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ANNA STUART
Bonnie and Stan
About Anna Stuart
I wanted to be an author from the moment I could pick up a pen and was writing boarding-school novels by the age of nine. I made the early mistake of thinking I ought to get a 'proper job' and went into Factory Planning – a career that gave me some wonderful experiences, amazing friends and even a fantastic husband, but didn't offer much creative scope. So when I stopped to have children I took the chance to start the 'improper job' of writing. During the baby years I wrote in the brief gaps provided by sleeps, playschools and obliging grandparents, publishing short stories and serials in all the women's magazines.
A little plotting on holiday!
Me with The Beatles!
But my ultimate aim was to write longer fiction and several years ago I published a series of successful historical novels under the pseudonym Joanna Courtney. I will continue to publish under that name but am delighted, as Anna Stuart, to also be able to write contemporary fiction. Bonnie and Stan, a true to life romance set in both the present day and sixties Liverpool, will be released in early 2019 with a second novel to follow in 2020.
Introducing Joanna Courtney
As Joanna Courtney I have published a trilogy The Queens of the Conquest about the three women fighting to be queen of England in 1066. It seemed to me inherently wrong that all we knew about this momentous year was from the male side of the story and I set out to redress the balance. My novels tell the events from the viewpoints of Edith of Mercia, wife of King Harold; Elizaveta of Kiev, wife of Harald Hardrada; and Mathilda of Flanders, wife of William the eventual conqueror.
I am now writing a new series called Shakespeare's Queens which explores the real histories of three of the great bard's best female creations - Lady Macbeth, Ophelia and Cordelia. The first of these, Blood Queen, will be released for kindle in September 2018 and in paperback in July 2019.
I wanted to write contemporary novels under the new name of Anna Stuart so that readers knew clearly what they were getting but do find out more about my historical work by visiting my Joanna Courtney site.
Joanna Courtney's site
I'd really love to hear from you with your thoughts on my books or any issues raised by them so do, please, get in touch.
anna@annastuartbooks.com
Happy couples don't make good stories – or do they?
Loving Liverpool…
The great drama of sport
How I came to find Bonnie and Stan (or different ways to sell a novel….)
Anna Stuart is born
Bonnie and Stan will be published by Trapeze, part of Orion books. Launched in October 2016 and already the home of 9 Sunday Times top 10 bestsellers, Trapeze is on a mission to publish books that start conversations. Visit their fabulous twitter page for more.
Copyright 2020 Anna Stuart
Do NOT follow this link or you will be banned from the site! Scroll To Top


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Charles Deering and Ramón Casas / Charles Deering y Ramón Casas
Trade Cloth – $60.00
Isabel Coll Mirabent (Author)
Jennifer Croft (Translator)
Lindsay King (Preface by)
A Friendship in Art / Una amistad en el arte
Isabel Coll Mirabent
This lavishly illustrated, bilingual art book presents drawings by Ramón Casas in the Charles Deering McCormick Library of Special Collections at the Northwestern University Library and oil paintings by Casas from private collections and the Art Institute of Chicago.Charles Deering and Ramón Casas follows the development and dramatic dissolution of a three-way friendship that connected the Spanish painter Ramón Casas (1866–1932); the Chicago industrialist Charles Deering (1852–1927), who was a collector and admirer of Casas's work as well as a patron of Northwestern University; and the Spanish artist Miguel Utrillo (1862–1934), Casas's lifelong friend and the father of the French painter Maurice Utrillo. Casas introduced Deering to Sitges, a beach town near Barcelona, Spain, where the latter created a palatial estate with a museum to house his art collection. Miguel Utrillo served as director of the museum. The text explores the treasures housed at Maricel and what happened among the three men that led Casas to abandon Utrillo and Deering to depart Spain, taking his art collection with him.
Isabel Coll Mirabent is a professor in the Department of Art History at the University of Barcelona.

redpajama_set_name: RedPajamaCommonCrawl


Finely handcarved late Georgian style candlesticks with twisted columns interspersed with beaded detail. Stiff leaf decoration to the base and drip pan. Patinated brass pricked fittings.
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What are the rules for live streaming events on Facebook?
Earlier this year Facebook entered the world of live streaming with the launch of its Facebook Live feature, making it possible for users to broadcast video content in real time to a large audience. Facebook live streaming is simple to use, easily accessible and a great way to interact with viewers in real time, so it's no surprise that individuals have seen the potential for using this technology at events.
As with all content that is transmitted via Facebook, there are reporting tools in place to enable content believed to violate community standards to be reported and removed. Before you think about live streaming an event, it's important that you take care protect yourself or your organisation from violating these standards.
Getting permission is key. When it comes to live streaming any type of event, always be aware of getting permission from the relevant people. Is the event private? If so, then the reasonable expectation of privacy may apply. This means that you are not permitted to record, or live stream, someone without their consent if the event is taking place on private property. For instance, if the event is taking place in a private meeting room or a personal office and not a public space then you cannot live stream without the permission of those present. Ensure that you have all the appropriate permission, including written consent from featured individuals, ahead of time before live streaming any event.
Even if the event is taking place in a public space, to be on the safe side, it is a good idea to include a release form in the event registration process as well as placing a crowd release notices around the event venue. This way you can make attendees aware that there will be a live stream taking place and give them the opportunity to leave if they don't wish to be involved. It is also worth noting that if you're live streaming an event as an attendee; the first thing you should do is ask the event organisers and ensure you have their permission to do so.
You should also be aware of rules and restrictions that apply to copyrighted or trademarked material to protect yourself from liability when live streaming at an event. We've all seen programmes on TV where a logo on a product or item of clothing has been pixelated because the producers don't have the permission of the copyright owner to show it. Sadly, when it comes to live streaming there is no option to go back and edit the content so you need to avoid capturing any content of this nature in the stream without permission. To guard yourself against copyright infringement, check the area where your stream will be taking place for any posters, artwork, logos and screens and ensure that your stream will not interfere with any keynote speeches, performances, music or video clips that may be subject to copyright laws or cover sensitive material.


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Barryville, NY
Best Events Near You in Barryville, New York
Intro To Photoshop
Saturday, Feb 8, 2020 at 9:00am
Bethel Woods Center for the Arts
Bethel, NY
Sunday, Feb 9, 2020 at 9:00am
Friday, Mar 27, 2020 at 9:00pm
Monticello, NY
Barryville Top Searches
Afternoon With Young Peoples Chorus Of New York City
Sunday, Apr 26, 2020 at 3:00pm
Wednesday, May 6, 2020 at 8:00pm
Thursday, May 7, 2020 at 8:00pm
Shawn Colvin: Steady On 30th Anniversary Tour
Thursday, May 14, 2020 at 8:00pm
The Big Sip
Saturday, May 23, 2020 at 1:00pm
Zac Brown Band: Roar with the Lions Tour presented by Polaris
Thursday, Jun 25, 2020 at 7:00pm
Steely Dan with Special Guest Steve Winwood
Saturday, Jul 11, 2020 at 7:00pm
Wednesday, Jul 15, 2020 at 8:00pm
Thursday, Jul 16, 2020
Bethel Woods 2020 Country Megaticket
Thursday, Jul 16, 2020 at 6:59pm
Lady Antebellum Ocean 2020 Tour
Apple Pond Farm and Renewable Energy Education Center
80 Hahn Road
Callicoon Center, NY
About The Farm Apple Pond Farm is about two hours from New York City, situated in Sullivan County and surrounded by the Catskill Mountains. &nbs...
200 Hurd Road
About Us Bethel Woods Center for the Arts is approximately 90 minutes from New York City at the site of the original 1969 Woodstock festival in Be...
Delaware Highlands Conservancy
120 Segar & Rosenberg Road
Kauneonga Lake, NY
The Delaware Highlands Conservancy is an accredited land trust dedicated to conserving the natural heritage and quality of life in the Upper Delawa...
Forestburgh Playhouse
39 Forestburgh Road
Forestburgh, NY
The Forestburgh Theatre Arts Center is a 501(c)3 not-for-profit corporation which operates the Forestburgh Playhouse.As specified in the Articles o...
Harness Racing Museum And Hall Of Fame
Mission The Harness Racing Museum & Hall of Fame is dedicated to comprehensive, active and authoritative support and promotion of the Standardb...
Knolliewood Skate Park
Campbell Hall, NY
Liberty Museum And Arts Center
Liberty, NY
The Liberty Museum & Arts Center Building was first constructed as a hotel in 1894. The Poellman House, as the hotel was known, contained 30 ro...
5281 Route 44-55
Kerhonkson, NY
About Us:Located in Ulster County, NY Minnewaska State Park Preserve is situated on the dramatic Shawangunk Mountain ridge, which rises more than 2...
Oakland Valley Race Park
305 Oakland Valley Road
Cuddebackville, NY
Oakland Valley Race Park is a premier Kart and Supermoto racing facility in the Northeast U.S. We are a great place for beginners and seasoned vete...
Port Jervis, NY
Features: - Playground/Tot Lot - Roller Hockey Rink - Skateboard Park - Basketball Courts - Ball Field - Band Stand a...
Sam's Point Preserve
400 Sam's Point Road
Cragsmoor, NY
Considered a National Natural Landmark, one of the Earth's Last Great Places, and an Important Bird Area, this awe inspiring preserve along the Sha...
Shadowland Stages
Ellenville, NY
CURTAIN TIMES & BOX OFFICE Box Office Tues - Wed : 12pm - 5pm Thurs - Sat : 12pm - 10pm Sunday : 12pm - 5pm Reservations: 24-hour recorde...
Sugar Loaf Art and Craft Village
Chester, NY
About Us: The hamlet of Sugar Loaf is a community of artisans, boutique shops, dinning and a world class performing arts center all nestled under ...
The Neversink Valley Museum of History and Innovation
26 Hoag Road
Mission The mission of the Neversink Valley Area Museum, a non-profit organization, is to preserve, document and interpret the history of the Never...
The Open Eye Theater
MISSION The Open Eye Theater is dedicated to creating, developing, and producing plays for a diverse audience in the Catskill Mountains; providing...
Memorial Park Drive
Warwick, NY
Features: - DPW Complex - Skateboard park - Waywayanda Creek - Chlorine Building - Grandstand - "Over 30 Field" ...


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Article Free access | 10.1172/JCI13463
High-affinity T helper epitope induces complementary helper and APC polarization, increased CTL, and protection against viral infection
Jeffrey D. Ahlers,1 Igor M. Belyakov,1 Elaine K. Thomas,2 and Jay A. Berzofsky1
1Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA2 Immunex Corporation, Seattle, Washington, USA
Address correspondence to: Jay A. Berzofsky, Molecular Immunogenetics and Vaccine Research Section, Metabolism Branch, National Cancer Institute, Building 10, Room 6B-12 (MSC#1578), National Institutes of Health, Bethesda, Maryland 20892-1578, USA. Phone: (301) 496-6874; Fax: (301) 480-0681; E-mail: berzofsk@helix.nih.gov.
Find articles by Ahlers, J. in: JCI | PubMed | Google Scholar
Find articles by Belyakov, I. in: JCI | PubMed | Google Scholar
Find articles by Thomas, E. in: JCI | PubMed | Google Scholar
Find articles by Berzofsky, J. in: JCI | PubMed | Google Scholar
First published December 1, 2001 - More info
Published in Volume 108, Issue 11 on December 1, 2001
J Clin Invest. 2001;108(11):1677–1685. https://doi.org/10.1172/JCI13463.
© 2001 The American Society for Clinical Investigation
First published December 1, 2001 - Version history
Received: June 7, 2001; Accepted: October 1, 2001
Natural viral proteins do not always make optimal vaccines. We have found that sequence modification to increase epitope affinity for class II MHC molecules (epitope enhancement) can improve immunogenicity. Here we show first that a higher-affinity helper epitope-enhanced HIV vaccine not only induces more cytotoxic T lymphocytes (CTLs), but also skews helper cells toward Th1 cytokine production and protects against HIV-1 recombinant vaccinia viral challenge. Furthermore, we elucidate a novel mechanism in which the higher-affinity vaccine induces dramatically more effective helper cells with a higher level of CD40L per helper cell and more positive cells, which in turn more effectively conditions dendritic cells (DCs) for CTL activation in a second culture. The improved helper cells also induce much greater IL-12 production by DCs, accounting for the reciprocal T helper polarization to Th1, and increase costimulatory molecule expression. Thus, increasing affinity for class II MHC results in a complementary interaction in which T helper and antigen-presenting cells polarize each other, as well as increase CTL, and provide greater vaccine efficacy against viral infection.
Viral proteins have not evolved to be optimal vaccines. We have demonstrated previously proof of principle for the approach of "epitope enhancement," whereby modifying the amino acid sequence of a Th epitope in the HIV-1 envelope to enhance binding to a class II MHC molecule can increase immunogenicity (1, 2). Attaching this modified Th epitope to a cytotoxic T lymphocyte (CTL) epitope enhanced CTL induction in vivo (2). Genetic mapping showed that the enhanced CD8+ CTL response was due to an improved CD4 + cell response. We (3) and others (4–6) have shown similar epitope enhancement for peptides binding to class I MHC molecules. However, the molecular mechanisms by which increased affinity for class II MHC translates into increased efficacy remain unclear. Is the enhanced peptide simply active at lower concentrations? Is the difference only quantitative, or are there qualitative differences in the response? One might have expected a peptide with higher affinity for MHC to be more immunogenic, but not necessarily to induce a qualitatively different response.
Here we focus on the mechanism of epitope enhancement for class II MHC–restricted helper T cells in augmenting a CTL response. We and others have found an essential role for CD4+ T helper cells in induction of CTL responses to peptide vaccines (7–9). The mechanism of CD4+ cell help for CTL induction appears to involve an intermediate step requiring conditioning of the APC via CD40 ligation by CD40L (10–13), but the downstream effects of this ligation have not been determined. Although an upregulation of essential costimulatory molecules on the antigen-presenting cells (APCs) has been suggested as a possible mechanism of help for CTL induction, the identity of the specific costimulatory molecules involved and the mechanism enhancing CD8+CTL activation remain unclear. Also, it was not known how the modified peptide increases help; we show here it acts by increasing CD40L expression. Furthermore, it was not known whether an increase in APC conditioning by augmentation of help would simply result in more effective antigen presentation or would qualitatively alter the APC to induce a more polarized response.
All of these questions are addressed in the current study. We find that modification of a Th cell epitope to increase binding to MHC causes quantitatively more T cells to undergo upregulation of CD40L and to express higher levels of CD40L per cell, which interacts with CD40 to condition the APC for CTL induction. The primary measurable effect of the improved APC conditioning by the higher-affinity peptide is upregulation of IL-12 production, along with some difference in B7-1 and B7-2 expression. Thus, unexpectedly, the APC becomes more polarizing toward a Th1 response. Furthermore, the conditioned APCs, removed from the Th cell, are more potent stimulators of CTL in vitro. Thus, epitope enhancement to increase peptide affinity for MHC class II results in a qualitative as well as quantitative change in help, through complementary interactions in which increased CD40L on the helper cells induces more polarizing APCs that act back on the T helper cell to produce a Th1-polarized response, as well as higher levels of CTL and protection against viral infection. Corollaries are that one mechanism to polarize APCs is to increase the stimulation through CD40 by CD40L and that peptide affinity for class II MHC can be used to mediate this effect for more effective vaccines.
Animals. BALB.A10 and A.AL mice were bred in our animal facility (BioCon Inc., Rockville, Maryland, USA) and used between 6 and 10 weeks of age. Both strains possess the class II molecule Ek, which bind Th peptides T1 and T1(A) and also express the class I Dd molecule, which presents the HIV-1 CTL epitope P18IIIB.
Bone marrow–derived dendritic cells. Dendritic cells (DCs) were derived from bone marrow cultures according to the procedure used previously (14), with slight modification. Bone marrow cells were plated in Nunc flasks at 2 × 106 per 100 mm/10 ml in complete medium (2) plus GM-CSF (30 ng/ml; Peprotech Inc., Rocky Hill, New Jersey, USA) ± IL-4 (200 U/ml; Life Technologies Inc., Grand Island, New York, USA). Day 7–9 cells were depleted of B cells, granulocytes, T cells, and NK cells using an excess of anti-B220 coated (Dynal Inc., Lake Success, New York, USA) and biotinylated anti-CD3, R6B, and pan NK(DX5)-streptavidin–coated magnetic beads. Buoyant cells after centrifugation on Lympholyte M (CedarLane Laboratories Ltd., Hornsby, Ontario, Canada) were about 80% CD11c+. In some experiments CD11c+ cells were positively selected using magnetic beads (Miltenyi Biotech, Auburn, California, USA).
Antigen. Multideterminant cluster peptides (15) containing overlapping T helper epitopes were colinearly synthesized with P18IIIB, an immunodominant CTL epitope in mice (16) to form a synthetic peptide vaccine construct (17). The constructs used for immunization were PCLUS 3-18IIIB, KQIINMWQEVGKAMYAPPISGQIRRIQRGPGRAFVTIG, composed of HIV-1 IIIB envelope amino acid residues 421–444 according to Los Alamos data base numbering (18), linked to P18IIIB shown in bold (amino acid residues 308–322), and the corresponding construct with an epitope-enhanced helper portion, PCLUS 3(A)-18IIIB, KQIINMWQAVGKAMYAPPISGQIRRIQRGPGRAFVTIGK. The helper peptide PCLUS 3 contains the native Th helper epitope, T1 KQIINMWQEVGKAMYA, whereas PCLUS 3(A) contains the modified Th epitope, T1A, KQIINMWQAVGKAMYA, created by replacing Glu with Ala. T1(A) has higher affinity for the I-Ek (1). Peptides (synthesized by Multiple Peptide Systems, San Diego, California, USA) were single peaks as seen by HPLC and by mass spectrometry.
Viruses. Recombinant vaccinia virus vPE16 expressing HIV-IIIB gp 160 was a kind gift of Bernard Moss and Patricia Earl, National Institute of Allergy and Infectious Diseases, NIH (19). Virus was injected intraperitoneally into female mice in protection experiments, and 5 days later, ovaries were homogenized and sonicated and virus titered by plaque assay on BSC-1 cells (20).
Generation of T helper line. Spleen cells from BALB.A10 congenic mice (H-2Ek) immunized twice subcutaneously with PCLUS 3-18IIIB in Montanide ISA-51 (a kind gift of Jean-Pierre Planchot, Seppic Inc., Fairfield, New Jersey, USA) were stimulated twice with T1(A) under Th1-polarizing conditions: IL-12 (10 ng/ml) and anti–IL-4 (10 μg/ml). CD4+ cells were positively selected with anti-CD4 beads (Dynal Inc.) and cloned by limiting dilution. Th1 clones expressing IL-2 and IFN-γ, but not IL-4, were selected. Proliferation was assayed by [3H] thymidine incorporation of 3 × 104 to 5 × 104 Th cells stimulated with T1(A) and 5 × 105 irradiated syngeneic spleen cells in 96-well plates for 96 hours.
Cytotoxicity assay. A.AL (I-Ek, Dd) and BALB.A10 (I-Ek, Dd) mice were immunized subcutaneously with vaccines (20 nmol) in Montanide ISA-51. Spleen cells (107) were stimulated with 107 peptide-pulsed (0.5 μM P18IIIB for 1.5 hours) naive irradiated (30 Gy) spleen cells in 12-well plates (Corning Costar Corp., Acton, Massachusetts, USA). Twelve hours later Rat T Stim (Collaborative Biomedical Research, Bedford, Massachusetts, USA) was added as a source of IL-2 (final concentration 7.5%). Nonadherent cells 4 days later were centrifuged over mouse Lympholyte M (CedarLane Laboratories Ltd.), replated with 2.5 U/ml rmIL-2, and assayed on day 7–8 in triplicate in 96-well round-bottom plates on 5,000 target cells, either 18Neo (3T3 fibroblasts neoR) or P815 P18IIIB pulsed or unpulsed in a 5-hour assay. Spontaneous lysis was less than or equal to 10%. CTL data were normally distributed based on the Shapiro-Wilk test, and means are plotted as arithmetic means plus or minus SEM. Error bars smaller than the symbols could not be shown. Lysis without peptide was less than 10%, except where indicated.
RT-PCR. Total RNA was isolated from CD4+ T cells 12–20 hours after in vitro restimulation, mRNA was reversed transcribed as described (21), and PCR was performed under nonsaturating conditions using primers described previously (21) for the housekeeping gene HPRT and murine cytokines and controls: IL-2, IL-4, IL-10, and IFN-γ (CLONTECH Laboratories, Palo Alto, California, USA).
ELISA. Duplicate samples of 100 μl of 48- to72-hour culture supernatants from 5 × 105 CD4+ cells stimulated in vitro with peptide and 5 × 105 DCs, were tested in an IFN-γ ELISA assay (Life Technologies Inc.). Forty-eight–hour culture supernatants were tested for IL-12 with an IL-12 (p40 and p70) set (OPTEIA; PharMingen, San Diego, California, USA).
DC conditioning cultures. Peptide T1, or T1(A), or no peptide was added to 5 × 105 DCs and 5 × 104 peptide-specific CD4+ Th cells in 12-well plates and incubated for 15–18 hours. T cell–DC conjugates were disrupted by washing with HBSS with 1% BSA, 25 mM HEPES, and 15 mM EDTA. CD4+ T cells were removed with an excess of anti–CD4-coated magnetic beads (Dynal Inc.), and the remaining conditioned APCs were pulsed with the CTL epitope P18IIIB (0.5 μM) for 1 hour. Pulsed DCs were irradiated and replated at 5 × 105 per well in 12-well plates with 5 × 106 CD8+ cells negatively selected from spleen cells from animals primed in vivo more than 1 month earlier with PCLUS 3-18IIIB. Control DCs were incubated with GM-CSF only, or Th cells without peptide, or with murine trimeric CD40L as a positive control (3 μg/ml; Immunex Corp., Seattle, Washington, USA). In blocking experiments, Ab's were added to DC-CD8+ cell cultures at the initiation of culture and on day 3.5. Blocking mouse Ab's used at 10–15 μg/ml — anti–B7-1 (16-10A1), anti–B7-2 (GL1), anti-ICAM (3E2), anti-CD44H (TM-1), anti–IL-12 (C17-8) — were obtained as no azide/low endotoxin preparations from PharMingen or dialyzed overnight at 4°C against PBS; murine anti-41BBL was a kind gift from Immunex Corp.
FACS analysis. T1-Th1 cells (3 × 104) were incubated in 96-well round-bottom plates with 3 × 105 DCs or purified naive splenic B cells (B220+) and T1 or T1(A) peptide. Phycoerythrin-conjugated (PE-conjugated) rat anti-mouse CD40L was added initially and again 20 hours later. FITC-labeled rat anti-mouse CD4+ cells were examined for CD40L expression by FACScan (Becton Dickinson Immunocytometry Systems, San Jose, California, USA). Cultures of DCs unconditioned or conditioned by CD4+ T1-specific Th cell clones or CD40L (Immunex Corp.) for 24–36 hours were stained with PE-CD11c+ and FITC class I (Dd), FITC anti–class II (Ek, 14.4.4), FITC B7-1, B7-2, ICAM, CD44H, CD11b, CCR5, and CD40 (PharMingen), and anti-41BBL (Immunex Corp.). PE-conjugated H-2Dd/I-10 peptide tetramer staining was performed as described previously (22) in the presence of Fc block (anti-CD16/CD32).
Statistical analysis. Means were plotted as geometric or arithmetic based on the distribution of data determined by the Shapiro-Wilk test. Δ means represent the experimental minus no-antigen control mean of triplicate samples. A two-way ANOVA was applied to CTL data, thereby averaging the respective values for each curve over all the effector/target (E/T) ratios. Means were compared using Tukey's multiple comparison test as described (23), and significance was determined at α = 0.05. IFN-γ and IL-12 ELISA data were treated similarly or by the Student t test at P = 0.01.
Modification of Th epitope in Th-CTL vaccine construct elicits enhanced CD8+ CTL response. To determine the effect on the CTL response of improving the helper epitope without altering the CTL epitope, we immunized BALB.A10 mice with PCLUS 3-18IIIB and the construct containing the modified helper epitope, PCLUS 3(A)-18IIIB, and measured CTL responses after in vitro stimulation. Immunization with PCLUS 3(A)-18IIIB resulted in greater CTL induction with an eightfold horizontal shift in E/T ratio after a single immunization (Figure 1a). This increase in CTL is confirmed by a doubling of the specific CD8+ cells binding P18-I10/H-2Dd tetramers in draining lymph nodes after 4 days of stimulation with P18 peptide, from 4.7% to 9.2% after immunizing with PCLUS 3-18IIIB versus PCLUS 3(A)-18IIIB, respectively, with nonspecific background 1.4% in unimmunized mice subtracted (Figure 1b).
Epitope enhancement of Th epitope in Th-CTL peptide vaccine construct elicits enhanced CTL response. (a) BALB/c mice were immunized subcutaneously with 20 nmol native vaccine construct PCLUS 3-18IIIB or construct with the modified helper epitope PCLUS 3(A)-18IIIB. Spleen cells were restimulated in vitro 14 days later with irradiated P18IIIB-pulsed syngeneic spleen cells, and 7 days later tested in a 51Cr-release assay on P815 targets pulsed with 0.5 μM P18IIIB. Open symbols represent nonspecific lysis of target cells without peptide. Curves are significantly different at α = 0.001 by Tukey multiple comparison test. (b) Draining lymph node cells from immunized animals were stimulated 4 days with P18IIIB and stained with anti-CD8 and P18-I10/H-2Dd tetramer. (c) Draining lymph node cells from immunized animals were tested immediately ex vivo without in vitro stimulation 3 days after immunization, in a 5-hour CTL assay. (d) Animals were challenged intraperitoneally with different doses of vPE16 14 days after two immunizations (primary immunization subcutaneously and boost intraperitoneally 14 days later). Four days after challenge, viral titers in homogenized ovary tissue were determined. Each column represents the mean of three mice.
In a separate experiment, when the CTL activity was measured ex vivo without restimulation using draining lymph node cells 3 days after immunization, a significant CTL response was already evident in animals immunized with PCLUS 3(A)-18IIIB, but not in those immunized with PCLUS 3-18IIIB (Figure 1c). Thus, the modified Th epitope with higher affinity for MHC class II resulted in enhanced help for class I–restricted CTL induction as determined by greater numbers and activity and induction of CD8+ CTL without in vitro stimulation. Thus, help was indeed limiting for CTL induction.
To determine if this enhanced CTL response was more protective in vivo, we challenged immunized animals intraperitoneally with increasing doses of virus. We have found this model to be strictly dependent on the presence of activated CD8+ CTL at the site of viral challenge. Complete protection was achieved with both vaccine constructs at low-dose viral challenge; however, at the intermediate dose complete protection was seen only in animals immunized with the enhanced (T1A)-vaccine construct (Figure 1d). Furthermore, at the highest challenge dose, virus was still inhibited by greater than 99% (100-fold) compared with 95% (20-fold) with the unmodified vaccine. Thus, greater induction of CD8+ CTL by immunization with the modified helper epitope-CTL vaccine construct correlated with improved protection from viral challenge.
Epitope modification polarizes the Th response to Th1 phenotype. To determine the nature of enhanced helper function for induction of CD8+ CTL and to determine if the difference was purely quantitative or also qualitative, we measured cytokine mRNA in CD4+ cells from draining lymph nodes 3 days after immunization. Unexpectedly, a clear difference in cytokines was observed. The native Th epitope, T1, polarized CD4+ cells toward a Th2 phenotype as early as day 3, whereas the construct containing the higher-affinity binding Th peptide T1(A) skewed the response toward Th1 (Figure 2a). In three separate experiments, IFN-γ mRNA was at least 2.5-fold greater in animals immunized with the enhanced construct. In addition, IL-4 expression was either absent or decreased approximately threefold in CD 4+ cells immunized with T1(A), and IL-10 expression was also reduced (Figure 2a). In contrast, we did not observe a significant difference in proliferation or IL-2 expression (Figure 2a and data not shown).
Higher-affinity binding peptide T1(A) skews T cells toward a Th1 response. (a) Inguinal and periaortic lymph node cells were harvested from animals immunized with PCLUS 3-18IIIB (PC3-18) or PCLUS 3(A)-18IIIB [PC3(A)-18] and stimulated overnight with the immunizing peptide at 3 μM. Total RNA was isolated from positively selected CD4+ T cells 20 hours after in vitro stimulation and semiquantitative RT-PCR performed under nonsaturating conditions to compare relative cytokine mRNA levels. These results were repeated with similar results in two additional experiments. (b) Stimulation of a T1 CD4+ T cell line with peptide T1(A) elicited greater proliferation than when stimulated with T1. A similar level of proliferation was obtained at a 30-fold lower concentration of peptide T1(A); compare proliferation at 0.1μM T1(A) versus 3μM T1. (c) IFN-γ production over 48 hours as measured by ELISA from a T1-specific CD4+ T cell clone stimulated with peptide T1 or T1(A).
The difference between the two peptides in vivo was also reflected in vitro in an established T1-specific Th1 clone for which the proliferative dose-response curve was shifted 30-fold, and IFN-γ production significantly increased when stimulated with the higher-affinity peptide T1(A) (Figure 2, b and c). Because we have observed recently that selection of IFN-γ–producing CD4+ cells require a higher threshold of signaling through the TCR (24), the increased strength of signal delivered by the higher-affinity peptide could, in part, explain these results. We hypothesized that the higher-affinity peptide T1(A) may produce a stronger CD40L signal to activate DCs to produce IL-12, producing a more polarizing APC. Subsequent experiments were designed to test this hypothesis.
Differential upregulation of CD40L on CD4+ T cells stimulated with high-affinity peptide T1(A). To examine the mechanism of improved help for CTL, we stimulated a CD4+ Th1 cell clone specific for peptide T1 with either T1 or T1(A) and examined expression of CD40L over a range of 2 logs antigen dose (Figure 3a). At the highest dose tested (5 μM), all CD4+ cells expressed CD40L regardless of stimulating peptide used; however, levels of expression were markedly higher on T cells stimulated with the higher-affinity peptide, T1(A). In addition, at decreasing doses of antigen, more CD4+cells were activated to express CD40L (at 0.05 μM peptide 68% versus 26%), and these expressed more CD40L per cell when stimulated with T1(A). The dose for inducing CD40L expression was reduced by greater than or equal to 1 log when T1(A) was used. Furthermore, this increase in CD40L expression was found when using either DCs (Figure 3a) or purified splenic B cells (Figure 3b) as APCs. These results were highly reproducible in four additional experiments. Thus, we show unexpectedly that increasing the affinity of the helper epitope for the class II MHC molecule results not only in increased numbers of activated helper T cells, but also in qualitatively different help, with more CD40L per cell and polarized to Th1 cytokines.
Qualitative and quantitative upregulation of CD40L on CD4+ T cells stimulated with T1(A) and concordant increased activation of DCs to stimulate CTL. (a) CD40L expression on T1-specific CD4+ T cells incubated overnight with different concentrations of T1 (dashed line) or T1(A) (solid line) and DCs, or without peptide (shaded area). (b) CD40L expression on gated CD4+ T1-specific T cells stimulated 36 hours with indicated concentrations of T1 or T1(A) peptide and naïve A.AL splenic B cells (B220+ selected). The bar in the left panel indicates background CD40L without peptide. (c) Percentage CD4+/ CD40L+ cells plotted versus peptide concentration for Th1 (5 × 104 CD4+) cells incubated 24 hours with 5 × 105 BALB.A10 DCs and T1 or T1(A) peptide, or no peptide (open diamond). (d) 5 × 106 DCs of the same preparation as in c were incubated with different concentrations of peptide and 5 × 105 of the same CD4+ cells, but in 12 well plates. CD4+ cells were depleted by magnetic beads 24 hours later. Remaining DCs were pulsed with 0.5 μM CTL epitope P18IIIB, irradiated, and 5 × 106 added to 5 × 106 purified CD8+ cells from spleens of 3 mice immunized 8 weeks earlier with PCLUS3-18IIIB, and rmIL-2 (10 U/ml) was added on day 0. Specific lysis 7 days later on P18IIIB-pulsed P815 targets at a 50:1 E:T ratio is plotted versus Th peptide concentration used in conditioning cultures, or without Th peptide (open diamonds). Similar results were obtained in 2 other experiments. (e) Level of CD40L expression on the Th cells as a function of peptide concentration, using less rested Th cells which were 93% CD40L+ even without stimulation. (f) P18-I10/H-2Dd tetramer staining of CD8+ cells stimulated with 1 μM P18-pulsed DC conditioned with Th cells from the same experiment as in (e) (less control 3.3% with unconditioned DCs).
Enhanced helper function through conditioning and polarizing of APCs. To determine if enhanced T helper CD40L expression was responsible for the enhanced CTL induction in vivo, we did a two-step mixing experiment in which peptide-specific CD4+ Th1 cells were stimulated overnight with different concentrations of peptide and DCs, and then the resulting DCs were used to stimulate CTL. The first stage, CD40L expression on gated CD4+ cells 20 hours after stimulation with T1 and T1(A), is shown in Figure 3c. Stimulation with the modified Th epitope T1(A) elicited more total CD40L+ T cells at all antigen concentrations (e.g., 72% vs. 50% at 1 μM peptide). There was a clear dose-response effect. Parallel cultures of the same cell preparation were then depleted of CD4+ T cells to recover the DCs, which were then pulsed with the CTL epitope peptide P18IIIB, irradiated, and mixed with CD8+ T cells enriched from spleens of animals immunized with PCLUS 3-18IIIB. The level of CTL activity induced by these conditioned DCs (Figure 3d) closely paralleled the level of CD40L expression induced on the Th cells used to condition the DCs (Figure 3c). In a separate experiment using less-rested Th cells already 93% CD40L positive, the level of CD40L per cell was increased significantly more by T1(A) than by T1 (Figure 3e), and the resulting DCs induced twice the percentage of CD8+ cells that were P18-I10/H-2Dd tetramer-positive (Figure 3f). Furthermore, blocking CD40L on the Th cell stimulated with peptide T1(A) abrogated IL-12 production by DCs (Figure 4) and reduced the CTL response (Figure 3f and see Figure 6a), formally establishing a link between the enhanced helper function and CTL induction. Thus we conclude that the epitope-enhanced helper peptide is more effective at inducing CTL because it better upregulates CD40L on the Th cell, which then more effectively conditions APCs to better activate CTL after the Th cells have been removed.
Induction of IL-12 production by DCs is dramatically increased by Th cells stimulated with higher-affinity peptide and is blocked by anti-CD40L. Triplicate cultures of 5 × 104 T1-specific CD4+ Th cell clone were stimulated with 5 × 105 DCs, and 50 hours later culture supernatants were assayed for IL-12 by ELISA. These results were highly reproducible in three separate experiments.
Blocking both B7-1 and B7-2 on DCs inhibits the CTL response. BALB.A10 DC cultures (2.5 × 10 6) conditioned for 24 hours with Th cells and peptide, T1, or T1(A) (0.5 μM), or no peptide, were depleted of CD4+ cells, pulsed for 1 hour with the CTL epitope P18IIIB (0.5 μM), irradiated, and added to 5 × 106 negatively selected splenic CD8+ cells from BALB.A10 mice primed 1 month earlier subcutaneously with PCLUS 3-18IIIB (20 nmol). (a) DCs conditioned with Th cell and T1(A) elicited higher levels of CTL than DCs conditioned with either Th cells and T1 or no peptide, or overnight treatment with trimeric murine CD40L (3 μg/ml). Lytic activity was determined on day 7 in a 5-hour 51Cr assay on 0.5 μM P18IIIB-pulsed P815 target cells (E/T = 10:1). (b) DCs were conditioned with Th cells and T1(A) (0.5 μM) for 24 hours and CD4+ cells removed. Cells were pulsed for 1.5 hours with P18IIIB, irradiated, and used to stimulate immune CD8+ cells. Anti–B7-1/B7-2 (15 μg/ml each), anti–IL-12 (15 μg/ml), anti-CCR5 (10 μg/ml), or anti-41BBL (10 μg/ml) were added to pulsed DCs at the initiation of CTL culture and on day 3. Similar results were obtained in two additional experiments.
CD40L expression on CD4+ cells stimulated with higher-affinity binding peptide T1(A) correlates with enhanced IL-12 production by DCs, resulting in polarizing DCs. To determine the molecular differences in the DCs that made those conditioned by T1(A) more effective at inducing CTLs, we examined cytokine production and surface expression of costimulatory molecules. T1-specific CD4+ Th cells were stimulated in vitro with DCs and either T1 or T1(A) and IL-12 production determined 48 hours later by ELISA for IL-12 (p40 subunit; with similar results for IL-12 p70 heterodimer, not shown). IL-12 production was increased more than threefold in cells stimulated with T1(A) compared with T1 at the optimum proliferative dose (0.5 μM peptide) (Figure 4) and significantly increased at a log lower dose (0.05 μM peptide). This effect was completely blocked by the addition of anti-CD40L Ab. Therefore, the induction of IL-12 production by DCs is dependent upon CD40L-CD40 interaction, and epitope enhancement leads to reciprocal cross talk between the T cell and DC, increasing CD40L expression on the CD4+ Th cell and IL-12 production by the DC, thus skewing Th cell phenotype toward Th1. A further implication is that one mechanism to generate polarizing APCs skewed toward Th1 induction is to increase the helper signal with a higher-affinity peptide.
Differential upregulation of costimulation on DCs by higher-affinity Th epitope T1(A). We also wanted to determine if enhanced CD40L expression contributed to an upregulation of any costimulatory molecules on the APCs. Bone marrow–derived DCs were incubated with peptide T1 or T1(A) and the T1-specific Th clone for 24 hours. Cells were gated on class II or CD11c+ and examined for costimulatory molecules, B7-1, B7-2, ICAM, CD44H, 41BBL, CCR5, and CD40. In contrast to class II expression, B7-1, B7-2, and CD40, were upregulated in cells conditioned with Th and either peptide, but B7-1 expression was greater with the higher-affinity peptide T1(A) (Figure 5a). In a subsequent experiment (Figure 5b), when we gated on CD11c+ cells, we found a marked increase in the level and numbers of cells expressing both B7-1 and B7-2 in DCs conditioned with the Th stimulated with T1(A). Although we always observed an upregulation of both ICAM and CD44H in cultures incubated with peptide compared with those without, increases were similar with either peptide, as was true for 41BBL or CCR5 (data not shown).
Differential expression of costimulatory molecules. Eight-day A.AL DC cultures (5 × 105 cells/well of 96-well round-bottom plate) grown with GM-CSF (10 ng/ml) were conditioned for 20 hours by incubation with 5 × 104 CD4+ Th cells and 0.5 μM peptide T1 or T1(A). (a) Gated class II+ cells were examined for expression of CD40 and costimulatory molecules B7-1 and B7-2 after stimulation with T1(A) (solid heavy line) compared with T1 (light line). DC cultures incubated with the Th cell without peptide are shown as the shaded area and are similar to cultures incubated without Th cells (not shown). (b) Eight-day BALB.A10 DCs were conditioned similarly for 36 hours. Gated CD11c+ shown in the first panel as the shaded area compared with PE-isotype control (dashed line) were examined for costimulatory molecules B7-1, B7-2, ICAM, and CD44H — DCs conditioned with Th cells stimulated with T1(A) (solid heavy line) versus T1 (light line). The shaded area represents DC cultures incubated with resting Th cells not stimulated with peptide. CD40 was maximally expressed under all culture conditions in this experiment. For B7-1 and 2, T1 and T1(A) are shown in separate panels for clarity. Similar results were observed in five additional experiments.
We performed blocking experiments to determine whether these molecules were necessary for the enhanced CTL induction. DCs conditioned overnight with T1(A) and T1-specific Th cells were depleted of CD4+ cells, pulsed with P18IIIB, and added to purified CD8+ cells from mice immunized with PC3-18IIIB. DCs conditioned with the T1(A) or CD40L elicited significantly higher levels of lysis than those conditioned with T1 or with Th-cells only, without peptide (Figure 6a). Furthermore, the enhanced CTL response could be blocked by the addition of anti-CD40L (10 μg/ml) during the initiation of culture, down to the level found with DCs preincubated with Th cells only, without peptide. (The high background with DCs that were not conditioned by CD4+ Th cells and peptide may be due to in vitro maturation of DCs). The addition of both anti-B7-1/B7-2 significantly decreased CTL induction (Figure 6b), where anti–IL-12, anti–41BBL, or anti–CCR5, partially reduced CTL. In six separate blocking experiments we could reproducibly inhibit CTL induction with the combination anti–B7-1/B7-2. We could not inhibit CTL with Ab to B7-1 or B7-2 alone, ICAM, CD44H (alone or in combination), or reproducibly inhibit CTL by greater than 40% with anti–IL-12, anti-41BBL, or anti-CCR5 (Figure 6b and data not shown). Thus, the major effect of the CD4+ Th cell in conditioning DCs for enhanced CTL induction occurs through CD40L-CD40 interaction and is dependent on upregulation of both B7-1 and B7-2. Upregulation of IL-12 production appears to be another major effect of conditioning, although blocking by anti–IL-12 was only partial. The increased IL-12 expression at least accounts for the DC's becoming more Th1 polarizing, but may be in sufficient excess for CTL induction that Ab blockade was incomplete. Thus, we conclude that the enhanced peptide with higher affinity for MHC class II acts by upregulating CD40L on the T helper cell, which in turn acts through CD40 on the DC to upregulate both B7-1/B7-2 and IL-12 production, thus making a more potent DC for CTL induction and also a more polarizing one for Th1 induction (Figure 7).
Mechanism of enhanced CTL induction and Th1 polarization by high-affinity peptide. Schematic representing the reciprocal interaction between the Th1 cell stimulated with either the native peptide T1 or the enhanced binding peptide T1(A) and the DC leading to induction of CTL, and to Th1 polarization.
These experiments define a mechanism of enhanced CTL induction by modification of a CD4+ T helper cell epitope. We have found not only quantitative but also qualitative improvement in the immune response through epitope enhancement and have dissected each step of the process (Figure 7). The improved peptide increases T helper activation by augmenting CD40L expression, which in turn increases activation of DCs and alters the DCs qualitatively, making them more Th1 polarizing through increased IL-12 production. The conditioned DCs, through higher IL-12 and B7-1 and B7-2, more effectively activate CTL and induce better protection against viral infection, but also feed back on the helper T cells to polarize them more toward Th1 phenotype.
We have shown that CD4+ T cells are essential for induction of CD8+ CTL responses to peptide-based vaccines (2, 7, 23), and other groups have shown a similar requirement for CD4+ help to many cell-based antigens (9, 13, 25, 26). However, it was not expected that CD4 help would be the limiting factor in improving a CD8+ CTL response, such that altering the helper epitope affinity for class II MHC, without modifying the CTL epitope presented by class I MHC, would greatly augment CTL induction. It was also not known whether such increased help would increase protection against viral infection, as we now show. These findings have important implications for vaccine design.
The mechanism of CD4+ T cell help for CTL has been clearly shown to involve activation of immature DCs to a fully mature competent state through CD40L-CD40 interaction (10–12), but the effect of the CD40L on the conditioned DC that results in increased activity has not been determined. Upregulation of costimulatory molecules has been assumed, but not yet demonstrated. In this study, we find that increasing the affinity of a Th epitope for class II MHC more efficiently upregulates CD40L on CD4+ Th 1 cells both quantitatively and qualitatively; i.e., both the number of CD40L+ cells and the level per cell. Furthermore, the level of CD40L expression was shown to correlate directly to induction of IL-12 by DCs and blocking CD40L on the Th-cell abrogated IL-12 production. Although it was known that signals delivered to DCs through CD40 can induce IL-12 production (27, 28), it was unexpected that augmentation of help through increasing the density of CD40L on helper T cells would generate polarizing DCs that would in turn skew the helper response to Th1. This may be a general approach to produce such polarizing APCs. The biological significance is supported by a recent study showing that protective anti–tumor immunity required CD40-mediated activation of DCs (29).
Thus, a key effect of enhanced Th epitope affinity is increased IL-12 production. IL-12 acts in driving the selective expansion of Th1 cells (23, 30, 31), enhances CTL proliferation (32), and may further upregulate costimulation (33). In fact, we observed that the higher-affinity peptide clearly skewed the Th phenotype to Th1. Thus, the strength of TCR signal determines levels of CD40L, which in turn determines IL-12 production by APCs and selection of a Th1 phenotype. In a two-step culture, the upregulation of CD40L on the T helper cells in the first culture directly correlated with the ability of DCs conditioned in this culture to stimulate CD8+ CTL activity in a second culture. Blockade of increased CTL induction in the second culture by treatment of the helper cells in the first culture with anti-CD40L (Figure 6a) suggests that this correlation represents cause and effect. These results complement and help to explain the mechanism by which altered peptide ligands have been shown to skew Th phenotype in the opposite direction, toward Th2 (34–38). Thus, both complementary results strongly support the conclusion that strength of signal contributes to T helper polarization.
DCs activated by CD4+ help stimulated with the higher-affinity peptide T1(A) induced somewhat higher levels of expression of B7-1 and B7-2, but not other costimulatory molecules, ICAM, CD44H, or 41BBL (39), and the resulting increase in CTL was inhibited by blocking both B7-1 and B7-2. However, the increase in B7-1 and B7-2 was modest compared with that of IL-12 production. Conditioning thus appears to represent predominantly an increase in IL-12 production and secondarily an increase in B7-1 and B7-2. IL-15 production by activated DCs may also play a role in CTL proliferation (40, 41), which we can investigate as reagents for detecting murine IL-15 become available.
The ability of DCs to stimulate antigen-primed CD8+ CTL independent of CD4 cells is dependent on the state of activation and maturation of DCs (42). Besides CD40L-CD40 interaction, inflammatory stimuli can also activate DCs for induction of CTL (13, 43, 44). Thus, DCs may be activated and CTL elicited in both CD4–dependent and CD4-independent pathways (45). A recent study (46) showed that although CD8+ CTL-mediated resolution of acute lymphocyte choriomeningitis virus infection occurred in CD40L-deficient mice, chronic infection was not controlled, suggesting a need for CD40L-CD40 interaction and a polarized CD4+ Th cell response in resolving chronic viral infections.
In summary, while exploring the mechanisms behind the epitope enhancement effect, we discovered several novel basic mechanisms by which T helper cells contribute to CTL induction and can generate polarizing APCs. Epitope enhancement to increase the affinity of a class II MHC binding peptide can dramatically enhance the efficacy of a Th-CTL peptide vaccine construct for CTL induction and protection against viral infection, both quantitatively and qualitatively. The enhanced peptide shifts the quality of the response toward Th1 by greater upregulation of CD40L on the helper T cells, which then more effectively condition and polarize the DCs by upregulation of B7-1 and B7-2 expression and IL-12 production, which improves CTL induction and skews the cytokine profile. Better understanding of the mechanism of epitope enhancement may lead to the rational design of more effective second-generation vaccines.
We thank Alfred Singer and Alan Sher for critical reading of the manuscript and helpful discussions; Patricia Earl and Bernard Moss for the recombinant vaccinia virus; Jean-Pierre Planchot for the Montanide ISA 51; and Immunex Corp. for the CD40L.
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Roddy Zambrano, the referee who took charge of the controversial Brazil v Argentina Copa America semi-final, gave an interview to 'Radio Super K-800' to explain his decisions. I wasn't called to look at the Otamendi incident, he stated.
Everything you need to know about the Liga de Expansión MX - Apertura match between CD Tapatío and Alebrijes de Oaxaca (21 September 2020): Summary, Stats, Lineups and Scores
Find all the latest news about PS Tira signings: new additions, departures, loans, reinforcements and possible signings at Besoccer.com.
Liga Bulgaria - Play Offs Descenso. Liga Bulgaria - Play Offs Descenso 2016 - Bulgaria - Live scores, results, fixtures, tables, statistics and news
Everything you need to know about the Copa Libertadores match between Guaraní and Racing Club (08 April 2015): Summary, Stats, Lineups and Scores
Watford's record-signing revealed his spectacular back tattoo on his Twitter earlier this month and the artwork features many key figures in civil rights history.
Discover the latest news from Real De Minas, the players that make up your team, live results, Table, Stats , transfers, photos and much more at BeSoccer
T. Davies (Everton). Biography, Age, team, best goals and videos, Injuries, photos and much more at BeSoccer
Everything you need to know about the S. America WC Qual. match between Chile and Bolivia (08 June 2021): Summary, Stats, Lineups and Scores
Everything you need to know about the Primera Division match between Banfield and Lanús (12 June 1949): Summary, Stats, Lineups and Scores
Buducnost Podgorica team details, competitions and latest matches
Deportivo Pereira team details, competitions and latest matches
Toulouse and Bordeaux at Club Friendlies soccer league. - Tuesday 4th August 2020
FK Zemun team details, competitions and latest matches
Angers and Reims at Ligue 1 soccer league. - Sunday 28th April 2019
Xoilac tin cap nhat
Tv101 ty le keo bong da seagame
Vatvo trực tiếp bóng đá việt nam hôm nay vtv6
Zamora FC team details, competitions and latest matches
Marseille team details, competitions and latest matches
Cercle Brugge and Standard Liege at First Division A soccer league. - Sunday 17th January 2021
Jocoro FC team details, competitions and latest matches
Torino and Bologna at Serie A soccer league. - Sunday 12th January 2020
Gent and Royal Antwerp at First Division A soccer league. - Sunday 17th January 2021


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Open Access Repositories
Open-access (OA) literature is digital, online, free of charge, and free of most copyright and licensing restrictions. What makes it possible is the Internet and the consent of the author or copyright-holder. OA is entirely compatible with peer review, and all the major OA initiatives for scientific and scholarly literature insist on its importance.
Read more about the three most important open access declarations at:
Budapest Open Access Initiative (2001)
Berlin Declaration (2003)
Bethesda Statement of Open Access Publishing (2003)
OA literature is not free to produce, even if it is less expensive to produce than conventionally published literature. The question is not whether scholarly literature can be made costless, but whether there are better ways to pay the bills than by charging readers and creating access barriers. Business models for paying the bills depend on how OA is delivered.
by Peter Suber
Depositing your work in an open access repository is another way to share your research. Be careful to comply with copyright and licensing restrictions when uploading your published work (see the "Know your Copyrights" tab in this guide). Repositories may be institutional, like Cornell's eCommons, or discipline-specific.
UVicSpace is an open access learning and research repository for published and unpublished digital scholarly works by the UVic community and its partners.
ArXiv.org is a fully automated electronic archive and distribution server for research papers for physics and related disciplines, mathematics, nonlinear sciences, computational linguistics, and neuroscience.
PubMed Central is a full-text, open access database administered by the National Library of Medicine.
Directory of Open Access Repositories. This directory provides a searchable database of academic open access repositories. Here you can search either for repositories or for repository content.
Social Science Research Network
Social Science Research Network (SSRN) is a coagulation of specialized network of scholarly publications in the social sciences covering disciplines that include accounting, economics, legal scholarship, and management, negotiation and marketing. The SSRN eLibrary consists of an abstract database of scholarly papers, that includes forthcoming publications and an ecollection of full text documents.
Cogprints
Cogprints is an open access archive of documents in the fields of psychology, neuroscience, linguistics, computer science, philosopy, biology and several scientific disciplines.
This is a very popular pre-print archive for the biological sciences and is modeled after ArXiv, the well-known physics pre-print repository. Authors wishing to submitt their pre-prints can do so by exporting their publications from UVicSpace.
<< Previous: Literature Review and Systematic Reviews
Next: Royal Society of Chemistry - Gold for Gold >>


We here at Website Cake believe that you can have your cake and eat it too, especially when it comes to your website. Our goal is to help you get the most out of your website or blog. We will help teach you how to do so through tips on blogging, web design, web hosting, and lots of other topics related to getting the most out of your sites.
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HC comes to Dal lake s rescue
till recently, restoring the pristine beauty of the famous Dal, Nagin and Aanchar lakes seemed an impossible task. But a recent court directive on the cleaning of these water bodies has rekindled hopes.
In a significant judgment, a two-member division bench of the Jammu and Kashmir High Court, comprising Chief Justice B C Patel and Justice Muzaffar Jan, directed the state government to take specific measures to give a new lease of life to the three lakes.
The court has banned all construction in the vicinity of the lakes. Washing of vehicles is also prohibited in and around the lakes. The inspector general of police (traffic) has been accorded the responsibility of taking action against those violating the order.
The Srinagar Municipality and other developmental authorities have been directed by the court to take steps to prevent the use of polythene in and around the lakes. In addition to this, the bench has also ordered the state pollution control board (pcb) to monitor the functioning of small industrial units around the lake
Dal Lake
Public Interest Litigation (PIL)
Nepal State of Environment Report: The Monthly Overview, November, 2014
Bombay High Court ultimatum to Cidco to protect mangroves
Bangladesh State of Environment Report: The Monthly Overview, April 2014
Residents wary of a 'chowpatty' coming up along Powai waterfront
Dead fish tell tales...


I completed my undergraduate degree in Biology at a small, private school, Howard Payne University, in Brownwood, Texas. I loved the small classes and the personal attention students got from their professors. By the time I finished my sophomore year, professors from several classes were encouraging me to consider graduate school. I was a little intimidated by the level of commitment required to complete a Ph.D., but it seemed to me like my Biology professors had the best job in the world and I was determined to join their ranks in academia.
At Howard Payne I double-majored in Biology and Chemistry because I was having a hard time deciding which field I would pursue for my Ph.D. I applied for an undergraduate research grant from the Chemistry Department at Baylor University for the summer after my junior year to help me make a more informed decision. I was enrolled in Vertebrate Zoology the semester I applied for the chemistry grant. By the end of the semester, I had been accepted into the summer program in chemistry, but was pretty certain that my heart was in biology. In Vertebrate Zoology we learned how to capture and identify amphibians, reptiles, and various rodents and bats. I learned so much from working in the field, from seeing the habitat of the different species first hand and trying to understand their behavior well enough to capture them. I followed through on my commitment to the chemistry research program and made some great friends in the Chemistry Department at Baylor, but the experience only further convinced me that I wanted to study biology in the field for the rest of my life, not work at a lab bench.
I attended Baylor University for my graduate work. Although not considered a large research university when compared to UT-Austin or Texas A&M, Baylor far outstripped Howard Payne in the number of professors (22 full-time professors and instructors in the Biology Department compared to 5 full-time professors in Biology at Howard Payne) and minimum class size. The largest class I ever attended at Howard Payne had less than 50 students and most classes didn't exceed 20. The limit for Baylor's freshman Biology course for majors was 125! Fortunately the labs I taught as a graduate teaching assistant never had more than 24 students.
Being at Baylor confirmed for me that I wanted to teach at a smaller school where every undergraduate has the opportunity to find a professor they connect with who can encourage them to dream big dreams and help them along the path to success. I made sure I taught a diversity of lab sections, from Plant Taxonomy to Mammalogy, so I would be prepared to teach a wider range of courses once I was professor at a small school.
I began teaching at UMHB in the fall of 2004, one year before I actually graduated at Baylor. My efforts to extend my versatility paid off; I teach a diverse range of courses including Plant Taxonomy, Ornithology, Vertebrate Zoology, and Marine Biology. I feel so blessed to be here at UMHB because most of my classes are field courses and our department and administration still support the idea of taking students into the field as much as possible. Every semester I have the opportunity to take students on field trips where they may develop the same sense of wonder and amazement I first experienced as an undergraduate at Howard Payne.


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Q: Symfony Translation using YAML files I'm currently facing a problem with translations in symfony2.
Some translations are loading, some not.
My messages.de.yml:
form:
    label:
        save: Speichern
status:
    daten_mangelhaft: Die Daten scheinen etwas mager zu sein, bitte überprüfen.

Some twig file:
{{ 'form.label.save'|trans }}

outputs (as expected):
Speichern

whereas (same twig file):
{{ 'status.daten_mangelhaft'|trans }}

outputs:
status.daten_mangelhaft

If I add it into the messages.de.yml as follows it works:
status.daten_mangelhaft: Die Daten scheinen etwas mager zu sein, bitte überprüfen.

I really don't get it, it works in some cases but not in others.
I tried:

*

*Clearing Cache

*omitting special chars (ä, ö, ü) in translations

*omitting under score in identifier

*Wrapping translations in quotes or single quotes

to no avail.
Has anyone ever had this problem before? How did you solve it?

A: Is this still accurate?
However: did you check your fallback language in your config.yml?
framework:
    translator: {fallbacks: ['de']}
    default_locale: 'de'

You can also use the locale parameter from your parameters.yml file and have:
framework:
    translator: {fallbacks: ['%locale%']}
    default_locale: '%locale%'

A: Try running php app/console translation:update after having deleted your translations (makesure you saved them). It should make a list of your needed translations.
With that you would be sure of where and what to write.

redpajama_set_name: RedPajamaStackExchange


text:
August: 5, 10 & 15 Years Ago in Corporate Governance
Corporate Governance Publisher's Note: Yes, you'll find many broken links in the material referenced below. After 5, 10 and 15 years, the internet moves on. Many of the organization's linked have since gone under. We're just glad to still be here, offering our readers a sense of the history we have shared. More about the WABAC machine.
Five Years Ago in Corporate Governance
CalPERS is believed by many, and for good reason, to be a paragon of virtue with regard to its advocacy of good corporate governance. Yet, their own election process had long been criticized as making it nearly impossible to unseat incumbents. At one point, the Board voted in favor of regulations prohibiting criticism of the Board in candidate statements, which were to be strictly limited to biographical information. To help remedy that problem I shelled out $500 to rent a hall, holding the first ever forum of CalPERS candidates. An expected winner who failed to show lost. Members finally had an opportunity to question candidates on their qualifications and their positions on the issues. These days, CalPERS is holding the forums in their auditorium. The next one is scheduled for September 16. See page 3 of Candidate Statement Booklet. For some of the latest issues, see CalPensions.
"When the banks did well, their employees were paid well. When the banks did poorly, their employees were paid well. And when the banks did very poorly, they were bailed out by taxpayers and their employees were still paid well." — New York Attorney General Andrew Cuomo explains the approach to 'performance-related' pay amongst banks. (PIRC Alerts, 8/4/09)
In an effort to increase transparency to beneficiaries, invested companies and investor peers, the SBA posts proxy voting records on its website. This real-time vote disclosure occurs in advance of all annual shareowner meetings, normally within a few hours of the proxy vote being cast. Voting information is fully searchable based on date, calendar range, company name, and SBA portfolio. Voting data covers every publicly traded equity security for which the SBA retains voting authority (which excludes most foreign securities). The SBA's current and historical proxy votes can be viewed here. Five years later and Florida SBA is still years ahead of most.
Years after one of my last contests to try to get on the CalPERS Board, the FPPC and the press are finally paying attention. In an exclusive to the Sacramento Bee, Andrew McIntosh notes Chuck Valdes' bankruptcies and $38,600 in campaign donations from companies and individuals doing business with the pension fund after the 2005 contest. "Valdes, who was chairman of the multibillion-dollar pension fund's investment committee at the time, has never had to account for how – or whether – the funds were spent because of an exemption in state regulations for retirement board members and candidates…." (FPPC probes campaign donations to CalPERS board member with money woes, 8/21/09)
Ten Years Ago in Corporate Governance
Nearly nine out of 10 (88%) companies said creating employee ownership through an ESOP (employee stockownership plan) was "a good decision that has helped the company." Asked to quantify how the presence of an ESOP improved business performance, 65% of survey respondents indicated a better performance in 2003 relative to 2002, according to the Employee Ownership Foundation's 13th Annual ESOP Economic Performance Survey.
Beginning on August 31st mutual funds are required to disclose their votes. "To help prepare investors for the new disclosure of fund voting practices," Pax World Funds, home to America's first socially responsible mutual fund, issued the following list of five key shareholder resolution categories that investors should follow (on some, we're still waiting):
Annual election of directors.
Separation of chairman of the board and CEO positions.
Risks associated with global warming.
Independent auditors.
Board diversity.
CalPERS, the biggest pension fund in the US, should take a page from its own guidelines and open a dialogue with its members on large issues. This will ensure the board doesn't stray too far from the will of its members, will help the Board solidify its base, and will better guard against political backlash… Increasing its dialogue with members on these major issues and others may just add an ounce of prevention when CalPERS directors are accused of putting their own political or personal considerations ahead of their fiduciary mandate. Of course, directors risk not getting the answers they want but reformulating a few policies will hone their skills and will ensure more wide-spread support.
In a powerful essay (Politics and money: a volatile mix, Financial Times 8/9/04) Stephen Davis, of Davis Global Advisors, calls on shareowners to form "an investor-class version of MoveOn.org, the powerful, web-based mobiliser of grassroots political activism. Without it, director election reform is jammed at the SEC."
Fifteen Years Ago in Corporate Governance
Two proxy guidelines that don't make sense. This editor has repeatedly praised Domini Social Investments (Domini) and the California Public Employees Retirement System (CalPERS) for the leading roles each has played in the field of corporate governance. Both, for example, recently began to publish their proxy votes online and both have published their voting guidelines for some time. There are widely divergent opinions among shareholder activists concerning the value added by many policies so it doesn't surprise me to find guidelines by Domini or CalPERS which I disagree with. However, two stand out: Domini's policy to vote against cumulative voting and CalPERS' policy to vote against proposals requiring director statements in support of candidacy.
Since Domini's April challenge to the fund industry for transparency in proxy votes, there is little sign that other funds are rushing to the Internet with their own voting decisions. Michael Collins of CBS MarketWatch says Investors should know how their fund votes.
Christian Science Monitor reports that 42,000 top corporate managers own a fifth of corporate America. Perhaps the Berle and Means problem is over for the smaller firms where this shift in ownership has occurred (management continues to own a little over 5% at the largest 10% of firms). Berle and Means feared diffused stockownership would bring management's control for management's benefit. Now that it appears to have happened, researchers seem to be only celebrating the alignment of shareholder and management interests. (see The boss's cut of the pie, 8/4/99)
August, CalPERS, Domini, employee ownership, Florida SBA, Pax World, PIRC, Stephen Davis
Deal Professor Equates Filing Proxy Proposals with Terrorism
United Natural Foods (UNFI) & Hain Celestial (HAIN): Win or Loss?


text:
Momentum for Tax Credit Revamp
by Peter Moreira | Jan 07, 2019
Senator Colin Deacon:
The investment tax credit discussion in Atlantic Canada has been intensifying lately, and now Senator Colin Deacon is taking up the cause.
These provincial taxation measures give private investors a tax break if they invest in approved companies. All four Atlantic provinces now offer these credits, as do most Canadian provinces and American states.
Players in the startup community have been pushing for improvements for ages – a position I've advocated for in this column several times over the years. Those calls are now intensifying. In June, a group headed by Atlantic Canada Opportunities Agency exec James Curry issued a discussion paper recommending these credits be harmonized and be offered to investors outside a company's home province. And now Deacon, a serial entrepreneur named to the Senate last summer, is pushing for similar changes.
"A good startup ecosystem needs a good angel group," said Deacon in an interview last week. "Equity tax credits are a great way for governments to leverage their investment in startups . . . through private investments."
He argues that equity tax credits (the name of the Nova Scotian program; each province has its own name for them) help to generate recurring waves of new companies year after year. That ensures the startup community keeps growing.
The main problem is that each province grants the credit only to taxpayers who live in that province. This means the credits can attract only a small pool of investors – exceptionally small in dollar terms given that there is less wealth in Atlantic Canada than in other parts of the continent.
Traditional Businesses are Linking up with Startups, to their Mutual Advantage
Curry's paper – co-written by BioNova head Scott Moffitt, Ogden Pond CEO Sean Sears, and Grant Thornton tax partner Keith MacIntyre – recommends that Atlantic Canada "regionalize" investment tax credits. That means the rules would be uniform across the region. (New Brunswick currently has the most generous tax credit by a long shot.) And it would mean investors in one province could receive a credit for funding a company in another province.
Deacon and the paper's authors even want the credits to reward investors who live outside the region. Some American states such as Arkansas and Minnesota already allow external investors to benefit from their tax credits. The thinking is that it's more important to get money into growing companies than be concerned about where the investor is based.
So far, the talk about enhanced credits is just that – talk. But there is more talk than a few years ago. (BioNova and Grant Thornton will continue the discussion with an information session in Halifax on Wednesday at noon at the Innovacorp Enterprise Centre on Summer Street.) Deacon says the discussion is intensifying because the region's startup community has proven it can generate wealth, exports and jobs, and policy makers are looking for ways to perpetuate that success.
It's hard to say what will happen. One possibility is that nothing will change – finance departments usually prefer not to give up revenue. The four provinces could harmonize and enhance their tax credits. Or possibly, the federal government could offer a tax credit, which would erase the provincial constraints.
Deacon said the long-term goal is a national tax credit.
"I'm a big believer that the federal government should have an equity tax credit nationally," said Deacon. "This is crucial to our economy."


text:
DVD Corner's blog
News, dvd and blu-ray reviews
About DVD Corner
Critters Attack! Blu-ray Review
"Critters Attack!" is the worst installment in the franchise so far.
Not to be confused with the recent Shudder mini-series "Critters: A New Binge (which was basically an episodic movie), "Critters Attack!" is a new 89 minute feature-length R rated sequel which revolves around a young woman Drea who is babysitting 2 kids for someone on the board of admissions at the local college alongside her alien obsessed brother Philip. While out on a picnic, the group discovers a cute and kind black and white creature. Shortly thereafter, they also discover the nasty, man-eating Crites have also landed in their small college town. The group is not the only ones to notice the Crites are back as the mysterious woman Aunt Dee is also on their trail.
With "Critters: A New Binge" and now "Critters Attack!," Critters fans are sure getting their fill this year. However, while 'Binge' turned out to be a surprisingly fun and tongue-in-cheek installment (hopefully that gets a home video release soon), "Critters Attack!" does not fare so well. Directed by Bobby Miller and written by Scott Lobdell, this new entry in the sci-fi-horror-comedy franchise is devoid of originality and feels slapped together. The plot is virtually non-existent, the deaths are uninspired, the dialogue is stilted, the new Crite is essentially a rip-off of "Gremlins," the cinematography is overly dark, the cast of largely no-name actors is as bland as can be, the movie looks and feels cheap, and the movie's greatest sin is that it's flat-out boring.
"Critters Attack!" does have two things going for it. For one, it's nice to see practical FX still (including the beloved Crite ball). Two, Dee Wallace (who starred in the original) returns to the franchise. Strangely though, she's barely in the movie. Even stranger, she's playing the same character but they have different names (don't ask me).
Video/Audio:
Presentation: 1.78:1 1080p. How does it look? The cheap looking movie looks cheap even in hi-def.
Audio Track: 5.1 DTS-HD MA. How does it sound? An adequate 5.1 track at best.
* DVD copy
* Digital copy
* WB trailers
* 2 featurettes titled "Engineering Gore: Designing Critters" and "Critters: An Out-Of-This World Experience" which contain behind-the-scenes look at the Crite puppets and gore FX, interviews, film discussions, set footage, etc.
* "The Critter Ball"- A featurette on the critter ball.
* "A Scene Specific Audio Commentary With Bobby Miller and Marty Krite"- A seriously goofy commentary on the climactic battle.
July 18, 2019 - Posted by	nicklyons1 | Blu-Ray review	| Crites, Critters, Critters Attack!, Critters Attack! Blu-ray Review, Dee Wallace, Horror Comedy, Sci-Fi Horror
DVDCorner.net's blog contains reviews of the latest DVD, 4K and Blu-ray discs, disc news, as well as reviews of other media items such as graphic novels, books, board/card games, and more. DVDCorner has been around for nearly 17 years now and this is our latest evolution. We are not affiliated with any other website that contains DVDCorner in its title nor do we sell DVDs or Blu-rays. We only review titles sent to us from different studios. Thanks for dropping by from all of us at DVDCorner.net.
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text:
PLN associate editor quoted in article re protest against Wells Fargo bank
Nashville Business Journal, Jan. 1, 2011. http://www.bizjournals.com/nashville/news/2011/...
PLN associate editor quoted in article re protest against Wells Fargo bank - Nashville Business Journal 2011
CCA, Wells Fargo target of demonstration in downtown Nashville
Nashville Business Journal - by Brian Reisinger, Staff Writer
Date: Friday, July 1, 2011, 1:52pm CDT
A small group of activists rallied in front of Wells Fargo bank in downtown Nashville today, calling on people to withdraw money because of ties to the private prison industry.
The protest — which drew about a dozen activists and caught the attention of passers by over the lunch hour — slammed the bank for what critics say amounts to an investment in the private prison industry. Also among the targets were Corrections Corp. of America (NYSE: CXW), the publicly traded Nashville company that is the largest player in the private prison industry.
"I encourage people to shop around and bank responsibly," said Alex Friedmann, a local critic of CCA and the private prison industry and associate editor of Prison Legal News.
The protest also included activists affiliated with the Tennessee Immigrant and Refugee Rights Coalition. Activists held signs and handed out flyers decrying the private prison industry for profiting off of incarceration, particularly immigrants who are detained.
Local Wells Fargo representatives provided material on the company's community outreach, and in a statement the bank emphasized its service to Hispanics and other immigrants. The statement also said that Wells Fargo does not directly own shares of CCA or its major competitor, Geo Group, noting that transactions tied to investment clients can "give the incorrect impression" of the bank owning stock.
"As a diversified financial services company, Wells Fargo invests on behalf of clients and customers in thousands of domestic and foreign companies," the bank said.
Activists still consider the investments the bank's responsibility, and displayed signs reading "Human Rights @ Wells Fargo? Don't bank on it!" and other criticisms.
CCA spokesman Steve Owen said private prison companies and governments are working to create "safe, humane and cost-effective" methods to deal with the country's incarceration problem "that respect the dignity of the detainees and inmates." He also said CCA does not lobby for laws affecting sentencing or immigration.
"We respect the group's right to protest, but this does nothing to help find solutions to a challenge that only grows in cost and complexity," he said in a statement.
The group said that it knew of more than $220,000 that activists would remove from Wells Fargo accounts, mainly from one large undisclosed client.