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Research | Open | Published: 19 June 2015
Patient-derived hepatitis C virus inhibits CD4+ but not CD8+ T lymphocyte proliferation in primary T cells
Sonya A. MacParland1,2,
Annie Y. Chen1,
Christopher P. Corkum1,
Tram N.Q. Pham1,3 &
Tomasz I. Michalak1
Virology Journalvolume 12, Article number: 93 (2015) | Download Citation
Hepatitis C virus (HCV) can replicate in cells of the immune system and productively propagate in primary T lymphocytes in vitro. We aimed to determine whether exposure to authentic, patient-derived HCV can modify the proliferation capacity, susceptibility to apoptosis and phenotype of T cells.
Primary total T cells from a healthy donor were used as targets and plasma-derived HCV from patients with chronic hepatitis C served as inocula. T cell phenotype was determined prior to and at different time points after exposure to HCV. T cell proliferation and apoptosis were measured by flow cytometry-based assays.
The HCV inocula that induced the highest intracellular expression of HCV also caused a greatest shift in the T cell phenotype from predominantly CD4-positive to CD8-positive. This shift was associated with inhibition of CD4+ but not CD8+ T cell proliferation and did not coincide with altered apoptotic death of either cell subset.
The data obtained imply that exposure to native HCV can have an impact on the relative frequencies of CD4+ and CD8+ T cells by selectively suppressing CD4+ T lymphocyte proliferation and this may occur in both the presence and the absence of measurable HCV replication in these cells. If the virus exerts a similar effect in vivo, it may contribute to the impairment of virus-specific T cell response by altering cooperation between immune cell subsets.
Hepatitis C virus (HCV) is a single-stranded RNA virus that affects 130–150 million people worldwide as a chronic infection [1]. In HCV-infected patients, viral replication has been demonstrated not only in hepatocytes but also in cells of the immune system, such as B cells, monocytes, and CD4+ and CD8+ T lymphocytes [2–6]. In this laboratory, it was uncovered that primary T lymphocyte cultures, generated by ex vivo treatment of peripheral blood mononuclear cells (PBMC) from healthy individuals with a T cell-inducing mitogen phytoheamagglutin (PHA), are susceptible to wild-type (patient-derived) HCV and capable of supporting its replication at a level comparable to that of in vivo infected lymphoid cells [6, 7]. Furthermore, these cells were able to produce infectious virions that de novo infected lymphocytes [7]. It was also uncovered that patient-derived HCV is significantly more infectious to primary T cells than laboratory-derived clonal strains of HCV [8]. As well, HCV infection of T cells requires surface expression of CD5, and transfection of HCV non-susceptible T cell lines with CD5 renders these cells susceptible to infection [9].
HCV infection causes chronic hepatitis C (CHC) in up to 85 % of those afflicted, while acute HCV infection is thought to spontaneously resolve in 15-25 % of cases [1]. Resolution of hepatitis C appears to be a result of a robust HCV-specific T cell-mediated response. In HCV-infected chimpanzees, representing the closest animal model of human HCV infection, recovery from hepatitis C and a drop in plasma HCV loads to levels undetectable by clinical assays requires the activity of both CD4+ T helper cells and CD8+ cytotoxic T cells [10, 11]. In patients with CHC, T cells display markers of exhaustion and are defective in their ability to produce interferon-γ (IFN-γ) and interleukin-2 (IL-2) [12]. In vitro, the functional consequences of T cell infection in Molt-4 and Jurkat T cell lines and primary T cells have been investigated only using laboratory-adapted clonal strains of HCV [4, 13, 14]. It was found, among others, that infection with a HCV SB strain of Molt-4 cells suppressed IFN-γ signalling through the STAT-1 pathway [4]. This virus strain was also able to infect primary CD4+ T cells and this infection was associated with a decrease in their proliferation [14].
Stemming from the previous observation that infection of naïve lymphoid cells with patient-derived HCV can lead to changes in the CD4+ to CD8+ T cell ratio [7], we asked in the current study whether the virus can differentially alter CD4+ or CD8+ T cell proliferation and/or their apoptosis resulting in a shift in the T cell phenotypic characteristics. We found that the exposure of T lymphocytes to a naturally occurring HCV, although not necessarily molecularly evident active virus replication in these cells, can be sufficient to alter the CD4+ to CD8+ T cell ratio. This phenotypic change was due to the selective inhibition of CD4+ but not CD8+ T lymphocyte proliferation and was not related to differential apoptotic death of either T cell subset.
Plasma-derived HCV inocula and target cells
Plasma from patients C26/F (CHC-1), C33/M (CHC-2), N28/F (CHC-3) with clinically and serologically documented CHC served as the source of naturally occurring HCV. The inocula contained HCV of different genotypes and viral loads (Table 1). CHC-1 carried a mixture of genotype 1a and 1b at 1.1 × 106 virus copies, also termed virus genome equivalents (vge)/mL, CHC-2 had genotype 3a at 2 × 104 vge/mL, and CHC-3 carried 2b genotype at 2.4 × 106 vge/mL. These HCV plasma donors had no laboratory evidence of hepatitis B virus (HBV) or HIV type 1 (HIV-1) infection, as testing of their sera by standard clinical assays indicated, and clinically apparent chronic morbidity other than CHC.
Table 1 Virological characteristics of individuals with CHC providing plasma serving as HCV inocula and HCV RNA loads in T cells infected with these inocula
Lymphoid cells serving as targets for in vitro HCV infection experiments were isolated from a single healthy donor who had no clinical history or molecular indication of HCV exposure, as confirmed by testing for antibodies to HCV (anti-HCV) and examining serum and PBMC by highly sensitive HCV-specific RT-PCR/nucleic acid hybridization (NAH) assay (sensitivity of <10 vge/mL or <2.5 IU/mL) [2]. The donor was also serum HBV DNA and HIV-1 RNA nonreactive and had normal alanine aminotransferase (ALT) level, as determined by conventional clinical assays.
In vitro HCV infection
De novo infection of lymphoid cells with HCV was carried out following the method reported before, including monocyte depletion to enhance viral replication in lymphocytes [7]. Briefly, monocyte depletion was carried out by plastic adherence for 4 h. This led to a three-fold decrease in CD14+ monocytes, as measured by flow cytometry (Additional file 1: Figure S1). Previously, we have shown that intermittent stimulation of PBMC exposed to HCV ex vivo with phytohemagglutinin (PHA) in the presence of human recombinant interleukin-2 (IL-2) leads to HCV propagation [7]. However, these conditions also augmented lymphocyte proliferation and led to a relatively high rate of lymphocyte apoptosis (data not shown). These outcomes were likely related to the repeated stimulation with PHA. To minimize this effect, which potentially masked the influence of HCV on cell proliferation and apoptosis, we stimulated lymphoid cells with PHA only once prior to infection in the current study. Thus, monocyte-depleted lymphoid cells from a healthy donor were treated with 5 μg/mL PHA (Sigma-Aldrich, Mississauga, Ontario, Canada) for 48 h [7]. Following stimulation, 1 × 107 cells were exposed to 2.7 × 105 vge from CHC-1 or CHC-3 or to 500 μL (1 × 104 vge) of plasma from CHC-2 in 9.5 mL of culture medium. In addition, the same number of target cells was exposed to three 500-μL samples of normal healthy plasma (NHP) from 3 different healthy donors (mock infections). As another control, target cells were cultured with 9.5 mL of medium alone (NP, no plasma). In all cases, inocula or NHP were removed after 24 h and the cells washed thoroughly prior to suspension in 9.5 mL of medium, as described [7]. Cells were cryopreserved for analysis prior to and after PHA stimulation (time 0) and at 1, 4, 7 and 10 d.p.i., unless otherwise indicated. In addition, cells were collected at each of the above time points to determine cell phenotype and apoptosis (see below).
Inhibition of HCV infection in T lymphocytes by Telaprevir
Telaprevir (TLP or VX-950), an HCV NS3-4A protease inhibitor, was purchased from Vertex Pharmaceuticals (Cambridge, Massachusetts, USA). TLP had shown capability of complete inhibition of HCV replication in de novo infected Molt4 T cell line [9] and naturally HCV-infected PBMC (Chen et al., manuscript submitted). At concentrations equal to or below 4 μM, TLP is not toxic to human lymphocytes, as assessed before [9]. We applied the previously established treatment conditions with TLP to determine whether the shift in CD4+ T cell proliferation can be normalized in the absence of detectable virus replication in the cells previously exposed to HCV. Briefly, approximately 5 × 106 cells were incubated in duplicate with CHC-1 or CHC-2 plasma under conditions described above in the presence or absence of 4 μM TLP in 0.5 % DMSO. The cells were harvested after 10 d.p.i. for evaluation of expression of HCV RNA positive and negative strands, as described above, and the CD4 and CD8 T cell frequency determined by flow cytometry. In parallel, lymphocytes exposed to the same amounts of CHC-1 or CHC-2 HCV alone and those incubated in culture medium supplemented with NHP in the absence of TLP served as infection controls.
HCV RNA positive and negative strand detection
HCV RNA positive strand in de novo infected lymphoid cells as well as in patients' plasma was determined by HCV-specific real-time RT-PCR (sensitivity 100 vge/mL) [2]. Expression of HCV RNA negative strand in T cells was detected by strand-specific RT-PCR/NAH using rTth DNA polymerase as described before (sensitivity ~100 copies/μg total RNA) [2, 9].
Determination of cell phenotype
Phenotype of the cells exposed to HCV inocula, NHP or cultured in medium alone was assessed by incubation of 1 × 105 cells per reaction with a cocktail containing an anti-CD3 monoclonal antibody (mAb) conjugated with Alexa Flour 488 (Alexa 488) (BD Pharmingen, San Diego, California, USA), an anti-CD4 mAb labelled with peridinin chlorophyll protein complex (PerCP) (BD Pharmingen) and an anti-CD8 mAb conjugated to allophycocyanin (APC) (Ebiosciences, San Diego, California, USA), an anti-CD14 mAb labeled with phycoerythrin (PE) (Ebiosciences) or with a control cocktail of the appropriate isotype mAb controls (Ebiosciences or BD Pharmingen) for 30 min at 4 °C. Using forward versus side scatter, lymphocytes were separated from debris by flow cytometric analysis (gate R1; see Fig. 1b). Then, lymphocytes were sub-gated on Alexa-488-positive CD3+ T cells (gate R2; see Fig. 1b) to enumerate in the next step APC-positive CD8+ cells located in the lower right (LR) quadrant and PerCP-positive CD4+ cells located in the upper left (UL) quadrant (see Fig. 1b). Quadrant markers were set up on background staining with appropriate isotype controls.
Phenotypic characterization of cultured lymphocytes after exposure to HCV. Lymphoid cells from the same healthy donor were either exposed to medium alone (no plasma, NP), normal human plasma (NHP 1–3) or HCV inocula (CHC 1–3). Cells collected at the time points indicated (d.p.i.) were stained for CD3, CD4 and CD8 or with isotype controls and analysed by flow cytometry. a Graphical representation of flow cytometry data showing phenotype of T cells for each infection condition tested. CD4+ T cells (solid black bars) and CD8+ T cells (hatched bars) are displayed as percentage of total CD3+ T cells at 4 different time points. The detection of HCV RNA positive strand was shown for 1, 7 and 10 d.p.i. For HCV RNA positive strand detection, (+++) indicates >5,000 vge/μg RNA, (++) between 500 and 5,000 vge/μg RNA, (+) between 50 and 500 vge/μg RNA, and (+/−) below 50 vge/μg RNA. For negative strand detection, (+) indicates detection and (−) no detection of the strand. Boxes indicate cultures in which the CD4+ to CD8+ ratio was altered when compared to cultures exposed to NP or NHP. n.a.- not applicable, n.t.- not tested. b Determination of T cell phenotype in cell cultures exposed or not to HCV and cultured for 10 d.p.i. Using forward versus side scatter, lymphocytes (gate R1) were separated from cellular debris. Lymphocytes were sub-gated on Alexa-488-positive CD3 T cells (gate R2) for enumeration of CD4+ and CD8+ T cells by detecting APC-positive CD8+ T cells found in the lower right (LR) quadrant and PerCP-positive CD4+ T cells in the upper left (UL) quadrant. Numbers in the UL and LR quadrants indicate percentages of positive cells
Flow cytometry-based T cell proliferation assay
To quantify proliferation of lymphocytes exposed to HCV or NHP, cells were stained with carboxyfluorescein succinimidyl ester (CFSE), as previously reported [15]. Briefly, ~2 × 107cells were incubated with CFSE (Molecular Probes, Eugene, Oregon, USA) at the pre-tested nontoxic concentration of 1 mM for 10 min at 37 °C and washed twice with 10 mL of 5 % fetal calf serum (FCS; GIBCO-Invitrogen Corporation, Auckland, New Zealand). To carry out triplicate infection with each of the HCV inocula tested, NHP or NP control, CFSE-labelled lymphocytes were suspended at 1 × 106 cells in one mL of medium containing 10 % FCS in 6-well plates. As an additional control, CFSE-labelled cells in medium alone were included. Cell proliferation was measured in triplicate wells before stimulation with PHA and at 0, 1, 4, 7 and 10 d.p.i., unless otherwise stated. The data were analyzed with Cellquest Pro (Becton Dickinson, San Jose, California, USA) or ModFit LT (Verity Software House, Topsham, Main, USA) software. Using forward versus side scatter, lymphocytes (gate R1) were separated from debris. Proliferation was presented as the mean percentage of CFSE-low cells with standard error of mean (SEM) from triplicate cultures using percentage of CFSE-low cells from cultures maintained in medium alone as the baseline. In the proliferation experiments, CFSE-labelled lymphoid cells were additionally stained with a cocktail of APC-anti-CD3, PerCP-anti-CD4 and PE-anti-CD8 mAbs, as outlined above. Using forward versus side scatter, lymphocytes were separated from debris, gated on CD3+ T cells, and sub-gated on CD4+ or CD8+ T cells to determine the proliferation of individual T cell subsets. Mitogen non-stimulated controls were routinely included, as described above. Dilution of CFSE fluorescence in the cells was measured using the proliferation wizard module of ModFit LT software revealing daughter cell generations. Proliferation index (P.I.) was determined using non-stimulated cells to define the parent generation.
Annexin V-PE-/7-AAD assay for detection of cell apoptosis
To determine the degree of apoptosis after exposure to HCV inocula or NHP, cells were stained with annexin V conjugated with phycoerythrin (PE), or 7-aminoactinomycin D (7-AAD), as previously established [16]. Briefly, 1 × 105 cells were washed with annexin buffer containing 10 mM HEPES (Invitrogen), 5 mM NaCl, 5 mM KCl and 2 mM CaCl2 and centrifuged at 1,500 rpm for 10 min at 4 °C. Then, a cocktail containing 50 μg/mL of 7-AAD (Invitrogen) and 50 μg/mL of annexin-V-PE (Invitrogen) were prepared in annexin buffer. Cells were suspended in 100 μL of apoptosis cocktail and kept on ice for 30 min. After staining, cells were washed once with annexin buffer, spun down and suspended in 500 μL of annexin buffer to be analyzed by flow cytometry [16].
Quantification of cytokine expression
In cultures in which numbers of recovered cells permitted, RNA was extracted, treated with DNase [17] and reversely transcribed [2]. Due to very limited numbers of cells available for RNA extraction, quantification of expression was feasible only for IFN-γ, IFN5-α (IFN-5α), tumor necrosis factor-α (TNF-α), and IL-2. This was done using cDNA (50 ng RNA equivalent) and amplification conditions previously reported [17]. Amplification reactions were performed using the following primers: sense primer 5′-TCAGCTCTGCATCGTTTTGG and antisense primer 5′-TGTTTTAGCTGCTGGGCACA for IFN-γ, sense primer 5′-CAGCCTGAGTAACAGGAGGA and antisense primer 5′-GCAGATGAGTCCTTTGTGCT for IFN-5α, sense primer 5′-TCTTCTCGAACCCCGAGTGA and antisense primer 5′-CCTCTGATGGCACCACCAG for TNF-α, and sense primer 5′-CCCAAGAAGGCCACAGAACT and antisense primer 5′-TGCTGATTAAGTCCCTGGGTCTTA for IL-2. Expression of the genes was normalized to β-actin.
Data were analyzed and differences between HCV-exposed cultures and those not exposed to virus were determined using GraphPad Prism 4 software (GraphPad Software Inc., San Diego, California, USA). Statistical significance was evaluated by two-tailed Mann–Whitney test. P values equal to or lower than 0.05 were considered as significant.
HCV expression in de novo infected lymphoid cells
HCV RNA positive strand was detected in T cell cultures following exposure to all three inocula tested (Table 1), but not in those exposed to NHP or medium alone (Fig. 1). The level of HCV RNA detection varied depending on inoculum and time of cell collection. HCV RNA load ranged from <50 to 8.7 × 103 vge/μg total RNA (Table 1). HCV RNA negative (replicative) strand was detected at 7 and 10 d.p.i. in cultures exposed to CHC-1 inoculum that also gave the highest level of HCV RNA positive strand expression among the inocula tested.
T cell phenotype after exposure to HCV
In preliminary experiments and in the study previously reported [7], cells exposed to HCV were maintained in culture in the presence of IL-2 under alternating stimulation with PHA. After 14 days of culture, more than 98 % cells were CD3-reactive and CD8+ T cells were evidently more prevalent than CD4+ T cells in cultures exposed to HCV [7]. Similarly, the current study, exposure of normal lymphoid cells to 2 of 3 HCV inocula tested, i.e., CHC-1 and CHC-2, resulted in a change in the CD4+ to CD8+ T cell ratio in that CD8+ T cells became more prevalent compared to cultures exposed to NHP or cultured in medium alone (Fig. 1). Figure 1a shows relations between numbers of the CD4+ and CD8+ T cells in cultures exposed to HCV, NHP or medium alone during the 10-d.p.i. investigation period. Thus, at 1 d.p.i. the ratio of CD4+ T to CD8+ T cells was approximately 4.4 to 1 (with CD8+ T cells taken as 1) and was not noticeably influenced by whether the cultures were exposed or not to HCV. At 4 d.p.i., the ratio of CD4+ to CD8+ T cells changed on average to 1.2 to 1, with the exception of culture exposed to CHC-1 inoculum in which CD8+ T cells were enriched and the CD4+ to CD8+ ratio shifted to 0.6 to 1. At 7 d.p.i., a change in the T cell subset distribution was also observed for cells inoculated with CHC-2. At this time point, the CD4+ to CD8+ T cell ratio for CHC-1 was 0.6 to 1 and for CHC-2 1.1 to 1, while for CHC-3, NHP-1, NHP-2, NHP-3 and NP the ratio was close to 1.5 to 1. At 10 d.p.i., the CD4+ to CD8+ ratio for T cells exposed to CHC-1 and CHC-2 were 1.3 to 1 and 1.2 to 1, respectively, while the control cultures and that exposed to CHC-3 had CD4+ to CD8+ ratios of approximately 3.5 to 1 (Fig. 1a). Thus, in contrast to CHC-1 and CHC-2, CHC-3 inoculum had no effect on T cell subset phenotype and behaved, in this regard, as control NHP. This inoculum induced a very low, unquantifiable level of HCV RNA in lymphocytes throughout the culture period (Table 1 and Fig. 1a), which may suggest very limited uptake of the virus carried in this inoculum by PBMC. Taken together, these findings suggested that a change in T cell subset distribution could be related to the cellular level of HCV RNA expression.
Apoptosis of lymphoid cells following exposure to HCV
To determine whether the observed enrichment in CD8+ T cells after exposure to HCV was due to enhanced apoptosis of CD4+ T cells, the degree of lymphoid cell death was determined in triplicate cultures after exposure to a given HCV inoculum or NHP. As shown in Fig. 2, at the time points examined, the degree of apoptosis was approximately similar and not noticeably related to whether the cells were cultured in the presence of medium alone, NHP or HCV (Fig. 2a and b). Thus, under the conditions tested, the virus did not increase the rate of T cell apoptotic death. To assess whether the alteration in phenotype may result from selective apoptosis of CD4+ or CD8+ T cells, the extent of apoptotic death of these two subsets was examined separately. We found that exposure to HCV did not differentially alter apoptosis of these cells (data not shown).
Quantification of lymphoid cell apoptosis by annexin-PE/7-AAD flow cytometry assay. a Graphical representation of degree of apoptosis detected in PHA-treated lymphoid cell cultures and exposed to NP, NHP 1–3 or CHC 1–3. Bars indicate the percentage of total cells in each phase of apoptosis. Graphs present data from triplicate experiments and are shown as mean ± SEM at 1, 4, 7 and 10 d.p.i. Bars with diagonal lines represent viable cells, checkered bars, early apoptotic cells, bars with horizontal lines, late apoptotic cells and vertical lines, necrotic cells. b Flow cytometric determination of stage of apoptosis and percentage of T cells in cultures exposed to NP, NHP or HCV at 10 d.p.i. after staining with annexin-PE and 7-AAD. Each quadrant represents a different phase of apoptosis: lower left (LL), viable cells; lower right (LR), early apoptotic cells; upper right (UR), late apoptotic cells and upper left (UL), necrotic cells. Numbers indicate percentages of cells in each quadrant
T cell proliferation after exposure to HCV
To determine the rate of T cell proliferation after exposure to HCV or NHP, triplicate cell cultures were evaluated using the flow cytometry-based CFSE proliferation assay. In preliminary experiments, T cells which had been stimulated more than once with PHA proliferated rapidly irrespective of HCV exposure (data not shown). However, in the current study, where cells were activated only once with mitogen prior to HCV exposure, the infection resulted in significant (P = 0.03) inhibition of proliferation in the total T cell population (Fig. 3a and Additional file 2: Figure S2). Upon further examination, it was established that CHC-1 and CHC-2 inocula inhibited T cell proliferation, while CHC-3 as well as NHP1-3 did not exert any remarkable anti-proliferative effect (Fig. 3b and Additional file 2: Figure S2).
Measurement of T cell proliferation after exposure to HCV or control normal human plasma. a Overall comparison of proliferation in total T cell population exposed to NHP 1–3 or CHC 1–3. The data represent the mean percentage CFSE-low cells measured in triplicate cultures at 4 time points throughout the culture period (also see Additional file 2: Figure S2 for details on identification of CFSE-low cells). b Graphical representation of lymphoid cell proliferation after exposure to HCV inocula or NHP. Proliferation represented by the mean percentage CFSE-low and SEM measured in triplicate at 1, 4, 7 and 10 d.p.i
Proliferation of T cell subsets in cultures exposed to HCV
To evaluate whether the observed inhibition in proliferation of the total T cell population could be limited to a particular T cell subset, the proliferation rates of CD4+ and CD8+ T cells were measured. When comparing cultures exposed to HCV to those treated with NHP, a significant (P = 0.0003) decrease in CD4+ T cell proliferation was found (Fig. 4a). In contrast, the proliferation of CD8+ T cells was not affected regardless of whether the cells were exposed to HCV or NHP. Upon further evaluation, it became apparent that CHC-1 and CHC-2 inocula caused a significant decrease in the proliferation of CD4+ T cell compared to the cells exposed to CHC-3 inoculum or NHP (Fig. 4b). Overall, a statistically significant decrease in the CD4+ T cell proliferation rates was seen in cultures which readily expressed HCV RNA, while no difference in CD8+ T cell proliferation was seen between the cultures exposed to HCV inocula or NHP (Fig. 4b).
CD4+ and CD8+ T cell proliferation rates after exposure to HCV. Lymphoid cells from the same healthy donor were stained with CFSE, PHA-stimulated and infected as described in Materials and Methods to measure proliferation of CD4+ and CD8+ T cell subsets. a Graphical representation of proliferation of T cell subsets after exposure to either NHP or HCV inocula. Proliferation represented by the mean percentage CFSE low ± SEM was measured at 4, 7 and 10 d.p.i. b The rates of T cell subset proliferation in cultures exposed to individual HCV inocula or NHP. Proliferation is represented by the mean percentage CFSE-low ± SEM measured at 3 different time points during the 10-d.p.i. culture
CD4+ and CD8+ T cell frequencies in HCV-exposed cultures treated with TLP
To examine the impact of HCV replication on the relative CD4+ T cell decrease and CD8+ T cell enrichment observed in earlier experiments, HCV-exposed PBMC were cultured in the presence or absence of TLP for 10 d.p.i. As expected, TLP treatment led to undetectable HCV RNA positive and negative strands in cultures exposed to CHC-1 (data not shown). For PBMC exposed to CHC-2, HCV RNA negative was undetectable either with or without TLP treatment, which was expected considering the data previously obtained (see Fig. 1). Interestingly, TPL treatment did not impact the relative decrease in the CD4+ T cell frequency caused by exposure to CHC-1 and CHC-2 inocula (Fig. 5). This suggests that uptake of virus alone, in the absence of detectable active replication, might be sufficient to impair CD4+ T cell proliferation. This may explain why we observed decreased CD4+ T cell proliferation after exposure to CHC-2 in the absence of detectable replication.
Phenotypic characterization of lymphocytes after exposure to HCV in the presence or absence of TLP. Lymphoid cells from a healthy donor were exposed in duplicate to CHC-1 or CHC-2 or to normal healthy human in the absence (untreated) or presence of TLP (TLP treated). Cells were collected at 10 d.p.i., stained for CD3, CD4 and CD8 or with isotype controls, and analyzed by flow cytometry. Percentage CD4+ T cell decrease and percentage CD8+ T cell increase was calculated based on CD4+ and CD8+ T cell frequencies (taken as 100 %) in control cultures exposed to normal human plasma
Cytokine expression in T cell cultures exposed to HCV
To recognize the effects of HCV on the T cell cytokine expression, levels of IFN-γ, TNF-α, IFN-α5 and IL-2 mRNA were quantified in the cells treated with HCV inocula or control NHP. Due to very limited amounts of RNA, evaluation of these 4 cytokines in all cell cultures at different time points was not always feasible. In cells stimulated with PHA for 48 h prior to infection, there was an evident upregulation in IFN-γ transcription (data not shown). After PHA stimulation, IFN-γ induction achieved a peak at 1 d.p.i. in cultures exposed to 2 of 3 NHP and to all 3 HCV inocula tested. Then, the cytokine expression declined in the cultures treated with HCV but not in those exposed to NHP. Nonetheless, differences in the IFN-γ mRNA level between the cultures exposed or not to HCV did not achieve statistical significance at any of the time points examined. For TNF-α, the cytokine expression sharply declined to undetectable level at 1 d.p.i. in cultures exposed to CHC-1 and CHC-2, but not in those exposed to NHP 1–3 or CHC-3 inoculum. It is of note that cultures which lost temporarily TNF-α transcription were those in which the highest levels of cellular HCV RNA were detected (Table 1). Further, IFN-α5 expression was not detected in cells either exposed to HCV or NHP. In regard to IL-2, the cytokine mRNA level tended to be upregulated at 1 d.p.i. in cultures exposed to NHP-1 and NHP-2, but not in those treated with HCV. At later time points, IL-2 transcription level progressively declined in cultures exposed to HCV inocula but not in those treated with NHP, although the difference was statistically insignificant at the end of follow-up (10 d.p.i.).
In this study, we examined the effect of authentic HCV on the proliferative capacity, apoptosis and phenotype of T lymphocytes in primary cultures. Exposure of lymphoid cells to 2 of 3 HCV inocula caused a decrease in the frequency of CD4+ T cells compared to virus-untreated cultures with a relative increase in CD8+ T cells. This relative CD8+ T cell enrichment was a result of a significant reduction in CD4+ T cell proliferation compared to the cultures exposed to normal healthy plasma. On this note, the effect appeared to correlate with HCV cell uptake, since the shift in the CD4+ to CD8+ ratio was highest in the cultures in which virus exposure lead to quantifiable levels of HCV RNA positive strands and, in the case of CHC-1, negative strands in test cells (Table 1). This was consistent with the observation that in cultures exposed to CHC-1 inoculum, which induced the highest levels of HCV RNA positive strand and detectable HCV RNA negative strand, the inhibition in CD4+ T cell proliferation appeared earlier, i.e., 4 d.p.i. In the cells exposed to CHC-2 inoculum, T cell proliferative and phenotypic changes were seen in the absence of detectable HCV replicative intermediate and after 10-fold lower multiplicity of infection compared to cells exposed to CHC-1. In the case of CHC-3, while the plasma viral load was higher than CHC-2 and comparable to CHC-1, there did not appear to be much virus uptake by cells, as indicated by undetectable HCV RNA by our high sensitivity assays. This may suggest that the amount of virus taken by cells exposed to CHC-3 was minuscule. HCV genotype may also play a role in the anti-proliferative effects observed, as CHC-1 carried an HCV genotype 1a/1b mix, CHC-2 genotype 3a, and CHC-3 genotype 2b. It is of note that there was no impact of TLP treatment on the change in the CD4+ and CD8+ T cell frequency, which suggests that exposure to HCV and uptake of the virus by cells rather than replication itself may be driving these phenotypic changes (Fig. 5).
Previously, infection of T cell lines and primary T cells with laboratory-derived HCV clones led to the impairment of T cell proliferation and IFN-γ production [4, 14]. In the current study, we show an anti-proliferative effect using patient-derived HCV as a virus source. The biological impact of HCV lymphotropism on immune cell function is a topic that is still not well understood. However, it was shown that HCV infection can led to disruption of immunological activity of different types of immune cells, including T cells, B cells, dendritic cells, monocytes and macrophages [4, 14, 18, 19].
In cultures exposed to CHC-3 inoculum, the level of HCV RNA positive strand detected during the 10-d.p.i. culture period was unquantifiable by a real time RT-PCR assay (below 50 vge/μg total RNA). However, HCV RNA signals were detected, as confirmed by NAH analysis of amplicons produced by real-time RT-PCR (data not shown). In this situation, detection of virus RNA negative strand would not be feasible, and, therefore, was not attempted (Table 1). As indicated, CHC-3 inoculum did not induce changes in T cell proliferation or phenotype, similarly as NHP-1-3, suggesting that regardless of the viral load in the inocula, if the virus cannot be taken up by cells, then the cells' proliferation is not affected. We have previously shown that not all patient-derived HCV inocula are infectious to ex vivo pre-stimulated lymphoid cells [7, 20]. The viral and host factors determining infectivity of T cells by wild-type HCV have yet to be recognized and are under investigation in this laboratory [8, 9, 21].
In experiments leading to this study, alternating stimulation with PHA and IL-2 was applied to upregulate lymphoid cell susceptibility to HCV infection and to augment virus replication [7]. In the current work, repeated stimulation with PHA after the initial 48-h treatment was not employed to prevent excessive cell activation that may mask virus pro- or anti-proliferative or apoptotic effects. The removal of this periodical stimulation resulted in a decreased HCV replication and, therefore, a reduced detection of both virus RNA positive and negative strands. This finding is consistent with previous observations in regard to HCV replication in lymphoid cell cultures [22] and in in vitro infections with other viruses [23–25].
It is well recognized that HCV-specific CD4+ T cell response in individuals chronically infected with HCV is poor or absent [26]. In HCV-infected patients who transiently control viremia and have fluctuating plasma viral load, including periods of apparent HCV RNA negativity, this transient viral control is accompanied by increased virus-specific CD4+ T cell response [26]. In patients with CHC, T cells have been found to be impaired in the production of IFN-γ and IL-2 [12]. As well, studies investigating CD4+ T cell function during a symptomatic persistent HCV infection have shown a significant loss of IL-2 secreting cells compared to individuals who spontaneously resolved viremia, as well as a weak IFN-γ production by HCV-specific CD4+ T cells upon stimulation [27]. In the latter study, HCV-specific IFN-γ production by CD4+ T cells was rescued after in vitro culture with exogenous IL-2, but the effect of IL-2 on the CD4+ T cell proliferation was not measured. These findings may suggest that impaired proliferation of CD4+ T cells observed in our study could be due to weakened IL-2 production. However, only CD4+ T cells but not CD8+ T cells were affected. Due to limitations in cell numbers we were not able to evaluate whether there was indeed impaired IL-2 and/or IFN-γ cytokine production by in vitro infected T cells. This issue requires further investigation. Preliminary data obtained in this regard in the present study indicate that there were no significant differences between T cells exposed or not to HCV in terms of IFN-γ, IFN-5α and IL-2 mRNA expression. However, TNF-α transcription appeared to be transiently but severely down-regulated shortly (i.e., 1 d.p.i.) after exposure to CHC-1 or CHC-2. This finding warrants further examination. Nonetheless, it needs to be taken under consideration that the level of cytokine gene expression may not accurately reflect the cytokine protein production.
The results of this study should be interpreted with certain limitations taking under consideration that PBMC exposed to authentic HCV were isolated from a single healthy donor. The observations made might be limited to this particular donor and experiments with cells from other healthy individuals are needed to confirm the findings. However, we have previously reported that a comparable shift in the CD4+ to CD8+ T cell ratio also occurred after exposure to native HCV of T cells from another healthy donor [7].
Overall, our findings imply that exposure to HCV can modify the T cell phenotype due to a relative decrease in the proliferation capacity of CD4+ T cells. Furthermore, exposure to naturally occurring HCV in vitro did not appear to differentially augment apoptotic death of lymphocyte subsets, although apoptotic effects of a HCV clone on T cells have been described previously [18]. In this context, it needs to be determined whether primary CD4+ and CD8+ T cells derived from patients with CHC display similar characteristics. The results from the current study raise a possibility that HCV may exert a direct effect on the overall T cell phenotypic properties and, in consequence, on the function of T cells as a whole in HCV-infected patients.
WHO. Hepatitis C Fact Sheet No, 164, Updated April 2014. [Cited Feb 03, 2015]. Available from: http://www.who.int/mediacentre/factsheets/fs164/en/
Pham TNQ, MacParland SA, Mulrooney PM, Cooksley H, Naoumov NV, Michalak TI. Hepatitis C virus persistence after spontaneous or treatment-induced resolution of hepatitis C. J Virol. 2004;78:5867–74.
Pal S, Sullivan DG, Kim S, et al. Productive replication of hepatitis C virus in perihepatic lymph nodes in vivo: implications of HCV lymphotropism. Gastroenterology. 2006;130:1107–16.
Kondo Y, Sung VMH, Machida K, Liu MY, Lai MMC. Hepatitis C virus infects T cells and affects interferon-gamma signaling in T cell lines. Virology. 2007;361:161–73.
Radkowski M, Bednarska A, Horban A, et al. Infection of primary human macrophages with hepatitis C virus in vitro: induction of tumour necrosis factor-alpha and interleukin 8. J Gen Virol. 2004;85:47–59.
Pham TNQ, King D, MacParland SA, et al. Hepatitis C virus replicates in the same immune cell subsets in chronic hepatitis C and occult infection. Gastroenterology. 2008;134:812–22.
MacParland SA, Pham TNQ, Gujar SA, Michalak TI. De novo infection and propagation of wild-type Hepatitis C virus in human T lymphocytes in vitro. J Gen Virol. 2006;87:3577–86.
Sarhan MA, Chen AY, Russell RS, Michalak TI. Patient-derived hepatitis C virus and JFH-1 clones differ in their ability to infect human hepatoma cells and lymphocytes. J Gen Virol. 2012;93:2399–407.
Sarhan MA, Pham TNQ, Chen AY, Michalak TI. Hepatitis C virus infection of human T lymphocytes is mediated by CD5. J Virol. 2012;86:3723–35.
Shoukry NH, Grakoui A, Houghton M, et al. Memory CD8(+) T cells are required for protection from persistent hepatitis C virus infection. J Exp Med. 2003;197:1645–55.
Grakoui A, Shoukry NH, Woollard DJ, et al. HCV persistence and immune evasion in the absence of memory T cell help. Science. 2003;302:659–62.
Semmo N, Krashias G, Willberg C, Klenerman P. Analysis of the relationship between cytokine secretion and proliferative capacity in hepatitis C virus infection. J Viral Hepat. 2007;14:492–502.
Hall CHT, Kassel R, Tacke RS, Hahn YS. HCV+ hepatocytes induce human regulatory CD4(+) T cells through the production of TGF-beta. PLOS ONE. 2010;5:e12154.
Kondo Y, Ueno Y, Kakazu E, et al. Lymphotropic HCV strain can infect human primary naive CD4(+) cells and affect their proliferation and IFN-gamma secretion activity. J Gastroenterol. 2011;46:232–41.
Gujar SA, Michalak TI. Flow cytometric quantification of T cell proliferation and division kinetics in woodchuck model of hepatitis B. Immunol Invest. 2005;34:215–36.
Gujar SA, Jenkins AK, MacParland SA, Michalak TI. Pre-acute hepadnaviral infection is associated with activation-induced apoptotic death of lymphocytes in the woodchuck (Marmota monax) model of hepatitis B. Dev Comp Immunol. 2010;34:999–1008.
Pham TNQ, Mulrooney-Cousins PM, Mercer SE, et al. Antagonistic expression of hepatitis C virus and alpha interferon in lymphoid cells during persistent occult infection. J Viral Hepat. 2007;14:537–48.
Kondo Y, Shimosegawa T. Direct effects of hepatitis C virus on the lymphoid cells. World J Gastroenterol. 2013;19:7889–95.
Kondo Y, Ueno Y, Shimosegawa T. Biological significance of HCV in various kinds of lymphoid cells. Int J Microbiol. 2012;2012:e647581.
MacParland SA, Pham TNQ, Guy CS, Michalak TI. Hepatitis C virus persisting after clinically apparent sustained virological response to antiviral therapy retains infectivity in vitro. Hepatology. 2009;49:1431–41.
Sarhan MA, Chen AY, Michalak TI. Differential expression of candidate virus receptors in human T lymphocytes prone or resistant to infection with patient-derived hepatitis C virus. PLOS One. 2013;8:e62159.
Pham TNQ, MacParland SA, Coffin CS, Lee SS, Bursey FR, Michalak TI. Mitogen-induced upregulation of hepatitis C virus expression in human lymphoid cells. J Gen Virol. 2005;86:657–66.
Braun RW, Kirchner H. T lymphocytes activated by interleukin 2 alone acquire permissiveness for replication of herpes simplex virus. Eur J Immunol. 1986;16:709–11.
Chatterjee R, Gupta P, Kashmiri SV, Ferrer JF. Phytohemagglutinin activation of the transcription of the bovine leukemia virus genome requires de novo protein synthesis. J Virol. 1985;54:860–3.
Hyypia T, Korkiamaki P, Vainionpaa R. Replication of measles virus in human lymphocytes. J Exp Med. 1985;161:1261–71.
Gerlach JT, Diepolder HM, Jung MC, et al. Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C. Gastroenterology. 1999;117:933–41.
Semmo N, Day CL, Ward SM, et al. Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection. Hepatology. 2005;41:1019–28.
The authors thank Dr. C.S. Coffin from the Liver Unit, Division of Gastroenterology, Faculty of Medicine, University of Calgary, Calgary, Alberta and Dr. S.B. Reddy and Ms. D. King, a hepatology nurse practitioner, from the Gastroenterology Clinic, Eastern Health, St. John's, Newfoundland and Labrador for providing clinical samples. They also thank Mr. Adam K.M. Jenkins for help with evaluations of T cells.
This work was supported by the research operating grant MOP-126056 to TIM from the Canadian Institutes of Health Research (CIHR). SAM was a recipient of graduate fellowship awards from the National CIHR Research Training Program in Hepatitis C (NCRTP-HepC) and the Canadian Liver Foundation. TNQP was supported by postdoctoral fellowship awards from the NCRTP-HepC and the Canadian Association for the Study of the Liver/Hoffmann-La Roche/Astellas Pharma Canada. TIM is supported by the Canada Research Chair Program and funds from the CIHR and the Canada Foundation for Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript.
Molecular Virology and Hepatology Research Group, Division of BioMedical Sciences, Faculty of Medicine, Health Sciences Centre, Memorial University, St. John's, Newfoundland and Labrador, Canada
Sonya A. MacParland
, Annie Y. Chen
, Christopher P. Corkum
, Tram N.Q. Pham
& Tomasz I. Michalak
Present address: Department of Immunology, Medical Sciences Building, University of Toronto, Toronto, ON, Canada
Present address: Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montreal (IRCM), Montreal, QC, Canada
Tram N.Q. Pham
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Correspondence to Tomasz I. Michalak.
SAM participated in the design and conducted majority of experiments, analyzed the data and wrote a draft of the manuscript; AYC contributed to infection experiments and performed treatments with telaprevir; CPC, contributed to acquisition and analysis of flow cytometric data; TNQP contributed to analysis of the data and assisted with writing; TIM conceived the study, supervised its execution and helped to draft the final manuscript. All authors read and approved the final manuscript.
Additional file 1: Figure S1.
Monocyte depletion prior to cell infection with HCV. Monocytes were depleted from PBMC via plastic adherence for 4 h and their frequencies in total PBMC and monocyte-depleted PBMC were determined after staining with anti-CD14 antibody by flow cytometry.
Flow cytometric determination of T cell proliferation after exposure to medium alone (NP), NHP 1–3 or HCV inocula CHC 1–3 at 7 d.p.i. Using forward versus side scatter, lymphocytes (gate R1) were separated from debris. Percentage CFSE low was determined based on unstimulated control cells cultured with medium alone. Low left (LL) and low right (LR) quadrants represent cells with CFSE low (dim) and CFSE high (bright) reactivity, respectively. Dilution of CFSE fluorescence was analyzed with ModFit LT showing daughter generations. P.I. values were determined by using non-stimulated cells to define parent generation.
HCV lymphotropism
HCV infection of T cells
T cell proliferation
T cell apoptosis
T cell cytokine expression


For the love of Poetry, Prose and other Artful things.
Carlflame was created primarily out of my love for writing – painting words in black and white and the written. Yea, written words are cool! While spoken communication is awesome, written expressions are beyond beautiful – the swirl, twists, turns and magic that might have been impossible or quite difficult to deliver at length and design in speech, are easily rendered in writing. It's beautiful. Really.
Aside the above, it affords me an opportunity to coagulate the essence of the many pixies that fly about in my head. It serves as an open bank or log of some sort to squirrel away the produce of my imagination, thoughts and experiences. So far, it has been a nice experience. I hope it is for you too.
Welcome to Carlflame; La Vallée de Mots – The valley of words. I hope you enjoy the grains that grow here.


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Reminder! Temporary Changes to Parades and Shows: Due to construction work, some Magic Kingdom entertainment offerings will be impacted certain dates throughout February and March. The Main Street Trolley Show and Move it! Shake it! Dance and Play it! will be adjusted March 9, 11 and 15. On these dates, the Trolley Show will use the Main Street fire truck and Move it! Shake it! Dance and Play it! will be presented at 12:30 and 15:40.
New! Dates Announced for 2016 Epcot International Food & Wine Festival: The 21st Epcot International Food & Wine Festival kicks off September 14 and runs through November 14 this fall. That means almost two additional weeks – from 53 days to 62 – of deliciousness, foodie fun, culinary events, celebrity chefs, seminars, music concerts and more.
New! New Pieces at the Kinsey Exhibit: "Re-Discovering America: Family Treasures from the Kinsey Collection" is a world renowned collection of rare art, unique artifacts, and documents on display at the American Adventure Pavilion at Epcot. Some intriguing new pieces have been added to the exhibit that takes you on a stroll through the pages of America's history and showcases the influence of African-American culture. Touch screens and guest-activated lanterns with narration by Whoopi Goldberg, Diane Sawyer and a host of other celebrities also help bring the history to life.
New! 2016 Garden Rocks Concert Series: Celebrating its 23rd year, the Epcot International Flower & Garden Festival at Walt Disney World Resort begins March 2 and will run for over 90 days this year. This special spring event is filled with Disney character topiaries, colorful flowers, exceptional food and live music at the America Gardens Theatre. Thirteen bands will hit the stage at the Garden Rocks concert series during the 2016 Epcot International Flower & Garden Festival. The full lineup (subject to change) is listed below:
March 4 – 6 – The Orchestra starring former members of ELO – "Evil Woman"
March 11 – 13 – Starship featuring Mickey Thomas – "We Built This City"
March 18 – 20 – Little River Band – "Reminiscing"
March 25 – 27 – Village People – "Y.M.C.A."
April 1 – 3 – Herman's Hermits starring Peter Noone – "I'm Henry the VIII, I Am"
April 8 – 10 – The Guess Who – "American Woman"
April 15 – 17 – Blood, Sweat & Tears featuring Bo Bice – "Spinning Wheel"
April 22 – 24 – Bad Company, former lead singer Brian Howe – "Can't Get Enough"
April 29 – May 1 – George Satellites – "Keep Your Hands to Yourself"
May 6 – 8 – Gin Blossoms – "Hey Jealousy"
May 13 – 15 – Darlene Love – "Wait 'Til My Bobby Gets Home"
May 20 – 22 – Night Ranger – "Sister Christian"
May 27 – 30 – The Spinners – "Working My Way Back to You"
New! New Star Wars Entertainment Coming in Spring and Summer: Disney's Hollywood Studios is unveiling even more Star Wars-themed entertainment, starting this spring – including one of the most elaborate fireworks and projection shows in the park's history. April 4 will bring the arrival of the new Star Warslive entertainment experience Star Wars: A Galaxy Far, Far Away (a stage show) and a menacing First Order stormtrooper procession lead by Captain Phasma. To kick off summer, Disney's Hollywood Studios is launching a new nightly fireworks and projection show called Star Wars: A Galactic Spectacular that will light up the sky with Star Wars-themed fireworks, lasers, light projections and other special effects combined withStar Wars-themed music and iconic characters and scenes from throughout the saga. The show will be punctuated with a tower of fire and spotlight beams, creating massive lightsabers in the sky. This new show will be the most elaborate fireworks display to take place in the history of Disney's Hollywood Studios.
New! Additional Details Announced for Star Wars-Themed Lands: During the "Wonderful World of Disney: Disneyland 60" special that aired on ABC-TV on Feb 21, Disney shared some exciting details about the Star Wars-themed lands planned for Disneyland and Walt Disney World Resorts. Guests will be able to step aboard The Millennium Falcon and actually pilot the fastest ship in the galaxy, steering the vessel through space, firing the laser cannons, in complete control of the experience. And with the arrival of the First Order to the planet, visitors will find themselves in the middle of a tense battle between stormtroopers and Resistance fighters.
New! Toy Story Mania Closed March 15: Due to work related to the additional track at Toy Story Mania coming this summer, the attraction will not be available on March 15. Guests who had made a FastPass+ selection for Toy Story Mania on that day will be provided with a FastPass+ selection for another experience at Disney's Hollywood Studios.
New! Baby Wallaby at the Oasis: There's a new tammar wallaby joey at Disney's Animal Kingdom! You can find the joey and wallaby family in the Oasis area of the park. This joey, the name for a young wallaby, is only about five months old, so it hasn't left mom's pouch yet. Last week, the keepers saw the joey peeking out of mom's pouch. At about seven months, the joey should completely emerge from the pouch with a full body of fur.
Reminder! Princess Promenade at Disney's Grand Floridian: Cinderella host the Princess Promenade that starts in M. Mouse Mercantile shop at Disney's Grand Floridian Resort & Spa. Guests ages 3–12 are invited to stroll down the grand staircase with Cinderella and participate in a waltz in the main lobby.
New! Blizzard Beach Closed Due to Cold Weather: Disney's Blizzard Beach Water Park will be closed February 25-27, 2016 due to forecasted low temperatures.
New! Typhoon Lagoon Refurbishment Extended: The Disney's Typhoon Lagoon Water Park refurbishment has been extended and the park will reopen on March 20.
New! Atlanta Brave Spring Training Starting Soon: ESPN Wide World of Sports Complex is home to the 2016 Atlanta Braves Spring Training Season for the 19th consecutive year. In true Disney fashion, there are entertainment opportunities for everyone in the family while at the ballpark. For the younger fans, at each game one child is selected at random to live out their broadcasting dream. The "junior announcer" has the opportunity to announce player introductions to the entire stadium for a full inning! You can savor some tasty ballpark food concoctions created especially for Spring Training. Adults can even enjoy craft beer from the out-of-market club. Take a look at some of the brews fans can look forward to.
New! New Act at La Nouba: Ekaterina and Alexander Abramova are real-life partners who dazzle Disney Springs audiences with their daring aerial bamboo performance. Practically a lost art, aerial bamboo, a skillful mix of acrobatics and storytelling that takes place high above the stage, is rarely seen on modern stages.
February 4 – 29: Representatives from Precious Moments dolls will be on hand for the release of their new Valentine inspired collection, among other fan favorites, throughout the day, each day, at Marketplace Fun Finds in Disney Springs Marketplace. Tip: Doll Designer Linda Rick will make a special appearance February 19 – 21.
February 24 – 28: Check out the latest releases from artist Greg McCullough appearing at Art of Disney from 1 – 8 p.m., each day.
« Around the Mouse: Walt Disney World News, February 17, 2016
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redpajama_set_name: RedPajamaCommonCrawl


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Get a SanDisk Extreme Pro 4TB SSD for a ridiculous price today
By Phil Nickinson November 28, 2022
Here's an insane Cyber Monday deal that perhaps doesn't look like that much of a deal at first glance, given that we're talking about a $350 hard drive. But look a little closer and you'll see that B&H Photo has this on sale for something like 65 percent off.
Phil Nickinson/Digital Trends
That's still a chunk of change, but those who know, know. This is the sort of SSD that's a lifesaver if you're often moving around large chunks of data, like photos or video, from one place to another. And if you're in the photo or video business, that's you. It'll significantly speed up your Lightroom or Premiere Pro workflow for a few reasons.
First off, it's an SSD. That means its read/write speeds are going to be much faster than a platter-based spinning hard drive. (We're talking four or five times faster.) Just be sure to either use the included USB-C cable or something rated for USB 3.2 "Superspeed." If you just grab any old USB-C cable you have lying around, you're probably going to be wondering what's broken. (I speak from experience; that works great for me.)
Best Cyber Monday iPad Deals: iPad Air, iPad Pro, iPad Mini
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Cyber Monday: get this 24-inch Smart TV for under $100 today
It's also super-portable — a decent amount smaller than a deck of cards. And if you really want to get snazzy, you can use the built-in carabiner hook. Or just slip it in your pocket. Your call. There's also a decent amount of ruggedness built into this SSD, with an aluminum chassis and IP55 protection against dust and water.
If you make any money off taking photos or shooting video, this little SSD is a lifesaver. As soon as I'm back from a shoot it's the first place I put my pics after moving them off the SD card. Redundancy is key, and you'll get much more into the habit of backing things up from the start if it doesn't take all day to do so.
And having a whopping 4 terabytes of storage at your disposal means you'll be able to keep all those precious files on hand — and on a speedy platform — much longer before having to clean things off.
Get these JBL earbuds and 4 months of Music Unlimited for $25
Get this 50-inch 4K TV for $230 at Best Buy for Cyber Monday
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The Chittagong Stock Exchange () is a stock exchange based in the port city Chittagong, Bangladesh. It is one of the twin financial hubs of the country, alongside the Dhaka Stock Exchange. Established in 1995, the exchange is located in the Agrabad business district in downtown Chittagong. It has a combined market capitalization of over US$ 38 billion as of 2020.

Timeline

1 April 1995 CSE incorporated as a company.
10 October Floor trading started in cry out system.
4 November 1995 formally opened by then former Prime Minister Begum Khaleda Zia.
30 May 2004 Internet based Trading system opened.
8 July 2015 CSE launched new brand logo.

Trading hours

Market opens at 09:30 am local time.

Market closes at 01:50 pm local time.

2010-11 crash

The bullish market turned bearish during 2010, with the exchange losing 1,800 points between December 2010 and January 2011. Millions of investors have been rendered bankrupt as a result of the market crash. The crash is believed to be caused artificially to benefit a handful of players at the expense of the big players.

See also

 Chittagong Tea Auction
 List of South Asian stock exchanges
 List of stock exchanges in the Commonwealth of Nations

References

External links

 Bangladeshstockmarket

Companies based in Chittagong
Agrabad

Chittagong
Economy of Chittagong
Financial services companies established in 1995
1995 establishments in Bangladesh
Finance in Bangladesh

redpajama_set_name: RedPajamaWikipedia


People are like mirrors, always reflecting the same attitudes that they think others have towards them.
The best way to favorably impress someone is not by telling them marvelous things about yourself, but by letting them know that you are favorably impressed by them.
You love whom you truly believe love you. The secret to winning someone's love is to first convince him or her of your real, genuine love for them.
5. Suggest the possibility of getting together for some sort of date.
Taking a visible interest in the other person is the first step to winning their heart. It immediately fills their need for attention and paves the way for future progress.
As soon as you have gained someone's attention by showing enough interest, it's time to begin building a relationship. You need to spend time together with the one you want.
A date is simply an opportunity to interact with the one you want on a personal level, one to one.
The first date can be very uncomfortable for both parties. You are getting to know the other person, and learning about their likes, dislikes, or other things that you didn't know before.
Be yourself. Leave the other person a warm feeling of having spent an interesting and enjoyable evening with you.
Something magical happens when human beings spend time together. They grow on each other. There is a psychologically binding effect.
However, don't reveal your anxiousness in asking for more dates. It is dangerous to ask for more than one date at a time. This can frighten off a potential sweetheart before the binding effect has even began. At the same time, don't settle for less than a real date. A relationship can't grow just by seeing someone everyday at class, work, or in some other neutral setting. This won't provide enough electricity to adequately charge the relationship.
Set your sights on that first date. Work for it until you succeed in getting it. Then after completing a date and giving it a little time to be digested, go for another one. Don't push for too much commitment at once. By focusing on one date at a time, you will actually be building the relationship.
In developing a relationship, you must spend time with the person, one on one, to build it properly. This requires many dates, but only ask for one date at a time.
If the one you want seems hesitant to grant you time even for a single date, be creative. Think of something you can ask the person to do with you, even if it involves as little as fifteen minutes. Little by little, you will surely reach your goal.
By allowing ourselves to remain open and vulnerable to another human being, even in the face of rejection and feeling foolish, we show our sincerity and true greatness of character. This will persuade others to become similarly open with us.
On the other hand, becoming defensive when someone hurts you by not responding to your attention will only reinforces their distant attitude and makes the other person feel justified in their initial standoffishness towards us. No matter who you are, you are going to experience some degree of rejection in trying to win someone's heart.
By exhibiting a sort of "If you don't want me then I don't want you" type of attitude, we demonstrate very clearly that our ability to be a true friend is shallow.
Make the effort to achieve unconditional friendship and stick with it, even if the going gets tough. Don't get defensive just because the sincerity of the love you extends doesn't seem to sufficiently impress itself upon someone's heart when you first offer it.
Allowing yourself to become vulnerable to another human being shows that you care more about the person than their actual reactions to you. Over an extended period of time, this type of treatment is guaranteed to product positive changes in that person's behavior towards you.


The 33rd Annual Red Mountain Eagle Pow Wow will be held on November 2-3, 2019 at the Salt River Baseball Field on Longmore just north of McDowell in Scottsdale, Arizona. Come join singers, dancers and vendors from across North American in this celebration of Native American Culture.
The Red Mountain Eagle Pow Wow and SRPMIC will not be held responsible for any damages, injuries, thefts, or losses to any person, vehicle or personal belongings. Alcohol, firearms and drugs are not permitted on or near pow-wow grounds.
Mark your calendar today so you won't miss the Red Mountain Eagle Pow Wow on the first weekend of November (Saturday – Sunday) at the Salt River Baseball Field in Scottsdale, Arizona.
NE corner of McDowell & Longmore Roads.


Baharat Spice Blend | Nomad Spice Co.
The name Baharat simply means "spice" in Arabic. However, it's a widely-used blend throughout the Middle East and Africa.
Our handcrafted version of Baharat Spice Blend is a warming mix of sweet, spicy, full-flavored seasonings packed with cumin, coriander and cardamom with spicier ingredients like paprika and black peppercorns for this signature blend.
Cinnamon, cumin, coriander, cardamom, nutmeg, cloves, black peppercorns and paprika.


Vitality STAR Qualified properties and apartments use considerably less Strength than typical new households and apartments though providing superior convenience, excellent, and longevity.
I also wonder how perfectly they will cope with hard water eventually – the extremely substantial costs of heat flux throughout quite compact warmth exchangers would seem to be at risk of scaling.
But, sometimes, tankless isn't The easiest method to go, as well as a tank water heater is a more prudent decision, especially when there are actually units like Sunnovation's Aquanta readily available. consider our tutorial to tank vs. tankless water heaters for almost everything you have to know.
mixture boilers demand less House than traditional tanked programs, and so are appreciably less expensive to set up, since water tanks and associated pipes and controls are not expected. Yet another advantage is the fact multiple unit could possibly be used to produce different heating zones or a number of loos, supplying larger time and temperature Regulate.
A big tank water heater that stores 40 to 60 gallons of hot water at a time will maintain your showers, laundry, along with other scorching water wants appropriately glad for really some time. modern-day tanks reheat supplemental supply really rapidly also, so you'll very likely never run outside of very hot water after you will need it.
These are pretty substantial and require a little bit of space in just your own home. But, should you take care of to deplete what exactly is within the tank, You will need to hold out till your water heater creates far more very hot water.
Disconnect source line from aged water heater. you'll likely have some water still left in the road Regardless that your principal is shut off. Place a bucket underneath water bib to capture any spills.
you should definitely examine the Instructions diligently. Some models involve the installer be certified to put in the device to ensure that the warranty to get valid.
naturally that you are accurate that tankless water heaters use less energy than tank-style water heaters. having said that, anyone who is considering decreasing their usage of fossil fuels or minimizing their carbon footprint nonetheless requires to look at regardless of whether any particular retrofit measure is definitely worth the financial investment, mainly because we don't have an unlimited number of dollars to invest.
make improvements to fiscal overall performance and lower carbon emissions with EPA's strategic Strength administration tactic.
Tankless water heaters are all of the rage. They appeal to everyone, from your household hunter, for the conservationist, into the comfort and ease and comfort minded. It's No surprise – you recuperate performance and endless very hot water with a device that doesn't occupy Significantly extra space than a substantial suitcase.
I would usually prefer the greater energy-economical option, although the significant selling price is really an indicator which the tankless device likely has greater embodied Power. It could also require extra Recurrent upkeep, particularly when you may have difficult water.


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Nightlife/Drag
Photos by Joseph Stevens
Rogers Park leather and fetish bar Jackhammer held its grand re-opening the evening of Jan. 27 after a long renovation process. The longtime North Side watering hole opened in 1999, taking over the space previously occupied ...
Billy Masters: George Santos gets to be a real drag, and all the dish you need
"If you're an enemy of drag, you're an enemy of mine." —George Lopez criticizes Ron DeSantis' "War on Drag Queens." There are some stories I try to stay away from, and that includes George Santos—if I ...
ONYX Midwest forum tackles racist performance at Touche's anniversary party
In a response to the racist performance that took place at the leather, fetish and bear-focused Touche Chicago's 45th anniversary celebration on Nov. 1, 2022, ONYX Midwest hosted a community forum Jan. 21 at The Leather ...
NATIONAL Indiana's 'Don't Say Gay,' drag show, Citi, new LGBTQ+ terms
GOP lawmakers in Indiana have said that a proposal to restrict discussions of sexual orientation and gender identity in schools—modeled after Florida's "Don't Say Gay" bill—will be drafted in the new year, according to INTO. The ...
Billy Masters: All the retrospective you'll need to show 2022 the door
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-- From a press release
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SHOWBIZ 'Black Panther,' 'Horror Story,' Beyonce, video game
Video below
I May Destroy You actress Michaela Coel will play Aneka in Black Panther: Wakanda Forever, according to Out. In the comics, Aneka is a Wakandan warrior who identifies as queer and has a relationship with Ayo ...
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WORLD Vietnam's pro-LGBTQ+ move, Grindr kidnappings, Taiwan event
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Drag show finally takes place at UpRising Bakery and Cafe
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UpRising Bakery allowed to hold drag brunch, other events
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'Spiritual DragCon' debuting in Chicago on Oct. 7-9; speakers sought
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Man arrested for vandalism of LGBTQ+-owned suburban bakery
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Suburban bakery cancels drag event after controversy, vandalism [UPDATE]
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Touche holds fundraiser for Windy City Times
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Billy Masters
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Chicago Pride Fest adds new features to the outdoor celebration
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States might make having kids at drag events a felony; LGBTQ+ groups respond
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Gerber/Hart unveils Pride Month events with 'Drag,' 'Bad Gays,' sneak peek
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Dance Divas return to Baton July 31 and Aug. 1
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--From a press release
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Podcast attached below
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text:
African American Voices
Asian American & Pacific Islander Voices
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Interviews by Collection
Merry Demarest Oral History Interview, May 25, 2017
Interviewee: Merry Demarest
Interviewer: Justyn Jacobs, Lucy Hillenbrand and Meredith Bowers
Interview Date: May 25, 2017
Location: Demarest residence, Corvallis, Oregon
In the interview, Merry Demarest begins by discussing her youth, including her many relocations during childhood and young adulthood, and a tumultuous family dynamic after her mother remarried. Following this portion of the interview, Demarest focuses on her social justice activism through the years, which began fairly early in her life. Demarest discusses her involvement with the National Organization for Women (NOW), her positions within the organization, and her campaigning for the federal Equal Rights Amendment. After this, Demarest proceeds to a discussion of her work campaigning for Hillary Clinton by encouraging constituents in Texas to attend party caucuses for the Democratic nomination. After 8 is the next organization she discusses, including her involvement with that organization in fighting Corvallis Measure 02-06, a homophobic measure written by the Oregon Citizens Alliance. She then talks about campaigning for Bill Clinton in the 1984 gubernatorial election in Arkansas. Demarest's involvement with the Democratic Party of Oregon and her chairing of the Democratic Oregon State Fair booth for 12 years are the next topics she discusses. Demarest then describes the importance of Emily's List to her extended family because of their early involvement. Demarest then outlines how she became the founding chair of the LGBTQ+ organization Basic Rights Oregon after being a co-chair of the anti-Oregon Measure 9 campaign, and what the organization's goals were both when it was founded and at the time of the interview, in 2017.
In the latter portion of the interview, Demarest details her involvement with the Human Rights Campaign and the award she and her husband Harry received from the organization. She then returns to a discussion about campaigning in Utah for the Equal Rights Amendment, and her interactions with the women opening their doors to her. One of the last topics Demarest spends time on is her work campaigning for Hillary Clinton, and then for Barack Obama in the 2008 election. Demarest gives her thoughts on the 2016 presidential election and advice for today's young activists, and explains what her more recent involvement with the Benton County Democrats has been. Finally, Demarest discusses her current project: trying to encourage local music venues to refuse to book the Eugene, Oregon band Cherry Poppin' Daddies.
Merry Demarest
OSU Queer Archives Oral History Collection
Special Collections and Archives Research Center, Oregon State University Libraries
Justyn Jacobs, Lucy Hillenbrand, and Meredith Bowers
Born Digital
OH34-demarest-merry-2017
Oral History Item Type Metadata
Justyn Jacobs, Lucy Hillenbrand and Meredith Bowers
Demarest residence, Corvallis, Oregon
OHMS Object
Interview Format
Merry Demarest, "Merry Demarest Oral History Interview, May 25, 2017," Special Collections & Archives Research Center, accessed January 18, 2021, http://scarc.library.oregonstate.edu/omeka/items/show/31541.
Presented by the Oral History Program at the Special Collections and Archives Research Center, Oregon State University Libraries.
Powered by Omeka and the Oral History Metadata Synchronizer.


text:
Q: How does macOS identify the signing status of an app downloaded from the Internet? I downloaded an app from the Internet using Safari. Upon executing it, macOS notifies that the app is from an unidentified developer.
How is macOS able to determine this information? Where is the signing metadata for an app stored?

A: In fact the message "Unidentified developer ..." is the result of a combination of both tasks: checking the com.apple.quarantine attribute and the code signing resources (in  */Contents/_CodeSignature/CodeResources) while the actual signing status is solely determined by */CodeResources.
You can get the extended attribute by entering:
$ xattr -p com.apple.quarantine /Applications/*.app

The code signing information is accessed by entering:
$ codesign -dvvv /Applications/*.app

Example:
$ codesign -dvvvv /Applications/Google\ Chrome.app/
Executable=/Applications/Google Chrome.app/Contents/MacOS/Google Chrome
Identifier=com.google.Chrome
Format=app bundle with Mach-O thin (x86_64)
CodeDirectory v=20200 size=273 flags=0x800(restrict) hashes=3+3 location=embedded
OSPlatform=36
OSSDKVersion=658432
OSVersionMin=657664
Hash type=sha256 size=32
CandidateCDHash sha1=bc8e3dffe7d9d5242e09ea80a220ed365d46fdf2
CandidateCDHash sha256=d7ae6211906bb17d593ce9c215d190a81f37e658
Hash choices=sha1,sha256
Page size=4096
CDHash=d7ae6211906bb17d593ce9c215d190a81f37e658
Signature size=8949
Authority=Developer ID Application: Google, Inc. (EQHXZ8M8AV)
Authority=Developer ID Certification Authority
Authority=Apple Root CA
Timestamp=20. Mar 2018 at 07:23:20
Info.plist entries=36
TeamIdentifier=EQHXZ8M8AV
Sealed Resources version=2 rules=7 files=203
Internal requirements count=1 size=240

The most important part is the certificate chain (and the chain of trust):
Authority=Developer ID Application: Google, Inc. (EQHXZ8M8AV)
Authority=Developer ID Certification Authority
Authority=Apple Root CA

Here three different authorities are visible:

*

*Root certificate: Apple Root CA

*Intermediate Apple developer certificate: Developer ID Certification Authority

*Developer certificate: Google, Inc. (EQHXZ8M8AV)

This means that the app is signed with a developer certificate, published and signed by Apple's intermediate developer authority, which itself is published and signed by Apple's Root CA.
This chain qualifies Google (or more exactly: the team/unit Google, Inc. (EQHXZ8M8AV)) as an Apple identified developer.
You can also codesign apps either yourself or with a codesigning certificate from another intermediate/root authority but this won't overcome Gatekeeper.
Now four permutations are possible assuming Gatekeeper is enabled (but not considering self-signing/alien-signing):

*

*No com.apple.quarantine attribute/no Apple Developer certificate (Example: brew cask installed Apache CouchDB)

*No com.apple.quarantine attribute/an Apple Developer certificate (Example: brew cask installed Google Chrome)

*A com.apple.quarantine attribute/no Apple Developer certificate (Example: Apache CouchDB installed by downloading the zip from the website and copying the unzipped app to /Applications/)

*A com.apple.quarantine attribute/an Apple Developer certificate (Example: Google Chrome installed by downloading the dmg from the website and copying the app inside to /Applications/)

In the first two cases the app will simply launch. In the third case you will get the Unindentified Developer message. You can resolve this by removing the extended attribute xattr -d com.apple.quarantine * (=case 1). In the fourth case you will get the "* was downloaded from the Internet, are you sure you want to open it?"

A:
How is macOS able to determine this information? Where is the signing metadata for an app stored?

The signature is stored within the application binary itself, with some additional data in the application bundle (in the file Contents/_CodeSignature/CodeResources).
You can get some information on the signature using the command-line utility codesign -- for instance:
# codesign -dv /Applications/Preview.app
Executable=/Applications/Preview.app/Contents/MacOS/Preview
Identifier=com.apple.Preview
Format=app bundle with Mach-O thin (x86_64)
CodeDirectory v=20100 size=21314 flags=0x0(none) hashes=659+5 location=embedded
Platform identifier=4
Signature size=4485
Info.plist entries=35
TeamIdentifier=not set
Sealed Resources version=2 rules=13 files=2077
Internal requirements count=1 size=68

A: The information is stored within extended attributes, with additional information potentially embedded within an application.
Extended Attributes
This quarantine information is stored as an extended attribute. Use the xattr tool to view the attributes associated with a file or application.
The attribute causing this message is com.apple.quarantine.
See How do I remove the "extended attributes" on a file in Mac OS X? and App can't be opened because it is from an unidentified developer for more details.
Gatekeeper and Code Signatures
The extended attribute is used by Gatekeeper on macOS and is discussed in How to set (restore) the com.apple.quarantine attribute?
A optional digital signature embedded within an application is used to determine the application's developer. If a signature is not found, macOS presents the application as being from an unidentified developer.

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