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Differential Evolution and Stability of Epitope-Specific CD8+ T Cell Responses in EBV Infection
Michelle D. Catalina, John L. Sullivan, Katherine R. Bak and Katherine Luzuriaga
J Immunol October 15, 2001, 167 (8) 4450-4457; DOI: https://doi.org/10.4049/jimmunol.167.8.4450
Michelle D. Catalina
Department of Pediatrics and Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605
John L. Sullivan
Katherine R. Bak
Katherine Luzuriaga
Murine models of lymphocytic choriomeningitis virus infection suggest that the memory CD8+ T cell repertoire is reflective of the CD8+ T cell repertoire generated during acute infection. Less is known regarding the evolution of CD8+ T cell repertoires during human viral infections. We therefore examined epitope-specific CD8+ T cell responses in a large cohort of individuals with acute through latent Epstein-Barr virus infection. Using 16 of 20 published EBV epitopes restricted by HLA-A2, HLA-A3 or HLA-B7, we showed that lytic cycle-specific CD8+ T cell responses predominated during acute EBV infection. However, whereas HLA-A2+-restricted BMLF-1-specific CD8+ T cell responses were maintained through latency, HLA-A2+- and HLA-B7+-restricted BZLF-1, as well as HLA-A3+-restricted BRLF-1 CD8+ T cell responses, were generated but not readily maintained. Analyses of CD8+ T cell responses to EBV latent cycle Ags showed delayed detection and lower frequencies of latent epitope-specific CD8+ T cell responses during acute EBV infection, with maintenance of these responses 1 yr post-EBV infection. Early BMLF-1 and EBNA-3A epitope-specific CD8+ T cell frequencies did not correlate with their frequencies at 1 yr postinfection. Interestingly, populations of EBV-specific CD8+ T cells were stable during 20 mo in our long term EBV-seropositive populations, suggesting homeostasis between virus and the host immune system. This study demonstrates that CD8+ T cell repertoires generated during persistent viral infections are not simply reflective of the initial pool of CD8+ T cells and provides evidence that the generation of CD8+ T cell responses to a persistent infection is a dynamic process.
Epstein-Barr virus is a ubiquitous herpesvirus that infects more than 90% of the world's population. During acute EBV infection, the virus replicates in lymphoid cells and oropharyngeal epithelium with the expression of the full array of viral proteins (1). The infection of B cells results in a life-long EBV carrier state in which B cells primarily express latent proteins (2). Whereas primary infection is usually asymptomatic in infants (3), adolescents and young adults frequently experience acute infectious mononucleosis (4). Studies spanning the past decade have shown that CD8+ T cell responses to EBV are important for controlling EBV replication (5, 6, 7, 8). Because individuals with acute EBV infection may be readily identified on the basis of the heterophile Ab test and the presence of atypical lymphocytes, EBV is an ideal model for the study of the generation and maintenance of CD8+ T cells in response to a persistent human viral infection.
The use of HLA class I tetramers in the lymphocytic choriomeningitis virus (LCMV)3 mouse model has shown that very high levels of Ag-specific CD8+ T cells are generated and maintained into memory (9). The size of the CD8+ T cell memory pool is reflective of the initial clonal burst size during the acute LCMV response (10). The TCR repertoire of the primary antiviral CD8 T cell response to LCMV was shown to be similar to that of the memory pool (11). Altogether, these data suggest a stochastic selection of memory cells from the pool of CD8+ T cells activated during primary infection.
Similar conclusions were reached by Callan et al. (12) in a study of CD8+ T cell responses from acute through chronic EBV infection. However, recent evidence from human HIV-1 infection has shown that an HLA-A2-restricted response to an HIV-1 gag epitope found in a majority of HLA-A2 individuals with chronic HIV-1 infection was not present in 11 HLA-A2 individuals during acute HIV-1 infection (13). This suggests that for persistent viral infections the memory/effector pool may not be reflective of the initial pool of CD8+ T cells and that the generation of CD8+ T cells may not fit a stochastic model.
To examine the generation and maintenance of CD8+ T cell responses during a persistent human viral infection, we analyzed a cohort of individuals experiencing acute EBV infection using several EBV peptide epitopes restricted by common HLA alleles: HLA-A*0201 (A2); HLA-B*0702 (B7); and HLA-A*0301 (A3). By studying individuals with acute EBV infection, we were able to determine the frequencies of CD8+ T cell responses to previously defined lytic and latent epitopes during acute infection. We then followed these same individuals into latent EBV infection to characterize the evolution of EBV epitope-specific CD8+ T cell responses over time.
These studies were conducted in adolescents (17–24 yr old) presenting to the clinic at the University of Massachusetts Amherst Student Health Service (Amherst, MA) with clinical symptoms consistent with acute EBV infection (fever, rash, fatigue, hepatosplenomegaly). After informed consent, students presenting with acute-like symptoms gave five blood samples (50 ml each) at the following time points: at presentation with symptoms (V-1); and 1 wk (V-2), 2 wk (V-3), 6 mo (V-4); and 1 yr (V-5) after presentation. Inclusion in the study was based on a positive Monospot test and the presence of atypical lymphocytes. Acute EBV infection was confirmed through the detection of IgM for the EBV viral capsid Ag.
Healthy EBV-seropositive adults >30 yr of age were recruited for these studies from the UMMS research community. All of these individuals likely experienced EBV infection for a minimum of 10 yr before study. Prior EBV infection was confirmed through the detection of EBV capsid Ag-specific IgG Abs in the peripheral blood. After informed consent, study subjects provided blood samples (50 ml) at entry and every 3 mo thereafter. These studies were approved by the Human Studies Committee at the University of Massachusetts Medical School (Worcester, MA).
Molecular HLA class I typing
Molecular HLA class I typing was done on all study participants by Microdiagnostic (Nashville, TN).
Detection of cytokine-secreting CD8+ T cells after in vitro stimulation with EBV peptide
A modification of the method by Kern et al. (14) was used for the detection of cytokine-secreting CD8+ T cells. Whole blood (0.3 ml/test, heparin) was incubated for 1 h at 37°C with 2 μM EBV peptides. Because staphylococcal enterotoxin B (Toxin Technology, Sarasota, FL) is able to nonspecifically stimulate T cells to secrete IFN-γ, it was used as a positive control. None of the donors used in this study was HIV-1 infected; therefore, the HLA-A2-binding, HIV-1 gag SLYNTVATL peptide was used as a negative control. After 1 h incubation with peptide, Golgiplug (BD PharMingen, San Diego, CA) was added to the cells, and they were incubated for an additional 5 h. After incubation, the cells were incubated with 2 μM EDTA for 15 min with vigorous vortexing every few minutes. The cells were stained with combinations of the following Abs: IFN-γ APC, CD3 PerCP, CD69 PE, and CD8 FITC. The cells were incubated at room temperature for 30 min and then washed with 1% FBS in PBS; 100,000 events gated on the lymphocyte population were collected and analyzed immediately by 4-color flow cytometry. Appropriate isotype, negative, and positive controls were used to define positive and negative cell populations. Background IFN-γ production in this assay was 0.02 ± 0.01% for stimulation of individuals with either a peptide derived from HIV-1- or EBV-derived non-HLA-binding peptides. Significant IFN-γ production was considered to be greater than the mean of the background plus 3 SD, or 0.05% of CD8+ T cells.
Generation of HLA class I tetramers
HLA-A*0201 H chain (residues 1–275) and human β2-microglobulin (β2m) in the prokaryotic expression system pET R&D were obtained from D. Garboczi (National Institutes of Health, Bethesda, MD). The 3′ end of the HLA-A*0201 H chain was modified with a BIR A biotinylation site as published (15). HLA-B*0702 was a gift of Dr. E. Pamer (Memorial Sloan-Kettering Cancer Center, New York, NY). As previously described (16), HLA class I H chain constructs or β2m were grown to midlog phase and induced with 0.5 M isopropyl-β-thiogalactosidase. Inclusion bodies were purified and solubilized in 6 M guanidine-HCl, pH 8.2.
The H chain, β2m, and peptide were refolded by dilution as described (16). The A2 BMLF-1 peptide, GLCTLVAML, and the B7 EBNA-3A peptide, RPPIFIRRL, were synthesized by the University of Massachusetts Medical Center peptide core facility and purified before usage. Briefly, 1 μM H chain, 2 μM β2m, and 30 μM peptide were refolded by dilution (0.4 M l-arginine, 0.1 M Tris (pH 8.0), 0.002 M EDTA, 0.005 M reduced glutathione, 0.5 M oxidized glutathione, and 0.5 mM PMSF (pH 8.3)) and incubated for 24 h at 10°C with gentle stirring. The refolded monomer was concentrated using an Amicon Stirred Cell (Amicon, Beverly, MA). The 45-kDa refolded product was isolated on an ACTA fast protein liquid chromatograph using a Superdex 200 column (Amersham-Pharmacia, Piscataway, NJ). After gel filtration, the refolded monomer was biotinylated with biotin ligase (birA; Avidity, Denver, CO) and washed to remove excess biotin. The resulting biotinylated monomer was mixed with streptavidin-APC conjugate (BD PharMingen) at a molar ratio of 4:1 to form tetramers (15). To stain 100 μl whole blood, 100 ng tetramer were used. Percentages of tetramer-binding cells were expressed as a percentage of total CD8+ T cells. These tetramers stained non-A2 or non-B7 individuals with acute EBV infection and A2, B7 individuals who were EBV seronegative at 0.02 ± 0.01%. Tetramer staining greater than the mean of the background plus 3 SD, or 0.05% of CD8+ T cells, was considered significant. A total of 100,000 events gated on the lymphocyte population were collected and analyzed.
Four color flow cytometry for Ki-67 and annexin V expression
Four-color flow cytometric analysis was performed using whole EDTA blood for Ki-67 analysis and heparin preserved blood for annexin V analysis. Because annexin V binding is Ca2+ dependent, EDTA blood stained with annexin V was run alongside as a negative control. Samples were analyzed on a BD Biosciences FACSort with an added laser and FACSCaliber software (BD Biosciences, San Jose, CA). HLA class I tetramers can bind nonspecifically to non-T cell populations; therefore cells were first gated through both CD3 and CD8 to ensure that the tetramer positive cells were all CD8+ T cells. Cells were analyzed with a tetramer conjugated to APC, CD8 FITC (Sigma, St. Louis, MO), CD3 PerCP (BD Biosciences), and Ki-67 PE or annexin V PE (BD PharMingen, San Diego, CA). Permeabilization for staining with Ki-67 was done after tetramer staining and lysing of the cells. Permeabilized isotype controls were run alongside each analysis for Ki-67.
Means and SDs were calculated and compared with each other using the Student t test to determine p values. The Wilcoxon signed rank test, a nonparametric paired t test, was used to determine p values for the differences in the range of tetramer-staining CD8+ T cells between study time points for the group with acute EBV infection. The Mann-Whitney U test was used to determine p values between the range of tetramer staining cells between study time points for the acute EBV infection group and the long term latently infected group. To determine whether differences in the percentage of tetramer staining cells were significant, tetramer staining was performed on 12 identical samples for 14 individuals, and the means ± SD for those 12 samples were determined. These were then compiled for the 14 individuals to determine normal variations in tetramer staining. Differences >0.2 ± 0.2% were considered to represent a significant change in magnitude.
Differences in specificity of EBV-specific CD8+ T cells during acute and latent EBV infection
We first examined the frequency and magnitude of CD8+ T cell responses to previously defined EBV lytic protein epitopes (Table I⇓) during acute and latent EBV infection using an in vitro assay that detects single-cell production of IFN-γ (Fig. 1⇓). The three most prevalent HLA types in our acute population were HLA-A2+ (45%, n = 25), HLA-A3+ (16%, n = 10), and HLA-B7+ (23%, n = 12); 67% of our acute population had one or more of these alleles. We therefore prioritized evaluation of responses to these epitopes. Because HLA typing results were not available at baseline testing, we used all of the peptides listed in Table I⇓ during presentation with symptoms (V-1); once HLA typing was completed, peptide panels were individualized for later visits. We then followed these individuals with acute EBV infection into latent EBV infection and evaluated their CD8+ T cell responses up to 1 yr postpresentation (V-5). Fresh whole blood was used for all assays to avoid the possible loss of Ag-specific CD8+ T cells during Ficoll (Amersham Pharmacia Biotech, Piscataway, NJ) separation and cell cryopreservation.
EBV-specific production of IFN γ by CD8 T cells of an HLA-A2+,A3+ acute EBV patient at presentation with symptoms (V-1).
Table I.
Peptides used in this study
The frequencies and epitope specificity of peripheral blood CD8+ T cell responses were evaluated from presentation through 1 yr and were compared with those detected in long term latently infected individuals. Of the eight early or lytic protein epitopes evaluated, all but one were recognized by the majority of individuals tested (Fig. 2⇓). CD8+ T cell responses to an HLA-A2+-restricted BMLF-1 epitope were detected in the majority of HLA-A2+ individuals during acute and latent infection. An HLA-A2+-restricted gp85 epitope (gp85SV) was also recognized during the acute and the latent stages of infection. By contrast, CD8+ T cell responses to the six other lytic epitopes were more commonly detected during acute infection than during latent infection.
Frequency of responses to EBV lytic peptides in HLA-A2+, -B7+, and -A3+ individuals at presentation with acute EBV infection or at least 1 yr after presentation (latent). n values for each peptide are listed at the end of each bar. ∗, Significant difference (p < 0.005) between the frequencies of individuals responding between the acute and latent time points.
All HLA-A2+ individuals with acute EBV infection produced IFN-γ in response to stimulation with the A2 BMLF-1 peptide (range, 0.08–12%; median, 2.3%; Fig. 2⇑). The majority (25 of 27 = 93%) of latently infected individuals also produced IFN-γ in response to this peptide at frequencies ranging from 0.06 to 1.6% (median, 0.56%). Responses to the A2 BZLF-1 epitope were commonly seen during acute EBV infection (5 of 8 = 63% of individuals; range of IFN-γ production, 0–1.25%) but rarely detected during latent infection (1 of 8 = 12.5%; range of IFN-γ production, 0–0.15%). Eight of ten (80%) HLA-B7+ individuals responded to the B7 BZLF-1 peptide (range, 0–1.4%) during acute infection; the response to the B7 BZLF-1 peptide was detected in only 25% of individuals (2 of 8; range, 0–0.34%) during latent infection. Similar to the pattern seen for HLA-A2+- and HLA-B7+-restricted lytic epitopes, responses were also more frequently detected to the A3 BRLF-1 epitope during acute EBV infection (8 of 10 = 80%; range, 0–3.2%), than during latent EBV infection (1 of 8 = 13%; range, 0–0.03%).
We also examined the frequency and magnitude of CD8+ T cell responses to previously defined EBV latent protein epitopes (Table I⇑) in acute and latent EBV infection using the in vitro assay for the production of IFN-γ (Fig. 3⇓). Latent epitope responses were rarely detected in individuals presenting with acute EBV infection. Only 2 of 20 (10%) HLA-A2+ individuals responded to the A2 EBNA-3A epitope during acute EBV infection (range, 0.05–0.06%), but 14 of 20 (70%) latently infected HLA-A2+ individuals responded to the A2 EBNA-3A epitope (range, 0.05–0.41%); 3 of 20 (13.6%) latently infected HLA-A2+ individuals also responded to the A2 LMP-1 epitope (range, 0.09–0.5%). Whereas only 3 of 12 (25%) acutely infected HLA-B7+ individuals responded to the B7 EBNA-3A epitope (range, 0–0.44%), all 10 latently infected individuals responded to the B7 EBNA-3A epitope (range, 0.08–1.2%). A similar pattern was detected for the B7 EBNA-3C epitope. Only one HLA-A3+ individual (n = 8; 0.23%) responded to the A3 EBNA-3A epitope during acute EBV infection, and this response was not maintained into latent EBV infection.
Frequency of responses to EBV latent peptides in HLA-A2+, -B7+, and -A3+ individuals at presentation with acute EBV infection or at least 1 yr after presentation (latent). ∗, Significant difference (p < 0.005) between the frequencies of individuals responding between the acute and latent time points.
Early epitope-specific CD8+ T cell frequencies do not correlate with CD8+ T cell frequencies during latent infection
We detected IFN-γ responses to the A2 BMLF-1 peptide in all individuals with acute EBV infection and in all of our acute study individuals 1 yr post-EBV infection. Because IFN-γ production can underestimate the frequency of these responses, we developed HLA class I tetramers to more precisely determine the magnitude of the A2 BMLF-1-specific response (Fig. 4⇓a). At presentation with acute EBV infection, A2 BMLF-1 (lytic EBV gene) tetramer-binding CD8+ T cells were detected in all HLA-A2+ individuals (V-1, n = 25; range, 0.1–47% of CD8+ T cells; median, 2.31% (Fig. 4⇓b). One week postpresentation (n = 19), the percentage of A2 BMLF-1-specific T cells ranged from 0.2 to 15.9% (median, 2.35%); 7 of 19 (36%) HLA-A2+ individuals at this time point had increases in their percentages of A2 BMLF-1 tetramer-binding CD8+ T cells. The median frequency of A2 BMLF-1 tetramer-binding CD8+ T cells then decreased at 2 wk (V-3; n = 19; range, 0.14–9.76%; median, 1.08%), 6 mo (V-4; n = 11; range, 0.2–1.93%; median, 1.19%) and 1 yr postpresentation (V-5; n = 7; range, 0.08–1.4%; median, 0.23%).
a, HLA class I tetramer analysis using the A2BMLF-1 on two EBV seropositive donors. Percentages of tetramer-binding T cells are expressed as a percentage of total CD8 T cells. b, Percent of A2 BMLF-1-specific CD8 T cells over time after acute EBV infection. For presentation through 1 yr, each symbol represents the same individual. n values for each time point are in parentheses. EBV-seropositive donors at least 10 yr from acute EBV infection are designated latent.
A2 BMLF-1 tetramer-binding CD8+ T cells were also detected in 20 of 22 (91%) HLA-A2+ healthy long term EBV seropositive individuals studied (range, 0.05 to 1.95%; median, 0.32%). The frequencies of A2 BMLF-1 tetramer-binding CD8+ T cells in the acute EBV-infected individuals 1 yr postpresentation were significantly different than the frequencies of A2 BMLF-1 tetramer-binding CD8+ T cells detected in acute patients at presentation (p = 0.02), 1 wk (p = 0.02), and 2 wk (p = 0.05) but not different than the frequencies of A2 BMLF-1 tetramer-binding CD8+ T cells detected at 6 mo. The frequencies of A2 BMLF-1 tetramer-binding CD8+ T cells in the long term latent group were significantly different from the range of A2 BMLF-1 tetramer-binding CD8+ T cells in the acute patients at presentation (p = 0.001), 1 wk (p = 0.001), and 2 wk (p = 0.01) but not significantly different from the frequencies of tetramer-binding CD8+ T cells at 6 mo or 1 yr.
B7 EBNA-3A-specific responses were detected in only three of eight of the HLA-B7+ individuals with acute EBV infection but in all EBV latently infected individuals. We developed a B7 EBNA-3A tetramer to more precisely determine the magnitude of epitope-specific CD8+ T cells during acute and latent EBV infection (Fig. 5⇓a). B7 EBNA-3A tetramer-binding CD8+ T cells were detected rarely (3 of 9 = 33%) and at low frequencies in HLA-B7+-positive individuals with acute EBV infection at multiple time points within 2 wk of symptom onset (V-1, V-2, or V-3; range, 0–0.44%; Fig. 5⇓b). In the three individuals with detectable B7 EBNA-3A tetramer-binding CD8+ T cells during V-1, the changes in magnitude between presentation and 2 wk were not statistically different. B7 EBNA-3A tetramer-binding cells have been detected in all individuals 6 mo after entry into the study (V4; 8 of 8; range, 0.08–1.2%; median, 0.18%) and 1 yr after entry to the study (V5; 5 of 5; range, 0.16–0.46%; median, 0.24%). B7 EBNA-3A tetramer-staining cells have also been detected in all 6 HLA-B7+ EBV-long term-seropositive individuals studied thus far (range, 0.08–1.95%; median, 0.39%).
a, HLA class I tetramer analysis using the B7 EBNA-3A tetramer on two EBV-seropositive donors. Percentages of tetramer-binding T cells are expressed as a percentage of total CD8 T cells. b, Percent of B7 EBNA-3A-specific CD8 T cells over time after acute EBV infection. For presentation through 1 yr, each symbol represents the same individual. n values for each time point are in parentheses. EBV-seropositive donors at least 10 yr from acute EBV infection are designated latent.
During the first 2 wk after presentation with acute EBV infection, a decrease in the frequency of A2 BMLF-1-specific CD8+ T cells was noted (Fig. 4⇑b). By contrast, there was very little change in the percentages of B7 EBNA-3A CD8+ T cells for the three individuals analyzed (Fig. 5⇑b). We wanted to determine whether the initial burst size of CD8+ T cells determined the final magnitude of response detected 1 yr post-EBV infection. Comparison of the percentages A2 BMLF-1-specific CD8+ T cells in early acute infection with the percentage of A2 BMLF-1-specific CD8+ T cells at 1 yr for the same individual revealed no correlation between the initial and final magnitude of the response (Fig. 4⇑b and data not shown). Additionally, there appeared to be no detectable burst for the B7 EBNA-3A tetramer-binding CD8+ T cells; there were no significant differences between the initial and final magnitude of the B7 EBNA-3A response in the 3 HLA-B7+ individuals studied (Fig. 5⇑b).
Changes in magnitude of epitope-specific CD8+ T cell responses from acute through chronic infection
Because the early A2 BMLF-1-specific CD8+ T cell frequencies did not appear to predict CD8+ T cell frequencies at 6 mo or 1 yr, we wanted to determine the dynamics of loss or gain of CD8+ T cells during the year. Tables II⇓ and III⇓ show the fold loss (or increase represented by a number <1) over time in the frequencies of A2 BMLF-1 or B7 EBNA3A tetramer-staining cells. Large changes in the frequencies of A2 BMLF-1 tetramer-binding CD8+ T cells were observed; in most (6 of 10) individuals, the largest changes occurred between 2 wk and 6 mo postpresentation. By contrast, the frequencies of the B7 EBNA-3A tetramer-binding CD8+ T cells did not change much over the course of our study. All of the HLA-B7+ individuals who did not have detectable B7 EBNA-3A tetramer-binding CD8+ T cells during the first 2 wk of our study developed them between 2 wk and 6 mo of the study.
Table II.
A2 BMLF-1-specific CD8+ T cell frequencies over time
Table III.
B7 EBNA-3A-specific CD8+ T cell frequencies over time
Further analysis of the dynamics of loss or gain of A2 BMLF-1 and B7 EBNA-3A tetramer binding CD8+ T cells was done directly ex vivo on whole EDTA blood by four-color flow cytometry using Ki-67, a marker for cellular proliferation, or annexin V, a marker for cell death. Fig. 6⇓ shows analyses of the expansion of epitope-specific cells using Ki-67, an Ag expressed during all active stages of the cell cycle. At presentation with symptoms, 60 ± 12% of the A2 BMLF-1 tetramer-binding CD8+ T cells coexpressed Ki-67. A significant reduction in Ki-67 coexpression was noted at 1 (26 ± 16) and 2 wk postpresentation (5 ± 4%) and persisted through 6 mo to 1 yr postinfection (Fig. 6⇓a). Analyses of Ki-67 expression for 3 HLA-B7+ individuals with detectable B7 EBNA-3A-specific CD8+ T cells at V-1–V-3 showed similar Ki-67 expression (data not shown). The coexpression of Ki-67 on the A2 BMLF-1 tetramer-binding CD8+ T cells was not significantly different from the coexpression of Ki-67 in the whole CD8+ T cell population at all time points.
Percentages of A2BMLF-1 tetramer-binding CD8 T cells coexpressing the markers K1–67 (a) or annexin V (b) during presentation with acute EBV infection, 6 mo postpresentation, 1 yr postpresentation, and on long term EBV-seropositive donors (latent). ∗, Significant difference (p < 0.005) between the mean percentage of tetramer-binding CD8 T cells coexpressing Ki-67.
Annexin V staining can be used to detect the translocation of phospholipid phosphatidylserine from the inner to the outer cell membrane, an early stage in apoptosis. Analysis of A2 BMLF-1 tetramer-binding CD8+ T cells showed that at presentation, 7.43 ± 7.95% coexpressed annexin V (Fig. 6⇑b). The percentage of CD8+ T cells coexpressing annexin V appeared to decrease at 1 wk (3.59 ± 4.42%) and 2 wk (2.04 ± 2.47%) after presentation, but these differences were not statistically significant. Similar patterns of expression of annexin V were detected in B7 EBNA-3A tetramer-binding CD8+ T cells. No annexin V expression was detected in the A2 BMLF-1 or B7 EBNA-3A tetramer-binding CD8+ T cells from the 6-mo or 1-yr postacute EBV infection patients or for the long term-seropositive individuals.
EBV-specific memory populations remain stable over time in latently infected individuals
EBV is a chronic viral infection in which Ag persists during the lifetime of the host, and the effect of this Ag on the immune system over time is unknown. Selin et al. (27) have shown that the frequency of virus-specific CD8+ T cells can change after infection with heterologous viruses. Humans do not live in germfree environments, and we would expect our long term EBV-seropositive donors to come into contact with many different types of pathogens during a period of 2 yr. To determine the stability of the EBV-specific CD8+ T cell populations over time, we monitored eight long term EBV-seropositive individuals several times during a period of 20 mo to determine their change in frequency of A2 BMLF-1- or B7 EBNA-3A-specific CD8+ T cell responses. Fig. 7⇓ demonstrates that the frequency of both A2 BMLF-1- and B7 EBNA-3A-specific CD8+ T cell responses were stable over time.
Longitudinal analysis of A2 BMLF-1 (⋄) or B7 EBNA-3A (□) tetramer-binding CD8 T cells during 20 mo for 3 A2/B7 and 5 A2 long term EBV-seropositive donors.
This study is among the first to study the evolution of epitope-specific CD8+ T cell responses in a large cohort of individuals with acute through latent EBV infection. We have shown that several epitope-specific immune responses generated during acute EBV infection were not maintained into latent EBV infection while other epitope-specific CD8+ T cell responses that were not dominant during acute EBV infection were generated and/or maintained during latent EBV infection. Additionally, using HLA class I tetramers we have shown that early epitope-specific CD8+ T cell frequencies did not correlate with the magnitude of the response at 6 mo or 1 yr after EBV infection. Altogether, these data suggest that the CD8+ T cell repertoire generated during a persistent viral infection is not simply reflective of the initial pool of CD8+ T cells generated during acute infection. Interestingly, the percentages of A2 BMLF-1- and B7 EBNA-3A-specific CD8+ T cells in the eight long term EBV-seropositive individuals studied during a 2-yr period were constant, suggesting that an equilibrium between virus and the immune response had been reached.
Of the eight early or lytic protein epitopes evaluated, all but one (an HLA A2-restricted gp350 epitope) were recognized by the majority of individuals evaluated during acute EBV infection. By contrast, responses to eight previously defined EBV latent protein epitopes were infrequently detected during acute infection. Of the eight EBV latent protein epitopes evaluated, only one A2- and two B7-restricted EBNA-3 epitopes were well recognized during latency. When detected, the CD8+ T cell responses to EBV latent protein epitopes were of lower frequency (≤1% of CD8+ T cells) than CD8+ T cell responses to the lytic epitopes and remained stable during the 1 yr of follow-up. These results suggest the predominance of lytic epitope-specific CD8+ T cell responses during acute EBV infection and are in agreement with other recently published data from fewer individuals (23, 28, 29, 30). Although it has been suggested that the differential response of CD8+ T cells to lytic and latent Ags is due to different stages of the EBV lifecycle (13), all of the latent gene products are expressed during the lytic cycle (31). Lytic proteins also appear to be expressed periodically during latent EBV infection (32, 33). Recently, it has been reported that although the EBNA-3A protein is expressed by cultured BLCL lines in vitro, EBNA-3A expression was not detected in either the peripheral blood or tonsillar cells of latently infected healthy, EBV-seropositive donors (34). Although these data do not exclude intermittent or ongoing low level expression of EBNA-3A (particularly at a site outside the blood), they suggest that the differential expression of viral proteins does not fully account for the response to these Ags.
Of interest is that Callan et al. (12, 28, 29) have reported that HLA-B8+ individuals generate and maintain an HLA-B8+-restricted CD8+ T cell response to a BRLF-1 epitope. In all but one of our HLA-A3+ individuals, BRLF-1 epitope-specific immune responses were readily detectable during acute EBV infection, but not at 1 yr post-EBV infection. If the presence of a specific CD8+ T cell response were simply dependent on the presence of Ag, we would expect to see maintenance of BRLF-1 responses to both the A3 and the B8 epitopes. Our data demonstrating loss of an HLA-A3+-restricted CD8+ T cell response to BRLF-1 along with the data of Callan et al. demonstrating the maintenance of an HLA-B8-restricted response to BRLF-1 suggest that maintenance of the response to this epitope may be determined in a large part by the HLA type of the individual. Alternatively, cross-reactive responses present before antigenic challenge may lead to preferential expansion and maintenance of some responses compared with others. This has been demonstrated in the mouse model of acute LCMV infection, in which pre-existing responses to LCMV resulted in increased CD8+ T cell frequencies to heterologous viral epitopes when compared with naive mice (35).
Study of the dynamics of the generation and maintenance of BMLF-1 (lytic) and EBNA-3A (latent) epitope-specific CD8+ T cells using HLA class I tetramers showed that the early frequencies of CD8+ T cell responses to these epitopes did not correlate with their maintenance into the pool of memory CD8+ T cells. Previous studies in the mouse have shown a correlation between the initial burst size and the final magnitude of the response (10). Our results demonstrated a massive expansion of A2 BMLF-1-specific CD8+ T cells, but large expansion of B7 EBNA-3A-specific CD8+ T cells, or any of the other latent EBV-specific CD8+ T cells were not detected during any of the time points of this study. These EBNA-3A-specific CD8+ T cell responses were maintained at percentages similar to those of the A2 BMLF-1-specific CD8+ T cells in all of the HLA-A2+B7+ individuals. These data suggest that a large burst size is not necessary to provide the host with both effector and long term memory CD8+T cells and that the generation of memory CD8+ T cells in a persistent viral infection is not simply a stochastic process.
In contrast to the dynamic nature of epitope-specific CD8+ T cell responses during acute infection, the frequency of A2 BMLF-1- and B7 EBNA-3A-specific CD8+ T cells remained constant in all chronically infected individuals studied during 2 yr. Although these individuals likely experienced many immune challenges during the 2 yr of study, there were only minor perturbations in their frequencies over time. Data generated by Khan et al. (36) demonstrated that EBV viral DNA loads are stable in individuals over time periods spanning several years. Periodic reactivations of the EBV virus in the peripheral blood have also been demonstrated, suggesting that the stability DNA load over time is not that way because the virus is quiescent (32, 33). Our data demonstrating that CD8+ T cell frequencies to two different EBV epitopes are also stable over time suggest that during persistent viral infections a long term homeostasis exists between the virus and the immune system.
In summary, the study of EBV as a model for the generation and maintenance of epitope-specific CD8+ T cells in a human persistent viral infection has shown that the memory pool is not reflective of the initial pool of CD8+ T cells and that the generation of CD8+ T cell responses may not fit a stochastic model. The maintenance of epitope-specific CD8+ T cells to a persistent viral infection over time reflects a homeostasis reached between virus and the immune system. Perturbations in this homeostasis may in part explain EBV-associated diseases.
We thank Rose Cicarelli for her assistance with obtaining patient samples at the UMass-Amherst acute EBV clinic. We are also indebted to Drs. David Garboczi (National Institutes of Health), Marcelo Kuroda, and Joern Schmitz (Harvard Medical School) for helping to set up tetramer technology in our laboratory and to Dr. Eric Pamer (Memorial Sloan-Kettering Cancer Center) for the B*0702 HLA class I H chain construct. We thank Drs. Liisa Selin and Raymond Welsh for helpful discussions and review of the manuscript.
↵1 This work was supported by Grants AI 32391 and HD 01489 from the National Institutes of Health (to K.L.) and Grant IRG 93-033 from the American Cancer Society (to M.D.C.). K.L. is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric AIDS Foundation.
↵2 Address correspondence and reprint requests to Dr. Katherine Luzuriaga, Pediatrics/Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Suite 318, Worcester, MA 01605. E-mail address: katherine.luzuriaga{at}umassmed.edu
↵3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; A2, HLA-A*0201; B7, HLA-B*0702; A3, HLA-A*0301; β2m, β2-microglobulin.
Received April 10, 2001.
Copyright © 2001 by The American Association of Immunologists
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The Journal of Immunology October 15, 2001, 167 (8) 4450-4457; DOI: 10.4049/jimmunol.167.8.4450
IL-1 Transcriptional Responses to Lipopolysaccharides Are Regulated by a Complex of RNA Binding Proteins
PD-1 Expression during Acute Infection Is Repressed through an LSD1–Blimp-1 Axis
Elevated Choline Kinase α–Mediated Choline Metabolism Supports the Prolonged Survival of TRAF3-Deficient B Lymphocytes
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Eduardo Miranda is an Associate Professor in the Department of Civil and Environmental at Stanford University. He received his MS and PhD degrees in Structural Engineering from the University of California and Berkeley and his Civil Engineer degree from the National Autonomous University of Mexico (UNAM). His previous research work has included work in evaluation and upgrading of existing buildings, strength reduction factors, displacement modification factors, estimation of lateral displacement demands in structures. He has published over 100 papers in international journals and conferences. Current research interests include estimation of acceleration demands in buildings, seismic response of nonstructural components, loss estimation and performance-based design. Further information can be found here.


Cairns, Australia – it's a long way away from, well, almost anywhere. But with stunning landscape and unique beauty it's well worth the trip. That's why if you're making the hop across the ocean (or even just an Aussie wanting a weekend away somewhere beautiful) you want to make the most of the time you have and pack it to the brim with everything to see, do, taste, touch, feel… you get the picture.
Australia is so diverse – I mean it would be stretching across 7.69 million square kilometres – but arguably one of Australia's most beautiful destinations is coastal town Cairns in Tropical North Queensland. Situated in North East Australia, not only is Cairns the gateway to one of the seven wonders of the natural world – The Great Barrier Reef, which, even though it can be seen from Outer Space it's a damn sight nicer looking close up, it is also home to some spectacular waterfalls and natural beauty. And a tight schedule shouldn't mean you should miss out on any of it, below is the best way to soak it all in in just three fun packed days.
Cairns is a tourist hub, but don't let that put you off, sometimes it's not always the 'path less travelled' that makes all the difference – if two million people from all across the globe visit this region each year they are probably onto a good thing. Its popularity means there is a variety of accommodation options– from 5 star hotels to backpackers and everything in between.
For those on a budget – Gilligan's comes highly regarded. Popular with travellers and locals alike, Gillie's is a cheap, sociable and very clean backpackers, located in Cairns City and home to the most popular nightclub in Cairns. Although this may put off some people, the rooms are far enough away not to be disrupted and free entry into the club doesn't hurt. Either way – Cairns City is often described as a university campus with its relaxed vibe and interactive and friendly atmosphere – so even if you don't choose Gillie's anywhere in the city will set you up for an ideal holiday!
Arrive in Cairns fresh as a daisy and ready for the days ahead. Cairns is a part of the famous Great Dividing Range in Australia and therefore, there's no better way to start your trip than with a journey on the renowned Kuranda train. Leaving from the Cairns Central Train Station at 8.30 and 9.30am each morning, the train winds it's way up the mountains, past waterfalls and spectacular views (did someone say Instagram opportunity?) into the Kuranda Village, a small hippy town situated on top of the Kuranda Range – 27km away from Cairns.
The journey takes roughly two hours and costs AUS $50 pp (or $40 if you manage to con a local into buying a ticket for you – Cairns locals enjoy 20% off most attractions so if you pick one up in Gilligans it doesn't do you any harm to invite them on your adventures!). On arriving in Kuranda you can spend your morning wandering through the little village markets on everyday, enjoying the simpler side of life.
Once you've had your fair share, take the short stroll to the Kuranda Sky Rail where you begin your unique trip back into Cairns. Cable cars suspended high above the rainforest canopy deliver you back into the city – if you own a GoPro here is a great opportunity to selfie like you've never selfie'd before. Sneak your arm out of the carriage window and snap away. You'll get yourself, the carriage and all the Cairns Rainforest in the shot – it really makes a great picture! This journey costs AUS $50 pp and it is actually cheaper to buy the Skyrail and Train tickets separately rather than a combined package as advertised on the website!
Spend your afternoon further North by heading out towards Mossman Gorge and Port Douglas. Hiring a car is easily the best way to get around in Cairns and there are plenty of places to do so. Get out onto the road and travel towards Mossman and Port along the spectacular Great Barrier Reef Drive. It's like no other, and must be one of the most picturesque drives in Australia. Don't forget to get a quick snap at Rex Lookout as you pass it on your right half way through your journey.
It takes just over an hour to get to Mossman, but it is definitely worth the journey. Park your car at the Visitor's Centre, and hop on the short bus ride out to the actual Gorge (AUS $9.10 return fare). Mossman Gorge will take your breath away (probably literally if you take the plunge and dive in – it is very, very fresh). Situated within the Daintree Rainforest, which is thought to be one of the three oldest rainforests in the entire world, the water sparkles as it cascades over enormous pebbles and is almost unbelievably clear. You'd be forgiven for thinking you had fallen into a real life fairy tale.
Spend the rest of your afternoon wandering through the picturesque holiday town of Port Douglas just a mere 10 minute drive away from the Gorge. Keep your eyes peeled as this famous little town has seen the likes of Mick Jagger, John Travolta, Tom Hanks and other celebrities all wanting to experience a little bit of the Tropical North Queensland magic themselves. Then finish the day taking in the sunset under the palm trees of Port Douglas' Rex Smeal Park.
Today we head South and West. For those who fancy an early morning hike The Pyramid, just 30 minutes south of Cairns, is well worth the climb. At 922m, it is the highest freestanding pyramid in the world, which not only gives yourself great bragging rights when you return home, but also provides with a sensational view of Cairns and the surrounds. Heading down South past the Pyramid we are met with the beautiful Paronella Park.
Australia may not have as many ancient castles as countries like England, but we definitely do our best to make up for it with Paronella Park. Situated within the rainforest, the castle ruins are incredibly picturesque and a popular spot for weddings due to its beauty and romantic feel. Plus you have the opportunity to feed wild turtles on the grounds.
Heading past Paronella and towards the Atherton Tablelands we begin our Waterfall Circuit tour. There are three well-known waterfalls on the Tablelands, however, one not so well known, but arguably the most impressive of them all is Nandroya Falls. Situated in Wooroonooran National Park an easy twenty-minute walk leads you to a 50m free fall drop waterfall. Taking due care, it's possible to swim and get very close to the bottom of the falls, as despite the power of the water gushing down the pool at the bottom is quite shallow. Continuing towards the Atherton Tablelands, don't forget to keep your eyes open for the Nucifora Tea Plantation on your left, if you're lucky you should also spot a small wallaby or two.
Millaa Millaa Waterfall is our first stop and one you may recognise from a Herbal Essence advertisement. It's very fun to jump into the water, dip your head under and flick back quickly to get the same effect as seen in the shampoo advertisement – if you don't know the ad I'm talking about that doesn't matter- it makes a wicked photo either way. If you don't find yourself partial to an ad recreation you can easily climb across the rocks on either side to reach behind the waterfall. A sheet of liquid perfection, it's a very popular and normally quite a crowded waterfall.
If you continue on you'll find lesser known and subsequently quieter and more peaceful falls. Like Zillie Falls, a waterfall with two viewing platforms. Most visitors here view the magnificent falls from above; however, it is worth the moderate hike towards the bottom. Not only will you likely have the falls to yourself, it's hard to appreciate the sheer beauty and power behind Zillie Falls unless you view them from underneath.
Finally, you hit Ellinjaa Falls. A short pathway takes you to this beautiful cascading falls. One that is much calmer than the others previously mentioned and a convenient tree stump makes a great spot for photos in front of the falls. A great way to enhance this day is to bring a packed lunch and have a picnic at the stunning Millaa Millaa lookout – not far from the waterfall circuit. This lookout shows you as much as your eye could possibly see of the Atherton Tablelands and just how green this part of the world really is.
Finally, finish your afternoon back in Cairns down at the Esplanade and Cairns Lagoon. This popular spot for relaxation is littered with locals, family holidays and backpackers alike all soaking up the sun and good vibes that come from the frequent markets and live music. If you want to continue your night head on over to the Pier Bar or Salthouse along the ocean boardwalk for a few drinks before finishing your night at Gillies – added bonus if your bed is close!
Today is the day to finally experience the Great Barrier Reef in all its glory. There are SO MANY ways you can experience the reef from taking a helicopter ride, scuba diving, snorkeling at the Cays and visiting tropical islands. My absolute favourite place in the World is Fitzroy Island, just a 45 minute ferry ride away from Cairns. Its beauty is second to none when you think about a tropical paradise. If you want to see the Reef and the islands this is the place to go.
It's in competition with Green Island where most tourists are directed to, but while Green Island has its positives it cannot compare to Fitzroy (and it's probably a good thing that majority of tourists haven't discovered this yet!). Raging Thunder boats leave Cairns at 9am each morning and Fitzroy 4.30pm each afternoon and cost AUS $75 pp for a return trip. This day trip gives you quite a lot of time on the island, however, as there is always more that can be seen if you do fall in love with the place it can be wise to book accommodation and stay over night – not only do you get to see the sunset, the island will be less busy after everyone has headed back to the mainland.
Fitzroy Island gives you the opportunity to stand up paddleboard, snorkel, scuba dive, hike to the very top or just indulge in utter relaxation. There is even an ocean trampoline for those feeling slightly active or just excited by something as cool as an ocean trampoline. Just remember if you're hopping into the water between October and May to cover up with a stinger suit – Cairns is home to some nasty jellyfish called Irukandji that are almost invisible. Don't be alarmed though – covering up in the water is all that is typically required to protect yourself from stings!
For those not wishing to spend too much of their time snorkelling the reef you can stop by at Foxy's Bar for a sneaky midday cocktail before venturing off to Nudey Beach (no, despite its name it is not a Nudist Beach keep your kit on!). There are countless number of photo spots along the way and if you're lucky the tide will be low revealing more of the rocks along the shore that look perfectly picturesque with the blue skies and pebbled shell beaches. This island is to be spent exactly how you want to spend it and no other way – it's completely up to you. But there is one thing that is promised by it – you'll leave it dying to return. This is Cairns. It's one of a kind and will stay with you long after you leave.
We hope that this article has inspired you to visit Cairns, Australia. If you have any questions about the destination or have your own travel tips to share please leave these in the comments below.
Thanks for reading Jennifer, glad you enjoyed this post!
Hi I love your blog it is very inspiring I love traveling too I love to learn more about traveling your amazing travelers.
Thanks for reading and commenting Yulie! And thank you so much for your support and being part of the We Are Travel Girls community. So pleased to hear that you like the blog.
Thanks for reading and commenting Raquel, pleased that this post will be useful for your own trip. Have fun!
Love this! I'm heading out to Cairns in a couple of weeks and you girls mentioned places that no other blog has so thank you! 🙂 I've got Fitzroy Island on my list of must do's and cannot wait to go!
Hi there, this blog is just wonderful and clearly explains the things to do at Cairns. We would be visiting Cairns in December and would be visiting Kuranda for sure. I have one question though. Are there any public transport services available from Smithfield to Port Douglas? We were thinking we would go for the Scenic Railway from Cairns to Kuranda and take the Cablecar back down to Smithfield from Kuranda.
Thanks for this great information. We are planning a trip to Queensland and trying to decide how much time we need in Cairns and what the top attractions are to explore there.


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Plus: Another agency buy for CHR Group, new offices for Vibrant and Ariad and other news you might have missed.
Starting next year, Canadians will pick, pay and determine the fate of Canada's slew of specialty channels.
Media cos are venturing into the over-the-top video playground and hoping advertisers will join the game.
To win in digital, media cos have been busy beefing up catch-up services and launching original online content.
As the media maven prepares to begin a new chapter, we look back at how she reshaped the industry.
In part three of strategy's diversity series, we examine what brands and agencies are doing to address the issue in their senior ranks.


text:
Cherokee lawyers appointed to Comanche Nation Supreme Court
LAWTON – Cherokee Nation citizens and lawyers Robert Don Gifford and Casey Ross have been selected by the Comanche Nation to serve on the tribe's first Supreme Court.
Gifford is also chief judge for the Kaw Nati...
50-year Cherokee Nation Industries employee Duvall receives retirement sendoff
BY D. SEAN ROWLEY
STILWELL – It isn't uncommon for an employee to become known as "an institution" at a place of business...
Nagle creates podcast about Supreme Court case
TAHLEQUAH – Through the evolvement of Cherokee storytelling and the use of digital audio, Cherokee Nation citizen Rebecca Nagle created and hosts the podcast "This Land" to tell the story of a U.S. Supreme Court case involving a murder...
Rose named Oklahoma Policy Institute executive director
The Oklahoma Policy Institute advances fiscal respons...
Joy Harjo is first Native American named US poet laureate
NEW YORK (AP) – Joy Harjo, the first Native American to be named U.S. poet laureate, has been ready for a long time.
"I've been an unofficial poetry a...
Brits cite cultural learning as reason to retrace Trail
Most of the trip was in a canoe, but two Unite...

redpajama_set_name: RedPajamaCommonCrawl


Although you actually drink this as opposed to eat it, I felt cold brew coffee deserved some love. It's less acerbic than your typical hot-brewed counterpart, and because it's less bitter, it's also more smooth and tasty. It stays fresh in the fridge for up to 2 weeks, and it's perfect for iced coffee (it's already cold!)!
Don't worry, you can still get your morning cup of hot coffee by adding hot water to the concentrate.
I use medium roast beans (left), which have been heated to a lower temperature than dark roast beans (right). Medium roast tastes more like coffee beans and less like the roasting process.
I grind my beans fine. Most cold-brew recipes call for medium-coarse or coarse ground beans, but that's a mistake. The factors with the biggest impact on coffee extraction are water temperature, grind size, extraction time, and finally, agitation—in that order. Since I use room-temperature water I can scratch temperature off the list of variables and grind size becomes most important. Finer particles will release more flavor compounds than larger ones.
I combine room-temperature water (usually filtered, as my tap water doesn't taste great) and freshly ground coffee in a large French press. The press makes it's a snap to separate the concentrate from the grinds after brewing.
After about 10 minutes, a solid raft of coffee grinds will form on the surface. I find it important to stir this raft into the water to maximize contact with the ground coffee.
This is the one and only time I stir the cold-brew. As I mentioned before, agitation is last on the list of factors affecting extraction. I find it a nuisance to stir a batch of cold-brew multiple times over the course of a day; the good news is I don't have to.
Next, I cover the French press with plastic wrap and let it sit at room temperature for 24 hours (give or take an hour in either direction). I've done room-temperature brews as short as 12 hours and as long as 72 hours. Twenty-four hours is consistently the sweet spot.
After 24 hours, I remove the plastic wrap and press the grinds to separate the concentrate.
Then I pour the concentrate into a coffee filter-lined fine-mesh strainer set over a large measuring cup or pitcher. Some would say that this filtering step is optional, but I don't like silt and grit in my cup. Most of the concentrate will filter through unaided, but I find it helpful to gently clear the sediment with a rubber spatula to let the last few drops through.
Finally, I dilute the concentrate one-to-one with cold water and pour it into a glass with plenty of ice.
Now, instead of reaching for the sugar (which is unnecessary with super-smooth cold-brew) I stir in a pinch of Kosher salt. Just trust me on this one.


A unique Antai trip planner. Antai is an important city of China. This city is home to many famous tourist attractions such as Yuan Shuai Miao, Yan Fu Gu Ju, Er Mei Shu Wu, Chen Shi Zong Ci, Bing Xin Gu Ju, Xinhai Revolution Memorial Hall, Xin Hai Ge Ming Ji Nian Guan, Shang Lin Shu Yuan ( Fu Zhou ), Qin Nan Shu Yuan, Fu Jian Min Su Bo Wu Guan and lots more. It is a city which required 3 to 4 Days to visit. One can enjoy a comfortable stay in Antai which has a huge variety of hotels. While planning a vacation to China one can surely include Antai city in their travel itinerary. On this page, create your virtual travel plan of China including Antai city and other neighbouring cities. Find the travelling distance between Antai and other city, time taken to travel and authentic travel reviews by other travellers. Antai travel planning made easy with JustOrbit. Plan your trip to Antai by selecting the perfect places to see, tourist attractions to visit, things to do and hotels in the city. Antai trip planner for everyone.
Many cities are accessible from Antai. We have selected the most popular tourist cities that you can visit after Antai. Based on number of days in hand for vacation these cities have been listed and can be added to your travel itinerary by selecting "Add to Trip" displayed on the right of each city name.


Is the Palmetto Bug. A.K.A. The Flying Roach.
I can deal with a whole lot of things, and flying roaches are not on that list. They make me die a little inside every time one gets close to me. Which is unfortunate, since I come in close contact with them on a regular basis these days. I have the Orkin man on speed dial and he told me today that it was a battle we were going to have to fight together. They live outside, these are not the sort of roaches that inhabit the inside of your walls. These are the kind of roaches who might fly off with your dog if you leave her in the backyard for too long.
Although, I do have to say that these roaches have nothing on this sucker that lives in maggie's parents pond. I swear to the heat of the South Georgia summer that thing in the water is a giant catfish. Who Maggie swears wants to eat her dog.
"What about the first kitty cat they eat? Or the first little poodle? They'd love poodles, I imagine," he said.
Mike Dorcas, a professor at Davidson College in North Carolina, has sliced open pythons in Florida to find the remains of white-tailed deer, bobcats and large birds.
Pythons are masters of disguise — slippery and quick — and all but one of the serpents was invisible within minutes of being deposited into the pit.
So counting pythons in the wild is a daunting task. Scientists don't have an accurate estimate of how many pythons are in Florida.
"It's certainly in the thousands, or tens of thousands, if not hundreds of thousands," said Gibbons.
The other train of thought is the safety of the fish you catch in the Central Savannah River Area. I thought it was interesting that the parts of the river that do have advisories say – one meal per week. Is that one fish per week? Because, my meal is not the same as some other people's meal. Some people could eat four fish.
So, we do a lot of quiet title actions around here, which is how you clear title to a piece of property that has been sold at a sheriff's sale when the property taxes haven't been paid. Although quiet title actions do not normally involve any particularly complicated legal principles, the difficulty lies in finding all the people who have ever had a claim of title to the property and serving them, which often time includes adjacent land owners or the heirs of an estate. Which means I spend a lot of time trying to find people and figuring out how to serve them in Jamaica, New York, or Erie, Indiana, or, as the case was today – Denver, Colorado.
The process server I found in Denver is a fleet of bike couriers. How cool is that? This time my petition to quiet title is going to be delivered by bike! This really made my day. And the guy I talked to on the phone was so nice and friendly and it made me really wish I was in Colorado. I got really Colorado Home Sick. Or maybe I'm just, IT IS REALLY HOT HERE SICK.
Last night at tennis clinic I got to play singles with Jane. AGGHH. I LOVE singles. I love singles so much more than doubles. In doubles I feel like I'm in the way, or the wrong place, or I should have made that shot because I'm letting my partner down. In singles, I know where I'm suppose to be the whole time. Maybe I'm not always there, but the knowing is much more instinctive. It's been so much fun getting back into a sport. I haven't played a game in a really long time and I didn't even know how much I missed it.
Briscoe has been sleeping in the new big bed with me recently and every morning I wake up I feel the need to grab her toe and sing to her about catching a tiger by the toe. She fails to see the humor and normally either tries to bite me or kick me.


text:
Gesha coffee, sometimes referred to as Geisha coffee, is a variety of coffee tree that originated in the Gori Gesha forest, Ethiopia, though it is now grown in several other nations in Africa, Asia, and the Americas. It is widely known for its unique flavor profile of floral and sweet notes, its high selling price, and its exclusivity as its demand has increased over the years.

While there are many varieties labeled gesha (or geisha), the Panamanian Geisha has a distinct and verifiable genetic fingerprint.

After its discovery in Ethiopia, it was sent to the Tengeru (now Lyamungu) Coffee Research Station in Tanzania. In 1953, plants successfully cultivated at Lyamungu as VC-496 were taken to Centro Agronómico Tropical de Investigación y Enseñanza (CATIE) in Costa Rica and recorded as ascension T2722. From there, CATIE distributed it throughout Central America. In 2005, The Peterson family of Boquete, Panama, entered a Geisha coffee into the Best Of Panama competition. It won the competition and sold for a then-record price of /pound (/kilogram); in 2017, a lot of natural Geisha from Hacienda La Esmeralda established a new Best of Panama record of /pound (/kilogram).

Name
The name "Geisha" has been applied to the coffee since it was first collected by British officials. A 1936 letter from the British Consulate in Ethiopia discussing a trip to the "Geisha coffee area," where samples were collected for further study. The name "Gesha" comes from the transliteration of the Amharic name for the region, . (The local Kafa language in Gesha did not have a written form until the 1990s.) In most instances, Geisha/Gesha coffees from Ethiopia use the Gesha spelling while those from elsewhere use Geisha, based on how the cultivator was originally labeled, although some Latin American producers use Gesha to indicate a variety grown from original Ethiopian stock. Despite the origin of the Geisha name being a misspelling or phonetic rendering for Gesha, the increasing awareness of the variety has raised concerns about improperly linking the coffee with Japanese geisha entertainers.

History
Coffee production in Ethiopia dates back dozens of centuries; according to legend it has been harvested there since the 9th century. Ethiopia is the world's main storehouse of genetic diversity for Arabica coffee, and this diversity is manifest in the chemicals that produce flavour. Ethiopia has a complex climate and landscape, resulting in regional and local differences across the coffee growing landscape. The Geisha variety of Coffea arabica was identified in the 1930s, in the mountainous Gesha region of southwestern Ethiopia. After seeds were collected in 1936 by a British consul, the coffee was planted in Tanzania and Costa Rica. From there, cultivation spread to Panama in the 1960s, including the famed Boquete region.

Growing and Processing
Geisha coffee begins with picking the ripest cherries. Producers prefer hand-picked cherries as it helps ensure only properly ripe coffee cherries are harvested. The riper the cherry means the resulting coffee will have a sweeter flavor profile. The cherries then go through quality inspection. Some producers have the technology to put their cherries into a machine which determines the quality of the cherries. Some smaller producers have workers sort through the picked cherries by hand. The cherries are processed either by means of wet or dry methods. Washed coffees are often described as 'cleaner' and more delicate in the cup (i.e., during drinking), lighter-bodied, and with a brighter, better-defined acidity and brighter fruit notes. Washed coffees are also described as more balanced (i.e., lacking particularly strong tasting notes or biases). Dry-processed (natural) coffees tend to be fuller-bodied, fruitier, sweeter and less acidic, although in some cases the acidity is more pronounced and easier to define.

After those methods are complete, the producers end with a Geisha coffee bean.

Flavor profile
The flavor profile of Geisha coffee is one aspect that contributes to its renown. Geisha is known for its sweet flavor and aroma of floral notes, jasmine, chocolate, honey, and even black tea. These sweet floral notes, and complex flavor profiles are some of the many characteristics that Geisha coffee shares with most other African coffees. Additionally, this unique flavor is also one of the contributing factors for Geisha's high price tag and prestige.  Whilst it may be difficult to pinpoint a flavour profile to any specific coffee area, the differences between (and even identification of) many of the coffee areas can be made by experienced coffee cuppers. Gesha is the home of the world-famous Gesha (Geisha) coffee, and this origin is now starting to produce its own very high quality washed, semi-washed, and dry-processed coffees. Gesha (Geisha) coffee grown within and outside Ethiopia is noted for its outstanding aroma and flavour, with notes of jasmine, black tea, and tropical fruit, and for profound sweetness. For these reasons, Gesha (Geisha) coffee is sought-after and usually commands a high price.

Sales & auction prices
The Best of Panama Coffee Competition has a long-standing reputation for having high dollar coffee at auction, which is where Geisha asserted itself as one of the world's highest costing coffees. This occurred in 2004 when Hacienda La Esmeralda entered the coffee into the competition where it was found to have a distinctly unusual taste before being purchased at auction for a record price of $21 per pound. 15 years later, at the 2019 Best of Panama Competition and Auction, another Geisha sold for $1,029 per pound (unroasted). Earning the nickname "Elida Natural Geisha 1029," this Geisha was produced on the Lamastus Family Estates. 100 pounds of Geisha coffee sold for $100,000, ranking above the next closest coffee by $80,000. Geisha is perhaps the most valuable coffee in the world.

External links
 World Coffee Research Varieties Catalog: Geisha

References

Coffee varieties

redpajama_set_name: RedPajamaWikipedia


Note: Were you looking for information about appendicitis symptoms? Appendicitis symptons is a common misspelling of appendicitis symptoms.
Common appendicitis symptoms include loss of appetite, nausea, and vomiting. People who have appendicitis may not have all of the appendicitis symptoms. However, people who have possible appendicitis symptoms should see their doctor, because only a doctor can diagnose and treat the problem.


text:
import { get, defineProperty, computed } from '@ember/object';
import Mixin from '@ember/object/mixin';
import { isHTMLSafe, htmlSafe } from '@ember/string';
import config from 'ember-get-config';

const defaults = config.EmberHammertime || {};

const FocusableInputTypes = ['button', 'submit', 'text', 'file'];
// Set this to `false` to not apply the styles automatically to elements with an `action`
const TouchActionOnAction = defaults.touchActionOnAction === undefined ? true : defaults.touchActionOnAction;
// Remove 'onclick' if you do not want the styles automatically applied to elements with an `onclick`
const TouchActionAttributes = defaults.touchActionAttributes || ['onclick'];
// Remove whichever element types you do not want automatically getting styles applied to them
const TouchActionSelectors = defaults.touchActionSelectors || ['button', 'input', 'a', 'textarea'];
// The actual style string that is applied to the elements. You can tweak this if you want something different.
const TouchActionProperties = defaults.touchActionProperties || 'touch-action: manipulation; -ms-touch-action: manipulation; cursor: pointer;';

function touchActionStyle() {
  let style = get(this, 'touchActionProperties');
  let otherStyleKey = get(this, 'otherStyleKey');

  if (otherStyleKey) {
    let otherStyle = get(this, otherStyleKey);

    if (otherStyle) {
      if (isHTMLSafe(otherStyle)) {
        otherStyle = otherStyle.string;
      }
      style += otherStyle;
    }
  }

  return htmlSafe(style);
}

export default Mixin.create({
  touchActionOnAction: TouchActionOnAction,
  touchActionAttributes: TouchActionAttributes,
  touchActionSelectors: TouchActionSelectors,
  touchActionProperties: TouchActionProperties,
  ignoreTouchAction: false,

  init() {
    this._super(...arguments);

    const {
      tagName,
      ignoreTouchAction,
      click
    } = this;

    const hasClick = click && click.apply;
    const hasTag = tagName !== '' || (tagName === null && hasClick);
    if (!hasTag) {
      return;
    }

    let maybeApplyStyle = ignoreTouchAction === false;
    let hasClickHandler = ignoreTouchAction === false && hasClick;
    let shouldApplyStyle = false;

    if (maybeApplyStyle) {
      let isFocusable = this.touchActionSelectors.indexOf(tagName) !== -1;

      if (isFocusable && tagName === 'input') {
        isFocusable = FocusableInputTypes.indexOf(this.type) !== -1;
      }

      shouldApplyStyle = isFocusable;
    }

    if (hasClickHandler || shouldApplyStyle) {
      let newAttributeBindings = [];
      const bindings = get(this, 'attributeBindings');

      // don't override other style bindings if present
      if (Array.isArray(bindings)) {
        bindings.forEach((binding) => {
          if (binding === 'style') {
            this.otherStyleKey = binding;
          } else {
            let end = binding.length - 6;

            if (end > 0 && ':style' === binding.substring(end)) {
              this.otherStyleKey = binding.substring(0, end);
            }
          }
        });
        newAttributeBindings = newAttributeBindings.concat(bindings);
      }

      newAttributeBindings.push('touchActionStyle:style');
      this.set('attributeBindings', newAttributeBindings);

      let desc = this.otherStyleKey ? computed(this.otherStyleKey, touchActionStyle) : computed(touchActionStyle);
      defineProperty(this, 'touchActionStyle', desc);
    }
  }
});

redpajama_set_name: RedPajamaGithub


November 16, 2015 October 14, 2015 by laurapennington1, posted in FOOD!
This dinner is incredibly simple, but it is SO yummy and filling, and it makes for a perfect fall meal! I'm pretty sure I could eat this everyday with how delicious it is! Spaghetti squash is so fun to work with, and it it so tasty! Of course, the best part is that it is really good for you! The following pin lists some of its health benefits!
The spaghetti squash along with the other healthy ingredients in this dish make for a wonderful dinner! Without further ado, let's get to the recipe!
1.) Start by baking your chicken: Drizzle a baking dish with olive oil. Add the chicken breasts, sprinkle with garlic powder and sea salt. Flip the chicken breast over and sprinkle the other side with garlic powder and sea salt. Place in the oven and bake at 400 degrees for 40-50 minutes until cooked through.
2.) Prepare your spaghetti squash: Chop the ends off of the squash, then cut into rings about 1 inch thick. Run a knife in a circle around the center of each ring to remove the seeds and pulp.
3.) Bake your spaghetti squash: Prepare a baking sheet with parchment paper. Drizzle the parchment paper with olive oil. Place the squash rings on the prepared baking sheet, and sprinkle with garlic powder and sea salt. Flip the squash rings over and sprinkle the other side with garlic powder and sea salt. Place in the oven and bake at 400 degrees for about 30 minutes. Take out, set aside, and let cool for about 10 minutes.
4.) When the chicken is done, cut into small bite size pieces. Heat 1 Tbsp. olive oil in a large frying pan over medium heat. Add the chicken pieces, vinegar, and honey. Stir to coat. Add the spinach, and cook until wilted. Turn the heat off, but leave the pan on the burner.
5.) Take your squash rings, and peel off the skin, then use a fork to scrape the squash rings into spaghetti. Add the spaghetti squash to the pan, and stir to mix.
6.) Add the goat cheese and season with sea salt. Transfer to a plate or bowl and serve.


text:
Marketers need more sense to stem the tide of CMO firings
The combination of business, digital and purpose sense Diageo CMO Syl Saller has should act as an example for all marketers craving greater influence.
By Thomas Barta	 16 Sep 2019 7:06 am
When Marketing Week's editor, Russell Parsons, asked me to write an article to accompany the launch of Marketing Week's Top 100 Most Effective UK Marketers, sponsored by Salesforce, I was torn. What's to celebrate?
This year saw a record number of top marketers being fired. The CMO brand is under attack. More worryingly, as everybody seems busy with digital porn, marketers are losing C-suite traction. I don't want to sound overly negative, but this stuff is real.
Belkin, Beats, Citi, Coty, Cisco, Dropbox, EY, GAP, GNC, Intuit, Hitachi, JC Penney, Johnson & Johnson, Kellogg's, Kimberly-Clark, Kraft Heinz, Lyft, Marriott, McDonald's, Mitsubishi Motors, Mondelēz International, Netflix, Nissan, PayPal, Rite Aid, Samsung, Sephora, Sisheideo, Subway, Suntory, Spotify, Taco Bell, Ticketmaster, Tyson, Uber, Walgreens, Wendy's – these are just a few brands from consultancy Spencer Stuart's latest CMO movements list ('movement' is their term for firing).
I wish every skilled and customer-loving marketer would also develop digital sense, purpose sense and business sense.
Everybody knows CMOs don't last long, but this year things have got worse. "Transitions are happening at an unprecedented rate," according to Spencer Stuart consultants. Meaning: it's been a bloodbath.
The CMO title bingo is on. Top marketers are now getting labels such as chief growth officer (what else would a marketer do?), chief customer officer (what's new?), chief experience officer (WTF?). And here's the new 2019 thing: teams are now getting rebranded as 'growth marketing'. Really?
Mondelez scraps CMO role and installs chief growth officer
Growth marketing is as clever a title as performance marketing. It suggests the job previously wasn't about growth (or performance). Behind the relabelling stands a more serious CEO message: "I'm dissatisfied with your work." For the CMO brand, this could be the beginning of the end.
Things on the agency side aren't looking better.
Who's doing well? Tech firms – and everybody who knows how to book a Facebook campaign. Where are the truly strategic agencies? The people who once had the CEO's ear? The agencies that challenged marketers to think long term?
J Walter Thompson, the force behind brands such as Kraft Cheese and De Beers ('A Diamond is Forever'), just got subsumed under the digital outlet Wunderman. Ogilvy & Mather, the former boardroom power, has just ditched David Ogilvy's mantra 'We sell or else' in favour of 'We change or else'. As a client, I'd be losing my will to live.
Ogilvy's rebrand reveals an ad industry in confusion
As a marketer, why should you care? Why should you worry about what's happening up there in the C-suite? The answer is simple: this is about your future. When marketing's reputation erodes at the top, so will yours. When CMOs get kicked out of the boardroom, so will the customer's voice. When everybody in marketing gets a funny title, so will you (one day).
Sorry if I'm sounding a little dark. But these facts went through my mind that night when I was thinking about what to write. To distract myself, I watched this interview with Syl Saller, Diageo's long standing CMO. It came at just the right time and brought all my optimism back.
For the past 10 years, I've been spearheading marketing leadership research. In over 200 keynotes, I've made the passionate case that marketers must mobilise bosses and colleagues for change (rather than just doing campaigns). Watching Saller and looking over Marketing Week's Top 100 made me hopeful: there are great marketing leaders who cut through the digital clutter. Who get brand purpose right. Who deliver growth.
Digital sense
Marketing technology is awesome. But digital could easily become the shovel with which marketers are digging their own grave.
Let's be clear: martech is critical. I'm a big advocate. I'm even helping many tech firms and media brands to make the case. But digital media is a fraction of one of the four Ps (product, price, promotion, place). Marketers who only learn tactical digital campaigning might simply end up in sales support (that's already happening).
When Saller talks about martech's benefits, she lists marketing effectiveness and figuring out at what time of the year TV works best. Or take Adobe, currently one of the world's most successful martech firms. It spends a large chunk of its budget on analogue marketing: events.
A true marketing leader has digital sense. That's loving technology when it helps to solve a real problem.
Brand purpose sense
It makes me nervous when a marketing team tells me their brand is now all about solving the world's problems. The remarkable Paul Polman, Unilever's former CEO, is perhaps the most transparent leader when it comes to purpose. Polman truly cares for our world (he's even become a full-time activist). But he loses no opportunity to talk about the challenges of doing good with shareholders on your back.
It's more about tiny steps – and starts with paying your taxes (I agree with Mark Ritson on this). Consumers see though glossy purpose adverts anyway. Bailey's, for example, long tried to empower women, but women didn't really want Bailey's help on that front. The brand now positions itself as an 'adult treat' again – and it's growing.
Keen to boost brand purpose? Do real things that help real people. Diageo's gender-neutral parental leave policy and Saller's push for more female agency talent are perhaps doing more for women than any of their ads ever did.
Customers and companies love similar things: more and better stuff, at lower costs. Being a marketer sets people up for this intrinsic conflict. The art of marketing leadership is to play inside the 'value-creation zone', the space where customer and company overlap. That's a pretty demanding job.
Most marketers are pretty busy trying to figure out which colour, taste and content customers want. Adidas, for example, has trend scouts everywhere who do nothing but find new grassroots trends.
But there's the other stakeholder: the CEO. Marketing professor Kim Whitler has found that, more often than not, CEOs and top marketers aren't aligned. It's one of the reasons why CMOs don't last long.
Saller is undoubtedly a great marketer. But make no mistake: she and her team are only around as long as they deliver what Diageo's CEO cares for: profitable growth.
Here's my dream: I wish every skilled and customer-loving marketer would also develop digital sense, purpose sense and business sense. The entire marketing profession would make a big leap forward. For me, that's a goal worth aspiring to.
But don't take my word for it, take Syl Saller's.
Thomas Barta is a marketing leadership expert, speaker and the co-author of 'The 12 Powers of a Marketing Leader'. He has teamed up with Marketing Week to launch the Marketing Leadership Masterclass, a new CPD-accredited online course designed to equip marketers with everything they need to become a better leader. To find out more and book your place visit leadership.marketingweek.com. The next course begins on 17 September.
Opinion Marketing Leadership
How marketers can improve their impact and influence
With confidence in marketing hitting a low point, how can the industry change negative perceptions and reclaim customer trust?
9 Sep 2019 7:00 am
How empathetic leadership can help marketers regain their influence
Being an empathetic leader, committed to progressing the talent in your team, will go a long way towards helping marketers extend their influence within organisations.
Marketing Week Top 100: The most effective marketers in the UK
Marketing Week reveals the top 100 most effective marketers in the UK, as judged by some of the marketing industry's most respected names.


Four undrafted rookies entered the offseason as long shots to make the Titans' 53-man roster. They did so anyway.
Four undrafted rookies — guard Aaron Stinnie, defensive end Matt Dickerson, outside linebacker Sharif Finch and wide receiver Cameron Batson — entered the offseason as long shots to make the Titans' 53-man roster. They did so anyway.
Here's what the rookies had to say about making the team ahead of its season opener at Miami on Sunday (noon CT).
Batson was laying in bed when the call came at 9:26 a.m. Saturday.
"I looked at it," Batson said, "saw it was from Tennessee, and was like, 'Yo, I'm cut.'"
On the other end was Carly Mygrants, executive assistant to general manager Jon Robinson. She told Batson he needed to come to the Titans' practice facility.
"So I'm like, 'Do I need to bring my iPad?'" Batson said. "Because if you turn in your iPad, you're done. She was like, 'Uh, yeah, you can bring it.' I'm like, 'No, Carly, do I need to bring my iPad?' She was like, 'Yeah, just go ahead and bring it.' So that kind of psyched me out a little bit."
What followed was a total whirlwind, the 22-year-old said. Batson's 13-to-15-minute Lyft ride to the facility was "the longest 13 to 15 minutes ever," he said. "Of course Nashville had traffic."
Once he got to Robinson's office, the Titans GM asked him to recount something he told the team at the beginning of training camp.
"I really couldn't even remember because I was just so nervous," Batson said. "And then he told me that I made the 53-man roster. I was actually about to start pulling out my iPad."
Batson, a Texas Tech alum who showed his value on special teams this offseason, then bowed his head and began praying. Robinson followed his lead.
"He was like, 'That's the first time somebody's ever done that,'" Batson said.
Finch still does not have a permanent name plate above his locker at Saint Thomas Sports Park. His temporary name plate is Scotch taped on.
But what makes Finch's story remarkable — beyond his background as an undrafted rookie — is what he had to endure while earning a spot in the Titans locker room. His father, Gregory, died of a heart attack on July 30. He lost his mother, Wendy, to breast cancer three years earlier.
"Emotionally, it was a rollercoaster," the outside linebacker said of his offseason, wherein he proved effective as a pass rusher. "It's something I still have to deal with everyday."
The first people he reached out to after making the team were his siblings — all 21 of them. Yes, 21.
"We have this huge group text with like all of my brothers and sisters in one," the Temple product said. "Told everybody at the same time."
The Titans released a veteran in David King. They let go of Julius Warmsley, whose stock rose as the preseason rolled along. And they only kept five defensive lineman.
How's that for a vote of confidence, Matt Dickerson?
"It gives me almost a call to continue to work hard," the defensive end out of UCLA said. "They trust me enough to be here. I've just got to keep on improving my game."
Stinnie knows football is a business and that nothing was certain heading into Saturday's cut day.
The offensive lineman out of James Madison applied a similar mindset when he took up football for the first time not that long ago. He played for the first time as a junior in high school. Before that, he was a basketball player.
"Honestly, I wasn't even thinking about football growing up," Stinnie said. "Ended up giving it a try. Started to fall in love with the game."
He went into college as a defensive lineman and only began playing O-line for the first time as a sophomore.
"It's definitely wild when I think about my past and where I am now," he said. "They're kind of two separate ends of the plane."


It is impossible for this world to be destroyed by a flood! I would stake my life on that! Even so, it is impossible for God to be angry at me or rebuke me, IMPOSSIBLE! – I have staked my life on this.
I have entered into a Covenant of Peace, forever sealed by the Blood of Jesus.
Why not make peace with God today? Why not believe in the finished work on the Cross?
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This Saturday, May 28th, come on out and read YOUR WRITING or POETRY aloud at the Redwood Writers Open Mic. We invite all writers and poets to join us for this afternoon of literary treats.
The open mic will be held from 2 to 4 p.m. at Gaia's Garden restaurant, 1899 Mendocino Ave., Santa Rosa, CA.
We ask that your limit your reading to 5 minutes maximum for the open mic portion. Our two featured readers for the day will be poet Juanita J. Martin and writer Mary Lynn Archibald.
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