text:
Pathogenesis and Immunity
CD8 T-Cell Recognition of Multiple Epitopes within Specific Gag Regions Is Associated with Maintenance of a Low Steady-State Viremia in Human Immunodeficiency Virus Type 1-Seropositive Patients
Christof Geldmacher, Jeffrey R. Currier, Eva Herrmann, Antelmo Haule, Ellen Kuta, Francine McCutchan, Lilian Njovu, Steffen Geis, Oliver Hoffmann, Leonard Maboko, Carolyn Williamson, Deborah Birx, Andreas Meyerhans, Josephine Cox, Michael Hoelscher
Christof Geldmacher
Jeffrey R. Currier
Eva Herrmann
Antelmo Haule
Ellen Kuta
Francine McCutchan
Lilian Njovu
Steffen Geis
Leonard Maboko
Carolyn Williamson
Deborah Birx
Andreas Meyerhans
Josephine Cox
Michael Hoelscher
DOI: 10.1128/JVI.01847-06
The importance of HLA class I-restricted CD8 T-cell responses in the control of human immunodeficiency virus (HIV) infection is generally accepted. While several studies have shown an association of certain HLA class I alleles with slower disease progression, it is not fully established whether this effect is mediated by HIV-specific CD8 T-cell responses restricted by these alleles. In order to study the influence of the HLA class I alleles on the HIV-specific CD8 T-cell response and on viral control, we have assessed HIV-specific epitope recognition, plasma viral load, and expression of HLA class I alleles in a cohort of HIV-seropositive bar workers. Possession of the HLA class I alleles B5801, B8101, and B0702 was associated with a low median viral load and simultaneously with a broader median recognition of Gag epitopes compared to all other HLA alleles (twofold increase) (P = 0.0035). We further found an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R = −0.36; P = 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (amino acids [aa] 1 to 75 and aa 248 to 500) was associated with the maintenance of a low steady-state viremia, even years after acute infection.
It is generally accepted that the HLA class I-restricted CD8 T-cell responses contribute substantially to the control of human immunodeficiency virus type 1 (HIV-1) replication in infected individuals. The appearance of HIV-specific CD8 T cells is temporally associated with the decline of plasma viremia after acute infection (5, 20), and their presence is associated with the control of viral replication during the chronic phase of infection (17, 28). Nonetheless, the parameters underlying the efficient control of viral replication by CD8 T cells remain controversial, since several recent studies have not found any correlation between the magnitude or breadth of the HIV-specific CD8 T-cell response and the plasma viral load in chronically infected individuals (1, 4, 6). Interestingly, CD8 T cells recognizing different HIV proteins may vary in their respective antiviral efficiencies (23, 33). Responses targeting the proteins encoded within the gag open reading frame have occasionally been shown to be associated with viral control (10, 23, 27, 29, 30), whereas responses to Nef or Env had no effect on viral control or even positively correlated with the plasma viral load (4, 23, 27).
CD8 T cells recognize virus-derived peptides in the context of HLA class I molecules. Three highly polymorphic genes, HLA-A, -B, and -C, encode these HLA class I molecules, and the CD8 T-cell specificities found within one individual are largely dependent on the HLA class I alleles expressed. Particular HLA-B alleles are strongly associated with different rates of disease progression and have been shown to restrict the majority of HIV-specific CD8 T-cell responses (18). These observations suggest that the association observed between a particular HLA-B allele and the corresponding viral load may be mediated by HIV-specific CD8 T cells restricted by these alleles. HLA-B alleles such as B5701/03, B5801, and B4201 (2, 16, 18) have been associated with a low viral load and also with slower disease progression. Conversely, HLA-B alleles such as B5802, B4501, and B1510 are associated with a high viral load and more rapid disease progression (18). Despite these important findings, it is not completely established whether disparities in the HIV-specific CD8 T-cell responses restricted by these alleles are contributing to these different rates of disease progression.
In order to study the mechanism underlying the beneficial effect of protective HLA class I alleles, seropositive study subjects from a high-risk cohort in Southwest Tanzania were HLA typed (54 of 56 subjects), and the plasma viral loads and CD4 counts were determined. At the same time, the HIV-specific CD8 T-cell response of each subject was examined for responses to Gag, Nef, and Env on a single-peptide level using multiple sets of 15-mer overlapping peptides representative of the locally occurring subtypes A, C, and D.
Study subjects.The 56 individuals in this study are part of a larger high-risk HIV cohort of female bar workers enrolled in a prospective study of HIV-1 superinfection (HIV Superinfection Study [HISIS]) in the Mbeya region of Southwest Tanzania. Between September and December 2000, 600 women were recruited after giving informed consent, and each participant provided blood samples at enrollment and every 3 months after for a period of up to 4 years. During the study, all participants received health care that included treatment of all acute infectious diseases, screening and treatment for sexually transmitted diseases, and, since 2003, cotrimoxazole prophylaxis for opportunistic infections for women with CD4 T-cell counts below 200 cells/μl. Since 2005, antiretroviral treatment has been available for all participants with AIDS-defining symptoms or CD4 counts below 200 cells/μl. During the course of this study, all individuals were antiretroviral naïve. Their HIV-1 status was determined using two diagnostic HIV enzyme-linked immunoassay tests (Enzygnost Anti HIV1/2 Plus [Dade Behring, Liederbach, Germany] and Determine HIV 1/2 [Abbott, Wiesbaden, Germany]). Discordant results were resolved by using a Western blot assay (Genelabs Diagnostics, Geneva, Switzerland). Plasma HIV-1 RNA levels were measured by using the Amplicor HIV-1 Monitor assay (Roche Diagnostics, Indianapolis, IN). In April 2003, 56 of the HIV-1 positive participants were enrolled in the HISIS-Cytotoxic T-Lymphocyte (CTL) substudy. The study was reviewed and approved by the ethics committees of all partners in compliance with national guidelines and institutional policies.
HLA typing.HLA-A, -B, and -C class I alleles were typed by DNA sequencing using an ABI 3700 sequencer. Blood samples of 200 μl were extracted using a QIAGEN blood extraction kit for yields of high-quality DNA. Exons 2 and 3 (encoding the peptide binding groove) were amplified in a first-round PCR as previously described (31). The initial amplicon of exons 2 and 3 then served as a template for the nested and/or heminested amplification of exon 2. Exon 3 was similarly amplified in a nested fashion. Both the first-round and nested PCR primers are located in the introns flanking the amplified exon(s) (31). The nested PCR primers are alternately tailed with an M13 Universal 10 DNA sequencing primer sequence. Bidirectional M13 cycle sequencing of exon 2 and exon 3 was executed with BigDye V3.1. HLA DNA sequence typing analysis was completed with Assign SBT software (Conexio Genomics). DNA sequence ambiguities were resolved using the PEL-FREEZ SSP kit.
Synthetic peptides and peptide pools.Seven sets of peptides consisted of 15-mer peptides overlapping by 11 amino acids covering the entire Gag, Nef, and Env protein sequences of primary isolates 90CF402 (subtype A Gag) (GenBank accession no. AAB38823), DU422 (subtype C Gag) (accession no. CAD62240), 98UG57143 (subtypes D Gag and D Env) ( accession no. AF484514) , 92UG037 (subtype A Nef) (accession no. AAC97549), DU151 (subtype C Nef) (accession no. AAL05314), and 94UG114 (subtype D Nef) (accession no. AAC97574). Subtype C Env peptides consisted of 18-mers and were based on primary isolate Du179 ( accession no. AX457092). Peptides were synthesized using 9-fluorenylmethoxy carbonyl chemistry and standard solid-phase techniques with free amino termini. All peptides were >80% pure as determined by high-performance liquid chromatography, mass spectrophotometry, amino acid analysis, and N-terminal sequencing. Peptides were synthesized at the Natural and Medical Sciences Institute (University of Tuebingen, Germany), the Henry M. Jackson Foundation (Rockville, MD), and Anaspec Incorporated (San Jose, CA). The Gag peptide sets were closely related to HIV-1 isolates from the Mbeya Region (data not shown). Initial screening for T-cell responses was performed using the peptides in a matrix format. Subtype A, C, and D Gag peptide sets were pooled in an 11-by-11 format, subtype A and D Nef peptide sets were pooled in a 7-by-7 format, and the subtype C Nef peptide set was pooled in a 10-by-5 format.
ELISPOT assays.During follow-up 13 of the HISIS, freshly isolated peripheral blood mononuclear cells (PBMC) (viability of <95%) were screened for HIV-specific T-cell responses by stimulation with overlapping peptide pools representing Gag, Nef, and Env from isolates of subtypes A, C, and D. (Follow-up 13 is the 13th follow-up of the study, roughly 39 months after the beginning of the study.) Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays were performed by using Nova-Red substrate (Vector, Burlingame, CA) as previously described (24). The assay consistency was monitored on each plate using quality control PBMC recovered from frozen stocks and stimulated with a peptide pool containing optimal peptides from cytomegalovirus, Epstein-Barr virus, and influenza virus (CEF peptide pool). Confirmation of individual peptide responses, viral loads, and CD4 counts was done at follow-up 14, 3 months after the initial screening with peptide matrices. Recognition of a single peptide or pair of adjacent overlapping peptides was defined as a single epitope response. Responses with at least 70 spot-forming cells/106 PBMC and at least three times the negative control were scored as positive.
Whole-blood-format intracellular cytokine staining assay.Intracellular IFN-γ staining in whole blood was performed to determine whether the responses were mediated by CD4 or CD8 T cells. Briefly, 250 μl of heparinized whole blood was added into polypropylene tubes and stimulated with either individual peptides (2 μg/ml [final]), staphylococcal enterotoxin B (10 μg/ml [final]) (Sigma) as a positive control, or no added stimulant as a negative control. A solution containing 25 μg/ml of anti-CD28 and anti-CD49d monoclonal antibodies (Pharmingen, San Diego, CA), 0.5% dimethyl sulfoxide, and 25 μg/ml of brefeldin A (Sigma) was added to each tube. Tubes were incubated for 6 h at 37°C in the presence of 4.5% CO2. Samples were then processed by lysing, fixation, permeabilization, and staining according to the BD Bioscience standard whole-blood assay analysis. Cells were stained with a four-color panel of antibodies: anti-IFN-γ-fluorescein isothiocyanate (clone 4S.B3; Pharmingen), anti-CD4-phycoerythrin (clone RPA-T4; Pharmingen), anti-CD8-PerCp-Cy5.5 (clone SK1; BD Biosciences), and anti-CD3-allophycocyanin (clone UCHT1; Pharmingen). Data acquisition was performed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA), and data sets were analyzed using FlowJo software (version 4) (TreeStar, Cupertino, CA). Responses of at least three times the negative control and at least 0.05% of CD8+ T cells were scored as positive.
Analysis of single peptide responses.Associations between particular peptide responses and HLA class I allele expression were described previously (14). Optimal epitopes within the targeted peptides were inferred after analysis of the peptide sequence using the HIV Molecular Immunology Database (http://www.hiv.lanl.gov/content/hiv-db/ELF/epitope_analyzer.html). The potential B8101 epitope within peptide Gag83 was inferred using the HLA B8101 binding motif with proline as a second position anchor and methionine as the C-terminal anchor.
Statistical analysis.Data analyses were carried out using either GraphPad Prism software or SPSS software. Comparisons of two groups were performed using the Mann-Whitney test. The relationship between viral load or CD4 counts and CD8 T-cell responses was analyzed using Spearman's rank correlation test and linear regression analysis. Tests used for statistical analysis are described in the figure legends.
Expression of "protective" HLA class I B alleles correlates with a broad, Gag-specific CD8 T-cell response.The most common HLA-B allele within the HISIS-CTL cohort was B4201 (n = 12), followed by B5301 (n = 10), B5802 (n = 9), B8101 (n = 9), B0702 (n = 8), and B1510 (n = 8) (data not shown). The alleles B5801 and B5703, which have been associated with a low viral load in previous studies, were less common (n = 4). The association between HLA-B alleles and viral load is shown in Fig. 1A. B5801-expressing subjects had the lowest viral load (median, 17,250 RNA copies/ml), followed by individuals expressing B8101 (median, 19,360 RNA copies/ml), B0702 (median, 45,350 RNA copies/ml), B5703 (median, 67,250 RNA copies/ml), and B4403 (median, 85,800 RNA copies/ml). On the contrary, expression of B1503, B4501, B5301 was associated with median plasma viral loads that were well above 200,000 RNA copies/ml.
Expression of "protective" HLA class I B alleles correlates with a broad Gag-specific CD8 T-cell response. HLA class I expression and plasma viral load in chronically infected study subjects (n = 54) are shown in panel A. The line indicates the median viral load of the HISIS-CTL cohort (198,500 viral RNA copies/ml). (B) Shown is the number of recognized epitopes for Gag and Nef from study subjects who express either HLA-B alleles B0702, B8101, or B5801 (n = 16) (open circles) or other HLA-B alleles (n = 37) (closed circles). Gag peptide responses were determined with an IFN-γ ELISPOT assay. Recognition of two consecutive peptides was counted as one epitope response. The Mann-Whitney test was used to analyze the breadth of the Gag- and Nef-specific T-cell responses associated with these alleles.
Next, we examined whether subjects expressing HLA alleles that were associated with a median plasma viral load of below 50,000 RNA copies/ml (B5801, B8101, and B0702, referred to as "protective" HLA alleles for simplicity) differed from other subjects in their HIV-specific CD8 T-cell responses. The recognition of the immunodominant HIV proteins Gag, Nef, and Env was analyzed with an IFN-γ ELISPOT assay using fresh PBMC and HIV subtype A, C, and D overlapping peptide matrices. Cytokine flow cytometric analysis for IFN-γ production confirmed the peptide responses as detected with the ELISPOT assay and revealed that 89% of peptide-specific responses were from CD8+ T cells (data not shown).
Subjects (n = 16) expressing "protective" HLA-B alleles had a broader median recognition of Gag peptides, twofold higher than that of individuals expressing other HLA-B alleles (P = 0.0035) (Fig. 1B). The magnitude of Gag-specific CD8 T cells was not significantly different between these groups, although the median magnitude was twofold higher (2,840 versus 1,420 spot-forming cells/106 PBMC) (data not shown) for subjects expressing a "protective" HLA allele. Interestingly, five of the six outlier subjects, expressing B8101 or B0702 but with viral loads above 200,000 RNA copies/ml, coexpressed either B4501 (two subjects), B1801 (two subjects), or B1503 (one subject), all HLA-B alleles that have previously been shown to be associated with rapid disease progression in a subtype C-dominated epidemic (11, 18). On the contrary, the coexpression of two protective B alleles was associated with low viral loads (mean, 13,243 copies/ml) and a broad Gag-specific T-cell response. For Nef- and Env-specific CD8 T-cell responses, there were no significant differences between the two groups (Fig. 1B and data not shown). The HLA class I allele B57 was not associated with broad Gag or Nef epitope recognition (data not shown). In summary, individuals expressing the "protective" HLA class I alleles B5801, B8101, and B0702 have a significantly broader recognition of Gag epitopes, but the protective effect may be abrogated in individuals coexpressing B alleles associated with rapid disease progression.
Inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load.To determine whether a broad recognition of Gag epitopes protects HIV-infected individuals from rapid disease progression, we compared the breadth of epitope recognition and the total magnitude of protein-specific CD8 T-cell responses with the plasma viral load and the CD4 T-cell count. As shown in Fig. 2, there was a negative linear relationship between the number of recognized epitopes within Gag and the plasma viral load (P = 0.0016; R linear = −0.36) and a positive linear relationship between the number of recognized epitopes within Gag and the CD4 T-cell count (P = 0.013; R linear = 0.33). Neither Nef- nor Env-specific CD8 T-cell responses correlated with a low plasma viral load or with the CD4 cell counts. There was no significant inverse linear relationship between the total magnitude of Gag-specific CD8 T cells and the plasma viral load (data not shown).
Linear relationship between viral load and CD4 counts with the number of recognized epitopes within Gag but not within Nef or Env. Shown are (A) the linear regression analysis of the viral load and (B) the CD4 count versus the number of epitopes recognized per subject for Gag (left panels), Nef (middle panels), and Env (right panels). Recognition of two consecutive peptides was counted as on epitope response. Statistical analysis was performed using the Spearman rank test.
The majority of subjects with efficient control of viral replication recognize multiple epitopes within two specific regions of Gag.To identify particular regions within the Gag protein that are rich in potentially protective epitopes, we first stratified the cohort into two groups accounting for approximately one-third and two-thirds of the studied cohort, respectively: the low-viral-load group (LVL group) (<50,000 RNA copies/ml; n = 19) and the high-viral-load group (HVL group) (>50,000 RNA copies/ml; n = 36). We then compared the Gag responses between the two groups (Fig. 3). There were striking differences in the CD8 T-cell specificities between these two groups. The average LVL subject not only recognized more peptides within the whole Gag protein (1.4-fold greater) but also differed in the specific Gag regions targeted compared to the HVL group. In comparison to HVL subjects, LVL subjects recognized peptides within the N-terminal region of Gag (GagR1, amino acids [aa] 1 to 75, peptides 1 to 16) and within the C-terminal Gag region (GagR3, aa 248 to 500, peptides 62 to 120) almost twice as often (1.8-fold and 2.0-fold, respectively). Peptides within the middle region of Gag (GagR2, aa 76 to 247, peptides 17 to 61) were recognized equally between the groups. We found an inverse linear relationship between the number of recognized GagR1 and GagR3 (GagR1R3) epitopes and the viral load (Fig. 4A). Furthermore, recognition of more than one epitope within the GagR1R3 regions was strongly associated with a low viral load (P = 0.015) (Fig. 4B) and a higher CD4 T-cell count (P = 0.016) (data not shown). The importance of targeting multiple epitopes within the GagR1R3 regions at a population level is further supported by the fact that 16 of 19 subjects (86%) with a viremia below 50,000 RNA copies/ml targeted at least two epitopes within GagR1R3. Expression of the protective HLA class I alleles B5801, B8101, and B0702 was associated with a broad recognition of GagR1R3 epitopes (Fig. 4C), and breadth was significantly associated with a low viral load within subjects expressing these alleles (Fig. 4D).
Gag-specific CD8 T-cell epitope recognition of subjects with a plasma viral load below (n = 19) or above (n = 36) 50,000 RNA copies/ml. Shown is the magnitude of responses after smooth local average with Gaussian kernel weights and a standard deviation (bandwidth) of 1 peptide.
Relationship of the recognition of multiple epitopes within Gag regions at aa 1 to 075 and aa 248 to 500 (GagR1R3) with efficient viral control and protective HLA class I B alleles. Shown are (A) the linear regression analysis of the viral load and the number of GagR1R3 epitopes recognized and (B) the viral loads of subjects with a broad (n = 29) or narrow (n = 26) CD8 T-cell response against GagR1R3. The cutoff value was 2 epitopes recognized. The respective median is indicated. HLA B5703-expressing subjects are indicated as gray squares. (C) The numbers of recognized epitopes for GagR1R3 from study subjects who express HLA-B alleles B0702, B8101, or B5801 (n = 16) ("protective") and from those expressing other HLA-B alleles (n = 37,other) and within these subjects (D) show a comparison of the number of recognized GagR1R3 epitopes of subjects with a high viral load above 200,000 RNA copies/ml (n = 6) and those with a viral load below 50,000 RNA copies/ml (n = 9). Statistical analysis for B, C, and D was performed using the Mann-Whitney test (two tailed).
Interestingly, the conservation of some important epitopes within GagR1R3 is relatively limited. For two epitope responses that were most strongly associated with low viral load, RPGGKKKYML (aa 22 to 31, presented by HLA B0702 and B4201) and the inferred B8101 epitope GPGATLEEM (aa 338 to 346), one of the HLA anchor residues for each epitope (shown in boldface type) is highly variable (Fig. 5). Mutations affecting anchor residues are very rare in the more frequently recognized epitopes within GagR2 (http://www.hiv.lanl.gov/content/hiv-db/ELF/epitope_analyzer.html). Another frequently recognized epitope, HYMLNHIVW (aa 28 to 36), is the most variable of the immunodominant epitopes, and recognition of this epitope is overrepresented in the LVL group.
Variability of important immunodominant Gag epitopes. Shown is the Shannon entropy score (y axis) for each amino acid position of the epitopes (A) RL10 (aa 22 to 31), (B) HW9 (aa 28 to 36), (C) TL9 (aa 180 to 188), and (D) GM9 (aa 338 to 346). The entropy at amino acid anchor residues is indicated as striped bars. The epitope sequence included in the best recognized peptide variant is shown on the x axis. The entropy score analysis is based on HIV-1 Gag sequences of primary isolates from the Mbeyan Region (n = 41).
Recognition of multiple epitopes within Gag R1R3 is associated with the long-term maintenance of a low viral load after seroconversion.It was of further interest to determine whether the recognition of multiple epitopes within GagR1R3 could be associated with the maintenance of low steady-state viral load levels. To address this question, the course of plasma viremia was studied in 19 subjects who seroconverted during the HISIS (three HLA B57-expressing subjects were excluded, because B57 is often associated with the maintenance of a very low viral set point, low viral load, and an exceptional disease course) (2). In subjects (n = 12) recognizing two or more GagR1R3 epitopes during follow-up 14, the course of plasma viremia is typical for HIV infection (Fig. 6A). During the acute phase, the viral load rises to a median peak level of 286,000 RNA copies/ml and subsequently drops down to a steady-state level of 10,000 to 20,000 RNA copies/ml for the next 24 months. Only 2 of 12 subjects had a viral load above 50,000 RNA copies/ml at 24 months after seroconversion. The course of plasma viremia of B57-negative subjects recognizing zero or one epitope within GagR1R3 (n = 7) is strikingly different (Fig. 6B). The plasma viral load rises to peak levels with a median of 403,000 RNA copies/ml during acute infection and subsequently stays at a steady-state level of viremia of 150,000 to 350,000 RNA copies/ml over the next 24 months. At 24 months after seroconversion, none of this group had a viral load below 50,000 RNA copies/ml, and only one subject was below 100,000 RNA copies/ml. It appears that viral replication is never brought under efficient control in these subjects. This difference in viral load is likely to affect disease progression, and indeed, there was a strong tendency supporting this assumption. At 24 to 27 months after seroconversion, 6 of 12 subjects recognizing multiple GagR1R3 epitopes had CD4 T-cell counts above 500 cells/μl, whereas 6 of 7 subjects with narrow recognition of GagR1R3 had CD4 T-cell counts below 500 cells/μl (data not shown). These results indicate that long after the acute phase of peak viremia, the recognition of multiple epitopes within GagR1R3 may still contribute substantially to the maintenance of a low steady-state viremia.
Dynamics of the plasma viral load after seroconversion. Shown is the viral load since seroconversion of study subjects who (A) recognized two or more epitopes (n = 12) or (B) zero or one epitope (n = 7) within the Gag regions at aa 1 to 75 and aa 297 to 500. The median viral load is indicated in red. Gag peptide responses were determined between 15 and 39 months (follow-up 14) after seroconversion with an IFN-γ ELISPOT assay. Three HLA-B5703-expressing subjects were excluded from the analysis. Recognition of two consecutive peptides was counted as one epitope response. vRNA, viral RNA.
The main objective of this study was to reveal antiviral determinants of the HIV-specific CD8 T-cell response and to examine the influence of the host genetic (HLA) background on the HIV-specific CD8 T-cell response. The HISIS-CTL cohort is composed of 56 chronically infected female bar workers highly exposed to a mixed-subtype epidemic where subtype C predominates (14). Within the HISIS-CTL cohort, HLA-B alleles B5801, B8101, and B0702 were associated with a low viral load. This finding is consistent with a larger study in a cohort of subtype C-infected individuals previously conducted in South Africa (18). We have expanded upon these observations to show that within this cohort of high HIV-1 exposure, it is T-cell responses restricted by these alleles, particularly those targeting certain regions of the Gag protein, that mediate significantly better control of viral load.
The CD8 T-cell specificities found within an HIV-infected individual are largely dependent on the HLA class I alleles expressed, the sequence of the infecting virus (8, 11), and, partly, the stage of infection (1, 15). Additionally, HIV-specific CD4 T cells may also help to maintain CD8 T-cell responses of broad HIV specificity (22). It was first shown in 1995 that the Gag-specific CD8 T-cell response may play an important role in slowing down progression to AIDS (30). However, recent reports are contradictory with respect to a role for the Gag-specific CD8 T-cell response in controlling viral load. Some studies supported an association of a strong Gag-specific CD8 T-cell response with the control of HIV replication (10, 27-30, 34), whereas others showed no such association (1, 4, 6, 12). Our results suggest that the breadth of epitope recognition within specific regions of Gag strongly contributes to the antiviral efficiency of the CD8 T-cell response during the chronic phase of infection. The expression of certain HLA-B alleles, namely, B0702, B5801, and B8101, was associated with this particular pattern of CD8 T-cell recognition. This finding, and the inverse linear relationship between the number of recognized Gag (or GagR1R3) epitopes and plasma viral load, further supports the notion that the viral load-HLA associations are mediated by the CD8 T-cell response restricted by these HLA alleles. The hypothesis that the breadth of GagR1R3 recognition is an important correlate of the expression of these HLA B alleles is reinforced by the examination of six individuals that have a high viral load despite expressing the protective allele B5801, B8101, or B0702. The breadth of GagR1R3 recognition was significantly reduced in these individuals compared to the other subjects expressing these B alleles (Fig. 4D).
Is the antigen specificity of CD8 T cells important for the maintenance of antiviral efficiency? In contrast to the Gag response, there was a trend of a broad Nef-specific response with higher viral loads, resembling observations of previous studies (4, 23, 27). The observed differences between Gag- and Nef-specific responses support the notion that antigen specificity plays a role in maintaining antiviral efficiency of HIV-specific CD8 T cells during the chronic phase of infection.
The observation that the magnitude of Gag- or GagR1R3-responding CD8 T cells, measured by cumulative IFN-γ ELISPOT responses, did not correlate with a low viral load contrasts with most of the recent reports that have found a protective role of strong Gag-specific T-cell responses. One possible explanation for this difference is that the majority of the HISIS-CTL substudy subjects were infected for extended time periods and had high viral loads. An alternative explanation is that the usage of three different Gag peptide sets permitted a better detection of epitope breadth compared to other studies. We have shown previously that that the Gag and Nef peptide sets used during this study did afford better detection of the breadth of the T-cell response (7, 14).
Importantly, "protective" HLA B alleles, B0702, B5801, and B8101, were associated with a median plasma viral load below 50,000 RNA copies/ml, constituting a fourfold reduction in the median viral load observed within the whole HISIS-CTL cohort. This comparatively high median viral load of 198,500 RNA copies per ml most likely reflects the long time since primary HIV infection in the majority of the study subjects, 63% of which (35 of 56) were infected for more than 3.5 years. The remaining 21 subjects seroconverted during the study and had a median viral load of 40,600 RNA copies/ml at 24 months after seroconversion, which is in the range observed in most other studies (10, 18, 23, 27). Together, these results underline that all study subjects were in the chronic phase of HIV infection: 21 were in the postacute or early chronic phase (9 months to 3.5 years of infection), and 35 were in later stages of the chronic phase of HIV infection (>3.5years of infection).
It is also noteworthy that most previous studies (and the present study) used IFN-γ-based assays to detected HIV-specific CD8 T cells. IFN-γ is a sensitive marker for HIV-specific CD8 T-cell activation; however, virus-specific cytolytic activity and IFN-γ secretion upon restimulation do not necessarily correlate during chronic HIV infection (3, 9, 19). This dislinkage of cytolytic activity and IFN-γ secretion has been ascribed to a phenomenon known as the "partial functional exhaustion" of T cells. In a mouse model of chronic LCMV infection, CD8 T cells of different epitope specificities can vary in their respective functional exhaustion, which is partly dependent on the antigenic load and the time from infection (32). A similar phenomenon may explain the differences between the present study and previous studies of HIV-1 infection.
One example of a very strong, yet potentially ineffective, CD8 T-cell response during chronic HIV infection is the targeting of the Gag epitope TL9. This epitope is presented by the class I alleles B0702, B4201, and B8101 and is targeted by 41% of the subjects in this study cohort, and responses are among those with the highest magnitude (14). This epitope is highly conserved within a particular subtype, and there is no evidence supporting viral escape by an epitope point mutation. Additionally, the high magnitude of responding cells and the lack of mutational changes in the immediate epitope proximity within subtype C argue against abrogated epitope processing. Therefore, we believe that neither the epitope itself nor its presentation is changed in the course of chronic infection. Despite all of these parameters, previously assumed to be favorable, we did not find any evidence supporting a substantial role for this immunodominant response in viral control during the chronic phase of infection. It is noteworthy that the three subjects that possess protective allele B8101, but with high viral loads, also targeted this epitope. The persistence of unaltered yet targeted epitopes during the course of chronic HIV infection has also been demonstrated in two recent studies (9, 19).
Why may the breadth of Gag or GagR1R3 but not Nef or Env recognition be important for viral control during the chronic stage of HIV infection? One possible explanation is that a broad Gag or GagR1R3 response is more likely to select for viral escape mutants that are characterized by a reduced replicative capacity. Responding T cells would at the same time poorly recognize the mutant epitope(s), and a reduced T-cell receptor stimulation may in turn slow down the functional exhaustion of virus-specific T cells. Indeed, some of the most "protective" epitopes within GagR1R3 are highly variable, and it has been shown previously that mutational escape from an epitope-specific T-cell response can cripple the replicative capacity of the mutant virus (25), despite it being less well recognized by virus-specific T cells. Episodic epitope reversion may then contribute to the persistence of wild-type epitope-specific T cells (13, 21) and at the same time "conserve" the antiviral efficiency of these T cells.
It is commonly accepted that the steady-state viral load is a major parameter affecting the rate of disease progression (26). Within this context, perhaps the most striking observation during this study was the course of the viral load in 19 B5703-negative individuals after seroconversion. Most subjects targeting multiple epitopes within GagR1R3 had a "normal" course of viral load, whereas subjects with a very narrow GagR1R3 response had an unusually high median viral load steady-state level. Therefore, this pattern of HIV-specific T-cell recognition is likely to be associated with slow/normal disease progression, and to the contrary, its absence is likely to be associated with a more rapid progression. Importantly, the latter finding may affect not only survival but also HIV transmission rates. Taken together, our findings highlight the association between the host genetic (HLA) background and the quality of the HIV-specific CD8 T-cell response during the chronic phase of HIV infection. The results may help to explain differences in disease progression on a population level. During evaluations of candidate HIV-1 vaccines, it may be useful to include vaccine immunogens that are capable of stimulating broad and targeted Gag responses in individuals with certain HLA class I alleles in the population.
This work was supported by the European Commission, DG XII, INCO-DC (grant ICA-CT-2002-10048), and by a cooperative agreement between the Henry M. Jackson Foundation for the Advancement of Military Medicine and the U.S. Department of Defense. This study was conducted at the Mbeya Medical Research Programme.
We thank the excellent staff at the Mbeya Medical Research Programme that conducted the HISIS, especially Vera Kleinfeldt, Frowin Nichombe, Weston Assisya, and Clemence Konkamkula, and all of the HISIS participants. Furthermore, we thank Clive Gray (NICD, Johannesburg, South Africa) for critical advice on some experimental procedures and the subtype C peptides. Additionally, we thank Patricia D'Souza (Division of AIDS, NIH), Christian Brander (Massachusetts General Hospital), Steve Cate, and William Hildebrand (University of Oklahoma Health Science Center) for facilitating the HLA typing of the specimens.
The views and opinions expressed herein do not necessarily reflect those of the U.S. Army or the Department of Defense.
Received 24 August 2006.
Accepted 27 November 2006.
↵*Corresponding author. Present address: Vaccine Research Center, NIAID, Bethesda, MD 20892. Phone: (301) 594-8619. Fax: (301) 480-2779. E-mail: cgeldmacher{at}mmrp.org.
↵▿ Published ahead of print on 20 December 2006.
Addo, M. M., X. G. Yu, A. Rathod, D. Cohen, R. L. Eldridge, D. Strick, et al. 2003. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses but no correlation to viral load. J. Virol. 77:2081-2092.
Altfeld, M., M. M. Addo, E. S. Rosenberg, F. M. Hecht, P. K. Lee, M. Vogel, et al. 2003. Influence of HLA-B57 on clinical presentation and viral control during acute HIV-1 infection. AIDS 17:2581-2591.
Appay, V., D. F. Nixon, S. M. Donahoe, G. M. Gillespie, T. Dong, A. King, et al. 2000. HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function. J. Exp. Med. 192:63-75.
Betts, M. R., D. R. Ambrozak, D. C. Douek, S. Bonhoeffer, J. M. Brenchley, J. P. Casazza, R. A. Koup, and L. J. Picker. 2001. Analysis of total human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell responses: relationship to viral load in untreated HIV infection. J. Virol. 75:11983-11991.
Borrow, P., H. Lewicki, B. H. Hahn, G. M. Shaw, and M. B. Oldstone. 1994. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J. Virol. 68:6103-6110.
Cao, J., J. McNevin, S. Holte, L. Fink, L. Corey, and M. J. McElrath. 2003. Comprehensive analysis of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-secreting CD8+ T cells in primary HIV-1 infection. J. Virol. 77:6867-6878.
Currier, J. R., U. Visawapoka, S. Tovanabutra, C. J. Mason, D. L. Birx, F. E. McCutchan, and J. H. Cox. 2006. CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: use of multiple peptide sets increases the detectable breadth of the CTL response. BMC Immunol. 7:8.
Draenert, R., T. M. Allen, Y. Liu, T. Wrin, C. Chappey, C. L. Verrill, et al. 2006. Constraints on HIV-1 evolution and immunodominance revealed in monozygotic adult twins infected with the same virus. J. Exp. Med. 203:529-539.
Draenert, R., C. L. Verrill, Y. Tang, T. M. Allen, A. G. Wurcel, M. Boczanowski, A. Lechner, A. Y. Kim, T. Suscovich, N. V. Brown, M. M. Addo, and B. D. Walker. 2004. Persistent recognition of autologous virus by high-avidity CD8 T cells in chronic, progressive human immunodeficiency virus type 1 infection. J. Virol. 78:630-641.
Edwards, B. H., A. Bansal, S. Sabbaj, J. Bakari, M. J. Mulligan, and P. A. Goepfert. 2002. Magnitude of functional CD8+ T-cell responses to the Gag protein of human immunodeficiency virus type 1 correlates inversely with viral load in plasma. J. Virol. 76:2298-2305.
Frahm, N., P. Kiepiela, S. Adams, C. H. Linde, H. S. Hewitt, K. Sango, et al. 2006. Control of human immunodeficiency virus replication by cytotoxic T lymphocytes targeting subdominant epitopes. Nat. Immunol. 7:173-178.
Frahm, N., B. T. Korber, C. M. Adams, J. J. Szinger, R. Draenert, M. M. Addo, M. E. Feeney, K. Yusim, K. Sango, N. V. Brown, D. SenGupta, A. Piechocka-Trocha, T. Simonis, F. M. Marincola, A. G. Wurcel, D. R. Stone, C. J. Russell, P. Adolf, D. Cohen, T. Roach, A. StJohn, A. Khatri, K. Davis, J. Mullins, P. J. R. Goulder, B. D. Walker, and C. Brander. 2004. Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities. J. Virol. 78:2187-2200.
Friedrich, T. C., E. J. Dodds, L. J. Yant, L. Vojnov, R. Rudersdorf, C. Cullen, et al. 2004. Reversion of CTL escape-variant immunodeficiency viruses in vivo. Nat. Med. 10:275-281.
Geldmacher, C., J. R. Currier, M. Gerhardt, A. Haule, L. Maboko, D. Birx, et al. 2007. In a mixed subtype epidemic, the HIV-1 Gag-specific T-cell response is biased towards the infecting subtype. AIDS 21:135-143.
Goulder, P. J., M. A. Altfeld, E. S. Rosenberg, T. Nguyen, Y. Tang, R. L. Eldridge, et al. 2001. Substantial differences in specificity of HIV-specific cytotoxic T cells in acute and chronic HIV infection. J. Exp. Med. 193:181-194.
Hendel, H., S. Caillat-Zucman, H. Lebuanec, M. Carrington, S. O'Brien, J. M. Andrieu, et al. 1999. New class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS. J. Immunol. 162:6942-6946.
Jones, N. A., X. Wei, D. R. Flower, M. Wong, F. Michor, M. S. Saag, et al. 2004. Determinants of human immunodeficiency virus type 1 escape from the primary CD8+ cytotoxic T lymphocyte response. J. Exp. Med. 200:1243-1256.
Kiepiela, P., A. J. Leslie, I. Honeyborne, D. Ramduth, C. Thobakgale, S. Chetty, et al. 2004. Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA. Nature 432:769-775.
Koibuchi, T., T. M. Allen, M. Lichterfeld, S. K. Mui, K. M. O'Sullivan, A. Trocha, S. A. Kalams, R. P. Johnson, and B. D. Walker. 2005. Limited sequence evolution within persistently targeted CD8 epitopes in chronic human immunodeficiency virus type 1 infection. J. Virol. 79:8171-8181.
Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrews, G. McLeod, W. Borkowsky, C. Farthing, and D. D. Ho. 1994. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J. Virol. 68:4650-4655.
Leslie, A. J., K. J. Pfafferott, P. Chetty, R. Draenert, M. M. Addo, M. Feeney, et al. 2004. HIV evolution: CTL escape mutation and reversion after transmission. Nat. Med. 10:282-289.
Lichterfeld, M., D. E. Kaufmann, X. G. Yu, S. K. Mui, M. M. Addo, M. N. Johnston, et al. 2004. Loss of HIV-1-specific CD8+ T cell proliferation after acute HIV-1 infection and restoration by vaccine-induced HIV-1-specific CD4+ T cells. J. Exp. Med. 200:701-712.
Masemola, A., T. Mashishi, G. Khoury, P. Mohube, P. Mokgotho, E. Vardas, M. Colvin, L. Zijenah, D. Katzenstein, R. Musonda, S. Allen, N. Kumwenda, T. Taha, G. Gray, J. McIntyre, S. Abdool Karim, H. W. Sheppard, and C. M. Gray 2004. Hierarchical targeting of subtype C human immunodeficiency virus type 1 proteins by CD8+ T cells: correlation with viral load. J. Virol. 78:3233-3243.
Mashishi, T., and C. M. Gray. 2002. The ELISPOT assay: an easily transferable method for measuring cellular responses and identifying T cell epitopes. Clin. Chem. Lab. Med. 40:903-910.
Matano, T., M. Kobayashi, H. Igarashi, A. Takeda, H. Nakamura, M. Kano, et al. 2004. Cytotoxic T lymphocyte-based control of simian immunodeficiency virus replication in a preclinical AIDS vaccine trial. J. Exp. Med. 199:1709-1718.
Mellors, J. W., C. R. Rinaldo, Jr., P. Gupta, R. M. White, J. A. Todd, and L. A. Kingsley. 1996. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 272:1167-1170. (Erratum, 275:14, 1997.)
Novitsky, V., P. Gilbert, T. Peter, M. F. McLane, S. Gaolekwe, N. Rybak, I. Thior, T. Ndung'u, R. Marlink, T. H. Lee, and M. Essex. 2003. Association between virus-specific T-cell responses and plasma viral load in human immunodeficiency virus type 1 subtype C infection. J. Virol. 77:882-890.
Ogg, G. S., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard, et al. 1998. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279:2103-2106.
Ramduth, D., P. Chetty, N. C. Mngquandaniso, N. Nene, J. D. Harlow, I. Honeyborne, et al. 2005. Differential immunogenicity of HIV-1 clade C proteins in eliciting CD8+ and CD4+ cell responses. J. Infect. Dis. 192:1588-1596.
Riviere, Y., M. B. McChesney, F. Porrot, F. Tanneau-Salvadori, P. Sansonetti, O. Lopez, et al. 1995. Gag-specific cytotoxic responses to HIV type 1 are associated with a decreased risk of progression to AIDS-related complex or AIDS. AIDS Res. Hum. Retrovir. 11:903-907.
Turner, S., M. E. Ellexson, H. D. Hickman, D. A. Sidebottom, M. Fernandez-Vina, D. L. Confer, and W. H. Hildebrand. 1998. Sequence-based typing provides a new look at HLA-C diversity. J. Immunol. 161:1406-1413.
Wherry, E. J., J. N. Blattman, K. Murali-Krishna, R. van der Most, and R. Ahmed. 2003. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J. Virol. 77:4911-4927.
Yang, O. O., P. T. Sarkis, A. Ali, J. D. Harlow, C. Brander, S. A. Kalams, and B. D. Walker. 2003. Determinant of HIV-1 mutational escape from cytotoxic T lymphocytes. J. Exp. Med. 197:1365-1375.
Zuñiga, R., A. Lucchetti, P. Galvan, S. Sanchez, C. Sanchez, A. Hernandez, H. Sanchez, N. Frahm, C. H. Linde, H. S. Hewitt, W. Hildebrand, M. Altfeld, T. M. Allen, B. D. Walker, B. T. Korber, T. Leitner, J. Sanchez, and C. Brander. 2006. Relative dominance of Gag p24-specific cytotoxic T lymphocytes is associated with human immunodeficiency virus control. J. Virol. 80:3122-3125.
Journal of Virology Feb 2007, 81 (5) 2440-2448; DOI: 10.1128/JVI.01847-06
You are going to email the following CD8 T-Cell Recognition of Multiple Epitopes within Specific Gag Regions Is Associated with Maintenance of a Low Steady-State Viremia in Human Immunodeficiency Virus Type 1-Seropositive Patients


text:
Q: Why does JSON Decoder return nil when program was working fine before with no changes? Hello wonderful people of the internet, I am confused as to why my program does not work. It was working fine and then suddenly it started returning nil from the JSON decoder. Any thoughts on why this might be? Thank you for your help and have a wonderful day!
import SwiftUI

struct CommentFeedView: View {
    @State var comments = [Comment]()
    var body: some View {
        NavigationView {
            List(comments) { comment in
                // UI Code goes here
                }
                // More UI Code goes here
            }
            .onAppear {
                Timer.scheduledTimer(withTimeInterval: 5, repeats: true) { timer in
                    getComments()
                }
            }
            .navigationTitle("Comments")
        }
    }
    func getComments() {
        let url = URL(string: "https://www.reddit.com/r/GoForGold/comments.json?limit=25")!
        URLSession.shared.dataTask(with: url) { data, response, error in
            if let data = data {
                if let decodedComments = try? JSONDecoder().decode(CommentListing.self, from: data) { //decodedComments is nil
                    DispatchQueue.main.async {
                        let commentObjectsWrapper = decodedComments.data!
                        let commentObjects = commentObjectsWrapper.children
                        for commentObject in commentObjects {
                            self.comments.append(commentObject.data!)
                        }
                    }
                }
            } else if let error = error {
                print(error)
            } else {
                print("Request failed")
            }
        }.resume()
    }
}

redpajama_set_name: RedPajamaStackExchange


text:
Category / Physiology and Biophysics
First part of $30 million grant awarded to USC Team for stroke project by National Institutes of Health
Categories: Latest, Physiology and Biophysics, Research, Zilkha|
Researchers at Keck School of Medicine of USC move experimental drug closer to approval for clinical use By Jeremy Deutchman USC Neurologist Patrick Lyden, MD (Photo: USC/Don ... Read More »
Eight Keck School of Medicine departments rank in top 10 for NIH funding
Categories: Family Medicine, Latest, Neurology, Neurosurgery, Ophthalmology, Orthopaedic Surgery, Orthopaedics, Otolaryngology, Physiology and Biophysics, Urology|Tags: Alzheimer's Disease|
Three departments—the Department of Ophthalmology, Population and Public Health Sciences and the Catherine and Joseph Aresty Department of Urology—all came in No. 2 for their disciplines.
Keck School faculty well-represented on Clarivate's Highly Cited 2021 list of most influential researchers
Categories: Alzheimer's Therapeutic Research Institute, Alzheimers Initiative, Latest, Neurology, Physiology and Biophysics, Research, Zilkha|
The list recognizes "the true pioneers in their fields over the last decade, demonstrated by the production of multiple highly cited papers that rank in the top 1% by citations."
Keck School faculty honored during USC's spring awards season
Categories: Biochemistry and Molecular Medicine, Physiology and Biophysics, Stem Cell|
On April 12, the University honored outstanding faculty during its 40th annual Academic Honors Convocation.
Six KSOM departments make top 10 in NIH funding nationally
Categories: Latest, Neurology, Ophthalmology, Orthopaedics, Otolaryngology, Physiology and Biophysics, Population and Public Health Sciences|
Ophthalmology is No. 1 among med schools for the fourth year in a row, while Preventive Medicine is No. 2.
KSOM faculty named to Highly Cited 2020 list of world's most influential researchers
Categories: Alzheimer's Therapeutic Research Institute, Molecular Microbiology and Immunology, Neuroimaging and Informatics, Neurology, Physiology and Biophysics, Population and Public Health Sciences, Research, Zilkha|
Multiple KSOM faculty members were named to the list compiled by Clarivate Analytics.
Keck School's Ophthalmology, Physiology/Neuroscience departments top NIH funding list
Categories: Cell and Neurobiology, Featured, Neurology, Ophthalmology, Orthopaedic Surgery, Orthopaedics, Otolaryngology, Physiology and Biophysics, Population and Public Health Sciences, Recent, Urology, Zilkha|
Seven departments at Keck School of Medicine USC are in the top 10 nationally in grant funding from the National Institutes of Health, according to the rankings of ... Read More »
KSOM Research Cited in New Federal Vaping Policy
Categories: Featured, Featured People, Physiology and Biophysics, Population and Public Health Sciences, Uncategorized|
Adam Leventhal, Ph.D., Professor of Preventive Medicine and Psychology A recent study by Adam Leventhal, Ph.D., professor of preventive medicine and psychology at the Keck School ... Read More »
Physiology department rises in national rankings of research funding
Categories: News, Physiology and Biophysics, Zilkha|
Many researchers at the Zilkha Neurogenetic Institute are funded by at least one NIH investigator-initiated grant. When Berislav Zlokovic, MD, PhD, returned to USC in 2011 after ... Read More »

redpajama_set_name: RedPajamaCommonCrawl


The Reuseable Hot/Cold Gel Packs bring relief to painful bumps and bruises, conforms comfortably around wounds, and measures 4" x 6" size to cover skin surface. Includes 12 reusable hot/cold gel packs.
Having the right first aid products at the right time, in the right place is important. Developed for playground use, field trips or any outside excursion when you need first aid supplies. Perfect for desk drawer, computer bag briefcase or car! Red canvas bag includes minor emergency essentials. Fits around your waist for convenience. Includes (6) Adhesive Fabric Bandages, 2" x 4", (16) Adhesive Plastic Bandages, 1" x 3", (10) Alcohol Wipes, (1) Cold Pack, 4" x 5", (1) First Aid Guide, (4) Nitrile Exam Gloves, and (10) Sting Relief Wipes.
Pudgies Unscented Baby Wipes Use nothing but the best for your child with premium Pudgies Baby Wipes. Disposable Pudgies wet wipes are perfect for the home, in the car and on the go. These thick and soft wet nap baby wipes are also ideal for daycare centers, hotels, changing areas and more. Designed for extra comfort and strength. Gentle formula contains Aloe and Vitamin E. Tissue-like dispensing allows single-handed use. Pudgies Wet Wipes are hypoallergenic, alcohol- free and latex-free. Premoistened 6.6x 7.9 baby wipes.Pudgies Baby Wipes, Unscented, 80 Wipes per Pack.
Hard Case Pepper Spray features a quick release key ring allows for accessibility even when keys are in use. Greater protection at a safe distance. 25 bursts (up to 5x the competition) with a 10' (3M) range.
Black Campus Safety Gel with Quick Release Key Ring features a gel formula ideal for college campuses. Virtually eliminates airborne particles and wind blowback and is exceedingly safe to use near bystanders and indoors. Quick release key ring allows for accessibility even when keys are in use, plus finger grip helps increase accuracy and enhances control. Greater protection at a safe distance - 25 bursts (up to 5x the competition) at up to 12' (4M) away.
Compact Pepper spray with Quick Release Key Ring supports Kuros!, a company that provides pepper spray to women in developing countries for self-defense against sexual assault. Quick release key ring allows for accessibility even when keys are in use. Greater protection at a safe distance - 25 bursts (up to 5x the competition) with a 10' (3M) range.
Superior to other home security products, the premium white door alarm looks great inside your home. When door pressure is applied, the extremely loud 120 dB alarm helps wake/alert homeowner or renter, helps deter intruder and possibly notifies neighbors (audible up to 1,000 feet/300M away). Features a non-skid pad to prevent door from opening while alarm sounds. Ideal for bedrooms, hotels, apartments, dorm rooms, front doors, etc. No wiring needed; battery-operated (requires 1x9Vbattery). Provides battery status update with a low battery indicator.
The Door/Window Alarm features an extremely loud 120 dB alarm wakes/alerts homeowner or renter, helps deter intruder and possibly notifies neighbors (audible up to 750 feet/230M away). Offers 3 adjustable settings (off/alarm/chime); chime mode is ideal to alert you when children or guests are coming and going when you are at home. No wiring needed, plus sensor mounts on either side of alarm. Provides battery status update with a low battery test button; 4xLR44 batteries included.
The Mini Personal Alarm is a sleek, compact modern design keychain that attaches to backpack, purse or keys for convenient, instant access, plus greater value with built-in LED light and batteries included. The 110 dB alarm helps call for assistance in an emergency and helps deter attacker (audible up to 500 feet/150M away). Pull metal chain from device to sound 110 dB alarm. Push pin back in to stop sound and use again. Safe for children, and a self-defense option for any age.
Superior to other home security products, this premium white alarm looks great inside your home. The extremely loud 120 dB alarm helps wake/alert homeowner or renter, helps deter intruder and possibly notifies neighbors (audible up to 750 feet/230M away). Features 3 adjustable settings (off/alarm/chime); chime mode is ideal to alert you when children or guests are coming and going when you are at home. Easy installation; no wiring needed, plus sensor mounts on either side of alarm for easier set up; 4xLR44 batteries included. Provides battery status update with a low battery test button.
Bright, eye-catching label features flowers, hearts and butterflies. Designer Pepper Spray with Key Ring reduces wind blow-back thanks to powerful stream delivery. Greater protection at a safe distance - 25 bursts (up to 5x other brands) at up to 10 feet away.
Attractive Designer Pepper Spray with pink ribbons supports National Breast Cancer Foundation. Reduced wind blow-back thanks to powerful stream delivery. Greater protection at a safe distance - 25 bursts (up to 5x other brands) at up to 10' away.
Runners Pepper Spray with Adjustable black hand strap helps ensure pepper spray is readily available on the move. Gel formulation virtually eliminates wind blow-back. Greater protection at a safe distance - 35 bursts (up to 5x the competition) at up to 12' (4M) away.


One of the best things we've done in this humble home of ours is when we made the decision to install butcher block counter tops in our kitchen. You guys have been curious because I've been getting emails and direct messages with questions about them ever since. I thought today was a good day to share my thoughts on them with you all. Here's my butcher block counter top review one year later.
When we purchased our house the original 80's laminate counter tops where still there. They were in pretty good shape so we lived with them for a year. I went over all the counter top options, quartz, marble, etc. I loved the thought of marble but I didn't love the price tag or the thought of having to baby them. I'm not a huge granite fan and after looking at many historical colonial homes I realized that butcher block was very commonly used and I loved the character it added. They don't require too much care and they're affordable! Shortly after we moved in we removed the stove top from the island and relocated it. When we did, it left a hole in the island counter top. We thought it was the perfect time to install the butcher block on the island and take it for a test drive. I'm so glad we did because it didn't take us long to find out that we loved it! Months later we came across an amazing deal and purchased our counter tops a lot sooner than we had planned. You can see our kitchen's before and the complete makeover here. It's been well over a year since we installed them and here's how it's worked out.
Our counter tops are wood and were purchased from Ikea. Our particular counter top has since been discontinued bit they still have a great selection. I'm happy to report so far everything has been great! I can honestly say there has not been any staining, scratching or splitting. They look just as good today as they did the day we installed them. I believe that a lot of that is due to how we sealed them. Before we decided to install butcher block I did hours of research on the sealing process. I wanted to make sure that they were sealed correctly so that we'd have years of use from them. Almost everywhere I read the Waterlox was the sealer of choice. It's a professional grade sealer and cannot be purchased in store but is available online. This stuff doesn't mess around. It has a very strong smell. We cut and dry fitted our counter tops and moved them to the garage to seal them. The sealing process takes several days and can become a little annoying. The impatient side of me wanted to install them right away but in the long run it would have been a mistake. Take the time to complete the recommended amount of coats. It's worth the wait friends!
One question I get all the time is how do I take of of them? I certainly don't baby them. I do try to keep spills wiped up. When I'm done doing the dishes we always make sure to take a towel and soak up any standing water that might be beside the sink. Here's a look at the butcher block by the sink. This area is constantly damp and it's not showing any trace of water damage.
Don't worry though because they can take a punch. Short story, I left our instant popsicle maker out on the island to thaw and did not realize that it had a large water puddle under it. I tell myself it was a crazy week because that thing sat there for over three days before I realized it. Ah! I thought the counter top would be ruined for sure. I was pleasantly surprised once I dried up the water that the butcher block was perfectly fine. I could believe it! Back to my point, it's all in how you seal them. Do I cut on them? I do NOT cut on them. That's what cutting boards are for, friends.
When it comes to cleaning them I just use a damp soapy rag. I will use Clorox wipes from time to time but I try not to use any kind of harsh cleaner on them. Just good old soap and water does the trick! Here's my favorite soaps to use here and here. Not only do they smell fantastic, they're gentle and work great on butcher block.
I can honestly say that I love our counter tops. I love the look, price and durability. In a few years we plan on sanding and resealing to keep them in tip top shape. I might even stain them and go a shade darker. For now, they're just perfect! I couldn't be more pleased with how they look today. I hope if you've been on the fence about butcher block this helps you.
I am so glad that you posted this update. We are getting ready to put an island in our kitchen and I've really been agonizing over what top to put on it. I love the look of butcher block but wasn't sure how it would hold up. I think you've helped with my decision. I forwarded the info to my husband so that he'll know how to seal it. Your kitchen is beautiful.
We have IKEA butcher block counters also in our small galley kitchen. Like you, we cut and installed them ourselves. We love them! The stain I used is a bit darker and goes well with our white cabinets. Even if I could afford a more expensive counter top, I would still go with butcher block. To me, they are classic. I think our total cost was about $175! How can you beat that?
You really can't beat it! We lucked up and found these discontinued counter tops for only $30 a counter top. We jumped on it! I may eventually stain my a darker shade. I didn't stain these but I love the thought of a warmer look.
If you don't mind me asking, what stain color did you use on yours? We are looking to install butcher block and I can't pick a stain to save my life!
We didn't stain them. The sealer does give them a slightly darker color than their natural one. I may stain them with a warmer color down the road. I completely understand though. Those decisions can be hard. Good luck!
What color are your cabinets? Love the butcher block with that color!
Thanks Christine! They're painted in fieldstone by Benjamin Moore.
Absolutely beautiful kitchen…thank you for sharing. Can I ask what kitchen cabinet color you used? And it looks like a marble like backsplash subway tile? Love it!!
Thank you so much Shelley! The cabinets are painted in Fieldstone by Benjamin Moore and the back splash is marble. ????
I am curious as to how your White Diamond countertop performed. I did not see an update on that, only on the new butcher block cupboards. Does that mean the Giani did not hold up more than a year? Did you put two or three finish coats on it and would you recommend using the Waterlox on top of the sealer provided by Giani?
Hi Michelle, the Diamon counter top kit worked and held up great. We had planned on keeping it for a longer period of time but unexpectedly found our butcher block counter tops on clearance. At $30 a slab we jumped on them and installed them much sooner than we had planned. We had a great experience with the Giani kit. We had never planned to keep it permanently, only until we were at a place to purchase our permanent counter tops. As far as the Waterlox, I believe it's just a finish you want to use on wood products. The Giani kit does come with a heavy sealer they recommend.
This is exactly what I am wanting for out kitchen remodel. I am curious about your floors. What do you have? Could you post a picture. Thank you so much. Your kitchen is beautiful.
What kind of sink do you have? I'm looking at doing this in our kitchen and would like a sink that extends to the wall.


text:
Lego Power Miners è stata una linea di prodotti LEGO, basata sulle vicende di un team chiamato "Power Miners", che investigando una serie di terremoti anomali scopre l'esistenza dei "Power Crystal" e dei "Rock Monsters". La linea di prodotti, lanciata nel 2009, terminò nel 2010 con un totale di 16 set.

Collegamenti esterni

LEGO

redpajama_set_name: RedPajamaWikipedia


Writing as someone who left Hong Kong when he was 14 who still visit every couple years, so take what I write with a load of salt.
I really think HK is over, as in it will never return to its peak glorious days. Much of the prosperity of HK came from being a middleman between China and the rest of the world. That position is eroding because China is slowly opening up. For example, of the top 5 container ports, 3 are in mainland China and HK is #5. HK was the busiest as recently as 2004. Why ship to/from HK when you can ship directly to/from China?
Much of the ruling elites (many of whom are/were businessmen or have business ties) understand this, and realize China has a lot more soft power over HK than what the Basic Law guarantees. The end game is clear, HK will become just another Chinese city, the Establishment is hoping that by pandering to mainland, they can slow down the process and maintain their self-interest. The more positive way to think about it is, if China allows, HK can become the Shanghai of the south instead of a little brother to Shenzhen.
Meanwhile, the poor. I grew up quite poor in HK and a family of 4 shared one studio. The kitchen and bathroom were shared with another 4-5 families. 4 of us slept in ONE bunk-bed. Life got much better after I was 7, because we moved to public housing in the suburb. You see, there's always a big divide between the rich and the poor, but all the public subsidies made life bearable.
A big part of HK's government revenue come from land sale. And land is more valuable when it's not used for public housing. That and immigration means the wait to get public housing is getting longer and longer. Immigration is supposed to increase revenue too, but as mentioned because a big part of revenue come from land sale and immigration doesn't increase the amount of developable land, the overall effect is smaller.
Normally one way to deal with this is impose immigration quota, but I looked into this a few years back and apparently for family reunion, HK government does not control the quota and have to accept however many the mainland sends over. For obvious reasons many in HK have family-ties in China so politically having a smaller quota would not be popular either. I don't have numbers to prove this, but there's a general sentiment that infrastructure is not growing fast enough to accommodate population increase.
I don't know what HK can do honestly. Politically it's in an impossible situation.


Dr. Brad Duet is from the town of Galliano, Louisiana. He did his undergraduate studies at Nicholls State, and transferred to Louisiana State University for his doctorate in veterinary medicine. Dr. Duet graduated from LSU in 2011. With the guidance of Dr. Neal Hebert, he is now practicing veterinary medicine at Animal Kingdom Veterinary Hospital. Dr. Duet's medical interest include small animal internal medicine, soft tissue surgery, and laser therapy. On his down times, he enjoys running in both competitive and charity races.


text:
Apple Releases Safari 5.0.1 With Safari Extensions Gallery
2:40 pm July 28, 2010 By Roland Hutchinson
Apple [AAPL] has released a new version of its web browser, Safari 5.0.1 and the latest version now features the Safari Extension Gallery. Apple introduced extensions support back in June to allow developers to create extensions using HTML5, CS3 and JavaScript.
The Safari Extension gallery lets you easily find a range of extensions, including ones for Twitter, Microsoft's Bing and many more.
Extensions are a great way for you to add new features to Safari 5.0.1. Built by developers, Safari Extensions use the latest HTML5, CSS3, and JavaScript web technologies. And they're digitally signed and sandboxed for improved security. You can install extensions with one click — no need to restart Safari.
You can find out more about the Safari Extensions gallery over at extensions.apple.com or from the Safari tab in the latest version of Safari, versions 5.0.1.
via 9 to 5 Mac
Filed Under: Apple, Technology News


Today's post is written by Stephanie Van Hoesen, the lovely owner of Nest Interiors in Edgehill Village. We are sad to see this store close, but believe me, there are deals – serious deals – to be had this week as the store closes for good on Jan 9th. Be on the look out for Stephanie's next big adventure; we will be!
Do you have the Christmas decor blues? Are you mourning the sparkle your mantle once had? Does your house feel bare? Well, you're not alone.
Many people are sad the day the Christmas decorations go back to the attic and the house is left empty. So many wish they could keep the decorations up for a little while longer, but know the tree is a fire hazard and the neighbors are going to start complaining about the blinking Charlie Brown lights on the house. So, they have no choice but to put it all away and wait for spring.
Why wait till spring when you can decorate for winter. Yes winter, a magical time of year when the world slows down for a bit.
Call me crazy, but I actually love winter. I love any excuse to be at home more and enjoy the simple things. Winter is the time to cozy up to the fire, read a good book and sip on a hot toddy. It's a time when you bake bread and make your mother's famous vegetable soup. It's a time when you want to snuggle with the family instead of going out. Winter is a season of home.
Winter decor is all about texture, candle light, neutral colors and organic elements. Neutralize the colors in your living spaces and bring in texture for interest.
White. I love bringing in lots of white into the home during the winter months. It's fresh, calming and reminds me of snow.
Fill glass mason jars with artificial snow and place candles inside. They look lovely in a window.
Lanterns. Instead of ornaments, hang little lanterns throughout the house or place one large one in the entry way. I love seeing candle light bounce around in lanterns, makes me think of foggy nights in Cape Cod.
Place snowy branches or a simple strand of white twinkle lights on your mantle. Add glass jars or apothecary jars in varying height. Fill them with pine-cones, acorns and twigs…sprinkle a little artificial snow on top and voila! a winter mantle.
A stack of antique books, white pillar candles and a collection of antique finds makes for a beautiful coffee table display.
Place grapewood or a manzanita branches on your dining table and surround it with mercury glass votive holders.
Switch your Christmas wreath out with a rustic or woodland-inspired wreath for your front door.
Cover your backyard trees with twinkle lights, make an outside fire, grab your favorite throw and enjoy the winter night.
Burlap, linen, heavy cotton…layer the house in these wonderful materials to create texture. From bathroom and kitchen towels, to table runners, to rag balls piled in a pretty bowl.
Furs and Hides. Throw a sheep skin over the sofa or your dining table chairs. Put a cow hide under your coffee table or at the foot of your bed. Bring warmth and texture to your home in unexpected ways.
Take your empty hanging planters and string floral lights through them. It dresses up the front porch instantly!
If you have empty planters, create a floral arrangement with branches and hanging tea-light holders. Place the arrangement on an entry table or buffet.
Wood. Whether it's cut logs, tiny branches or a stack of firewood, wood brings a wonderful element to your home. Whether you burn the wood daily or you use it just for decoration, it adds a lot of wintry character.
Looking for winter decor? Lucky for you Nest Interiors is chock full of winter decor. We have snowy branches & garland, snowy pine-cones & other organic baubles, hanging lanterns, snowy birds nest, metallic silver and bronze pots, and an abundance of mercury glass in all shapes & sizes.


I have a confession to make and I am going to come out and say it: I hate rich people (Ok hate may be a strong word, maybe "get irritated" is better?).
There's something about people with too much of everything that just rubs me the wrong way. I've been struggling with this "hate" for a long time and after questioning my own motives I have come to a few conclusions. I often asked myself what it is about rich people that I hate? And although I was yet to realize how rich I really am… I just couldn't figure it out. Was it because I was jealous? Envious? I just could not pin point it.
I am a peace keeper, and even more so I am someone that generates harmony in society and around me. I love unity. I detest division with every part of my being. I therefore will obviously protest against anything that creates division and destroys the harmony. Things like greed, anger, impatience, selfishness, etc. If you had to ask me to combine all these bad words into one thing/being/term then it would be a filthy rich person. A rich person (grossly generalized) embodies all these things…greed, selfishness, division, pride, etc.
I know…I know. It's not always the case, I am generalizing, and I need to get over this issue. The one thing that will help is the realization that I am also rich. To the person without a job begging at the street corner I am just as rich as the person I myself view as rich.
I am the rich person. I am the same person I hate.


It may be their first outing at the Doreen Dalton Memorial premier onemen's softball tournament but that has not stopped Richmond Keas fromupsetting the form books on the opening day today at Fraser Park, LowerHutt.
In a tough schedule today the Christchurch based Keas side have anunbeaten record after three consecutive games. They opened the day withan 8-5 win over Northcote and then backed that up with a 7-0 win overHutt Valley Cardinals. To round out the day Keas then had a resounding18-3 victory against Johnsonville.
Keas coach Dave Pouaka was a happy man at the end of the days play andlooking forward day two and hopefully a continuation of the winning form.
"We have a young team and the main reason for coming up here was to keeplearning and keep progressing. Three from three is a good day for us butit is only the first day of the tournament so we have to keep it going,"said Pouaka.
Throughout the day power hitting has been a feature of the Keas' play asthey have scored runs consistently while they have also shown fightingspirit to come from behind in two of the games today.
Against Northcote after taking the early advantage they then foundthemselves behind and required a rally of runs late in game to take itout. They were also down early against Johnsonville but fought back withinterest to take a commanding victory.
Stand out performers for the Keas against Johnsonville were Aaron Riodenand Gus Leger with the bat. Rioden smashed two home runs in the gamewhile also picking up six RBI's. NZCT Black Sox outfielder Leger was alsostrong with the bat accumulating one home run and four RBI's.
On the mound Penese Iosefo completed the job for Keas as he kept a lid onthe Johnsonville hitters after a hiccup in the first inning. Johnsonvillescored three runs in the opening inning but after that they couldn'tmanage another hit. Iosefo picked up four strikeouts in the game and gaveup just two hits.
On the back of the results today Richmond are at the top of section Bwith an unbeaten record along with Porirua who have also had twovictories today.
Section A in the 14 team competition is not so cut and dried due to anumber of upsets in the section. Tournament hosts and national clubchampions Hutt City United have had a forgettable day and have recordedonly one win to go with two losses.
Poneke Kilbirnie sit at the top of section A with a three win one lossrecord. Their only loss came this morning against Giants as they wentdown 7-3 in a tie breaker. The scores had been all level at 1-1 afterregular innings then in the eighth inning Poneke Kilbirnie were the firstto score with two runs but Giants came back with interest smashing sixruns in the extra inning to win the game.
Round robin play in the tournament continues tomorrow with the key clashcoming in section B when the Richmond Keas take on Porirua. After roundrobin play the top four teams will contest the semi finals on Mondayahead of the final in the afternoon.
Poneke Kilbirnie 8 Stoke 1, HV Dodgers 4 HV Cardinals 1, Kapiti 3Ramblers 2, Richmond 8 Northcote 5, Porirua 8 Johnsonville 5, Giants 7Poneke Kilbirnie 3 (tie breaker), Miramar 4 Northcote 3, Richmond 7 HVCardinals 0, Papanui 6 Stoke 4, Porirua 6 Miramar 4, Ramblers 4 Hutt CityUnited 0, Richmond 18 Johnsonville 3, Ramblers 6 Giants 0, Hutt CityUnited 7 Papanui 0, Poneke Kilbirnie 7 Kapiti 1.


Creating an exciting digital platform for something as purely sensory as perfume might sound like a challenge but it's one that can be overcome through innovation, says The Perfume Shop [IRDX RPSH] managing director Gill Smith. Her team has focused on an increasingly integrated online experience to make it easier for customers to shop. "We try to link everything up," she says. "We want to provide that 360° experience so the customer can get anywhere." This approach has already led to its websites driving a greater number of sales.
A major challenge facing The Perfume Shop was how to overcome that sensory barrier. "Fragrance is very hard to sell online because you can't smell it and while that's a fundamental problem, we've tried to innovate beyond it," says Smith. To achieve this, The Perfume Shop makes sure the experience fits the product. Although the scent itself can't be communicated, the pictures of perfume bottles offer a visual element that's popular on social media.
The Perfume Shop has also continued to improve its in-store experience alongside the digital experience. Smith says its stores are built to convey a sense of expertise to customers, which is important when selling expensive fragrances that are bought infrequently. "We were founded on excellent customer service. For us, it's all about accessibility. Fragrance can be seen as an expensive, kind-of-snobby, inaccessible luxury. The solution is to give the customer the right information about each brand," she notes.
The success of the online experience for customers has involved combining the visual elements of products with a robust online experience. The inner working of this programme, Smith explains, has been about creating a "360° experience" so that customers can connect separate channels together, such as the blog and the website, to make a purchase. "The customer should be able to get us from anywhere," she notes.
The company was the first fragrance retailer to incorporate live web chat, alongside phone and email, for customer support. All channels are monitored day and night, and a research team from InternetRetailing found rapid and helpful responses across them all, including a response time of 1h 24min over Facebook. The company is also now integrating new payment methods such as Apple Pay and launching a new app to tie in with its loyalty card, though Smith says that all this is really just "keeping up" with rivals.
The Perfume Shop uses an in-house social media team and, says Smith, conveying a sense of "fun" with social is a priority. The team has separate feeds with separate functions, such as customer services and a main channel where they post competitions and quizzes – and they post a lot, generating nearly 25,000 tweets per month. "We love to be social and we love to share," says Smith.


text:
Occupation / Country
Usage Citation
Memorability Metrics »
Page views of Grigori Rasputins by language »
Among OCCULTISTS »
Contemporaries »
In Russia »
Among OCCULTISTS In Russia »
Grigori Yefimovich Rasputin (; Russian: Григорий Ефимович Распутин [ɡrʲɪˈɡorʲɪj jɪˈfʲiməvʲɪtɕ rɐˈsputʲɪn]; 21 January [O.S. 9 January] 1869 – 30 December [O.S. 17 December] 1916) was a Russian mystic and self-proclaimed holy man who befriended the family of Nicholas II, the last emperor of Russia, and gained considerable influence in late imperial Russia. Rasputin was born to a peasant family in the Siberian village of Pokrovskoye in the Tyumensky Uyezd of Tobolsk Governorate (now Yarkovsky District of Tyumen Oblast). He had a religious conversion experience after taking a pilgrimage to a monastery in 1897. Read more on Wikipedia
Since 2007, the English Wikipedia page of Grigori Rasputin has received more than 14,189,849 page views. His biography is available in 83 different languages on Wikipedia (up from 81 in 2019). Grigori Rasputin is the 3rd most popular occultist, the 17th most popular biography from Russia (up from 18th in 2019) and the most popular Russian Occultist.
Grigori Rasputin was a Russian mystic who was said to have healing powers. He was also said to have been a sexual predator.
Memorability Metrics
Page Views (PV)
Historical Popularity Index (HPI)
Languages Editions (L)
Effective Languages (L*)
Coefficient of Variation (CV)
Page views of Grigori Rasputins by language
Among OCCULTISTS
Among occultists, Grigori Rasputin ranks 3 out of 9. Before him are Nostradamus and Rajneesh. After him are Baba Vanga, John Dee, Aleister Crowley, Count of St. Germain, Helena Blavatsky, Alessandro Cagliostro, Anton LaVey, Heinrich Cornelius Agrippa, and Allan Kardec.
Most Popular Occultists in Wikipedia
Go to all Rankings
HPI: 88.63
Baba Vanga
Count of St. Germain
Alessandro Cagliostro
Anton LaVey
Heinrich Cornelius Agrippa
Among people born in 1869, Grigori Rasputin ranks 3. Before him are Mahatma Gandhi and Henri Matisse. After him are André Gide, Victor Emmanuel III of Italy, Neville Chamberlain, Charles Thomson Rees Wilson, Nadezhda Krupskaya, Emma Goldman, Karl Haushofer, Fritz Pregl, and Gustaf Dalén. Among people deceased in 1916, Grigori Rasputin ranks 2. Before him is Franz Joseph I of Austria. After him are Jack London, Ernst Mach, Yuan Shikai, Élie Metchnikoff, Henryk Sienkiewicz, Franz Marc, William Ramsay, Henry James, Sholem Aleichem, and Odilon Redon.
Others Born in 1869
Victor Emmanuel III of Italy
Charles Thomson Rees Wilson
Nadezhda Krupskaya
Fritz Pregl
Others Deceased in 1916
Franz Joseph I of Austria
Yuan Shikai
In Russia
Among people born in Russia, Grigori Rasputin ranks 17 out of 2,688. Before him are Alexander Pushkin (1799), Vladimir Putin (1952), Nicholas II of Russia (1868), Ivan the Terrible (1530), Nikita Khrushchev (1894), and Ivan Pavlov (1849). After him are Boris Yeltsin (1931), Igor Stravinsky (1882), Kim Jong-il (1941), Wassily Kandinsky (1866), Maxim Gorky (1868), and Ayn Rand (1905).
Others born in Russia
Nicholas II of Russia
Boris Yeltsin
Maxim Gorky
Among OCCULTISTS In Russia
Among occultists born in Russia, Grigori Rasputin ranks 1. After him are Nina Kulagina (1926).
Russian born Occultists
Nina Kulagina
Alexander Protopopov
Anna Vyrubova
Alexander Trepov
Boris Stürmer
Alexei Nikolaevich, Tsarevich of Russia
Last Heir Apparent Of The Last Imperial Family Of Russia (1904-1917)
Grand Duchess Maria Nikolaevna of Russia
Third Daughter Of Nicholas Ii Of Russia And Alexandra Fyodorovna
Russian Prince
Grand Duchess Anastasia Nikolaevna of Russia
Youngest Daughter Of Tsar Nicholas Ii Of Russia
Grand Duchess Olga Nikolaevna of Russia
Eldest Daughter Of Tsar Nicholas Ii Of Russia
Wife Of Nicholas Ii Of Russia
Grand Duchess Tatiana Nikolaevna of Russia
Second Daughter Of Tsar Nicholas Ii Of Russia
Occupations / Countries
Report Data Error


Consultation so you and your patients can thrive.
I've been practicing as a psychologist for more than twenty-five years, and I'm grateful to have received expert help, support, and consultation along the way.
One thing I learned early on is that the more good therapists there are, the stronger our communities are, and that helps us all.
We can meet once, occasionally, or on a regular basis, depending on your needs.
Need some help? We all do, at least from time to time. Let's see if we might be a good fit for moving your psychotherapy skills and your practice up to the next level. I'd be honored to help.


In 2006, I moved to Taos, New Mexico from Colorado to help create a ceramics community. This included a summer workshop program, two residencies, ongoing classes and variety of firing possibilities. in 2014 I handed over Taos Clay and I am currently pursuing making full time. Taos Clay helped me draw in ceramic artists from all over the world and has been the majority of my education and background in ceramics. It takes a community to fire many of the kilns we have built around Taos and Taos Clay has been a vital part of keeping the ceramics community aware of the trends of contemporary ceramics. I have a gallery in downtown Taos where i show the majority of my work. I also host workshops in Taos and around the country as well as showcase my work in hotels, galleries, boutiques through out the United States, The intention of my website is to not only create an avenue to sell work directly to my collectors but also share my story of creating a life as an artist, father and avid outdoorsman in rural New Mexico. Please visit my daily photo blog or if you have the opportunity, come visit me in my gallery and studio in Taos, New Mexico. Ceramics is a medium that requires patience and experience. In no other craft is the artists up against so many variables. The clay and the firing process are constantly educating and opening up new challenges. Working in clay has been a meditative, grounding journey. It is humbling yet, rewarding. You are an alchemist trying to recreate and refine the elements into something functional and inspiring.
Since moving from London to the U.S. in 1986, Elizabeth Rose has worked as a professional sculptor, showing in solo and group exhibitions in the U.S., Australia, Canada and the U.K. Recognized for her innovative works in ceramic, bronze and steel, her sculptures have been featured in the San Francisco Examiner, the Houston Chronicle, the Albuquerque Journal, the Sunset Western Garden Book and are on public display in the City Santa Fe. Her earlier works were brightly colored cut and folded steel figures, often with a kinetic element. Since 1995, Elizabeth has worked in ceramic and bronze, creating large and small-scale figures, vessels, wall-reliefs and abstract works. Presently she is developing smaller, touchable ceramic pieces reminiscent of archeological findings.
Elizabeth Rose has shown in museums and is in private and corporate collections. She was born in India and lives and works in Galisteo, near Santa Fe, New Mexico.
Originally from Houston, Texas, I now live in Santa Fe. After the Air Force (1960-1964), I attended the University of Houston (BFA, 1967) and the University of New Mexico (MA, 1969).
My work in ceramics, using animal imagery, was first inspired by the realities of hunting and fishing, and later by their power as symbols, surrogates and totems.
My first teaching position was at Nichols State University, Thibodaux, Louisiana. I taught at and contributed significantly to the programs of Louisiana State University in the 70s and 80s and Ohio University beginning in 1990. My later work continues an underlying eroticism inspired by Moche pottery in addition to an increased sense of socio-political content.
My work can be found in the collections of the Arizona State University Art Museum; Greenville Museum of Art, Greenville, SC; International Ceramics Studio, Kecskemet, Hungary; Los Angeles County Museum, Los Angeles, CA; Mint Museum of Art, Charlotte, NC (two works); and the San Angelo Museum of Art, San Angelo, TX.
I am a past president and a Fellow of NCECA, and I was awarded the NCECA Excellence in Teaching Award in 2006. Other awards include the Southeastern Center for Contemporary Art Fellowship, 1980, an SAF/NEA Fellowship, 1985, several university sabbatical awards, and a fellowship to the International Ceramics Studio (ICS) in Kecskemet, Hungary, 2004. The International Academy of Ceramics, Geneva, elected me a member in 2005.
I have been awarded a Fulbright to Ireland for 2011. I was at the National College of Art and Design in Dublin for 3 months. Work on this page comes from that the period in Ireland.
A decades-long association with Penland includes a term as Chair of the Board of Trustees. Visiting artist appointments have included the NY State College of Ceramics at Alfred, University of Georgia's Cortona Italy Program, Haystack School in Maine, Penland School in North Carolina, the Rhode Island School of Design, and Anderson Ranch Arts Center in Colorado.
My work and teaching has led to extensive travels through Latin America, Europe and China. In 2008 I returned to the ICS in Hungary and again in 2010.
Mono con manos – Stoneware. Wheel thrown pottery form, Monkey head is repoussé modeled from slabs. Wood fired ^9 in catenary kiln with cottonwood and reduction cooled at Red Lodge Clay Center, Montana. No applied glazes or slip.
JoAnne DeKeuster grew up near the small town of Waumandee, Wisconsin. She received a B.A. in Art Education from the University of Wisconsin – Eau Claire. Studying at Northern Arizona University in Flagstaff she received a Masters degree in fibers/ceramics. Coursework there included travel and study in Japan. After completion of her masters degree at Northern AZ University, Jo and her husband continued to spend summers in Flagstaff. They began working for the University as kiln assistants and eventually each taught the summer Noborigama Wood Fire class. In 1997 the couple bought property in Taos Canyon and began building their home, studio, and kiln area. In 2003 they moved to New Mexico full time.
professional, full-time pottery career that now centers my life. I moved to New Mexico in 1999 and, after 4 years in Santa Fe, now live in Dixon, a small rural farming village rich with its community of artistic spirits. My studio is a small adobe house nestled in the hills at the edge of high desert wilderness where I enjoy quiet, peace and simplicity.
I moved to Abiquiu, New Mexico full time two years ago. Knowing that the environment should inform one's artwork, I am bursting with new images, thoughts and inspirations. I am listening to the land and allowing my intuitive reactions to transfer the clay sculpture to a spiritual and meaningful level. I feel fortunate to be able to embrace this beautiful area. My days are spent in the studio working or teaching workshops. I am teaching week workshops at my studio in Abiquiu and will be teaching at Anderson Ranch and Taos Clay this summer. I also offer a class once a week. I encourage creative thinking with students.
My sculptural pieces are mainly coil built and are multiple fired to achieve a painterly quality with slips, oxides, underglazes and glazes. I want to feel mystery and to have questions in my finished work.
Joanne began her love of pottery when she took her first throwing class in Illinois thirty years ago, right after her daughter was born.
After that, the demands of motherhood, moving to New Mexico, volunteering in the community, and a teaching career, all took precedence over the pursuit of art. Now, years later, she has taken up her love of art and pottery once again.
For the past five years she has been playing with handbuilding and on the wheel. She calls her work Funky Functional because it is both useful and unique. Sometimes the pieces contain messages such as scripture or other inspiring, positive thoughts. Recently, she has begun to enjoy sculpting animals. She has participated in local arts and crafts fairs as well as shown work in her hometown in Illinois.
Joanne is also a watercolor painter who has been instructed by some very talented New Mexico artists. She has earned her art endorsement and is currently licensed by the state of New Mexico to teach art. She is an active member of the New Mexico Watercolor Society. She loves a challenge and some of her favorite subjects to paint are animals and her grandchildren. Some of her goals for this year are to bring her painting and pottery to the next level of development.
She has recently joined NMPCA in order to gain experience from all of the talented professional clay artists and to be a more active part of the art community. She believes that working with clay is very joyful and healing, almost a form of therapy. She looks forward to meeting and learning from the other members who share her passion for clay!
Terence studied at the University of Oklahoma, receiving a Fine Arts degree in Object Design. From the time he fired his first glazed piece, he knew clay was his medium of expression. However, following graduation, he applied his design expertise in graphic design working at Sandia Labs for many years. During that time he designed and constructed a studio and a large home for his family in Tijeras Canyon. This proved to be such a time-consuming task that he was unable to give much time to his ceramic studio. Since retiring from Sandia in 1989, he has at last been able to devote time to working with clay .
He has exhibited work in the New Mex. Potter's Assoc. Celebration of Clay, the Biennial Southwest Artist Series at the Albuquerque Museum, The "Internal Illumination" exhibit at The Foundry Art Centre in St. Charles, Missouri , and the 17th National Ceramic Competition at the San Angelo Museum of Fine Arts.
Although he has produced many functional pieces he prefers one-of-a-kind abstract sculptural items. Here are images of some of his work.
Terence has been an NMPCA member since 2002.
Growing up in New Mexico gave me the opportunity to experience the vast richness of modern and ancient art. I originally studied art with my mother, accomplished Southwestern oil painter Helen L. Minton who studied with four of the original founders of the Taos Society of Artist in 1932.
In 1933 she studied Southwestern Indian Art with artist and teacher Kenneth M. Chapman in Santa Fe. Exposure to this rich background provided me with an appreciation of ancient and contemporary art forms and informed much of my work. I got my first pottery wheel when I was 11 and have continued to work primarily in clay although as a multimedia artist I have worked in painting, jewelry, fiber, glass, metalwork, clay sculpture, graphics and woodworking. In my current multimedia work I combine ceramic art, painting and sculpture using clay and metal forms as the base. I am now focusing on two very different lines of work, functional stoneware with carved animal forms and clay wind bells with painted animal images.
My education and chosen career were in the field of electrical and computer systems engineering in which I spent forty-four years before retiring, December 2004. During the latter two years of employment I had, with the encouragement and support of my artistic wife, Penne, begun to, "play with clay". With study, focused attention and determination, I slowly learned to throw on the wheel.
The past three years, I have turned more toward hand building using my understanding of geometric relationships (envision the organized slices of the spectacular Sydney Opera House). I find that slabs of clay can be formed and molded into almost any shape imaginable. The fun comes from experimenting, discovering, and attempting to, "break out of the box!


text:
Connect(); Special Issue 2018
Volume 33 Number 13
[Editor's Note]
December Is for Developers
By Michael Desmond; Special Issue 2018
Microsoft Connect(); has emerged as one of the most important and impactful developer-oriented events on the Microsoft calendar. Since 2014, Microsoft has used Connect(); to evangelize its growing cross-platform and open source software development efforts, release important products like Visual Studio for Mac, and articulate strategies in areas like artificial intelligence (AI) and the Internet of Things.
This year Connect(); kicked off on Dec. 4, and the conference hit on all these notes and more. On the open source front, Microsoft and Docker announced the Cloud Native Application Bundle (CNAB)—an open source, cloud-agnostic specification for packaging and running distributed applications. Microsoft also announced the open sourcing of Windows Presentation Foundation, Windows Forms and the Windows UI XAML Library.
There was plenty of tool news on tap. Microsoft debuted Visual Studio 2019 Preview, and revealed new capabilities like Visual Studio Live Share for remote collaboration and Visual Studio IntelliCode, which expands language support to XAML and C++ and leverages AI for improved code insight. Also announced were the general availability of .NET Core 2.2, and the release of the public preview of .NET Core 3.0. Cross-platform development got a boost as well, with the Xamarin.Forms 4.0 Public Preview and the general availability of Xamarin.Forms 3.4.
Of course, AI and machine learning (ML) have been a huge area of focus at Microsoft. Connect(); this year provided a platform for some key announcements, including the general availability of Azure Machine Learning Service, which helps developers and data scientists quickly build, train and deploy ML models. Also released was the public preview of ML.NET, Microsoft's open source, cross-platform ML framework that helps developers infuse AI into their .NET applications.
This special issue of MSDN Magazine is dedicated to helping developers take full advantage of the tools and technologies featured at the Connect(); conference. Articles like Scott Hunter's "What's Coming in .NET Core 3.0," Julie Lerman's "Collaborative Development with Visual Studio Live Share" and Mads Kristensen's "What's New in Visual Studio 2019" will help you get a jump on the improvements to these flagship tools and frameworks.
DevOps has been a core message at Connect(); over the years and is the focus of the article "Deploy Your Code the Right Way with Azure Pipelines," written by Micheal Learned and Andy Lewis. And don't miss the pair of features exploring ML. James McCaffrey offers an introduction to the ML.NET framework ("ML.NET: The Machine Learning Framework for .NET Developers"), while Krishna Anumalasetty explores the automated ML capabilities of Azure Machine Learning and how they make AI accessible to more organizations ("Accelerate AI Solutions with Automated Machine Learning").
There's more still, including David Ortinau's article, "Exploring the Xamarin.Forms Shell," that shows exciting new capabilities coming in Xamarin.Forms 4.0, and Michael Crumb's productivity-minded piece titled "7 Tips and Tricks for Azure App Service." Finally, be sure to check out our bonus Web feature from Kevin Farlee, "Introducing Azure SQL Database Hyperscale," which explores the re-architected storage engine in the SQL database and how it enables a highly scalable service tier for databases that adapt to workloads on-demand.
Connect(); is part of a larger conversation between Microsoft and its developers. It's an opportunity for Microsoft to articulate its vision, and for developers to provide feedback that shapes the direction of that vision going forward. Learn more at microsoft.com/connectevent.
Michael Desmond is the Editor-in-Chief of MSDN Magazine.
Discuss this article in the MSDN Magazine forum


One of the most beautiful cities in Lombardy, Bergamo is divided into two parts, Bergamo Bassa (lower city) and Bergamo Alta (upper city).
Bergamo Alta, in particular, is characterized by a strong medieval mark, also accentuated by the mighty Venetian walls that make it a wonderful architectonic and historic jewel.
Piazza Vecchia (Old Square) is the symbol of the city, with the Palazzo della Ragione, the Civic Tower (also called Campanone), the Public Library and the Contarini Fountain (XVIII century).
On the Southern side of Piazza Vecchia we can admire the Cathedral, the Basilica di Santa Maria Maggiore - where the famous musician Gaetano Doninzetti is buried - the Colleoni Chapel and the Baptistry (1340).
Bergamo Bassa, modern and busy, houses the Theater, dedicated to Doninzetti, the Doninzetti's Museum, the Gallery of Modern and Contemporary Art, the Diocesan Museum, the History Museum, the Natural Science Museum, the Botanic Garden and the Matris Domini Museum.
This town web page has been visited 56,810 times.


text:
<resources>
    <string name="app_name">SplashScreenDemo</string>
</resources>

redpajama_set_name: RedPajamaGithub