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Kidney Disease Hospitalizations, Deaths Down in United States »
"Kidney diseaseâ"related deaths and hospitalizations have declined during the last 2 decades, according to the US Renal Data System (USRDS) annual report.
The report provides an annual snapshot of data from the USRDS, a national data re"...
Read the Kidney Disease Hospitalizations, Deaths Down in United States article »
(calcium acetate) Tablet
Each white round tablet (stamped "BRA200") contains 667 mg calcium acetate, USP (anhydrous; Ca(CH3COO)2; MW=158.17 grams) equal to 169 mg (8.45 mEq) calcium, and 10 mg of the inert binder, polyethylene glycol 8000 NF. PhosLo Tablets (calcium acetate) are administered orally for the control of hyperphosphatemia in end stage renal failure.
What are the possible side effects of calcium acetate (Eliphos, PhosLo Gelcap, Phoslyra)?
Get emergency medical help if you have any of these signs of an allergic reaction: hives; difficulty breathing; swelling of your face, lips, tongue, or throat.
Less serious side effects are more likely to occur, such as:
- nausea, vomiting, loss of appetite;
- constipation;
- dry mouth or increased thirst; or
- urinating more than usual.
This is not a complete list of side effects and others may occur. Call your doctor for medical advice about side effects. You may report side effects to FDA at...
Read All Potential Side Effects and See Pictures of Phoslo »
What are the precautions when taking calcium acetate tablet (Phoslo)?
Before taking calcium acetate, tell your doctor or pharmacist if you are allergic to it; or if you have any other allergies. This product may contain inactive ingredients, which can cause allergic reactions or other problems. Talk to your pharmacist for more details.
This medication should not be used if you have a certain medical condition. Before using this medicine, consult your doctor or pharmacist if you have: a high blood calcium level (hypercalcemia).
Before using this medication, tell your doctor or pharmacist your medical history.
During pregnancy, this medication should be used only when clearly needed. Discuss the risks and benefits with your doctor.
This medication passes into breast milk but is unlikely to harm a nursing infant. Consult your...
Last reviewed on RxList: 5/27/2009
This monograph has been modified to include the generic and brand name in many instances.
Phoslo FDA Prescribing Information
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Here is a collection of user reviews for the medication Phoslo sorted by most helpful.
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Chandradhar Sharma Guleri
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Chandradhar Sharma (1883–1922) was a writer and scholar of Hindi, Sanskrit, Prakrit and Pali from Jaipur, India. Described as a versatile genius,[1] he is known as the author of Usne Kaha Tha ( Hindi उसने कहा था ), first published in 1915, which is debated to be the first short story in Hindi.[2] He is also remembered for his efforts to preserve the Jantar Mantar Observatory in Jaipur.[3]
Academic career[edit]
Pandit Guleri graduated from Allahabad University and later headed the Department of Sanskrit at Mayo College, Ajmer. In 1922, he was appointed to the Manindra Chandra Nandi Chair in Ancient History and Religion at Banaras Hindu University.[4]
To read Usne kaha tha (in Hindi and English) go to external links
- Purani Hindi (Essays on Hindi language and literature; with a sampling of early Hindi literature). Publisher: Nagariprachirini Sabha, Kashi 1948.
- Guleri Rachnavali ( Selected works). Edited by Dr. Manoharlal. Publisher: Kitab Ghar. New Delhi 1991 ISBN 81-7016-084-7.
- Guleri Katha Kahani Samagra ( Collection of short stories). Edited by Sudhakar Pandey. Publisher: Nagariprachirini Sabha, Varanasi 1994.
- Pandit Chandradhar Sharma Guleri aur unki kavitayen ( Poems in Hindi and Sanskrit) Edited by Vidyadhar Sharma Guleri. Publisher: Chinmaya Prakashan, Jaipur 1981.
- Guleri patra-sahitya:antahsakshya dvara parichaya ( Insights from the correspondence of Pandit Guleri). Edited by Jhabarmal Sharma. Publisher: Gaurav Gaatha Sangam, Delhi 1988.
Commemorative volume[edit]
- Guleri Sahityalok (Articles on works and sampling of writings; published to mark Pandit Guleri's birth centenary) by Dr. Manoharlal. Publisher: Kitab Ghar, Delhi 1984.
Edited work[edit]
- Prithvirajavijaya (Poem in Sanskrit on Prithviraja III by Jayanaka in 12th century CE and commentary of Jonaraja). Edited by Gaurishankar Hirachand Ojha and Chandradhar Sharma Guleri. First published 1941. Publisher: Rajasthani Granthaghar, Jodhpur 1997.
Stage adaptation[edit]
- Buddhu ka Kanta ( Play based on short story by Pandit Guleri) Adaptation by Ashok Kumar Hans. Publisher: Rishabacharan Jain, New Delhi 1984.
- Buddha-charita (Hindi translation of Edwin Arnold's The Light of Asia). Translated by Ram Chandra Shukla and Chandradhar Sharma Guleri. Publisher: Nagaripracharini Sabha, Kashi 1938.
Popular culture[edit]
The story Usne Kaha Tha was brought to the big screen in 1960. Produced by Bimal Roy, the film starred Sunil Dutt and Nanda and directed by Moni Bhattacharjee.[2]
Further reading[edit]
- Chandradhar Sharma Guleri, vyaktitva aur krititva ( Articles on the life and works of Pandit Guleri ). Editor Prakash Atura. Publisher: Rajasthan Sahitya Academy, Udaipur 1983.
- Pandit Chandradhar Sharma Guleri ( Contributed articles on the works of Pandit Guleri ). Chief Editor: K R Bharati, Editor: Sudarshan Vashisht. Publisher: Nirmala Publications, Delhi 2000.
- ^ Roy, Ashim Kumar (1978), History of the Jaipur City. Manohar Publications Delhi
- ^ a b "Usne Kaha Tha 1961". The Hindu. 29 November 2008.
- ^ Srivastava V S Editor (1981), Cultural contours of India p43. Abhinav Publications Delhi ISBN 0-391-02358-6
- ^ News article on 125th birth anniversary celebrations at Himvani.com
Source material (publications)[edit]
- Columbia University Library catalogue
- University of Chicago Library catalogue

subset: commoncrawl


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5 dead in California fires
San Diego: Towering wildfires burned out of control across Southern California for a third day on Tuesday as 500,000 people fled the San Diego area, and firefighters made a desperate stand to save a mountain town ringed by flames.
More than a dozen fires blazed from the horse country north of Los Angeles to the Mexican border 240 km to the south, torching 1,500 houses and other buildings, blotting out the sun with smoke and raining ash on the streets.
Most of the destroyed homes were in the San Diego area, where three major wildfires burned unchecked and half a million people were ordered to leave in what may be the largest US evacuation since Hurricane Katrina hit the Gulf Coast in 2005.
At least five deaths were reported, three of them elderly evacuees from the San Diego area, and more than three dozen others had been injured, including 18 firefighters.
As the firestorms raged into the evening, Gov. Arnold Schwarzenegger asked President George W. Bush to upgrade California's wildfires to a "major disaster," which would trigger federal help.
Bush already issued a declaration of emergency early Tuesday. But Schwarzenegger told him in a new letter that "this disaster is of such severity and magnitude that effective response is beyond the capability of the state and local governments."
Schwarzenegger said 68,000 homes, from cabins to luxury villas, were threatened statewide and 6,000 men and women were working the fire lines. More than 300,000 acres have been blackened and the state government put economic losses in the hundreds of millions.
"We have had three things come together -- very dry areas, very hot weather and a lot of wind. This makes the perfect storm for fire," Schwarzenegger said at Lake Arrowhead, where blazes threatened two nearby mountain communities.
San Diego Mayor Jerry Sanders put the number of evacuees at half a million.
Bush planned to visit the fire-stricken area on Thursday.
Running Springs, a town of about 5,000 people nestled in the San Bernardino Mountains, was surrounded by fire by Tuesday afternoon as crews made a furious effort to save homes.
California power authorities issued a transmission emergency because downed power lines had left San Diego with only 60 percent of its usual supply of electricity.
The hot Santa Ana winds, which have fanned the flames as they blow in to Southern California from the desert, continued to gust up to 105 kmph and high wind warnings remained in effect for most of the region until Wednesday afternoon.
Officials were hoping that easing winds and an accompanying rise in humidity would help them gain the upper hand against the wildfires.


text: Art.Lebedev Releases Pics of Newly-Named Optimus Maximus Vaporware KeyboardCharlie White3/15/07 3:00pmFiled to: PeripheralsArt.LebedevCeBITKeyboardsOptimusOptimus MaximusVaporware31EditPromoteShare to KinjaToggle Conversation toolsGo to permalink It turns out that picture we showed you last week of the Optimus keyboard from Art.Lebedev Studio was not a fake, but was a picture of an actual model of a keyboard that may someday actually ship. The company rolled out its official portraits of the OLED-lit keyboard just before CeBIT opened in Germany, and the gallery includes pictures of the company's trade show booth which is proudly emblazoned with the new name, Optimus Keyboards. So now the official name of this much-delayed and over-hyped keyboard is Optimus Maximus. Too bad the damn thing will have a maximus price to go along with that naming conceit, spanking early adopters with a $1490 price tag, and now the company is saying it won't be available until December of this year. If ever. Sigh. GIF Optimus Keyboards [Art.Lebedev]Reply31 repliesLeave a reply


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A sophisticated cannabis factory that produced up to £4m worth of the drug has been discovered inside a derelict building in Dartford, Kent.
Up to 6,000 cannabis plants were found in the empty office block near the centre of town in West Hill, following a tip-off on Monday when a member of the public saw someone clambering in through a window.
Police estimate the drug factory, which included extensive lighting and ventilation equipment, produced cannabis estimated at between £2m and £4m. Ian Learmonth, the chief constable of Kent Police, said it was unclear whether the drugs were intended to be used on the county's streets or elsewhere.
As well as a number of mattresses on the floor, police discovered rooms piled high with leftover soil, empty soil bags and containers of chemicals required to cultivate cannabis on an industrial scale.
Mr Learmonth said the shutting down of the operation marked a major blow to drug dealers and users. No arrests have yet been made and it is not yet clear who owns the property.
"This is a major drugs supply operation and while we don't yet know whether these drugs were to be used on Kent streets or further afield, stripping down an operation of this size will undoubtedly make a big impact on the county's drug network," Mr Learmonth said.
Officers will remain on the scene for a number of days dismantling the factory and gathering evidence.
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text:
Security Habits Differ Among Millennials, Baby Boomers
While 95 percent of Baby Boomers and 85 percent of Millennials are concerned with their personal online security, Boomers are taking more steps to secure their mobile devices, according to a Webroot survey of 201 Millennials and 204 Baby Boomers in the United States.
Despite a reputation for being less tech-savvy than Millennials, 49 percent of Boomers reported they have antivirus solutions installed on their devices—10 percent more than Millennials (39 percent).
Grayson Milbourne, security intelligence director at Webroot, told eWEEK the most surprising finding from the study was that less than half of both Millennials and Baby Boomers use security software to protect their mobile devices.
"There are many web-based threats which users need to be protected from aside from malicious apps," he explained.
Despite a reputation for being less tech savvy than Millennials, 49 percent of Baby Boomers have antivirus solutions installed on their devices.
When asked what types of personal information they would be most concerned about if a mobile device was lost, both Millennials and Boomers (79 percent each) picked banking or credit card account information as their top choice, followed by social security numbers.
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Elizacass's Traveler Profile
I have traveled to 18 countries, posted 3,480 pictures and written 152 entries in 16 travel blogs
About Elizacass
- Profession
- flight attendant
- Favourite music
- Rock, Alternative, folk
- Birthplace
- New York City
- Languages
- Hello and Goodbye, and a few other important pharses in Various Languages
- Favourite quote
- My Favorite Place is where I haven't been
- Interests and hobbies
- Travel, Music, Hiking, Backpacking, Thailand, Live music, Sushi, Spicy Food, Getting Lost...
- Favourite book
- Anything by Lonely Planet
- My friends describe me as
- Optimistic, Adventurous, and Love going off the Beaten Path
my trip advisor
My hotel reviews from my travels!
My hotel reviews from my travels!
Recent Entries by Elizacass
The Journey of just getting there.... - Jul 19, 2012
Traveling standby in the summer....some in the business would never ponder such a thought, and to add to the challenge, pick a popular destination in Europe, on a day where unfortunately the day be...
Those steps look a bit dangerous..Climbing up Coba - Mar 13, 2016
I've always been a bit of an adventure junkie. Visiting tigers in Thailand, swimming with elephants, zip lining in Honduras, staying in tree houses in Laos. So when given the opportunity to climb a...
Eat to your Yogis heart content. - Mar 11, 2016
I was lucky enough to discover Tulum many years ago. Not when huts on the beach were less than $10( that's been at least 30 years ago) . But long enough ago when it took at least 10 minutes to walk...


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Deadly Premonition Coming to XBL Games on Demand Next Week
Written Tuesday, June 28, 2011 By Richard WalkerView author's profile
Deadly Premonition completely split the critics upon its release last year, winning awards and perfect scores in some instances, while getting completely lambasted and receiving low scores like a 2 out of 10 in others. Suffice to say, people didn't quite know what to make of it, but it managed to develop a cult following regardless.
You play as FBI agent, Francis York who must solve the ritualistic, brutal murder of a local girl, while maintaining his daily personal hygiene and nutritional requirements by shaving, showering and eating, lest the townsfolk think that you smell and react to you accordingly. Along the way, you'll follow a trail of clues and bodies to supernatural creatures, eccentric locals and a floklore killer dressed in a bloody raincoat. Deadly Premonition also features side-quests and mini-games like fishing and darts.
"Deadly Premonition is a quirky and unique title we are proud to have published," says Martin Defries, managing director, Rising Star Games. "We knew there was an audience for the title and we delivered them the game they wanted. Now Deadly Premonition is on Games on Demand it will give even more people a chance to marvel at this completely individual title and decide for themselves what they think of the most controversial title of 2010."
Deadly Premonition will be coming to Games on Demand from July 5th, 2011 for an as yet unspecified price.


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A Malaysian workers' rights group has urged Cambodia to stop supplying housemaids to Malaysia, claiming many are abused and made to work like slaves because of inadequate protection.
Malaysia has recruited thousands of Cambodian women since Indonesia barred its domestic workers from coming to Malaysia in 2009 following a string of assaults by employers. Officials estimate there are more than 50,000 Cambodian maids in Malaysia.
A Cambodian maid was found dead in Malaysia last month under mysterious circumstances, while another was rescued by police after she was allegedly abused and had her head shaved by her employer.
Irene Fernandez, a director of the rights group Tenaganita, said her organisation had helped 41 Cambodian maids, some as young as 15, after they were rescued or ran away from their employers in the past six months because of abuse or unpaid wages.
"Today, another nation of domestic workers goes through the same experiences of torture, control and denial of rights," Ms Fernandez said.
Tenaganita urged Cambodia to ban maids from coming to Malaysia until tougher laws were enacted.
Malaysia is among South-east Asia's top importers of foreign labour. Many households rely on maids to cook, clean and look after children and the elderly.Reuse content


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Defective CD4+CD25+regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-γ
DOI: 10.1186/ar1500
© Kelchtermans et al.; licensee BioMed Central Ltd. 2005
Received: 8 July 2004
Accepted: 20 December 2004
Published: 28 January 2005
Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4+CD25+ Treg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of Treg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4+CD25+ T cells in the central and peripheral lymphoid tissues. In addition, CD4+CD25+ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4+CD25+ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for Treg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both Treg cells and accessory cells. Our results demonstrate that the decrease in Treg cell activity in CIA is counter-regulated by endogenous IFN-γ.
Keywordsarthritis autoimmunity interferon-γ regulatory T cells
The adaptive immune system uses various potent effector mechanisms for the elimination of foreign pathogens. Because these mechanisms are potentially damaging to the host, an essential feature of the immune system is its ability to distinguish self from non-self antigens and to develop tolerance to the former. With regard to T cell tolerance, the immune system has evolved several strategies. Most autoreactive T cells are eliminated during (primary) maturation in the thymus, a process described as negative selection, resulting in central T cell tolerance. Autoreactive T cells that escape negative selection will nevertheless be prevented from being activated as they are confronted with auto-antigen in the periphery. Several mechanisms have been proposed to account for this peripheral tolerance. One of those is suppression by a subset of T cells that express both CD4 and CD25. Evidence for the important role of these cells is overwhelming [1]. For example, when CD4+ T cells isolated from peripheral lymphoid tissues of normal mice are depleted of CD4+CD25+ T cells and injected into nu/nu mice, the recipients develop a high incidence of organ-specific autoimmune disease [2]. Co-transfer of the CD4+CD25+ population prevents the induction of disease. CD4+CD25- and CD4+CD25+ T cells are therefore often designated as, respectively, Teff and Treg cells. CD4+CD25+ Treg cells are generated in the thymus. Their development is directed by relatively high-avidity interactions between the TCR and self-peptide ligands [3–5]. The CD4+CD25+ Treg cell population constitutes 5 to 10% of the mature CD4+ cell population in the adult thymus and the peripheral lymphoid tissue and blood.
In vitro, CD4+CD25+ Treg cells inhibit polyclonal T cell activation [6, 7]. The suppression is mediated by a cytokine-independent, cell contact-dependent mechanism that requires activation of the CD4+CD25+ cells via the TCR with specific antigen [8]. However, once stimulated, they are competent to suppress in an antigen-independent manner. Although the exact mechanism by which Treg cells exert their regulatory function is still unknown, there are indications that interaction of transforming growth factor-β (TGF-β) with its receptor [9–11], inhibition of IL-2 production [6] or downregulation of co-stimulatory molecules on antigen-presenting cells [12] could be involved.
Treg cells have proved to be important in various animal models of autoimmune diseases. Administration of anti-CD25 antibody in vivo induces organ-localised autoimmune diseases [13]. Inoculation of CD4+ T cells depleted of CD25+ cells in nu/nu mice results in autoimmune diseases such as gastritis, thyroiditis and insulitis [2]. Thus, transfer of Treg cells prevents autoimmune gastritis after neonatal thymectomy, and inhibits gastritis induced by H/K ATPase-reactive effector T cells [14]. MBP-specific CD25+CD4+ T cells prevent spontaneous autoimmune encephalomyelitis in TCR-transgenic mice deficient in the recombination activating gene RAG-1 [15]. Similarly, CD4+CD25+ Treg cells suppress central nervous system inflammation during active experimental autoimmune encephalomyelitis [16].
Collagen-induced arthritis (CIA) is a well-described animal model for rheumatoid arthritis. The disease is induced in genetically susceptible DBA/1 mice by immunisation with collagen type II (CII), and both T cell and B cell autoimmune responses are required for its development [17–19]. IFN-γ receptor knock-out (IFN-γR KO) mice have been found to suffer an accelerated and more severe form of CIA [20–23]. Moreover, knocking-out of the IFN-γ gene makes genetically resistant strains of mice susceptible to CIA [24, 25]. These data indicate that deletion of the IFN-γ response somehow disrupts an endogenous protective mechanism against CIA.
Morgan and colleagues [26] have recently demonstrated that CD25+ Treg cells are important in the pathogenesis of CIA. In the present study we confirmed the importance of Treg cells in the pathogenesis of CIA by rendering wild-type DBA/1 mice deficient in Treg cells by depleting anti-CD25 antibodies. Anti-CD25-treated mice developed a significantly more severe arthritis, comparable to the disease course in IFN-γR KO mice. Thus, we proposed that the higher susceptibility of IFN-γR KO DBA/1 mice to CIA might be ascribed to defects in the production (differentiation and homeostasis) or function of these CD4+CD25+ Treg cells. We therefore determined the numbers of Treg cells in central and peripheral lymphoid organs of IFN-γR KO and wild-type mice. We further investigated whether Treg cells of IFN-γR KO mice have defects in the ability to suppress TCR-induced in vitro proliferation of CD4+CD25- Teff cells.
Materials and methods
Mice and experimental conditions
The generation and the basic characteristics of the mutant mouse strain (129/Sv/Ev) with a disruption in the gene coding for the α-chain of the IFN-γ receptor (IFN-γR KO) have been described [27]. These IFN-γR KO mice were backcrossed with DBA/1 wild-type mice for 10 generations to obtain the DBA/1 IFN-γR KO mice used in the present study. The homozygous IFN-γR KO mice were identified by PCR as described [23]. Wild-type and IFN-γR KO DBA/1 mice were bred in the Experimental Animal Centre of the University of Leuven. The experiments were performed in mice 6 to 10 weeks old, but in each experiment the mutant and wild-type mice were age-matched within 5-day limits. The male : female ratio was kept between 0.8 and 1.3 in each experiment group, unless otherwise mentioned. All animal experiments were approved by the local ethical committee (University of Leuven).
Induction and clinical assessment of arthritis
Native chicken CII (Sigma-Aldrich, St Louis, MO, USA) was dissolved at 2 mg/ml in PBS containing 0.1 M acetic acid by stirring overnight at 6°C and emulsified in an equal volume of complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA) with added heat-killed Mycobacterium butyricum (0.5 mg/ml). IFN-γR KO and wild-type mice were sensitised with a single intradermal injection at the base of the tail with 100 μl of the emulsion on day 0. From day 0 after immunisation, mice were examined for signs of arthritis five times a week. The disease severity was recorded with the following scoring system for each limb: score 0, normal; score 1, redness and/or swelling in one joint; score 2, redness and/or swelling in more than one joint; score 3, redness and/or swelling in the entire paw; score 4, deformity and/or ankylosis.
Media, reagents and antibodies
All cells were grown in RPMI 1640 (Bio Whittaker Europe, Verviers, Belgium), supplemented with 10% heat-inactivated FCS (Gibco, Paisley, UK), penicillin (100 IU/ml; Continental Pharma, Brussel, Belgium), streptomycin (100 μg/ml; Continental Pharma), 2 mM L-glutamine, 10 mM Hepes (Gibco), 0.1 mM nonessential amino acids (ICN, Asse Relegem, Belgium), 1 mM sodium pyruvate (Gibco) and 50 μM 2-mercaptoethanol (Fluka, AG, Switzerland).
Anti-CD25 IL-2Rα monoclonal antibody was produced by hybridoma PC61 in an INTEGRA CELLine CL1000 (Elscolab, Kruibeke, Belgium) and is a rat IgG1 antibody. The hybridoma supernatant was purified by Protein G-Sepharose chromatography (Amersham Biosciences, Roosendaal, The Netherlands) for administration in vivo.
The hamster monoclonal antibody, directed against the mouse CD3 complex, was prepared from the culture supernatant of 145-2C11 hybridoma cells [28]. The antibodies were purified by affinity chromatography with Protein A-Sepharose (Amersham Biosciences). Batches of anti-CD3 antibody were tested for endotoxin content with the Limulus amebocyte lysate QCL-1000 kit (Bio Whittaker) and were found to contain less than 3 ng/ml endotoxin.
Cell purification
Lymph nodes (axillary, inguinal and mesenteric) and spleens were harvested from mice 6 to 8 weeks old. Lymph nodes and spleens were gently cut into small pieces and passed through cell strainers (Becton Dickinson Labware, Franklin Lakes, NJ, USA). Red blood cells were lysed by two consecutive incubations (5 and 3 min at 37°C) of the suspension in NH4Cl (0.83% in 0.01 M Tris-HCl, pH 7.2). Remaining cells were washed, resuspended in cold PBS and counted. Lymph node preparations were then enriched for CD4+ T cells with the Mouse T cell CD4 Subset Column Kit (R&D systems, Abingdon, UK). To purify CD4+CD25+ and CD4+CD25- cells, the enriched CD4+ T cells were incubated for 20 min at 4°C with FITC-conjugated anti-CD25 and phycoerythrin (PE)-conjugated anti-CD4 antibodies (10 μg per 108 cells) in PBS containing 2% FCS. They were sorted by flow cytometry on a FACS Vantage (Becton Dickinson, San Jose, CA, USA). The resultant purity of the CD4+CD25- population was 99%, whereas the purity of the CD4+CD25+ population varied from 96% to 99%. Alternatively, CD4+ T cells were labelled with PE-conjugated anti-CD25 monoclonal antibody, followed by incubation with magnetic-activated cell sorting (MACS) anti-PE beads (CD25 Microbead Kit; Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+CD25+ T cells were selected on an LS column in a magnetic field and the flow-through was collected as CD4+CD25- T cells. After removal of the column from the magnetic field, CD4+CD25+ T cells were flushed out by a plunger. The purity of the CD4+CD25- population was 99% and the purity of the CD4+CD25+ population varied from 90% to 95%.
T cell-depleted spleen suspensions were prepared by MACS (Miltenyi Biotec) and used as accessory cells (ACs). For MACS separation, the cell suspension was magnetically labelled with CD90 (Thy1.2) microbeads and passed through a CS separation column, placed in a magnetic field. The unlabelled CD90- cells ran through.
Flow cytometry
Single-cell suspensions (5 × 105 cells) were incubated for 15 min with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (CD16/CD32; BD Biosciences Pharmingen, San Diego, CA, USA). Cells were washed with PBS containing 2% FCS and stained with the indicated FITC-conjugated antibodies (0.5 μg) for 30 min, washed twice and incubated for 30 min with the indicated PE- or biotin-conjugated antibodies. For the biotin-conjugated antibodies, a third staining step with streptavidin conjugated with peridinin chlorophyll a protein (PerCP) was performed. After washing, propidium iodide (Sigma-Aldrich) was added at a final concentration of 4 μg/ml to distinguish dead cells from living cells. Biotin-conjugated anti-CD25 (7D4), FITC-conjugated anti-CD25 (7D4), FITC-conjugated CD69 (H1.2F3), PE-conjugated anti-CD4 (RM4-5) and PerCP-conjugated streptavidin were purchased from BD Biosciences Pharmingen. FITC-conjugated anti-CD62L (MEL-14) and anti-CD44-FITC (IM7.8.1) were from CALTAG Laboratories (Burlingame, CA, USA).
For intracellular staining with anti-CTLA-4-PE (UC10-4F10-11; BD Biosciences Pharmingen), 106 cells were first labelled with anti-CD25-FITC as described above. Then, cells were fixed, permeabilised and stained with anti-CTLA-4-PE using the Cytofix/Cytoperm™ Kit (BD Biosciences Pharmingen) according to the recommendations of the manufacturers.
Flow-cytometric analysis was performed on a FACScan flow cytometer with Cell Quest software (Becton Dickinson).
Proliferation assays
CD4+CD25- cells (5 × 104 per well) were cultured in U-bottomed 96-well plates (200 μl) with ACs (5 × 104 per well, 30 Gy γ-irradiated or treated with mitomycin-C (Sigma-Aldrich)), 3 μg/ml anti-CD3 and the indicated numbers of CD4+CD25+ cells for 48 hours at 37°C in 7% CO2. Cultures were pulsed for the last 16 hours with 1 μCi of [3H]TdR and harvested. The suppressive activity of the Treg cells can be presented by plotting the percentage of inhibition (100 × (Radioactivity in condition without Treg cells – Radioactivity in condition with Treg cells)/Radioactivity in condition without Treg cells) against the number of Treg cells.
Antibody administration
DBA/1 mice were immunised with CII in CFA; 13 days after immunisation, the mice were treated every second day with 0.25 mg of anti-CD25 (PC61) or control IgG antibodies, for 4 weeks (injected intraperitoneally).
Histological examination
Forelimbs and hindlimbs were fixed in 10% formalin and decalcified with formic acid (31.5% (v/v) formic acid and 13% (w/v) sodium citrate). The paraffin sections were stained with haematoxylin and eosin.
Measurement of serum anti-CII antibodies
Blood samples were taken from the orbital sinus and were allowed to clot at room temperature for about 1 hour, and at 4°C overnight. Individual sera were tested by ELISA for antibodies directed against chicken CII. In brief, ELISA plates (Maxisorb; Nunc, Wiesbaden, Germany) were coated overnight at 4°C with native CII (1 μg/ml; 100 μl per well) in coating buffer (50 mM Tris-HCl, pH 8.5, 0.154 mM NaCl), followed by incubation for 2 hours with blocking buffer (50 mM Tris-HCl, pH 7.4, 0.154 mM NaCl and 0.1% caseine) to saturate non-specific binding sites. Serial twofold dilutions of the sera in assay buffer (50 mM Tris-HCl, pH 7.4, 154 mM NaCl and 0.05% Tween 20) were added and incubated for 2 hours at room temperature. The plates were then incubated for 2 hours with peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Finally, the substrate 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) in reaction buffer (100 mM sodium acetate/citric acid, pH 4.9) was added for a 10 min incubation and absorbance was determined at 450 nm. Plates were washed five times between each step with PBS containing 0.05% Tween 20. A serial twofold dilution series of a purified standard was included to permit a calculation of the antibody content of each sample. The standard was purified by affinity chromatography from pooled sera obtained from various arthritic wild-type and IFN-γR KO mice.
Quantitative RT-PCR
Isolated CD4+CD25+ and CD4+CD25- cells were pelleted and directly used for total RNA isolation, using the Micro-to-Midi Total RNA Purification System (Invitrogen Life Technologies, Carlsbad, CA, USA). Total RNA (1 μg) was used for random primed cDNA synthesis with RAV-2 reverse transcriptase (Amersham, Aylesbury, Bucks., UK). The reaction mixture was incubated for 80 min at 42°C and the reverse transcriptase was inactivated by incubating the cDNA samples for 5 min at 95°C.
The cDNA samples were then subjected to real-time quantitative PCR, performed in the ABI prism 7700 sequence detector (Applied Biosystems, Foster City, CA) as previously described [29]. The sequences of the forward (-FW) and reverse (-RV) primers and probes (-TP) for β-actin and Foxp3 were as follows: β-actin-FW, AGA GGG AAA TCG TGC GTG AC; β-actin-RV, CAA TAG TGA TGA CCT GGC CG T; β-actin-TP, CAC TGC CGC ATC CTC TTC CTC CC; Foxp3-FW, CCC AGG AAA GAC AGC AAC CTT; Foxp3-RV, TTC TCA CAA CCA GGC CAC TTG; Foxp3-TP, ATC CTA CCC ACT GCT GGC AAA TGG AGT C; TGF-β-FW, TGA CGT CAC TGG AGT TGT ACG G; TGF-β-RV, GGT TCA TGT CAT GGA TGG TGC; TGF-β-TP, TTC AGC GCT CAC TGC TCT TGT GAC AG. Probes were dual-labelled with 5'-FAM and 3'-TAMRA.
All primers and probes were designed with the assistance of the computer program Primer Express (AB) and were purchased from Eurogentec (Seraing, Belgium). The 5'-nuclease activity of the Taq polymerase was used to cleave a nonextendable dual-labelled fluorogenic probe. Fluorescent emission was measured continuously during the PCR reaction. PCR amplifications were performed in a total volume of 25 μl containing 5 μl of cDNA, 12.5 μl of Universal PCR Master Mix, no AmpErase UNG (AB), each primer at 100 to 300 nM, and the corresponding detection probe at 200 nM. Each PCR amplification was performed in triplicate wells under the following conditions: 94°C for 10 min, followed by 40 or 45 cycles at 94°C for 15 s and 60°C for 1 min. cDNA plasmid standards, consisting of purified plasmid DNA specific for each individual target, were used to quantify the target gene in the unknown samples, as described [29]. All results were normalised to β-actin and/or hypoxanthine–guanine phosphoribosyltransferase (HPRT) to compensate for differences in the amount of cDNA in all samples. Results were similar whether β-actin or HPRT was used as the housekeeping gene.
Effect of treatment in vivowith depleting anti-CD25 antibodies on the development of CIA in wild-type DBA/1 mice
On day 35 after immunisation, the titres of collagen-specific antibodies in the sera were determined. No differences in antibody levels in sera of mice treated with anti-CD25 or control IgG could be detected (data not shown).
Number and phenotype of CD4+CD25+ Tregcells in IFN-γR KO and wild-type mice
Proportion of regulatory T cells to the total CD4+ T cell population in lymphoid organs of naive and immunised IFN-γ receptor knock-out and wild-type (WT) DBA/1 mice
Expt no.
IFN-γR KO
3.2 ± 0.6 (5)
2.2 ± 0.7 (5)
10.1 ± 0.9 (3) *
7.4 ± 0.2 (3)
Lymph nodes
6.9 ± 1.1 (5)
5.1 ± 1.1 (5)
14.4 (1)
9.9 (1)
Lymph nodes
9.1 ± 0.9 (4)
6.6 ± 0.7 (4)
Lymph nodes
11.2 (1)
7.0 (1)
3.5 ± 0.9 (3)
4.0 ± 1.6 (3)
11.0 ± 1.3 (2)
7.9 ± 0.7 (2)
Lymph nodes
10.2 ± 0.8 (6)
7.7 ± 0.6 (6)
12.1 ± 2.9 (3)
9.2 ± 0.8 (3)
Lymph nodes
12.9 ± 1.4 (4)
10.3 ± 1.1 (4)
Lymph nodes
13.4 ± 0.4 (4) *
9.2 ± 0.7 (4)
Thus, after immunisation, IFN-γR KO mice possessed a slightly higher percentage of CD4+CD25+ cells than wild-type mice. However, the actual Treg cells present in this population might be considerably less numerous or might be qualitatively different so as to express less Foxp3.
Reduced suppressive activity of CD4+CD25+ Tregcells in arthritic IFN-γR KO mice
In a separate set of seven experiments we investigated the suppressive effect of CD4+CD25+ cells from mice that had been immunised with CII in CFA. IFN-γR KO and wild-type DBA/1 mice were immunised on day 0, and CD4+CD25+ cells, Teff cells and ACs were isolated on day 21 after immunisation. The data of the individual experiments are plotted in Fig. 4b and the means of the seven experiments are shown in Fig. 4c. It can be seen that the capacity to suppress TCR-triggered proliferation of Teff cells was significantly lower in CD4+CD25+ cells isolated from immunised mice than in those of naive animals. Indeed, to obtain 40% inhibition of proliferation, 4.5 × 103 CD4+CD25+ cells from immunised wild-type mice were required, in comparison with only 1.5 × 103 CD4+CD25+ cells from naive wild-type mice. Moreover, CD4+CD25+ cells from immunised IFN-γR KO mice were significantly less suppressive than those of immunised wild-type mice: 104 CD4+CD25+ cells were necessary to decrease Teff cell proliferation by 40%. In an additional experiment we verified whether the deficit in inhibition by CD4+CD25+ cells from immunised IFN-γR KO mice could be corrected by adding excess CD4+CD25+ cells. However, with 2 × 104 and 4 × 104 CD4+CD25+ cells the inhibition on T cell proliferation was 64.6% and 65.8%, respectively, indicating that a plateau level of suppressive activity had been reached.
Normal levels of TGF-β in IFN-γR KO and wild-type mice
Several studies have shown the critical role of TGF-β in the induction of Foxp3 and the activity of Treg cells [10, 33, 34]. Because IFN-γ and TGF-β act antagonistically with each other (reviewed in [35]), it is possible that TGF-β is upregulated in wild-type mice as a homeostatic response to IFN-γ produced by their activated T cells, and similarly in IFN-γR KO mice the decreased Foxp3 levels and the decreased suppressive activity of Treg cells might be due to inadequate amounts of TGF-β produced in the co-cultures or in vivo in mice. We therefore analysed the expression of TGF-β by quantitative PCR in Treg cells as well as in co-cultures and in spleens of naive and immunised mice. The following results were obtained. First, the levels of TGF-β from the sorted CD4+CD25+ cells from immunised IFN-γR KO mice were not different from those of wild-type mice (normalised TGF-β mRNA levels were 179 ± 16 and 193 ± 22, respectively; mean ± SEM for three measurements). Second, because TGF-β might be produced by ACs (or Teff cells), quantitative PCR was performed on cells obtained from co-cultures (Treg plus Teff plus ACs) from immunised IFN-γR KO and wild-type mice. It was found that the levels of TGF-β were even increased in IFN-γR KO cells in comparison with wild-type cells (2,184 versus 1,574, respectively, in the condition of 2 × 104 Treg cells, in a pool of eight mice). Third, the TGF-β levels were also analysed ex vivo; that is, in spleen tissue from IFN-γR KO and wild-type mice at day 21 after immunisation (thus at a time point at which Treg, Teff and ACs were isolated). Here again, the TGF-β levels were found to be slightly increased in spleens from IFN-γR KO mice (816 ± 129 and 633 ± 40 for IFN-γR KO and wild-type mice, respectively). If these results are taken together, the defective activity of Treg cells from arthritic IFN-γR KO mice (in comparison with those from wild-type animals) seems not to be associated with a defective TGF-β production.
It was notable that the TGF-β levels were higher in immunised mice than in their naive counterparts (for example, 633 ± 40 and 205 ± 19 for immunised and naive wild-type mice, respectively). These data suggest that the differences in suppressive activity of Treg cells from immunised versus naive mice cannot be explained by differences in the TGF-β production.
Tregcells from immunised IFN-γR KO mice have the capacity to inhibit proliferation responses
As expected, when all cells in the reconstituted co-cultures were of IFN-γR KO mouse origin, suppressive activity was less than when all cells were of wild-type origin. In co-cultures of mixed composition, suppressive activity of IFN-γR KO-derived CD4+CD25+ cells was less than that of the wild type only when ACs were from IFN-γR KO origin, but not when they were of wild-type origin. However, such ACs of IFN-γR KO mice were unable to reduce the suppressive effect of wild-type Treg cells against wild-type or IFN-γR KO (data not shown) Teff cells. These data demonstrate that the defect in inhibiting CD4+CD25- Teff cells acquired the presence of Treg cells from immunised IFN-γR KO mice in combination with their autologous ACs.
We and others have previously demonstrated that IFN-γ(R) KO mice show an accelerated and more severe from of arthritis than their wild-type counterparts, indicating that endogenous IFN-γ acts as a protective factor in CIA [20, 21, 24, 25]. Because CIA has been defined as a Th1-driven disease (reviewed in [17]), the protective effect of IFN-γ in CIA constitutes an enigma that compromises the Th1/Th2 paradigm as a basis for explaining the regulation of autoimmune diseases. A clue to the enigma seemed to be the use of CFA in the induction procedure of CIA. In the absence of IFN-γ, CFA induces an extensive extramedullary myelopoiesis that goes together with an even more pronounced Th1 cytokine profile than in wild-type counterparts [22, 36]. The data suggest that IFN-γ can, under certain circumstances, be a strong Th2 inducer, a finding that has recently been confirmed by others [37]. Here, we tested the hypothesis that this protective action of IFN-γ is due to a stimulatory effect on Treg cells. Specifically, we addressed the following two questions. Are Treg cells important in modulating CIA? And, because we found that depletion of Treg cells in wild-type mice increased the severity of CIA, can the higher susceptibility of IFN-γR KO mice to CIA be explained by defects in the number or function of their Treg cells?
As to the first question, we found that administration of a Treg cell-depleting anti-CD25 antibody to wild-type DBA/1 mice after CFA-assisted immunisation with CII resulted in accelerated and more severe arthritis. In fact, the disease course in these mice was comparable to that in IFN-γR KO mice [20–23]. The actual depletion of Treg cells was monitored by flow cytometry, and the authenticity of arthritis was verified histopathologically. These results are in line with those of Morgan and colleagues [26], who showed that the administration of depleting anti-CD25 antibody before immunisation (days –28, –24, –21 and –14) hastened the onset of severe CIA. Because in our experiments antibodies were administered starting from day 11 or day 13 after immunisation, we can conclude that Treg cells are important in the pathogenesis of CIA, not only in the immunisation phase but also in the effector phase. In contrast to the findings of Morgan and colleagues [26], the accelerated and more severe course of arthritis was, in our experiments, not accompanied by a higher concentration of anti-collagen II antibodies, possibly due to the different regimen of anti-CD25 treatment. Indirect evidence for the involvement of Treg cells in the pathogenesis of CIA comes from data of Min and colleagues [38]. They found that the immune tolerance induced by oral feeding of CII before induction of CIA was mediated by IL-10-producing CD4+CD25+ T cells. Notably, in proteoglycan-induced arthritis, another model of autoimmune arthritis, it has been shown that CD4+CD25+ Treg cells might not have a critical role [39]. This might result from the use of a different auto-antigen.
To address the second question, we compared CD4+CD25+ cell numbers and Treg cell function in IFN-γR KO DBA/1 mice with those in wild-type mice. According to our hypothesis we expected numbers of Treg cells in IFN-γR KO mice to be lower. Counter to this expectation, in each of the six experiments done, we found a trend for a higher proportion of CD4+CD25+ T cells in the total CD4+ cell population. This was true for thymic, splenic and lymph node CD4+ cells, in both naive and immunised mice. Analysis of all data as one set revealed a significant difference of about 30% and 20% in naive and immunised mice, respectively. CD25 is not an exclusive marker of Treg cells: especially in immunised mice, part of the CD4+CD25+ population might be effector rather than regulatory T cells [40, 41]. Therefore, to exclude the possibility that we were comparing two completely different populations, we performed additional flow-cytometric characterisation studies on pre-sorted CD4+CD25+ cells.
Expression of CD44, CD69, CTLA-4 and CD62L in CD4+CD25+ cells from IFN-γR KO mice did not differ from expression in cells from corresponding wild-type mice, whether naive or immunised. However, because Treg cells display an activated phenotype, activation markers might not be adequate to distinguish Treg cells from activated Teff cells. According to Fontenot and colleagues [42] a specific marker for Treg cells is Foxp3, because it is highly expressed in CD4+CD25+ Treg cells and is virtually undetectable in both resting and activated Teff cells. We examined Foxp3 expression by determining mRNA levels with PCR. After immunisation, CD4+CD25+ cells contained lower levels of Foxp3 mRNA than those of their naive counterparts. Moreover, mRNA levels in immunised IFN-γR KO mice were less than one-third of those in their wild-type counterparts, indicating that IFN-γR KO mice have a smaller number of Treg cells or that expression of Foxp3 in each Treg cell is lower.
Recently, Bruder and colleagues [43] have shown linked expression of neuropilin-1 and Foxp3, thereby identifying neuropilin-1 as a specific surface marker for CD4+CD25+ Treg cells able to distinguish them from both naive and recently activated CD4+CD25+ non-regulatory T cells.
Nishibori and colleagues [44] demonstrated impaired development of Treg cells in naive signal transduction and activators of transcription (STAT)-1-deficient mice, associated with an increased susceptibility to autoimmune disease. Because IFN-γ is among the strongest activators of STAT-1, these observations seem to conflict with ours. However, several cytokines, other than IFN-γ, can also activate STAT-1, including IFN-α, IFN-β, IL-6, IL-9, IL-11, oncostatin M, leukaemia inhibitory factor and the chemokines RANTES and macrophage inflammatory protein 1α [45, 46].
To determine whether overall Treg cell activity would be lower in IFN-γR KO mice, we co-cultured increasing numbers of CD4+CD25+ T cells with fixed numbers of CD4+CD25- Teff cells and ACs in the presence of anti-CD3 antibody. We observed a dose-dependent inhibition of the proliferative responses by CD4+CD25+ Treg cells. By estimating numbers of CD4+CD25+ cells required to attain a selected level of suppression, we could compare suppressive activity in the different groups of mice. In naive mice, the inhibition curves were almost identical, whether the Treg cells were derived from wild-type or IFN-γR KO mice, indicating that endogenous IFN-γ is not an important regulator of the function of constitutive CD4+CD25+ Treg cells. In co-cultures of cells from immunised wild-type mice, the Treg suppressive capacity was about one-third of that in those from corresponding naive mice, and a further halving was noted in co-cultures of cells from immunised IFN-γR KO mice.
The observation that immunisation renders Treg cells less suppressive is in line with results of Pasare and Medzhitov [47], who found that microbial triggering of the Toll-like receptor (TLR) pathway by lipopolysaccharide or CpG, which are ligands for TLR4 and TLR9, respectively, blocked the suppressive effect of CD4+CD25+ Treg cells. Because mycobacteria also contain TLR ligands, immunisation with CFA can be expected to affect Treg cell activity similarly. The decrease in suppressive activity that takes place after TLR4 or TLR9 triggering was found to be dependent on IL-6 production [47]. It might therefore be of interest to note that in our experiments, IL-6 production was enhanced after exposure to CFA-assisted immunisation, and this effect was even more pronounced in IFN-γR KO mice (P Matthys, unpublished data). This could provide an explanation for the fact that CD25+ Treg cells are totally functional before immunisation but lose (part of) their function after immunisation. However, the most important observation is the lower Treg suppressive capacity in IFN-γR KO than in wild-type mice after CFA-assisted immunisation, because this supports our hypothesis that the protective effect of endogenous IFN-γ against CIA could be mediated in part by its stimulatory effect on Treg cells.
Because the disease is barely detectable in wild-type mice on day 21 after immunisation, we investigated whether the decreased suppressive activity in immunised wild-type mice was further downregulated at a later time point (namely, day 35 after immunisation, when most of the animals show symptoms of arthritis). However, suppressive activity was not further downregulated to the level seen in homogeneous IFN-γR KO co-cultures, but was comparable to that seen in co-cultures from immunised wild-type mice on day 21 after immunisation (maximal inhibition 60%; data not shown). This indicates that the low suppressive activity as evident in immunised IFN-γR KO mice is restricted to conditions under which IFN-γ is abrogated.
The implication is that the CIA immunisation schedule induces a decrease in Treg activity and that endogenous IFN-γ largely counteracts this decrease. It therefore becomes important to know by what mechanism, direct or indirect, IFN-γ influences Treg cell function. Addition of anti-IFN-γ antibody to the co-cultures failed to affect suppressive activity (data not shown), indicating that the relevant IFN-γ effect takes place in vivo before sampling of the T cells. To examine the role of the different cell components, we tested suppressive activity in mixed co-cultures. CD4+CD25+ cells from immunised IFN-γR KO mice, confronted with Teff cells and ACs from immunised wild-type mice, were not less suppressive than wild-type CD4+CD25+ confronted with wild-type or IFN-γR KO Teff cells and ACs. This suggests that lower levels of suppression in homogeneous IFN-γR KO cultures result in part from the presence of IFN-γR KO-derived ACs. And, indeed, when CD4+CD25+ and Teff cells from immunised IFN-γR KO mice were co-cultured with ACs from immunised wild-type mice, suppressive activity was not inhibited. Finally, ACs from IFN-γR KO mice by themselves were unable to downregulate the activity of wild-type Treg cells acting on wild-type Teff cells. We therefore conclude that the in vivo effect of endogenous IFN-γ that accounts for the greater suppressive activity in wild-type mice than in IFN-γR KO mice concerns reprogramming of both ACs and Treg cells.
Because CD4+CD25+ T cells from immunised IFN-γR KO mice were not less suppressive than those of immunised wild-type mice in co-cultures with Teff cells and ACs from immunised wild-type mice, we can refute the proposition that the lower expression of Foxp3 in the CD4+CD25+ population from immunised IFN-γR KO mice is due to a smaller proportion of Treg cells and a larger number of activated Teff cells. Indeed, if the CD4+CD25+ population from immunised IFN-γR KO mice contained a higher proportion of activated Teff cells, suppression by these CD4+CD25+ cells should be lower, irrespective of the origin of the Teff cells and ACs. Another argument is that the addition of more CD4+CD25+ cells failed to improve suppression in co-cultures of cells from immunised IFN-γR KO mice. Our data are therefore more in line with the proposition of a lower Foxp3 expression level per cell.
Expression of Foxp3 could be downregulated by the interaction of Treg cells with ACs. ACs might be source of TGF-β, which has been described to convert naive T cells into CD25+ suppressor cells by inducing Foxp3 expression [48]. Because IFN-γ and TGF-β act antagonistically with each other, the low levels of Foxp3 in arthritic IFN-γR KO mice might be due to inadequate amounts of TGF-β produced by ACs or other cells. However, quantitative PCR performed on isolated Treg cells, on cells obtained from co-cultures (Treg plus Teff plus ACs) and on splenocytes from immunised IFN-γR KO and wild-type mice does not support the concept that the defective activity of Treg cells in vitro or in vivo is due to defects in the production of TGF-β. ACs have also been shown to be able to reverse suppression by CD4+CD25+ cells through the GITR/GITR-ligand system [49]. GITR (glucocorticoid-induced tumour necrosis factor receptor) is expressed on CD4+CD25+ T cells; GITR-ligand is initially upregulated on activated APCs. It remains to be determined whether this process involves a downregulation of Foxp3 expression. This or a similar mechanism might take place during the interaction of Treg cells and ACs from immunised IFN-γR KO mice. Co-cultures with ACs of immunised wild-type mice might possibly normalise Foxp3 expression in the Treg cells of immunised IFN-γR KO mice, together with their Treg suppressive activity.
In conclusion, our experiments support a pathogenesis model that ascribes an important role to Treg cells as moderators of the disease course in CIA. In particular we show that Treg cells fulfil this role not only during the induction phase but also during the effector phase of the autoimmune response. Furthermore, we were able to refine the model by showing that, after the immunisation with CII in CFA, Treg cells lose part of their suppressive potential. This effect is more pronounced in IFN-γR KO than in wild-type mice, indicating that, in this system, IFN-γ acts as an upregulator of Treg activity, which might be part of the explanation for the well-known protective effect of endogenous IFN-γ. Finally, we present evidence that the mechanism underlying the effect of IFN-γ on Treg cell activity is exerted in part via ACs.
- ACs:
= accessory cells
= complete Freund's adjuvant
= collagen-induced arthritis
= collagen type II
= cytolytic T lymphocyte-associated antigen
= enzyme-linked immunosorbent assay
fetal calf serum =
= fluorescein isothiocyanate
= glucocorticoid-induced tumour necrosis factor receptor
- IFN-γ:
= interferon-γ
- IFN-γR KO:
= interferon-γ receptor knock-out
= interleukin
= magnetic-activated cell sorting
= phosphate-buffered saline
= reverse transcriptase polymerase chain reaction
= signal transduction and activators of transcription
- Teff:
= effector T
= transforming growth factor
= Toll-like receptor
- Treg:
= regulatory T.
We thank C Dillen, E Dilissen, W Landuyt and O Rutgeerts for excellent assistance and helpful discussions. This work was supported by grants from the Fund of Scientific Research Flanders (FWO Vlaanderen), from the Regional Government of Flanders (GOA Program), and from the Belgian Federal Government (Interuniversity Network for Fundamental Research, IUAP). PM and DB are postdoctoral research fellows of the FWO Vlaanderen, and HK holds a fellowship from the FWO Vlaanderen.
Authors' Affiliations
- Fehervari Z, Sakaguchi S: A paragon of self-tolerance: CD25+CD4+ regulatory T cells and the control of immune responses. Arthritis Res Ther. 2004, 6: 19-25. 10.1186/ar1037.PubMed CentralView ArticlePubMedGoogle Scholar
- Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M: Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995, 155: 1151-1164.PubMedGoogle Scholar
- Jordan MS, Boesteanu A, Reed AJ, Petrone AL, Holenbeck AE, Lerman MA, Naji A, Caton AJ: Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat Immunol. 2001, 2: 301-306. 10.1038/86302.View ArticlePubMedGoogle Scholar
- Itoh M, Takahashi T, Sakaguchi N, Kuniyasu Y, Shimizu J, Otsuka F, Sakaguchi S: Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance. J Immunol. 1999, 162: 5317-5326.PubMedGoogle Scholar
- Maloy KJ, Powrie F: Regulatory T cells in the control of immune pathology. Nat Immunol. 2001, 2: 816-822. 10.1038/ni0901-816.View ArticlePubMedGoogle Scholar
- Thornton AM, Shevach EM: CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med. 1998, 188: 287-296. 10.1084/jem.188.2.287.PubMed CentralView ArticlePubMedGoogle Scholar
- Takahashi T, Kuniyasu Y, Toda M, Sakaguchi N, Itoh M, Iwata M, Shimizu J, Sakaguchi S: Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol. 1998, 10: 1969-1980. 10.1093/intimm/10.12.1969.View ArticlePubMedGoogle Scholar
- Read S, Powrie F: CD4+ regulatory T cells. Curr Opin Immunol. 2001, 13: 644-649. 10.1016/S0952-7915(01)00273-4.View ArticlePubMedGoogle Scholar
- Green EA, Gorelik L, McGregor CM, Tran EH, Flavell RA: CD4+CD25+ T regulatory cells control anti-islet CD8+ T cells through TGF-β–TGF-β receptor interactions in type 1 diabetes. Proc Natl Acad Sci USA. 2003, 100: 10878-10883. 10.1073/pnas.1834400100.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen W, Wahl SM: TGF-beta: the missing link in CD4+CD25+ regulatory T cell-mediated immunosuppression. Cytokine Growth Factor Rev. 2003, 14: 85-89. 10.1016/S1359-6101(03)00003-0.View ArticlePubMedGoogle Scholar
- Liu H, Hu B, Xu D, Liew FY: CD4+CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-β, and CTLA4. J Immunol. 2003, 171: 5012-5017.View ArticlePubMedGoogle Scholar
- Cederbom L, Hall H, Ivars F: CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigen-presenting cells. Eur J Immunol. 2000, 30: 1538-1543. 10.1002/1521-4141(200006)30:6<1538::AID-IMMU1538>3.0.CO;2-X.View ArticlePubMedGoogle Scholar
- Taguchi O, Takahashi T: Administration of anti-interleukin-2 receptor alpha antibody in vivo induces localized autoimmune disease. Eur J Immunol. 1996, 26: 1608-1612.View ArticlePubMedGoogle Scholar
- Suri-Payer E, Amar AZ, Thornton AM, Shevach EM: CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells. J Immunol. 1998, 160: 1212-1218.PubMedGoogle Scholar
- Hori S, Haury M, Coutinho A, Demengeot J: Specificity requirements for selection and effector functions of CD25+4+ regulatory T cells in anti-myelin basic protein T cell receptor transgenic mice. Proc Natl Acad Sci USA. 2002, 99: 8213-8218. 10.1073/pnas.122224799.PubMed CentralView ArticlePubMedGoogle Scholar
- Kohm AP, Carpentier PA, Anger HA, Miller SD: Cutting edge: CD4+CD25+ regulatory T cells suppress antigen-specific autoreactive immune responses and central nervous system inflammation during active experimental autoimmune encephalomyelitis. J Immunol. 2002, 169: 4712-4716.View ArticlePubMedGoogle Scholar
- Brand DD, Kang AH, Rosloniec EF: Immunopathogenesis of collagen arthritis. Springer Semin Immunopathol. 2003, 25: 3-18. 10.1007/s00281-003-0127-1.View ArticlePubMedGoogle Scholar
- Ehinger M, Vestberg M, Johansson AC, Johannesson M, Svensson A, Holmdahl R: Influence of CD4 or CD8 deficiency on collagen-induced arthritis. Immunology. 2001, 103: 291-300. 10.1046/j.1365-2567.2001.01257.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Ranges GE, Sriram S, Cooper SM: Prevention of type II collagen-induced arthritis by in vivo treatment with anti-L3T4. J Exp Med. 1985, 162: 1105-1110. 10.1084/jem.162.3.1105.View ArticlePubMedGoogle Scholar
- Vermeire K, Heremans H, Vandeputte M, Huang S, Billiau A, Matthys P: Accelerated collagen-induced arthritis in IFN-γ receptor-deficient mice. J Immunol. 1997, 158: 5507-5513.PubMedGoogle Scholar
- Manoury-Schwartz B, Chiocchia G, Bessis N, Abehsira-Amar O, Batteux F, Muller S, Huang S, Boissier MC, Fournier C: High susceptibility to collagen-induced arthritis in mice lacking IFN-γ receptors. J Immunol. 1997, 158: 5501-5506.PubMedGoogle Scholar
- Matthys P, Vermeire K, Mitera T, Heremans H, Huang S, Schols D, Wolf-Peeters C, Billiau A: Enhanced autoimmune arthritis in IFN-gamma receptor-deficient mice is conditioned by mycobacteria in Freund's adjuvant and by increased expansion of Mac-1+ myeloid cells. J Immunol. 1999, 163: 3503-3510.PubMedGoogle Scholar
- De Klerck B, Carpentier I, Lories RJ, Habraken Y, Piette J, Carmeliet G, Beyaert R, Billiau A, Matthys P: Enhanced osteoclast development in collagen-induced arthritis in interferon-γ receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis. Arthritis Res Ther. 2004, 6: 221-231. 10.1186/ar1167.View ArticleGoogle Scholar
- Chu CQ, Song Z, Mayton L, Wu B, Wooley PH: IFNγ deficient C57BL/6 (H-2b) mice develop collagen induced arthritis with predominant usage of T cell receptor Vβ6 and Vβ8 in arthritic joints. Ann Rheum Dis. 2003, 62: 983-990. 10.1136/ard.62.10.983.PubMed CentralView ArticlePubMedGoogle Scholar
- Guedez YB, Whittington KB, Clayton JL, Joosten LA, van de Loo FA, van den Berg WB, Rosloniec EF: Genetic ablation of interferon-γ up-regulates interleukin-1β expression and enables the elicitation of collagen-induced arthritis in a nonsusceptible mouse strain. Arthritis Rheum. 2001, 44: 2413-2424. 10.1002/1529-0131(200110)44:10<2413::AID-ART406>3.0.CO;2-E.View ArticlePubMedGoogle Scholar
- Morgan ME, Sutmuller RP, Witteveen HJ, van Duivenvoorde LM, Zanelli E, Melief CJ, Snijders A, Offringa R, de Vries RR, Toes RE: CD25+ cell depletion hastens the onset of severe disease in collagen-induced arthritis. Arthritis Rheum. 2003, 48: 1452-1460. 10.1002/art.11063.View ArticlePubMedGoogle Scholar
- Huang S, Hendriks W, Althage A, Hemmi S, Bluethmann H, Kamijo R, Vilcek J, Zinkernagel RM, Aguet M: Immune response in mice that lack the interferon-γ receptor. Science. 1993, 259: 1742-1745.View ArticlePubMedGoogle Scholar
- Leo O, Foo M, Sachs DH, Samelson LE, Bluestone JA: Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc Natl Acad Sci USA. 1987, 84: 1374-1378.PubMed CentralView ArticlePubMedGoogle Scholar
- Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C: An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods. 2001, 25: 386-401. 10.1006/meth.2001.1261.View ArticlePubMedGoogle Scholar
- Holmdahl R, Jansson L, Andersson M: Female sex hormones suppress development of collagen-induced arthritis in mice. Arthritis Rheum. 1986, 29: 1501-1509.View ArticlePubMedGoogle Scholar
- Holmdahl R, Jansson L, Larsson E, Rubin K, Klareskog L: Homologous type II collagen induces chronic and progressive arthritis in mice. Arthritis Rheum. 1986, 29: 106-113.View ArticlePubMedGoogle Scholar
- Billiau A, Matthys P: Modes of action of Freund's adjuvants in experimental models of autoimmune diseases. J Leukoc Biol. 2001, 70: 849-860.PubMedGoogle Scholar
- Nakamura K, Kitani A, Strober W: Cell contact-dependent immunosuppression by CD4+CD25+ regulatory T cells is mediated by cell surface-bound transforming growth factor β. J Exp Med. 2001, 194: 629-644. 10.1084/jem.194.5.629.PubMed CentralView ArticlePubMedGoogle Scholar
- Nakamura K, Kitani A, Fuss I, Pedersen A, Harada N, Nawata H, Strober W: TGF-β1 plays an important role in the mechanism of CD4+CD25+ regulatory T cell activity in both humans and mice. J Immunol. 2004, 172: 834-842.View ArticlePubMedGoogle Scholar
- Billiau A: Interferon-γ: biology and role in pathogenesis. Adv Immunol. 1996, 62: 61-130.View ArticlePubMedGoogle Scholar
- Matthys P, Vermeire K, Billiau A: Mac-1+ myelopoiesis induced by CFA: a clue to the paradoxical effects of IFN-γ in autoimmune disease models. Trends Immunol. 2001, 22: 367-371. 10.1016/S1471-4906(01)01937-8.View ArticlePubMedGoogle Scholar
- Bocek P, Foucras G, Paul WE: Interferon γ enhances both in vitro and in vivo priming of CD4+ T cells for IL-4 production. J Exp Med. 2004, 199: 1619-1630. 10.1084/jem.20032014.PubMed CentralView ArticlePubMedGoogle Scholar
- Min SY, Hwang SY, Park KS, Lee JS, Lee KE, Kim KW, Jung YO, Koh HJ, Do JH, Kim H, et al: Induction of IL-10-producing CD4+CD25+ T cells in animal model of collagen-induced arthritis by oral administration of type II collagen. Arthritis Res Ther. 2004, 6: R213-R219. 10.1186/ar1169.PubMed CentralView ArticlePubMedGoogle Scholar
- Bardos T, Czipri M, Vermes C, Finnegan A, Mikecz K, Zhang J: CD4+CD25+ immunoregulatory T cells may not be involved in controlling autoimmune arthritis. Arthritis Res Ther. 2003, 5: R106-R113. 10.1186/ar624.PubMed CentralView ArticlePubMedGoogle Scholar
- Nelson BH, Willerford DM: Biology of the interleukin-2 receptor. Adv Immunol. 1998, 70: 1-81.View ArticlePubMedGoogle Scholar
- Wang HM, Smith KA: The interleukin 2 receptor. Functional consequences of its bimolecular structure. J Exp Med. 1987, 166: 1055-1069. 10.1084/jem.166.4.1055.View ArticlePubMedGoogle Scholar
- Fontenot JD, Gavin MA, Rudensky AY: Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003, 4: 330-336. 10.1038/ni904.View ArticlePubMedGoogle Scholar
- Bruder D, Probst-Kepper M, Westendorf AM, Geffers R, Beissert S, Loser K, von Boehmer H, Buer J, Hansen W: Frontline: neuropilin-1: a surface marker of regulatory T cells. Eur J Immunol. 2004, 34: 1498-10.1002/eji.200490008.View ArticleGoogle Scholar
- Nishibori T, Tanabe Y, Su L, David M: Impaired development of CD4+ CD25+ regulatory T cells in the absence of STAT1: increased susceptibility to autoimmune disease. J Exp Med. 2004, 199: 25-34. 10.1084/jem.20020509.PubMed CentralView ArticlePubMedGoogle Scholar
- Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW: Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene. 2002, 285: 1-24. 10.1016/S0378-1119(02)00398-0.View ArticlePubMedGoogle Scholar
- Wong M, Fish EN: RANTES and MIP-1α activate Stats in T cells. J Biol Chem. 1998, 273: 309-314. 10.1074/jbc.273.1.309.View ArticlePubMedGoogle Scholar
- Pasare C, Medzhitov R: Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science. 2003, 299: 1033-1036. 10.1126/science.1078231.View ArticlePubMedGoogle Scholar
- Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM: Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-β induction of transcription factor Foxp3. J Exp Med. 2003, 198: 1875-1886. 10.1084/jem.20030152.PubMed CentralView ArticlePubMedGoogle Scholar
- Tone M, Tone Y, Adams E, Yates SF, Frewin MR, Cobbold SP, Waldmann H: Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proc Natl Acad Sci USA. 2003, 100: 15059-15064. 10.1073/pnas.2334901100.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
iPhone App
- Status Closed
- Budget N/A
- Total Bids 12
Project Description
We would like to make an iPhone app that will be implimented in stages.
1. It is a simple slideshow app, that people will download and the content will already be in it. So, just show the photos that it comes with.
2. We want to sell modules of other "photo packs" that people will pay for and it will download into the app. So they are like add on photo galleries to the app.Get free quotes for a project like this
Skills Required
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text:
Well as you may know Pikmin 3 is the much-awaited sequel to the GCN's Pikmin and Pikmin 2. Many fans were disappointed that the franchise skipped over the Wii but has finally returned on the Wii U, with a release of Q2 2013. Many of you may be familiar with Metroid Prime Trilogy- a compilation of 3 titles in the Metroid series, 2 from the GCN and one from the Wii. Well when thinking of that past collection-which sold out in weeks and has subsequently been discontinued- does it really seem unreasonable that after a few months have gone by since Pikmin 3's release that they combine the three entries in the series-two on the GCN and one on wii u- into one collector's edition. In Metroid Trilogy we saw the updates of motion control, widescreen presentation and achievements in the first two games. Well the first two have already been updated with motion control and wide screen presentation, both were selected as New Play Control! titles which were released on the Wii. They could also use the first control scheme that they used with gamecube controllers and put that to use with the Wii U Pro Controller too. So all Nintendo would have to do is update it to HD and alter the graphics slighty, perhaps a few other minor adjustments (achievements, concept art,etc) and it is very feasible to create one product that encompasses the entire series. Nintendo could go even further and perhaps offer Gamepad controls to the first pikmin games as it did Pikmin 3 which would be stellar, so they could give the user the option of using between three different control devices-allowing them to switch at any time. In terms of memory it would also work, the maximum of gamecube games were 1.5 gigabytes, even if you add 500 megabytes lets say for added features thats only about 3 GB of memory, then lets say that Pikmin 3 is 20 GB-a fairly large size- that still leaves room for the other two games to fit onto the 25 GB optical storage disc, or they could sell it on the Nintendo eShop, easily fitting ontoany SD card or the memory of Deluxe set owners. So Nintendo could very well create a single product that offers the entire series with graphical and controller updates (if they choose to update the controls to also use the wii u gamepad) at say $60-$70 and easily make a large profit on it. Do you think this could happen? Will it happen? Would you like to see this collection? Tell me below.
Pikmin3-full HD glory
Blurry, dull Gamecube graphics would be great redone in HD
Three great control schemes to play the games, as we've already seen the gamepad and motion control go
great with Pikmin.
Could this finally happen?


text:
Natural Pro Bodybuilder Rich Knapp's training log.
View Single Post
Rich Knapp
Wheelchair Pro
Max Brawn
Join Date: Mar 2010
Location: Appleton, WI
Posts: 6,037
Training Exp: > 13 years
Training Type: Bodybuilding
Fav Supp: AAEFX's whole line.
Reputation: 148270
Originally Posted by
Hopefully its nothing serious. Pullin' for ya!
Any updates?
Originally Posted by
hope you get better soon mate. Heal well
Originally Posted by
Any updates on the Neck/Spine issue?
I was always a fast walker. Well since in the chair I hunch over to push it faster so I don't flip over backwards.
Add the lifting and muscles healing to the mix and my muscle healed and grew pulling my neck and spine cockamamie.
A bbers worsted nightmare. Stop lifting completely and let the muscle relax and atrophy a bit so the spine can realign. During this time I have to make a conches effort to sit up straight and not hunch over in my chair.
It's been a few months since my last workout. I sold my home equipment to keep the salt out of a open wound per say. I have been working with people as they ask me.
Been active with my RC helicopters to keep the mind busy. I just finished building my Blade 550X. ( Servo's are unplugged here for ESC set up.)
Doc. may have been onto something. My neck is showing improvement. It still may be a while before I can lift again, but at least there is a light at the end of the tunnel.
Now if the Neurologist would ever call with my appointment date.
DFAC Wheelchair Rep.
"3D Muscle Journey" Sponsored Athlete
"If you think your lean enough. Your 4 weeks out, keep going."
Rich Knapp
View Public Profile
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text:
Tranz Brite Limited (formerly known as Car Brite (NZ) Ltd) began trading as an automotive detailing supplier to the transport industry in 1998. Our name was changed in 2007, in order to symbolize our expanding range of capabilities within the entire transport industry.
Tranz Brite is a 100% New Zealand family owned company, with a team of the most qualified people in their field. Our management team, collectively, have over 50 years experience in the automotive industry. The Tranz Brite Team is devoted to increasing the bar of quality & technology for the professional detailing industry of New Zealand. With this in mind, we are in constant contact with the biggest players of Products and technology from all over the world.
With a product range ranging from top quality cleaning & dressing products, to the latest in technology in automotive detailing accessories to ensure that you get the best possible presentation. Working closely with guidelines implemented by ERMA (Environmental Risk Management Authority), you can be assured that we have not only your health and safety in mind, but also the health of the environment.
Our sales and technical support team works in the field, hand-in-hand with our customers to supply technical support & sales assistance. With a comprehensive Automotive Detailing Workshop, Technical Bulletins & MSD Sheets, we provide the end user with the best possible support.
We believe that in order to succeed in a 21st century business climate; a company must be able to deliver a promise of excellent quality products in a timely fashion and at a good value to its customers. Tranz Brite is pleased to be able to meet this challenge!


text: Tank ships operated by the Alaska Tanker Company (ATC), ALASKAN NAVIGATOR and the ALASKAN FRONTIER were outfitted with new anchors and are back in service. Two other tank ships, the ALASKAN LEGAND and ALASKAN EXPLORER each had a new temporary anchor installed and are operating under restrictions outlined by the Coast Guard. The decision to replace all the anchors on these vessels were made by ATC after it was discovered the original anchors were showing signs of metal failure. The Coast Guard, Washington Department of Ecology and Alaska Tanker Co. are still investigating the cause of the failure of the anchors.


text:
David, you and Linda have been on all of our minds. You will continue to be
on them I know, and we are all hoping and praying that you and Linda will
someday be able to have a moment or two of comfort and joy again. Sending
thoughts, prayers, good vibes, and love to you and Linda.
Andrea H
Beaufort, SC
> [Original Message]
> From: David Franzman <firstname.lastname@example.com>
> To: <email@example.com>
> Date: 4/15/2005 3:30:25 AM
> Subject: [CHAT] Thank you
> Hi folks
> I'm very sorry it's taken me this long to write to you all. Fact is that
> didn't know what to say. Everything is not alright but we're certainly
> as well as we can. I have buried myself in my plants and my business
> I find that helps me to keep my mind from dwelling where it's not healthy
> dwell. I think that Theresa has probably told you everything of
relevance so
> I think you are up to date. I truly wish there was something more to add
> there isn't. Hence my reason for not having anything to say except this
> very important message:
> I have never associated myself, in my long 4(almost 5)4 years of life
with a
> more giving, loving group of people than you folks are. We received your
> cards and letters and flowers and we've been overwhelmed by your caring
> thoughtfulness. This internet thing is still a mystery for me how I could
> care about somebody I've never actually met in South Carolina more than I
> about my neighbor who I've known for 8 years. And that goes for those in
> Indiana and Illinois and Florida, Maryland, Ohio, Tennesee, Texas,
> and New York and lets not forget Idaho and the list goes on.
> So, the only other thing I can say and the only thing that has meaning
for me
> and my wife Linda is to say thank you from the bottom of our hearts.
> David
> http://www.atouchofthetropics.net
> ---------------------------------------------------------------------
> To sign-off this list, send email to email@example.com with the
> message text UNSUBSCRIBE GARDENCHAT
To sign-off this list, send email to email@example.com with the
message text UNSUBSCRIBE GARDENCHAT


text:
Firefox extensions+jquery: replacing words with textfields
This project was awarded to majid4466 for $100 USD.Get free quotes for a project like this
Project Budget$30 - $250 USD
Total Bids4
Project Description
Firefox extensions using jquery for substituting a given list of words with a text input.
Hello. I would like to have a firefox extension which does the following:
1) load jquery
2) replace words with input element of type text
3) for every text field implement a little word-matching game.
1) Load jquery (version > 1.3). I am aware that loading Jquery could be a bit complicated for FF extensions, but I would like to reuse the projects in other contextes, and work on it again. That's why I really want to use JQuery.
More information about how to include jquery here:
[url removed, login to view]
[url removed, login to view]+extension+jquery
2) When the Extension is active everytime a new page loads a function will be called.
The function replaces every instance a given word (configurable) in the body of the html document with a input element of type text.
The list of words to replace should be configurable in code, a javascript array is all what I need.
In the attached example the list of words to replace contains just the word "browser", but it should work with more words.
3) For every replacement a new "game" has been created. When the user click enter after writing in a text field, the typed word will be matched with the word which have been replaced by the text field:
if the typed word is correct, than the text field will disappear, and the word will be printed like in the original text;
if the typed word is not correct, than a javascript alert will say "You didn't guess the hidden word. Try again".
I would like to have delivered the source code of the extension.
Awarded to:
Skills Required
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- Outline your proposal
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text:
Is Evita Peroni Concept Shop incorrect or missing key business information? Update your Hotfrog listing today.
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Is Evita Peroni Concept Shop in R2, Archeology, Power Plant Mall, Amorsolo Drive, Makati Metro Manila your business? Claim your listing and attract more leads by adding more content, photos and other business details.
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text:
Posts: 32
Registered: 02-13
Posts: 17
Registered: 06-07
Posts: 110
Registered: 07-11
Diverse said:
So... nothing has changed? What was point in posting this?
Posts: 1282
Registered: 02-12
Posts: 4428
Registered: 07-12
Z86 said:
Umm.. The point is that it uses fuel now... And If i saw correctly has a faster spin mode as well
Posts: 5826
Registered: 10-12
Posts: 2209
Registered: 05-12
joe-ilya said:
There should be an ability to turn off the chainsaw. and just hit with it.
Posts: 3207
Registered: 07-03
Posts: 5618
Registered: 03-00
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text:
This cotton jersey T-shirt features a vintage flocking laurel detail on the chest for maximum impact. The short sleeves add to the casual statement and the natural Fred Perry cool.
Thank you for visiting Fred Perry Vintage Flocked Logo T-Shirt, Vintage Steel on shoppingfind.co.uk
We bring the products from many websites to one website for you to search, save and compare.


text:
The Hammerhead Wireless Bluetooth Keyboard is a great accessory for an iPad, iPhone, iPod touch and any other device that supports a Bluetooth wireless connection. Using a physical keyboard with your portable Bluetooth devices can make it easier to type emails, long text messages or notes while using your device.
What's more, the design of the keyboard is sleek and simple to mirror that of the iPhone, iPad and iPod - ensuring an aesthetic pairing as well. The keyboard is wireless and easy to pair with any Bluetooth device and you can use it from up to 30 feet away. An added convenience is that the keyboard has shortcut keys for common iOS applications, so you can launch them directly by tapping a single key.
|US keyboard layout
|Enhanced Function Keys:
|Yes, for iOS


text:
Whitefish With Garlic Butter
|Dressed white fish/Red snapper (with head and tail)
|2 Pound (Use either fresh or frozen, cod or pike)
|1 Clove (5 gm)
|6 Clove (30 gm)
|Green onions
|1⁄4 Cup (4 tbs)
Thaw fish, if frozen.
Remove head and tail, if desired.
Rub the clove of minced garlic on inside cavity of fish.
Lightly grease cooking grill.
Place fish in center of the cooking grill.
Grill 30 minutes or till fish flakes when tested with a fork.
Meanwhile, for garlic butter, in a small saucepan saute halved cloves of garlic and green onions in butter over low heat 12 to 15 minutes or till tender and golden brown.
Arrange fish on a serving platter.
Spoon garlic butter over top.
Serve with lemon wedges.
Recipe Summary
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 1261 Calories from Fat 579
% Daily Value*
Total Fat 66 g101.3%
Saturated Fat 28.9 g144.6%
Trans Fat 0 g
Cholesterol 721 mg240.3%
Sodium 813.1 mg33.9%
Total Carbohydrates 13 g4.3%
Dietary Fiber 1.6 g6.3%
Sugars 0.9 g
Protein 163 g326.3%
Vitamin A 47.4% Vitamin C 36.4%
Calcium 57.2% Iron 5.9%
*Based on a 2000 Calorie diet


text:
Developer (Live Video): PHP, MySQL, Flash, Paypal, AWS, Wowza.
- Status Closed
- Budget $3000 - $5000 USD
- Total Bids 37
Project Description
We're wanting to work with a talented developer who has significant experience building highly scalable websites (desktop and mobile) with massive amounts of traffic, that include the following:
1) Live Video Streaming
- 1-to-1 private video chats (with text chat enabled), 1-to-1 video chats (with text chat enabled) with unlimited viewers. Both paid (pay per minute) and free.
- 1-to-unlimited (with text chat enabled) amount of viewers. Both paid (pay per view) and free.
2) Paypal Adaptive Payments
3) User Management
4) Hosting on Amazon Web Services
5) Wowza Media Server
= = =
To apply, please include responses to the following questions in your reply:
1) In regards to Live Video Streaming, how long have you been working with live video streaming? Please give examples (links work great) of projects you've worked on that incorporated Live Video Streaming (video chat with text chat, event streaming with text chat). Please note if the chats/events were free or paid.
2) How much traffic did the web projects you've built receive/handle? Any numbers such as active users, live audience size, average streaming times, would be helpful.
3) In regards to Paypal Adaptive Payments, how long have you been working with Paypal Adaptive Payments? Please give examples of projects you've worked on that incorporated Paypal Adaptive Payments. Please mention the ways in which you used Paypal Adaptive Payments - example: authorizations, charging, refunds, payouts, chained payments, etc.
4) In regards to User Management, please mention any features that you've built for the admin panel to manage the site.
5) In regards to Amazon Web Services, how long have you been working with AWS? Please mention all the services you are familiar with. Example: EC2, S3, CloudFront.
6) In regards to Wowza's Media Server, how long have you been using Wowza's server? Which server in particular do you have experience with? Which add-ons do you have experience with? Example: GoCoder, Transcoder. Have you used Wowza to stream live video to mobile devices (mobile browser, iOS app, Android app)? Have you used Wowza to record live video (using iOS app or Android app) and stream it to multiple platforms?
7) Have you worked closely with an iOS Developer before, to integrate the backend, with an iOS app?
8) Have you worked closely with a designer before, to integrate the design, with a website and mobile website?
9) Which programming languages are you familiar with? Please note number of years of experience, next to each language.
10) How big is your team? (If it's just you, that's fine)
11) What is your hourly rate?
12) What is your availability? Please include days, time and timezone.
13) How do you communicate with your clients? Example: Elance messaging, Email, Skype, Google Hangouts.
14) How do you document your code?
15) How do you manage projects? Example: Basecamp, Asana, Trello.
16) Are you on Github or Stackoverflow? Please include links to your profiles if you are.
= = =
Thank you for your interest!Get free quotes for a project like this
Skills Required
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text:
It only takes a minute to get started. Fill out our uncomplicated sign up form and login to the website.
This step is optional but will greatly raise your chances of receiving views from other daters on the site. Tell people about yourself, your pasttimes and upload a nice photo of yourself.
Use our website's advanced search to spot over 60s members who match your needs. Or just browse through listings and spot folks that you find attractive.
Located someone who looks suitable? Send an email to them or just a wink and see if anything develops. With a little luck you'll be on your way to uncover that special someone for you.
It only takes a few clicks


text: Amazing Scrabble Tips With David Kwong Featured 08/01/2013 Want to defeat your opponents in Scrabble or Words With Friends? Look no further, magician David Kwong shows you how. Autoplay On Next Video Share Tweet Stumble Email Pin It Embed: Use old embed code Use new embed code Tags: scrabble tricks tips NEXT VIDEO Timelapse Catches A Murder 74 Comments Login to Comment


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Patient Comments: Spondylolisthesis - Effective Treatments
What was the treatment for your spondylolisthesis?
Comment from: So, 55-64 Female (Patient) Published: May 30
I just discovered an undiagnosed L4 dislocation. I went to several orthopedic doctors. I had MRI. It wasn't until I went to this site that I realized what in truth is wrong. I suffered severe fall 5 and a half years ago. The hospital, after I arrived via ambulance, only concentrated on my upper body, multiple fractures, dislocated shoulder and head injury. Years later, back pain became disabling. I went back to trauma doctor. He x-rayed my lower back and there it was; spondylolisthesis. Now, after MRI, steroid injections - massive facet shots, and epidural shots I am in more pain than before. I was told I need severe open back surgery with caging and brace for a year. Looking for solutions. This site is great.
Comment from: Marleyparley, 35-44 Female (Patient) Published: March 21
I began having pain 4 years ago mostly in upper left thigh and my lower back. I have seen every consultant known to man, some more than once or twice. I am on several opiate medications, muscle relaxants, tried numerous anti-depressants all of which don't even take the edge of my pain. Just over a year ago I gave up my 2 walking sticks for a wheelchair as the pain from standing/ walking was just excruciating. I gave up my job 2 years ago as I was a liability, I can do very little for myself, I no longer drive, cook, socialize, etc., it's just not worth the pain. It hurts just to be. About 2 weeks ago in desperation I went to a chiropractor who sent me for an x- ray who then informed me I had spondylolisthesis L4-5 Grade 2, I am so glad to finally have a reason for this nonstop crippling pain.
Comment from: ddriver1123, 35-44 Female (Patient) Published: February 20
I was just diagnosed with L5 grade 1 spondylolisthesis. Mine was the result of a fall at work. The pain is so bad some days and then other days I can"t tell I have a problem. I have to wear a back brace and try injections and go to a chiropractor. Hope this will this get better in time.
Comment from: Celticlove, 45-54 Female (Patient) Published: May 20
I am 49 ½ and was diagnosed with spondylolethisis, anterior grade 4, along with spinal stenosis, and bone spurs. I'm deteriorating; it has caused scoliosis and I have now been diagnosed with neuropathy in my left leg. I was hit by a car at age 4, which broke my pelvis in two places and my left femur. I was in traction, a body cast, and had to learn to walk all over again. My left leg is ¾ inches longer then my right. I was never to run again and walked with a limp, yet ended up being a very strong top athlete. I've had three kids, whom I played with and trained in sports. I just stopped playing softball five years ago. I also biked, hiked, adventured raced, and did so many outdoor things. But eight months ago, all this came to an abrupt end. I fell down some stairs, which really did some damage. When my X-rays were read, the doctors discovered my condition. September 13th, 2012, began a very fast deterioration. The hip cramps at night keep me from sleeping, the pain in my left hip travels around to my quad to my knee, down my shin, to the top of my foot, where it sometimes leaves me in tears. It's so unbearable. I can hardly walk anymore; I'm losing motor control. It's taking a toll on my speech, my focus, and my attitude. Stretching does not help, nor does icing or adjustments. Only a high dose of pain meds help, but they make me violently ill and depressed, and I sleep for two days sometimes. I'm told I will more than likely be paralyzed by 60, and I'm 49. My nerve damage is getting so severe that I may not make it another 4-5 years. I have three grandbabies, and I can't even hold them, let alone play with them. I've read about the surgeries and I am getting to the point of desiring it. At one time, I was against surgery and tried to keep people away from it with the very positive and effective body work I've done. I want my life back so bad and I'm feeling resentful and suffering major depression. I read someone else's post about how other people in their life diminish the pain they suffer. Well, you are not the only one. It frustrates me that anyone can call what we suffer minimal, undermine us, or call us hypochondriacs. I'd love to see any one of them spend time in our bodies for just a day, or maybe from the time we have to get out of bed each day up to trying to get into the shower. And then they get upset with us for how cranky we are. I'm glad that I could vent how I'm feeling. Some days I cry out of frustration. From being an athlete to "nothing." Losing 30 pounds, suffering atrophy, and muscle loss everywhere. I've lost my butt muscles and none of my clothes fit. I've even been accused of being on meth! I want my life back.
Comment from: colm, 45-54 Male (Patient) Published: April 12
I am 47 and was diagnosed with Spondylolisthesis and Spinal Stenosis 10 years ago. Although the doctors tell me that it is mild spondylolisthesis, only grade one, I am mostly in some sort of pain daily. My biggest problem is the dreaded Sciatica, I have to be so careful and avoid at all costs lifting, pulling or pushing anything around, I am a farmer so a lot of heavy work is involved daily, but I have someone to help out with the heavy work. When the doctor uses the word mild I feel like slapping him across the face and asking him how mild does that feel. Pain is pain and living with it daily can be depressing, but I refuse to let it control me, which it used to do. I do not take any medication such as anti inflammatory, this are pure poison to anyone's stomach. I would rather suffer back pain than to partake in any type of anti inflammatory drug.
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