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Boone, David A. CP, MPH, PhD
Journal of Prosthetics and Orthotics
Many of our colleagues are taking a beating from audits of their practice. Some of these audits have motivations that are not even directly related to individual patients and their care. Their driving force is money, but as practitioners, we are paid professionals, and financial controls, whether implemented well or not, are a fact of life for us.
So we prepare ourselves. The good news is that the best of us can get better with our preparation. We all trained in a similar way and were probably barely able to practice when we left our training. But, with support of senior clinicians, we gained experience and with it confidence. Confidence led to more independence and maybe a little laziness. As soon as we are convinced that we know what we are doing, it gets harder to change what is "working" for us.
We know that objective information and complete documentation about the patient are critical to support our clinical decisions. But to document well is not about just filling in boxes of potential audit flags and to keep doing what works, but rather, it requires deep understanding. I believe that as a profession we have arrived at a crossroads long anticipated. The audit situations just point out that being confident within ourselves is no longer sufficient. If you have not already done so, it is time to renew your professional commitment to lifelong learning.
And all of this brings us to the Journal of Prosthetics and Orthotics (JPO). The JPO remains one of the very best media for constantly reconsidering our individual knowledge base. Read, consider carefully, challenge your own understanding with the viewpoint of another, and contribute your voice to these pages, and by doing so, we can all be better prepared to document and support our clinical decisions with evidence-based practice.
David A. Boone, CP, MPH, PhD
© 2014 by the American Academy of Orthotists and Prosthetists.
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Online Law Schools / Re: Alternative to University of London - Northumbria University« on: October 29, 2012, 07:33:31 AM »
My point is that the prudent thing to do is use Rule 4.33 to ensure that the proposed course of study is acceptable to the bar and register as a law student. I am an online law school grad and work in online education, students fail to realize that bar examiners are incredibly hostile to online education. The Bar is not your friend and if a rule can be construed against you, it often is. As for the California Bar being "liberal", it is different alright, but just as rule oriented as any other bar.


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Lissina, Annaa,*; Fastenackels, Solènea,*; Inglesias, Maria C.a,†; Ladell, Kristinb; McLaren, James E.b; Briceño, Oliviaa; Gostick, Emmab; Papagno, Lauraa; Autran, Brigittea; Sauce, Delphinea; Price, David A.b; Saez-Cirion, Asierc; Appay, Victora
aINSERM UMR S 945, Infections and Immunity, Université Pierre et Marie Curie-Paris6, Hôpital Pitié-Salpêtrière, Paris, France
bInstitute of Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff, Wales, UK
cInstitut Pasteur, Unité de Régulation des Infections Rétrovirales, Paris, France.
*Anna Lissina and Solène Fastenackels contributed equally to this writing of this work.
†Current address: Centro de Investigacion en Enfermedades Infecciosas, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico.
Correspondence to Victor Appay, INSERM UMR S 945, Infections and Immunity, Avenir Group, Université Pierre et Marie Curie-Paris6, Hôpital Pitié-Salpêtrière, Paris, France. Tel: +33 1 40 77 81 83; fax: +33 1 40 77 97 34; e-mail: firstname.lastname@example.com
Received 11 July, 2013
Revised 3 December, 2013
Accepted 3 December, 2013
Background: Although it is established that CD8+ T-cell immunity is critical for the control of HIV replication in vivo, the key factors that determine antiviral efficacy are yet to be fully elucidated. Antigen-sensitivity and T-cell receptor (TCR) avidity have been identified as potential determinants of CD8+ T-cell efficacy. However, there is no general consensus in this regard because the relationship between these parameters and the control of HIV infection has been established primarily in the context of immunodominant CD8+ T-cell responses against the Gag263–272 KK10 epitope restricted by human leukocyte antigen (HLA)-B27.
Methods: To investigate the relationship between antigen-sensitivity, TCR avidity and HIV-suppressive capacity in vitro across epitope specificities and HLA class I restriction elements, we used a variety of techniques to study CD8+ T-cell clones specific for Nef73–82 QK10 and Gag20–29 RY10, both restricted by HLA-A3, alongside CD8+ T-cell clones specific for Gag263–272 KK10.
Results: For each targeted epitope, the linked parameters of antigen-sensitivity and TCR avidity correlated directly with antiviral efficacy. However, marked differences in HIV-suppressive capacity were observed between epitope specificities, HLA class I restriction elements and viral isolates.
Conclusions: Collectively, these data emphasize the central role of the TCR as a determinant of CD8+ T-cell efficacy and demonstrate that the complexities of antigen recognition across epitope and HLA class I boundaries can confound simple relationships between TCR engagement and HIV suppression.
CD8+ T cells play a central role in the control of viruses that persist within the host, such as HIV [1,2] and its simian counterpart [simian immunodeficiency virus (SIV)] [3]. Despite considerable efforts to identify correlates of protective immunity against HIV, however, the precise attributes of CD8+ T cells that define antiviral efficacy are unclear. In part, this reflects the inherent difficulties associated with qualitative studies of CD8+ T-cell immunity, which include a requirement to control for inter-individual heterogeneity and the numerous parameters that govern target cell engagement. These latter variables include epitope specificity, T-cell receptor (TCR) affinity, TCR and CD8+ coreceptor density and valency, plasma membrane topography, differentiation status and regulatory molecule expression [4]. In addition, CD8+ T-cell recognition is influenced by the state of the antigen-presenting cell, its ability to process and present the relevant epitope, the nature and expression levels of the restricting major histocompatibility complex class I (MHCI) molecule and the stability of the peptide–MHCI (pMHCI) complex. In HIV infection, the situation is further complicated by various immune evasion mechanisms deployed by the virus, including Nef-mediated MHCI down-regulation and mutational escape, both of which can directly impact antigen processing and presentation [5–14].
It has been shown that the ability of CD8+ T cells to suppress HIV replication in vitro associates with favorable disease outcome [15]. This key observation demarcates a correlate of CD8+ T-cell efficacy in infected patients, yet the parameters that determine this functional property remain to be clarified. There is an emerging view that the functional avidity or antigen-sensitivity (AgS) of a CD8+ T-cell response greatly influences the control of HIV replication [16–18]. This qualitative attribute has been linked with TCR avidity, a compound measure of clonotype-dependent antigen engagement, although the supporting evidence is largely confined to studies of CD8+ T cells specific for the immunodominant Gag263–272 KK10 epitope presented by human leukocyte antigen (HLA)-B*2705 (HLA-B27 from hereon) [19–21], a molecule associated with superior control of HIV infection [22–24]. Moreover, there is no general consensus on the role of AgS as a determinant of CD8+ T-cell-mediated control of HIV replication. For instance, it has been suggested that high-avidity CD8+ T cells may not exert strong selection pressure on the virus because such cells were found to persist in late-stage disease despite the absence of mutations within the targeted viral epitopes [12]. It has also been reported that protective polyfunctional CD8+ T cells display low, rather than high, avidities for HIV epitopes [25]. Moreover, other studies suggest that HIV-specific CD8+ T-cell efficacy is related primarily to antigen specificity [26,27] and the kinetics of antigen expression [28]. These divergent studies prompted us to investigate the impact of AgS and TCR avidity on HIV-suppressive capacity.
To minimize the confounding factors that arise as a function of heterogeneity, we have worked previously with CD8+ T-cell clones isolated from HIV-1-infected patients as an alternative to studying polyclonal responses directly ex vivo. This approach facilitates a simpler view of the different parameters that affect CD8+ T-cell efficacy. However, these studies were limited to a panel of CD8+ T-cell clones specific for the HLA-B27-restricted Gag263–272 KK10 epitope [19–21]. As a direct continuation of this study, we proceeded to examine the relationship between AgS and antiviral efficacy using CD8+ T cells specific for other immunodominant epitopes derived from different HIV-1 proteins and restricted by an alternative HLA class I molecule. To this end, we generated CD8+ T-cell clones specific for either the Nef73–82 QK10 epitope or the Gag20–29 RY10 epitope, both restricted by HLA-A*0301 (HLA-A3 from hereon), from HIV-1-infected patients. Equipped with a panel of CD8+ T-cell clones incorporating a range of sensitivities across three distinct specificities, we were able to study the effect of AgS on HIV-suppressive capacity independent of antigen source (Gag/Nef), epitope sequence (QK10/RY10/KK10), restricting HLA class I molecule (HLA-A3/HLA-B27) and viral strain (LAI/JR-CSF/DH12).
Tetrameric antigen complexes for QK10/HLA-A3, RY10/HLA-A3 and KK10/HLA-B27, all conjugated to phycoerythrin, were generated as described previously [29,30]. Directly conjugated monoclonal antibodies (mAbs) were purchased from commercial sources as follows: anti-CD4-allophycocyanin-cyanine7 (APC-Cy7), anti-CD8-APC-Cy7, anti-CD45RA-V450, anti-CCR7-PE-Cy7, anti-CD107a-PE-Cy5, anti-IFNγ-Alexa700, anti-IL-2 APC and anti-TNF-PE-Cy7 (BD Biosciences, San Diego, California, USA); anti-CD3-ECD and anti-p24 Gag-PE (Beckman Coulter, Miami, Florida, USA); (iii) anti-CD28-Alexa700 (Biolegend, San Diego, California, USA); (iv) anti-CD8-Alexa405 (Life Technologies, Eugene, Oregon, USA); and (v) anti-MIP-1β-fluorescein isothiocyanate (FITC; R&D Systems, Minneapolis, Minnesota, USA). The amine-reactive viability dye Aqua (Life Technologies) was used to eliminate dead cells from the analysis. Staining with all reagents was conducted according to standard procedures [31,32].
Peptides corresponding to the HIV-1-derived epitopes QK10 (QVPLRPMTYK, Nef73–82), RY10 (RLRPGGKKKY, Gag20–29) and KK10 (KRWIILGLNK, Gag263–272) were synthesized commercially at above 95% purity (Biosynthesis Inc., Lewisville, Texas, USA). The HIV-1 viral isolates LAI, JR-CSF and DH12 (all clade B) were obtained from the National Institutes of Health AIDS reagent program and amplified on a mix of activated CD4+ T cells from three healthy donors.
CD8+ T-cell clones specific for the HLA-A3/A11-restricted Nef epitope QK10 and the HLA-A3-restricted p17 Gag epitope RY10 were derived from peripheral blood mononuclear cell (PBMC) samples obtained from four HLA-A3+ patients during the chronic phase of HIV-1 infection. All donors were asymptomatic in the absence of antiretroviral therapy with CD4+ cell counts greater than 500 cells/μl and plasma viral loads ranging from 1000 to 250 000 copies of HIV-1 RNA/ml. Briefly, single tetramer+ CD8+ T cells were sorted using a FACSAria flow cytometer (BD Biosciences) in a biosafety containment level III laboratory and expanded in microtiter plates by periodic stimulation in the presence of mixed irradiated allogeneic PBMCs, phytohemagglutinin [phytohemagglutinin (PHA); 1 μg/ml] and recombinant human interleukin (rhIL)-2 (200 IU/ml). CD8+ T-cell clones specific for the HLA-B27-restricted p24 Gag epitope KK10 were generated and expanded as described previously [19]. All CD8+ T-cell clones were cultured at 37°C/5% CO2 in R+ medium (Roswell Park Memorial Institute medium-1640 containing 2 mmol/l L-glutamine and antibiotics) supplemented with 5% human AB serum and 200 IU/ml rhIL-2. Primary HLA-A3+/HLA-B27+ CD4+ T cells were isolated by positive selection (MACS; Miltenyi Biotec, Paris, France) from healthy donor PBMCs cultured in R10 medium (R+ supplemented with 10% fetal calf serum) containing 200 IU/ml rhIL-2. HLA-A3+ Epstein–Barr virus (EBV)-transformed B-cell lines (HLA-A3+ LCLs) used to present exogenous antigen in interferon (IFN)γ enzyme-linked immunosorbent spot (ELISpot) assays were cultured in R10 medium. Unbiased molecular characterization of all expressed TCR gene rearrangements (i.e. clonotypic analysis) was performed as described previously [19,30,33,34].
T-cell receptor avidity was measured using tetramer dilution assays. Briefly, CD8+ T-cell clones were incubated with cognate tetramer at a range of concentrations (10–0.0015 μg/ml in 1/3 dilutions) for 30 min at 4°C, then stained for CD8 expression before fixation. The percentage and fluorescence intensity of tetramer+ CD8+ T cells at each tetramer concentration was determined by flow cytometry.
Each well of a 96-well polyvinylidene plate (Millipore, Paris, France) precoated with antihuman IFNγ capture mAb (Diaclone, Besançon, France) was subjected to 105 HLA-A3+ LCLs, 103 clonal CD8+ T cells and peptide at the desired concentration (ranging from 10−5 to 10−11 mol/l). Plates were then incubated overnight at 37°C/5% CO2 and developed according to the manufacturer's instructions (Mabtech, Nacka Strand, Sweden). Spots were counted using an automated ELISpot reader (Carl Zeiss MicroImaging Inc., Le Pecq, France). A PHA control (final concentration 1 μg/ml) was included for each CD8+ T-cell clone. All assays were performed in duplicate and normalized.
CD8+ T-cell clones and HLA-A3+ LCLs pulsed with the indicated concentrations of cognate peptide were incubated at an effector : target (E : T) ratio of 1 : 10 for 1 h with anti-CD107a and a further 5 h in the presence of monensin (2.5 μg/ml; Sigma-Aldrich, St. Louis, Missouri, USA) and brefeldin A (5 μg/ml; Sigma-Aldrich) at 37°C/5% CO2. Negative controls were processed likewise in the absence of peptide. Staining for intracellular markers was performed as described previously [19]. Data were acquired using a Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software version 9.4.4 (TreeStar Inc., Ashland, Oregon, USA). Doughnut plots were constructed using Excel software and polyfunctionality indices were calculated as described previously [35].
Primary HLA-A3+/HLA-B27+ CD4+ T cells were stimulated for 48 h with PHA (1 μg/ml) and cultured for 7 days in the presence of 100 IU/ml rhIL-2 to facilitate productive HIV infection. The cells were then plated out at 105 cells/well in a 96-well plate and infected with virus by spinoculation [15]. Infected CD4+ T cells were cocultured with clonal CD8+ T cells at different E : T ratios as indicated. Three days later, the cells were harvested and stained with Aqua. Surface staining for CD8 and CD4 and intracellular staining for p24 were then conducted to enable flow cytometric evaluation of HIV-infected target cell elimination.
Previously, we generated and characterized a set of CD8+ T-cell clones specific for the HLA-B27-restricted KK10 epitope (KRWIILGLNK, p24 Gag263–272) to investigate the determinants of HIV-suppressive capacity in vitro[19–21]. To extend these studies across different epitopes and HLA class I restriction elements, we generated CD8+ T-cell clones specific for two highly conserved HIV-1-derived epitopes restricted by HLA-A3: QK10 (QVPLRPMTYK, Nef73–82) and RY10 (RLRPGGKKKY, p24 Gag20–29). Nine clones with equivalent surface expression levels of CD3+ and CD8+ were established in total (A3-QK10, n = 5; A3-RY10, n = 4) from the peripheral blood of HIV-1-infected patients with the appropriate HLA class I genotype (n = 4).
The functional avidity or AgS of these A3-QK10 and A3-RY10 clones was assessed using dose titrations of exogenous cognate peptide in the presence of an HLA-A3+ target cell line and quantified as the EC50 value, defined as the concentration required to elicit a half-maximal response, in IFNγ ELISpot assays. A range of sensitivities was observed for both sets of clones (Fig. 1a). We also used tetramer dilution assays, which focus specifically on the TCR–pMHCI interaction and eliminate the confounding influences of other molecular interactions that can occur in contiguous membrane domains, to measure TCR avidity (Fig. 1b). This parameter takes into account the intrinsic binding strength (affinity) of the TCR and the role of the CD8+ coreceptor, as well as the density, topography and coordinate relationship of these antigen-binding receptors within the constraints of cell surface mobility. The percentage of tetramer+ cells was quantified by flow cytometry as a function of tetramer concentration and used to calculate TCR avidity (i.e. EC50 value) for both sets of clones (Fig. 1c).
A significant correlation was observed between measures of AgS and TCR avidity for the different clones (Fig. 1d). This indicates that TCR avidity is a major determinant of AgS, consistent with our previous study [19–21]. The EC50 values for both the A3-QK10 and A3-RY10 clones, despite their different specificities, spanned a narrow range (tetramer dilution: 0.029–0.002 μg/ml; IFNγ ELISpot: 1.55 × 10–7 to 1.49 × 10–8 mol/l). This is not entirely surprising as virus-specific CD8+ T cells generally display relatively high levels of AgS and TCR avidity, reflecting relatively high monomeric TCR–pMHCI affinities [36]. Nevertheless, this initial characterization led to the identification of CD8+ T-cell clones with distinct TCR avidities, thereby enabling us to examine a putative link between this parameter and HIV-suppressive capacity.
Next, we assessed the ability of CD8+ T-cell clones restricted by HLA-A3 or HLA-B27 to suppress HIV replication in vitro as a function of TCR avidity. For this purpose, we selected pairs of A3-QK10 and A3-RY10 clones to include high and low TCR avidity representatives. Two previously characterized B27-KK10 clones with markedly different TCR avidities were also included for comparison [19,20]. Clonotypic analysis confirmed that the selected A3-QK10 and A3-RY10 clones expressed unique TCRs (Fig. 2a). Consistent with our earlier findings in the B27-KK10 system [19–21], the functional profile of CD8+ T-cell clones restricted by HLA-A3 was intimately associated with TCR avidity (Fig. 2b). All clones exhibited a homogeneous CD28− CCR7− CD45RA− effector memory phenotype (Fig. 2c).
Dual HLA-A3+/HLA-B27+ primary CD4+ T cells were used as infected targets to enable meaningful comparative analyses of HIV-suppressive capacity across restriction elements. These cells were infected with the X4-tropic LAI laboratory strain of HIV-1, which was titrated in the absence of CD8+ T cells to achieve an infectivity of 10–20%, determined by intracellular p24 staining [9,37]. Target cells infected with HIV-1 LAI were combined with each CD8+ T-cell clone at E : T ratios ranging from 0.01 : 1 to 1 : 1. Infected targets alone were incorporated as a reference for infectivity in the absence of CD8+ T cells. No alloreactivity was detected in these assays. The percentage of p24 staining obtained at each E : T ratio was then used as a quantitative measure of HIV-suppressive activity, with the most potent CD8+ T-cell clones achieving the lowest p24 levels at the lowest E : T ratios (Fig. 3a).
For each specificity, the higher TCR avidity CD8+ T-cell clone consistently suppressed HIV replication more efficiently than its lower TCR avidity partner (Fig. 3b). Across specificities, however, the relationship between TCR avidity and HIV-suppressive activity was less robust (Fig. 3c). For instance, although the A3-RY10 clones displayed higher TCR avidities and higher levels of AgS than the corresponding A3-QK10 clones, they were consistently less efficient at suppressing HIV replication. Thus, the relationship between TCR avidity and HIV-suppressive capacity only held true within a given specificity. These observations suggest that the restricting HLA class I molecule and/or the cognate epitope delimit the antiviral efficacy of CD8+ T cells.
In further experiments, we examined CD8+ T-cell-mediated suppression of the R5-tropic JR-CSF and X4R5-tropic DH12 laboratory stains of HIV-1. Again, HLA-A3+/HLA-B27+ primary CD4+ T-cell targets were spinoculated with titrated doses of each viral strain to achieve a final infectivity of approximately 20% (Fig. 4a). Individual CD8+ T-cell clones displayed marked differences in their ability to suppress HIV replication across different strains (Fig. 4b). For instance, the B27-KK10 and A3-QK10 clones suppressed LAI effectively, JR-CSF modestly and DH12 poorly. In contrast, the A3-RY10 clones suppressed all three viruses suboptimally.
Epitope sequence differences provide a potential explanation for these discrepancies in antiviral efficacy (Fig. 4c). In particular, the reduced suppressive activity of the A3-QK10 and B27-KK10 clones against DH12 likely reflects the presence of point mutations in the targeted epitopes (e.g. L268M for KK10 and V74I for QK10). Instead, recognition of the RY10 epitope on DH12-infected CD4+ T cells was presumably less affected due to the lack of antigenic sequence variation. The L268M mutation in KK10 can impair TCR recognition at the cell surface and represents a clonotype-specific escape variant for the high-avidity B27-KK10 clone used in this study [20]. The V74I mutation in QK10 could act similarly, affecting TCR–pMHC interaction, thus reducing CD8+ T-cell clone efficacy to inhibit DH12 replication. Indeed, the A3-QK10 clone 4G3 displayed impaired recognition of the V74I peptide, with a 1 log lower functional avidity for the variant peptide compared to the wild-type peptide (data not shown), consistent with the viral suppression data (Fig. 4b). Moreover, the epitope-flanking mutations present in JR-CSF and DH12 might impede antigen processing [38], thereby contributing to the reduced antiviral efficacy of A3-QK10 clones against these isolates.
In this study, we used CD8+ T-cell clones with distinct epitope specificities and HLA class I restriction elements to investigate the relationship between AgS, TCR avidity and HIV-suppressive capacity. Consistent with our previous observations in the B27-KK10 system [20], we found that TCR avidity and AgS were directly correlated across panels of HLA-A3-restricted CD8+ T-cell clones specific for the HIV epitopes QK10 and RY10, thereby reinforcing the central role of the TCR as a determinant of functional potency. Furthermore, within each specificity, CD8+ T-cell clones with higher TCR avidities consistently suppressed HIV replication more efficiently than their lower TCR avidity counterparts. However, this association did not hold between specificities. Moreover, the hierarchy of antiviral CD8+ T-cell efficacy varied across different HIV-1 strains, even when the cognate epitope sequence was conserved. Consequently, it is difficult to draw a general parallel between antiviral efficacy and either AgS or TCR avidity, which explains the lack of consensus in the current literature.
The use of peptide antigens or single viral strains to determine antiviral CD8+ T-cell efficacy is intrinsically limited. In a recent study, Dong and colleagues examined the activity of CD8+ T-cell clones specific for the HLA-B8-restricted Nef90–97 FL8 epitope (FLKEKGGL) in response to target cells infected with different HIV-1 strains or recombinant vaccinia viruses expressing different isolate-derived Nef proteins [39]. Despite conservation of the FL8 sequence across all viruses tested, only 23% were recognized. This suboptimal response rate was attributed to epitope-flanking region polymorphisms in the majority of viral isolates, which impaired cognate antigen generation. Our data further highlight the potential bias associated with noninclusive in-vitro systems.
Additional factors beyond sequence variation can affect antigen presentation and confound comparisons between antiviral efficacy and either AgS or TCR avidity. For example, the Nef protein is expressed early in the HIV replication cycle and processed/presented before synthesis of the structural proteins, such as Gag [40]. In addition, although the majority of HLA class I-restricted epitopes are processed by the proteasome or immunoproteasome, the Nef-derived QK10 epitope is unusual in that it is generated by an aminopeptidase enzyme called tripeptidyl peptidase II (TPPII) [41]. Taking both factors into account, the QK10 epitope may therefore exhibit different and likely more rapid processing/presentation kinetics compared to the RY10 and KK10 epitopes. Together with competition for binding to the HLA-A3 molecule, these kinetic differences could explain why the A3-QK10 clones outperformed the A3-RY10 clones in HIV-suppression assays despite lower overall TCR avidities.
The nature of the restricting HLA class I molecule is also noteworthy. In the present study, we performed all HIV-suppression assays using a single target cell line expressing both HLA-A3 and HLA-B27 to standardize our approach across restriction elements. Nonetheless, we cannot account for the relative densities of these molecules on the target cell surface. Techniques such as high-affinity TCR microscopy [42,43] or micro-Raman spectroscopy [44] would be required to compare HLA-A3 and HLA-B27 expression levels directly. Furthermore, these HLA class I molecules may be differentially susceptible to Nef-mediated down-regulation. Indeed, it was recently demonstrated that HLA-A molecules are down-regulated by Nef to a greater extent than HLA-B molecules [45]. Irrespective of TCR avidity, such differences could explain the greater potency of HLA-B27-restricted clones compared to HLA-A3-restricted clones. In line with these considerations, epitope expression kinetics and Nef-mediated HLA class I down-regulation have been reported previously to influence the HIV-suppressive capacity of CD8+ T cells [46,47].
Finally, it is important to consider the possibility that interclonal differences in functional potential, for example, due to epigenetic regulation [48], could affect comparisons of antiviral efficacy irrespective of other parameters. In addition, the differential sensitivity of viral isolates to nonlytic suppression mediated by soluble factors, such as CC-chemokines and IL-16 [49,50], might explain some of the observed variability in our system.
To conclude, our data show that the relationship between HIV-suppressive capacity and either AgS or TCR avidity is blurred by the multiple factors that can affect CD8+ T-cell recognition of cognate pMHCI molecules on the target cell surface. Thus, the correlation between TCR avidity, AgS and antiviral activity holds within, but not necessarily between, antigen specificities. Collectively, these observations emphasize the importance of the TCR as a key determinant of CD8+ T-cell efficacy and help resolve purported discrepancies in the field.
We thank Catherine Blanc for sorting viable tetramer+ cells at the Pitié-Salpêtrière Flow Cytometry Platform, and Martin Larsen for help with polyfunctionality index calculations.
Funding: This work was supported by Sidaction, the French Agence Nationale de la Recherche sur le SIDA (ANRS) and the ANR (Project ANR-09-JCJC-0114-01). DAP is a Wellcome Trust Senior Investigator.
The authors declare that they have no competing financial interests.
antigen-sensitivity; CD8+ T lymphocyte; HIV epitope; TCR avidity; viral suppression
Copyright © 2014 Wolters Kluwer Health, Inc.
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Blessings For Women
by Larson, Susie;
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Blessings For Women
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Daily Blessings to Lift a Woman's Soul. Begin your day with a reminder of God's love and purpose. This beautiful devotional book is filled with words of comfort, encouragement, and wisdom that will bring joy to your soul. Each blessing and related Scripture is specifically aimed to nourish a woman's heart. The blessings take only moments to read but are packed with biblical promise and perspective.. Several years ago Susie began posting daily blessings on Facebook, and since then thousands have responded and forwarded the blessings on to others. This collection of new blessings, presented with inspiring Scriptures and beautiful photos, will be treasured by anyone seeking peace and encouragement or looking to encourage those they love.
Blessings For Women by Larson, Susie was published by Christian Audio in September 2016. The ISBN for Blessings For Women is 9781633899643.
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Re: Do you really think is a good idea to give a mic to every PS4
buckallmighty wrote:
Thats just my opinion though. You dont believe these things are possible more power to you. I personally dont know if they are or not but ive learned over the years that technology always has weaknesses, security flaws, and all around instability. No reason to call someone paranoid or anti social, its just a opinion and a "chance" im not willing to pay for.
To each their own though. You should embrace that quote yourself and just do you.
I thought you're first comment was sarcasm, but now i'm not so sure. Honestly, you're entire argument at this point is moot, since you don't have to use a headset with the PS4.
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I don't work for Sony. Just a gamer helping others.
Re: Do you really think is a good idea to give a mic to every PS4[ Edited ]
chickenbonetom wrote:
al2009man wrote:
chickenbonetom wrote:
Yeah Xbox live is basically their baby sitter.
...and that's why Xbox Live Gold is the best Online Service than PSN's Service
I didn't quite understand that last part lol.
They are joking that, because parents know that their kids can play XBL for hours, that it sold better as a babysitting aid, rather than a gaming system.
As for the earlier post about the headset being used as a monitoring device, that's a weak argument. Like Nova said, unplug the mic, and don't buy a PS4 camera. If you have concerns about the PSN, then abstain from it until you see evidence that the security is robust enough to protect you (which is one of the things that we are all hoping comes along with the promised upgrades when PS4 comes out). Oh, and don't own a cell phone, car, PO Box, passport, driver's license, or computer. Complaining of a lack of privacy via the internet is quite possibly to most hilarious thing one can read while connected to it.
Re: Do you really think is a good idea to give a mic to every PS4
ExMimic wrote:
FiveFourteen wrote:
chickenbonetom wrote:
RatchetH22 wrote:
Agreed. Muting someone only takes the press of a button. It isn't that difficult.
That's gunna be A LOT of muting though lol. I can't even imagine all the spoiled little brats talking on mic with every PS4 game online. One shutters at the thought.
I can't imagine all kids using the mics. Parents won't want them to I know I wouldn't want my kid talking to stangers over PSN
Have you played xbox live? lots of parents just don't care.
yes and a lot of the kids when you explain that continuing to act like they are will result in everyone muting / ignoring them in games and then they will be left with no one to talk to, do calm down. If not hit mute it was not that hard to do, also the amount of kids you have to deal with changes with the games you play.
And acting annoying on the mic is not limited to kids.
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text: As you are probably aware by navigating here, a SYN Flood' or SYN Attack is a DoS and/or DDoS method of attack which takes advantage of the three-way handshake in TCP/IP networking. In this guide, we aim to help system administrators mitigate the effects of and even fully prevent against SYN flooding. This guide is especially useful for firms and individuals who cannot afford services like CloudFlare or others that purport to stop DDoS.


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French Ornaments Framed Wall Art by Tim Burney are mixed media works featuring ornate antiqued emblems surrounded by a collage of letters. The style of the shadowboxes is cream with beaded aged gold finish edging. Perfect for displaying in your living room or office.
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They cannot reproduce without infecting an income cell and turning that host cell in a little virus factory. There's an array of reality contest signifies that focus on creative pursuits like designing clothing, cooking elaborate meals, or producing art. Took my prescribed antiviral medicine with this recommended dosage and frequency. I put lotion on my own butt to soothe the irritation caused by the shingles. Until that period, your "discards" are highly infectious.


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— By Sam Bartos |
It's that time of year again—when we celebrate a week of local music in the Bay Area by boasting Noise Pop: the Bay Area's premiere indie music festival. Truly, it's an exciting time to be alive. Bands will influx. Tickets will sell out. The Bay Guardian will do a puff piece-turned-cover story on up-and-coming bands that are on the middle of the marquee list of Noise Pop artists. All those bands will share their newfound exposure on their facebooks, and the wheels of blogocracy will keep turning.
Except, here's the thing about Noise Pop: it's just a week of San Francisco shows. We live in a major metropolitan area with vast appeal and resources, any given week of shows is good, and Noise Pop makes a random week of the year out to be, somehow, a thing. To call this week of shows somehow spectacular (or even, particular), is a bombastic and perplexing tradition that serves little tangible meaning in the Bay Area music scene. It's a waste of energy that distracts—not highlights—from the terrific and unwavering musical draw and appreciation the Bay Area has.
Perhaps the greatest misdemeanor Noise Pop commits is that the so called 'Festival' suffers from a complete deficit of noteworthy headliners. For example, Noise Pop 2013 headliners include:
Toro Y Moi: Don't get me wrong, Toro Y Moi is a great and tremendously relevant band that does a terrific live show, but fall short of being considered the flagship headliner of a music festival, especially considering they've done three Bay Area shows in the last two years.
Body/Head: You run a real gamble paying money to see a Kim Gordon noise project in concert: you might witness one of the most profound and honest pieces of musical performance art in the modern era, or you might see a totally self-indulgent spectacle of humiliation and shame. There is a good chance, actually, that the concert will be both. As cool as it may or may not be, it is a guaranteed salting of our collective wounds that Sonic Youth will probably never get back together, and that children (us) are the real losers in divorce.
Rogue Wave: Playing the Noise Pop Festival in a rare Bay Area live appearance.
Amon Tobin: That's cool. Amon Tobin is cool, except, he's playing a DJ set. Not putting the 'DJ set' in parentheses next to your music festival's feature headliner is kind of like padding your bra in the giant middle school that is San Francisco.
!!! (chk chk chk): !! was the most bloggable band of 2005, immediately before people began to realize the psychedelic jam rock they were listening to was the very same psychedelic jam rock they don't care for very much at all. Almost immediately after their plateau, the band would become largely marginalized by a new influx of electronic bands. A more accurate name for this band today would be '…', which will also be your reaction when you realized you payed $23 to see this band in concert.
Look, no offense to Portland or Los Angeles or New York, but fuck those cities. We live in the greatest city in America. We don't need to create imaginary music festivals to make ourselves cool. Go to foopee.com—a website that outlines every show happening in the Bay Area on any given night, for weeks into the future—and you'll find that every week in San Francisco is a relentless deluge of awesome shows both with big and small bands.
Spend a few dollars to go see some shitty band's first show at Bottom of the Hill. Sell some jorts at Buffalo Exchange so you can get $12 to go support that new venue on Valencia. Don't do it under the guise of a festival, do it because it's a fucking Wednesday. We don't need to waste our time making a big deal about a Thao And The Get Down Stay Down show, pretending it's part of a some big art and culture festival. It isn't. So before you spend $150 on a Noise Pop lift pass that gets you into (some of) the Noise Pop shows, remember: Noise Pop is all in your head.
Comments (30)
alright | [Permalink]
Applause. Could not have said it better myself. Cheers.
Hey There. I found your blog using msn. This is a really well written article.
I'll make sure to bookmark it and come back to read more of your useful information. Thanks for the post. I will certainly comeback.
EW77 | [Permalink]
totally agree. if anything, I attend less shows than normal during the week of Noise Pop.
Herr Doktor Professor Deth Vegetable | [Permalink]
Naw. NoisePop is awesome.
I didn't so therefore I won't | [Permalink]
Huge bowl of crack ready for the tokin'= check!
Jackson 5 greatest hits lp spinning on the record player= check!
This white entitled misogynistic pepperoni pizza lover being blissfully lifted away to paradise= PRICELESS!
Sam Bartos | [Permalink]
I didn't so therefore I won't | [Permalink]
Wanna go to Noise Pop together? I'll even spring for a bucket of Popeye's.
Ted | [Permalink]
Great post, and pretty much exactly how I feel.
This is the sanest thing I've ever read on this blog.
cliff notes | [Permalink]
http://showli.st you can have an advert free listing, and listen to the bands on spotify if you haven't heard of them.
cliff notes | [Permalink]
ps good article
Nick | [Permalink]
DIIV is playing. Whatever brings them here is OK by me.
scum | [Permalink]
I have a very good friend that works for Noise Pop. That being said, it blows large amounts of shit through tiny venues like a rectal tsunami. Even when I got into the shows for free I found myself walking out shaking my head. The Goblin Cock and Warship gig a few years back was awesome. My taste tends to run to heavier anyway.
I don't think before I type | [Permalink]
Uh, isn't Amon Tobin a Ninja Tune DJ? It stands to reason that he would DJ at Noise Pop unless you're disappointed that he's not bringing out the entire ISAM2.0 setup.
I don't get it.
plumpy | [Permalink]
Amon Tobin is a Ninja Tune recording artist. Sure, he DJs sometimes, but he also makes tons and tons of his own music. (Eight albums worth.) And he can certainly perform it without the full ISAM setup; I've seen it several times back in the day.
I completely agree, but I can also get behind the attempts of Noise Pop's promoters (who are mostly good people who care about the Bay Area music scene) to lure bands to the area by calling it a "festival." I have no idea about the history of SXSW, but presumably it started small and grew over time… maybe some day Noise Pop will become significant enough to draw a critical mass of bands from outside the area? One can hope, anyway.
I don't think before I type | [Permalink]
You could say this is an apples-to-oranges comparison, but if we're going to relate the two, SXSW is only 6 years older than Noise Pop and has been a bigger deal for longer than that time difference.
Whatever, having SXSW in town would probably be pretty fucking annoying.
Turn down that Noise Pop | [Permalink]
One of the good things about Noise Pop is that it's not SXSW. We don't need one. SXSW get's pretty miserable and SF doesn't need one more event that spikes the city's population for a week.
EW77 | [Permalink]
I think the point is that there is no need to 'lure' bands to SF. There are great shows happening all the time. This is already a huge tour destination given our cultural history and the fact that we are 1 of 4 major US cities on the West Coast (Seattle, PDX, SF/Bay Area, LA). If anything, Noise Pop is way behind the curve of what is happening here all the time. I've managed to catch 4 shows in the past week with 2 more this week as a mid-30s married dude with a day job (noting that there's a lot more I'd like to see if I had the time and energy).
Ben | [Permalink]
If you're already the type of person who sifts through show listings on a regular basis, then yeah there's nothing that significant about NoisePop - congrats, you're already cool. There are way more people out there with a casual level of involvement.
trazmick | [Permalink]
could not agree more. good post.
buff tuff | [Permalink]
I've gotta say for rookie sf music scenesters its still a good bet. I saw some good and weird stuff when I came to this city when I didn't know shit. That said, I still don't know shit and you are right on the money for those who know what they want to see and hear.
Erik | [Permalink]
It's funny how DJs pretend to be musicians.
tastr | [Permalink]
Sam, I was with you all the way up until you made fun of Thao & the Get Down Stay Down. Now you can shit all over this made up festival and how they promote shows that would already happen here anyway. But Thao is a San Francisco artist we should be celebrating. Maybe her genre isn't your favorite, but she makes honest music and attempts to be a part of the community, it's not cool to make that your parting line in this shitfest. If you claim to be such in the know scenester, you should celebrating Thao.
Kevin Montgomery | [Permalink]
Again, it's less making fun of Thao as an artist and more saying people's access to her & band is already fairly high (never mind she did Noise Pop a few years back), so why pretend like NP is providing a unique opportunity to see her perform?
Ben | [Permalink]
Maybe it's helpful to think of it as something like Bike to Work day? It makes more accessible something that plenty of folks are interested in, but maybe intimidated by or cautious about. I mean, you can dismiss it with a "lol bro I ride my bike every day just fuckin' man up and start riding" attitude and that probably seems reasonable if your circle runs heavy on the bike stuff already. It's just a fallacy to assume that everyone else who might be interested already shares your level of involvement.
NoisePop is the same sort of deal with music for the set who isn't already intimately familiar with what venues usually provide a good time, what bands are worth seeing, yadda yadda. It's a guided tour, but you can still have a good time if you put the cynicism in check.
I don't think before I type | [Permalink]
Here are bands I'm pumped to see and don't come around often: Diiv, OBN III and Fuzz (Ty Segall's new band), Body/Head, Damien Jurado, and Rouge Wave. This years seems to be missing a big headliner though (e.g., last year's Flaming Lips). Putting that aside, the quality of music during noise pop week is pretty solid. I can only imagine early to mid-April being a better time (when all the coachella bands swing though).
ughhhh | [Permalink]
change the headline of this to "Boring White Boy Discovers At Early Age That Having Edgy, Cynical, Opinions Will Get Him Attention and Shield Him From Criticism"


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In Bringer of Chaos: the Origin of Pietas, a clash of willls between Six and Pietas results in an impasse. Will Pietas apologize to an infuriating human? Never!
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Eagles go 1-1 in doubleheader
NEW ULM?- In a rare soccer doubleheader, the New Ulm boys finished one game strong as that momentum carried over into the second.
The end result of a day in which the temperature was in the high 80s was a 1-1 record on the afternoon for the Eagles. They lost the season opener to South Central Conference rival Waseca 5-2 and came back to defeat Southwest Christian 6-3 in the second game.
At one point in the day, New Ulm scored eight straight goals between the two games. Waseca led 5-0 late in the game before New Ulm's Josh Koelpin scored a couple of goals late for the final 5-2 margin.
Koelpin then scored the first goal in the second game as the Eagles jumped out to a 6-0 lead and cruised to the 6-3 victory.
Despite the first-game loss to a team that usually gives the Eagles fits, they were happy with how the day went overall.
"I think that the first game is kind of tough to see where everyone is at on the field," Koelpin said. "I thought our communication was good in the second game and I think we had a little bit better passing I thought."
New Ulm came out a bit sluggish in the first game and watched the Bluejays jump out to a big lead. Koelpin's first goal in the game came off of an assist from Christian Linan. Jacob Rakoczy helped on Koelpin's second score.
That late-game momentum led to an early goal by Koelpin in the second game at the 36:32 mark off of an assist from Noah Webb. After a couple close calls from the Eagles, they took a 2-0 lead with 3:08 left in the half when Seth Wiechmann scored on an assist from Broderick Brown.
Getting that early momentum was big for the Eagles, who struggled at times last year, especially when they got down.
"It's huge, it's really important at the beginning of the game, and it carries you on for the whole entire season," Brown said of the early lead.
While the lead was slim at halftime, the offense picked it in the second half, netting four goals to take over.
Rakoczy scored on an assist from Linan and Brown (assisted by Justus Morschauser) made it 4-0 with 19:59 left in the game. New Ulm got goals from Nicolas Garnter (assisted by Brown) and Jonathan Sehloff (unassisted).
"When you start scoring goals, your confidence overall as a team is raised," Koelpin said.
Southwest Christian did score three goals late in the game. The first goal ended the shutout attempt for goalkeeper Alexander King with 4:25 left. The next two goals came within a span of about a minute. However, it wasn't really a frustrating finish for the Eagles.
"It did not bother me at all, I knew that we had it locked up, once it hit three minutes I knew that there was no way that we could lose," Brown said.
New Ulm (1-1) gets back into action at 5 p.m. Thursday at New Prague.


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