, Refugee Week has celebrated the various contributions, creativity and resilience of refugees across the UK. Migration Matters Festival will celebrate this and more in the city-wide event and will take place across an extended list of venues across Sheffield, spreading beyond the city centre.
The line-up for this year will include the Sheffield Carnival at Norfolk Park, the Ubuntu Fashion Show and Food Event, the award-winning father of 'dub poetry' Linton Kwesi Johnson, and the Arab Puppet Theatre Foundation from Lebanon. There'll also be six exhibitions running throughout the festival, as well as numerous workshops covering topics including storytelling and activism.
The opening party on 19 June will offer a snapshot of just what you can expect from the week, including Kuo-Shin Chuang Pangcah Dance Theatre from Taiwan, delicious food from local charity Open Kitchen Social Club and live music from The Turbans at the festival's hub, Theatre Delicatessen.
Fancy getting involved? You'll find the full line-up and more at www.migrationmattersfestival.co.uk. All events are pay as you feel so that the festival is open and available to all. Tickets can however be reserved for most events through Tickets for Good.


text:
Getafe shocks Real Madrid
Sports Network
Abdelaziz Barrada scored the match-winner in the 75th minute as Getafe shocked Real Madrid, 2-1, on Sunday at the Coliseum Alfonso Perez in La Liga.
Gonzalo Higuain fired Real in front after 26 minutes, but Juan Valera pulled a goal back in the 53rd and Barrada followed in the 75th as Getafe pulled off a huge upset in the second week of the season.
Madrid tied its opener against Valencia last week, 1-1, and lost the first leg of the Spanish Supercup to Barcelona, 3-2, midweek. Higuain converted a pass from Angel Di Maria inside the half-hour mark as Real eyed its first win.
Barrada set up the first goal with a pass to Valera, who scored to the bottom- left corner. Adrian Colunga provided the final pass on the winning goal, and Barrada made no mistake from 16 yards with a high finish to the middle.
Fabio Coentrao received his marching orders for a straight red from the bench, as Real Madrid was left frustrated and on one point - already six behind rival Barcelona - after just two weeks.
Getafe won its first match of the season.
Barcelona 2, Osasuna 1
Navarra, Spain - Lionel Messi scored two goals in a five-minute stretch late in the second half as Barcelona rallied for a 2-1 win over 10-man Osasuna on Sunday at Estadio El Sadar in La Liga.
Messi opened the season with a brace in a 5-1 win over Real Sociedad, and with his club on the verge of a surprise loss, rescued a win with goals in the 75th and 80th minutes.
Joseba Llorente put Osasuna in front in the 17th minute, and Nino almost added a second on a breakaway in the 71st but slotted just wide of the right post.
Messi took over from there, slipping a shot into the bottom left from the edge of the six-yard box in the 75th for his first goal and a shot from 16 yards to the bottom left in the 80th.
Valencia 3, Deportivo 3
Valencia, Spain - Abel Aguilar scored a brace to cancel out two goals from Roberto Soldado, and Pizzi converted the tying penalty kick in the 76th minute as Deportivo rallied for a 3-3 draw against Valencia on Sunday.
Roberto Soldado scored a brace inside the half-hour mark and Sofiane Feghouli also scored in the first half as Valencia built two two-goal leads in the first half at the Mestalla.
But Aguilar scored his second goal in the 59th to pull Deportivo within one goal for the second time, and Pizzi converted from the spot after Ricardo Costa earned his marching orders.
Valencia held on for a point with 10 men, and sits on just two points after its first two matches. Valencia tied Real Madrid in its opener. Deportivo won its opener, and has four points.
Granada 1, Sevilla 1
Granada, Spain - Alvaro Negredo scored just before halftime as 10-man Sevilla escaped Los Carmenes with 1-1 draw against Granada in La Liga on Sunday.
Diego Lopez was sent off in the 34th minute, but although Guillherme Siqueira missed the ensuing penalty, Moreno Mikel Rico put home a second chance to give the hosts a lead.
Negredo split the points when he scored two minutes before half,.
https://www.foxnews.com/sports/getafe-shocks-real-madrid
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text:
Black and White are twins with a powerful connection. After their parents' separation, though, White's father took him out of the country, their family effectively severed in two. It isn't until fifteen years have passed that White-now grown-returns to Thailand.
One day, seemingly from nowhere, White feels an agony like death. He recovers in the hospital, but they're unable to determine the cause. Then he picks up a call from a childhood friend, Tod. Tod tells him that Black is in a coma following a vicious attack and he learns that Black is in a motorcycle gang. Unable to endure the harm done to his brother in silence, White disguises himself as Black to root out the traitor.
But White doesn't know his brother as well as he thought; he's not behaving like the Black Sean knows, distrustful and uncaring about the feelings of others. Sean's suspicious. The difference gets under Sean's skin and makes him feel things he shouldn't.
Main | Episodes | Cast
Action, Crime, Thriller
GMM One, Thailand at 8:30 pm +07
Sun Jan 16, 2022
Countdown:
4 days 6 hours
Jumpol Adulkittiporn
as Sean
Atthaphan Phunsawat
as White
as Black
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