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Elle Macpherson Quotes
Top 33 wise famous quotes and sayings by Elle Macpherson
It's always been very clear to me that I was going to be a mother and that my purpose in life was to give life.
You know, Mamet is not a huge writer of female parts. Most of his movies don't even have women in them, so I'm lucky I'm in it at all.
I don't have a problem with nudity. I never have. I was born naked. I'd like to buried naked. It's a way of life in Australia.
I choose movies, I never choose roles. I look at the script. I look at the director. I look at the other actors - and then the role.
I come from a country where you don't wear clothes most of the year. Nudity is the most natural state.
I like colourful knickers, but most importantly a great pair of knickers should be taken off with more joy than they were put on.
I feel beautiful when I'm at peace with myself. When I'm serene, when I'm a good person, when I've been considerate of others.
As long as people want to see me in movies, and as long as I find it interesting, I'll keep working.
I try to meditate every morning. It relaxes me, clears my mind, and sets my day off on the right foot before things get too manic.
I think that natural beauty is very charismatic ... I just believe in enhancing who we are rather than trying to manipulate or change it too much.
Being a public person doesn't necessarily mean you're a piece of meat for everybody.
I wanted so badly to study ballet, but it was really all about wearing the tutu.
But I do know focusing on the exterior doesn't make me happy. If I want peace and serenity, it won't be reached by getting thinner or fatter.
G-strings are uncomfortable. Girls want real knickers now.
Everything I do is about women honouring themselves, treating themselves and taking care of themselves.
I eat like a horse!
A few pounds show a lot on my body. But it's okay. I believe women look good with a bit of softness to them.
It was like a big family, since so many of us brought our kids. We all went surfing. It was so relaxed and easy.
I don't look at myself in the mirror. I'll flash past a mirror in the morning to check how I'm dressed, that's it.
For a while I was on the cover of every Sports Illustrated swimsuit issue, which was regarded as the pinnacle of success in America.
The birth of my sons. The birth of new emotions ... what a privilege. What a blessing.
For 15 years, I've been playing the same character-which is myself-and I'm bored with 'myself'.
Anyway, I believe you don't fix the inside by putting something on the outside.
I don't like everybody knowing what I'm doing, unless it's related to a movie. I don't like speaking to the press. I never did.
A celebrity name is never enough for an intelligent mass market ... truly successful businesses are born of passion and heartfelt interest.
I don't believe we own our children, they're their own souls who make their own choices, we just guide and support them.


text: Whether you're looking to expand the storage space on your smartphone, camera, or gaming system, today's deal from Sandisk could have precisely the card you're looking for, especially if you need to store 4K video or other especially large files. Several MicroSDXC cards are currently on sale at Amazon, including 256GB and 400GB cards discounted to their lowest prices yet ($28 and $64, respectively) and a 1TB card that's available for only $208 — which is just a few dollars off its best-ever pricing. Read MoreSandisk MicroSD one-day sale drops select cards to all-time lows (up to $50 off) was written by the awesome team at Android Police.

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March 5, 2020, 9:30 AM - 7:30 PM
Celebrate World Book Day with us! Come by to create your own bookmark and maybe even check out a new read.
This event is self-guided and open to all ages. All bookmark making materials are provided.
March 5, 2020, 5:00 PM - 6:30 PM
The public is invited to join us as we celebrate our recent award, being named a Texas Library of Excellence which places Converse Public Library in the top 10% of public libraries statewide!


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Your individual Medicare Supplement Open Enrollment period is the period of time in which you can purchase a Medicare Supplement from any insurance company, without having to qualify medically, pay a higher rate due to any current or former?health conditions, or wait for coverage. Open Enrollment is Federally-mandated, so every company has to abide by it.
When Is It?
This initial?enrollment period begins for each individual on the first day of the month in which they are both age 65 or older and enrolled in Medicare Part B. It lasts for a period of six months from that date.?For example, if your birthday is March?3 and you enroll in Medicare Part B?to start on March 1 of that same year, your Open Enrollment period would begin on March 1 and last through the month of August.
One thing that causes confusion in understanding when the Open Enrollment period falls is the fact that many Medicare Supplement companies also extend a six month period prior to the start of one's Open Enrollment. During this period, companies allow you to sign up for coverage that is not effective until the actual Part B?effective date (often the same as the first day of the month in which you turn 65). So in that case, an individual would have a 12-month period in which he/she could sign up with no medical underwriting.
If you have employer group health insurance, it is sometimes wise to delay enrollment in Medicare Part?B until if and when you lose or terminate that group coverage. What this does is guarantees you the ability to have an Open Enrollment period at the later time that you do sign up for Medicare Part?B.
How Does It Work?
During this period, you do not have to answer any medical questions, take a physical or discuss medications with the Medicare Supplement insurance companies.
Essentially, your application is void of medical information. This allows underwriting to be quick, and you can, in?most cases, get the coverage in a?timely manner with little?complication.?
Under no circumstances can the insurance company decline you coverage?or force you to pay a higher rate based on your past or current health.?
It is imperative that you sign?up for a Medicare Supplement plan during Open Enrollment.?Failure to do so can cause problems?for you later, if you wish to sign up then.?At that point, you would have to qualify medically (i.e. be in reasonably good health) in order to be eligible for a plan. Signing up during your initial Open Enrollment period is the only sure way to avoid that dilemma and secure?a financial future of minimized health care expenses.
About the Author: Read more about this and other topics related to sustainable living


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Yes, I just finished bumping many posts that had zero responses basically all from week before last. There were almost twice as many posts to bump than last week. If you have questions or bug reports that haven't been answered and I didn't bump, you'll need to bump it yourself - I only scan posts with no responses.
Orthodox Bishop Hilarion Alfeyev: "To be a theologian means to have experience of a personal encounter with God through prayer and worship."


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CONTEST – Find the Fake
Somewhere in each of the TownVibe magazine issues (Bedford, Berkshire, Fairfield , Litchfield, Norwalk, Ridgefield Wilton) is a fake ad. Find it and fill out the form and be entered to win! Winner receives a gift certificate to an area business.
Deadline for entry for the Holiday issue is Jan 31.
Recent Winners
More than 200 people took part in our Fake Ad Contest in the Nov-Dec issues and Winter issue for Berkshire Magazine. We have a winner for each magazine. Congratulations to everyone who picked Ken's Overalls (see below.) The winner, selected randomly from a list of all those who entered and provided the correct answer is:
1) Coni Guhl of Bedford.
2) Bryan Burns of Fairfield.
3) Mac Rand of Litchfield.
4) Paul Stoogenke of Ridgefield.
5) Sung Pak of Wilton.
Nov-Dec FAKE AD
Berkshire is Amanda Jones of Berkshire.


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Possibly aston's most important car yet. how good is it?
3y ago
"The DB11 is the most important car in Aston Martin's history" says Aston CEO Andy Palmer. That, is a statement of great importance. This Aston Martin DB11 begins what Aston call their ''Second Century Plan'' and it replaces the DB9 which has been with us for around a decade.
Absolutely mesmerising to look at. Fine design, Mr Reichman.
Aston Martin has created a new unit for the DB11 and no doubt other Aston's to come. It is a new, 5.2 lire V12 unit with 48 valves and an alloy construction. Aston says goodbye to natural aspiration through the application of two turbochargers to this new V12. It puts out 600bhp and 515lb/ft. A lot. Other than the One-77, this is Aston's most powerful car ever. 0-60mph is over in 3.7 seconds and the top speed of the DB11 is 200mph. Not slow, then. The DB11 will end up having a Mercedes 4.0 V8 option in its life cycle, but not yet.
New face, new goal. Will the DB11 be a great successor to the DB9?
The DB11 is out of this world, for me. I think that the design is appropriate to the decade rather than being something that was, now, almost a decade old. The car is like a ballet dancer, presenting itself gently and without screaming out for attention. Instead, it's just there and puts on a fantastic performance for the eyes of pedestrians or other drivers. It's unreal. Due to the different dimensional changes from the predecessor of the DB11 that is the DB9, the design has revolutionised. The wheelbase of the DB11 has increased by 65mm. The car is 28mm wider than the DB9 so not a worrying increase for those buyers who may fear narrow road traffic. These little dimension changes have allowed for Marek Reichman to go absolutely over the moon with the design process. He's done a fine job of exterior design.
Plush and mostly well made interior. Very German...
Aston Martin always had decent interiors. The old DB9 had something similar to the current Mercedes C-Class, a sort of sloping console. It was a very fine piece of interior design but then the buttons and switches mounted upon this console weren't all that beautifully played around with. The new DB11 sources some equipment from Mercedes, Aston's new partner. This includes the infotainment, switchgear and other controls within the cabin. If you've recently sat in a Mercedes, you'll recognise that this partnership is no bad thing because Merc produce very high quality stuff. The shame comes to other materials found in the cabin, though, because not everything is so fantastic. Some plastics are very unattractive and simply not nice while other bits and bops like the seat controls or the vast array of buttons that just don't seem to be all that intuitive and 'nice'. Generally though, decent work.
This angle is perhaps my favourite. Mesmerising.
The DB11 is a bit of a mixed character. Firstly, it scores 10/10 for driving fun. The V12 makes fine noises, the car pushes you thanks to RWD and all in all, the experience is very sporty. There's little to no body roll and the steering is very well adapted. It's sharp and precise. The car can feel twitchy and slightly daunting to drive hard in worse conditions but you wouldn't want to push a 600bhp RWD car in heavy rain anyway. The other side to the DB11 is its comfort. Even on low profile tires, things are managed well throughout because the seats themselves are exquisite and noise insulation is very well dealt with (Apart from some wind noise at higher speeds). After all, this is a GT car which means it needs to be a relaxing cruiser a great deal of the time. It only becomes a fine beast when you want it to and that's what's great about this class of car.
Want to be scared? Floor it.
You'll need to know something. This car is not practical. Advertised as a 2+2? Yes. Is it a 2+2? Not really. The rear seats do have ISOFIX points so are definitely somehow acceptable for children. Very small children, though. Even a junior of age 5-8 would struggle in the rear seats. Not fantastic. At least front space is sound and applicable to tall drivers. The boot is enough for a few cases but nothing of large volume. The boot only has 270 litres...
Perfect propotions thanks to careful adjustment.
I think that this new DB11 is a great car. It's a great companion that won't lose its value as quickly as some rivals and offers mixed personalities. You can drive this car comfortably and cruise to beautiful music on the Italian Riviera or thrash it around amazing roads like the Transfagarasan Highway if you so choose. The choice is yours and because of this fantastic driveability, practicality and minor quality issues can be ignored because you pay for a fantastic power train that is protected by an absolutely gorgeous body. A fine investment, Mr Palmer. Good job Aston Martin.


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January 14, 2017
While we were in New York we ran into an old friend. Bela Horvath. Bela was born in Hungary and arrived in New York 15 years ago to study with Pinchas Zukerman at the Manhattan School of Music. As he has friends in Sweden we met in Stockholm in 2006 when he was over to teach at some of the music high schools there. It coincided with Mr Zukerman's latest performance in Stockholm.
Let's hope we get the chance to play together in the near future, Bela!
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Search for: "Eric D. Morton, Attorney" Results 1 - 13 of 13
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6 Jan 2019, 6:20 pm
Attorney Eric D. [read post]
21 Mar 2020, 5:15 pm
This is a recent presentation I gave for the Attorney Action Club of San Francisco (via Zoom). [read post]
25 Oct 2011, 10:29 pm
Mike and Kellie will add considerable depth to the Law Offices of Eric D. [read post]
Illinois Appellate Court Sends Case Back to Trial Judge for Further Disposition on Relation-Back Doctrine
22 Jun 2017, 8:00 am
In response, Owens argued that under Section 2-616(d) of the Code (735 ILCS 5/2-616(d)), plaintiff's amended complaint related back to the filing of his original complaint. [read post]
UPDATE - Manhattan DA Takes a Bite Out of Crime: Steakhouse Credit Card Fraud & ID Theft Ring Served by Waiters, Grilled by Prosecutors
17 Nov 2011, 7:20 pm
D felony of Criminal Possession of a Forged Instrument in the Second Degree. [read post]
Prosecutorial Misconduct in Texas Alert: Ethical Rules Held to Have Broader Duty Than Brady to Turn Over Exculpatory Evidence to the Defense
30 Dec 2015, 9:34 am
And what are the attorneys who work for the state supposed to do? [read post]
11 Aug 2011, 12:40 pm
Obama and Attorney General Eric Holder decided earlier this year that the law, commonly known as DOMA, is unconstitutional on equal protection grounds and that the administration would no longer defend it in court. [read post]
9 Mar 2011, 6:51 am
On Digital Detectives, co-hosts Sharon D. [read post]
3 Jul 2012, 6:18 am
Attorney Rudolf Guliani put Michael Milken (another Master of the Universe) in Master of the Universe Prison. [read post]
6 Feb 2011, 1:04 pm
However, ICE Director John Morton remains undeterred"This swift action by our Homeland Security Investigations New York special agents and analysts sends a clear message to website operators who mistakenly believe it's worth the risk to take copyrighted programming and portray it as their own. [read post]
25 Nov 2013, 7:22 pm
The professors propose the following six general reforms: To discourage weak claims of patent infringement brought at least in part for nuisance value, we recommend an increase in the frequency of attorneys' fee awards to accused patent infringers who choose to fight, rather than settle, and ultimately defeat the infringement allegations levelled against them. [read post]
4 Jan 2010, 2:22 pm
Eric Affeldt, chief executive, ClubCorp Inc. [read post]
17 Jun 2019, 2:17 pm
If there is the appearance of a conflict of interest, then we will also withdraw from representation, unless both parties consent to the representation. d. [read post]


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The OtterBox has launched a new wireless portable, stackable charging base called OtterSpot. Basically, it's a wireless charging system, and each layer of the OtterSpot is a portable 5,000mAh capacity battery. Moreover with its own Qi charging coil and support for USB-C wired charging. The charging base is designed to be stackable, it means it allows you to pile up to three puck-like OtterBox Wireless Charging Batteries and a device on top of it and charges all of them at once.
While, coming to the price, it costs $129.99 and it ships with a charging base and one wireless charging battery. Besides that, each additional battery costs $69.95.
OtterSpot Charging Base
Other than these charges, OtterBox is also offering a Wireless Charging Pad, $49.95, and a Charging Stand, $59.95. The charging devices will work with Qi-enabled phones like the iPhone XS, Samsung Galaxy S10, Google Pixel 3 and others.
Since Apple canceled AirPower, there has not been an innovation but OtterBox seems to change the trend. It comes up with new modular wireless charging system for iPhone and other Qi-enabled devices. While it doesn't offer multi-device charging in the way that AirPower was shooting for, it offers what looks like a sharp experience by blending a home wireless charger with wireless portable chargers that stack and juice up all in one place.
Most of people are interested in wireless charging and since plenty of popular phone models support Qi charging, like the iPhone XS, Samsung Galaxy S10, Google Pixel 3, LG G8 ThinQ, and others. However, it's something most people only do at home or at the office, due to the fact that most chargers require a wall adapter to get the fastest charging speeds. OtterSpot aims to solve this by allowing each battery to charge at 10W speeds, if your device supports it.


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Pension committee members administer the pension plan together. Operating rules are necessary for the pension committee's business to run smoothly. These rules must be provided for in the internal by-laws adopted by the committee to facilitate effective operations.
At a minimum, the operating rules must cover the following 5 subjects:
In addition to operating rules, the pension committee's internal by-laws must include governance rules that mainly cover risk management and internal controls.
Instalment 3 of the collection Administering a pension plan well
See our Frequently Asked Questions for details on the temporary easing measure we have implemented regarding LIF withdrawals during the COVID-19 pandemic.
Please note that our LIF Quick Calc tool, which makes it possible to determine the amounts that you can withdraw from an LIF, does not take into account the measure and will not be modified during the outbreak.
See the Frequently Asked Questions for details regarding the temporary easing measures for supplemental pension plans within the context of the COVID-19 pandemic.


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Download AIA Guide to Chicago eBook
by Alice Sinkevitch
The AIA Guide to Chicago does far more than merely catalog structures of stone, steel and glass. It opens a portal on the city's soul, revealing how architects came to create the classy urban sightscape that so defines the everyday experience of Chicago.
The AIA Guide to Chicago does far more than merely catalog structures of stone, steel and glass. I found it devilishly useful. -Erik Larson, author of Devil in the White City. Chicago is justly celebrated as the birthplace of modern architecture, and the AIA Guide to Chicago has long been the premier guide to the city.
AIA Guide to Chicago Paperback – June 15, 1993. by Alice Sinkevitch (Author). From Library Journal. Even the taxi drivers in Chicago talk about architecture. But only now, a century after the World's Columbian Exposition, the city has an architectural guide on a par with the AIA Guide to New York City (Harvest: Harcourt, 1988), long the standard for this type of work.
AIA Guide to Chicago book. The most wide-ranging guide to Chicago's built environment, written under the auspices of the american Institute of Architects, the Chicago Architecture Foundation, and the Landmarks Preservation Council of Illinois. Index; black-and-white and duotoned photographs and maps. In collaboration with: American Institute of Architects, Chicago Architecture Foundation, and Landmarks Preservation Council of Illinois.
Watch the book trailer for AIA Guide to Chicago. Laurie McGovern Petersen is a writer for Chicago Architect magazine. Geoffrey Baer is the host of WTTW-TV's popular television specials about Chicago architecture and history. AIA Guide to Chicago. Perry Duis is professor emeritus of history at the University of Illinois at Chicago and the author of Challenging Chicago.
Four hundred photos and thirty-four specially commissioned maps make it easy to find each of the one thousand-plus featured buildings, while a. comprehensive index organizes buildings by name and architect.
PRAISE FOR AIA GUIDE TO CHICAGO "Excellent. An exhaustive source of information about local architecture
PRAISE FOR AIA GUIDE TO CHICAGO "Excellent. An exhaustive source of information about local architecture. A many-voiced celebration of the rich flavors of Chicago architecture. CHICAGO TRIBUNE, Even the taxi drivers in Chicago talk about architecture.
More than a thousand individual buildings are featured, along with more than four hundred photos-many taken expressly for this volume-and thirty-five specially commissioned maps. The book is arranged geographically so that the user, whether Chicago citizen or visitor, can tour each area of the city as conveniently as possible


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I get very enthusiastic when watching. I dont like pretty boys Norwa waiting for some BI mans to play with my male and me from time to time. Fire chiefs' failures led to more Grenfell Tower deaths: Scathing official report slams brigade for 'stay View Gallery View Gallery 9 Photos. Celebs all Most Read Most Recent. How come no one is asking Kising about the letter he signed in asking the then President of Ukraine to make changes Steinkjer vedo sex the Prosecutors office Who What Wear.
More plans and pricing. Edit with RF Editor.|The photograph, obtained by MailOnline, is being used in Massage rosemount Molde prevention classes in Norway to teach asylum seekers appropriate sexual behaviour following the recent attacks in Cologne and Sinnliche massage Sandefjord in Europe.
In classes which may be replicated across the continent, male pupils, mostly of Arab and North African origin, are taught that skimpy clothes do not provide an excuse for rape or abuse. Scroll down for video. Migrant men attending rape prevention classes in Norway are shown this photo of an attractive woman sitting in a short skirt to teach them this is not an invitation to have sex.
Instruction: Linda Hagen address Syrian and Sudanese asylum seekers during a courses to prevent violence against women in Ha, near Stavanger, Norway. The headline reads: 'perception of women and cultural codes'. Speaking Picture man woman kissing in Norway to MailOnline, Linda Hagen, the leader of the course, has revealed the challenges she faces in trying to change the mindset of migrant men who have perpetrated sexual assault.
Participants in the course often make comments like 'she kissed him, it's an invitation to have sex', and Bodo latin escorts she wants come Stavanger massage soi twilight my place, that means she's consenting. In response, Ms Hagen explains that Western Picture man woman kissing in Norway are different to those found in some Islamic countries.
Norway has been offering these courses for several years, but in the wake of the New Year's Eve mass sex attacks in Cologne, Germany, other countries affected have expressed an interest in. In Cologne, police reports were made, including for Picture man woman kissing in Norway Porsgrunn personals services that police have blamed on Arab and North African men.]Download this Couple Kissing On The Preikestolen In Norway photo.
And search more of iStock's library of royalty-free stock images. David Beckham defied the trolls and Piers Morgan to kiss daughter England beat Norway to make it to the semi-final of the Women's Speaking on an Instagram live, the former Man United ace said: 'I kiss all my kids on the lips.
Harper cheered on the Lionesses (Picture: Dave Shopland/BPI/REX). Sami girl kisses boy. norwegian funny humor Kizsing www.potamkinusa.com Download this stock image: Norway, mature woman kissing man forehead - XBWR


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Dry weather forecast for city
BAHAWALPUR: The Met Office has forecast a dry weather for Bahawalpur during next 24 hours.
The highest maximum temperatures 33 Centigrade and the lowest minimum 17 Centigrade degrees were forecast for next 24 hours.
The Met Office has also forecast a dry weather for other parts of the region during next 24 hours.


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How long do we have to wait until driverless race cars are a reality? Not too long, as these futuristic autonomous driverless machines are coming – and they're coming quick!
Designed by Daniel Simon, who created many lit sci-fi cars for Hollywood, it's called the Robocar. It is the world's first driverless race car and is set to compete against other Robocars in an all-electric racing series taking place next to the Formula E championship.
The cabin-free, high-downforce racer comes equipped with some very clever tech and makes the new generation of Formula One cars look like massive fails!
Weighing just 975kg, the carbon fibre prototype is 4.8 metres long and 2 metres wide – not too crazy. The crazy part comes in the form of the four electric motors (300kW each) and a 540kW battery that creates enough buzz to get the Robocar to 320 km/h while running on Michelin road tyres. Maybe it's safer that no one is on board driving!
While normally the smarts of a race car come from the fleshy human controlling the madness onboard, here the 'brain' is the Nvidia Drive PX2, a digital 'engine' capable of up to 24 trillion artificial intelligence operations per second! Working in sync with five light detectors, two radars, 18 ultrasonic sensors, two optical speed sensors, and six AI cameras, the PX2 helps the Robocars from smashing into each other – or the spectators!
All races will last about an hour and will have ten teams running two cars each. The competition element comes from the Robo teams programming their cars with different software to 'drive' differently and make them the quickest on track day!
Bringing robots and humans together for racing could be sci-fi good or Terminator bad. We love watching humans burning rubber to rubber, but seeing these machines programming the hell out of each other on the track would be cool – as long as they don't short circuit and chase us!


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Reality Television
Does anyone have a list of production companies or managers who specialize in reality television. I have a concept I want to pitch, but don't know the proper channels to follow for something like this. Thanks in advance.
Re: Reality Television
There are thousands of reality ideas being pushed at any one time so emphasize your point of difference and universal entertainment value. I'm not aware of managers who specialize in this area - there may be. Not many prodcos specialize just in reality. You may also want to look at major reality franchisers such as Freemantle.
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Innate immunity relies on pattern recognition receptors to detect the presence of infectious pathogens. In the case of Gram-positive bacteria, binding of bacterial lipopeptides to TLR2 is currently regarded as an important mechanism. In the present study, we used the synthetic bacterial lipopeptide Pam3CysSK4, a selective TLR2 agonist, to induce meningeal inflammation in rodents. In a 6-h rat model, intrathecal application of Pam3CysSK4 caused influx of leukocytes into the cerebrospinal fluid (CSF) and induced a marked increase of regional cerebral blood flow and intracranial pressure. In wild-type mice, we observed CSF pleocytosis and an increased number of apoptotic neurons in the dentate gyrus 24 h after intrathecal challenge. Inflammation and associated neuronal loss were absent in TLR2 knockout mice. In purified neurons, cytotoxicity of Pam3CysSK4 itself was not observed. Exposure of microglia to Pam3CysSK4 induced neurotoxic properties in the supernatant of wild-type, but not TLR2-deficient microglia. We conclude that TLR2-mediated signaling is sufficient to induce the host-dependent key features of acute bacterial meningitis. Therefore, synthetic lipopeptides are a highly specific tool to study mechanisms of TLR2-driven neurodegeneration in vivo.
The innate immune system recognizes bacterial molecules and generates an inflammatory response to invading pathogens. In recent years, important progress has been made in understanding how specific receptors of the immune system recognize pathogen-associated molecular patterns to induce immune response (1, 2). A highly relevant class of pattern recognition receptors is the family of TLRs, of which 12 have been found in mammals (2). In the case of Gram-positive bacteria, components of the bacterial cell wall interact with TLR2. Although the exact molecular motif required for binding has not been identified, it appears that bacterial lipopeptides and teichoicated peptidoglycans (PGs)4 are the major ligands. Controversy exists whether TLR2 is also capable of sensing lipoteichoic acid-depleted PG. However, other components of Gram-positive bacteria may be recognized by other TLRs, e.g., bacterial nucleic acids via TLR9, or they may bind to non-TLRs, e.g., the muramyl dipeptide of PGs to NOD2 receptors (3, 4). Diversity at the level of the intracellular adaptor proteins adds further complexity. Most of the evidence discussed above was gathered in vitro, mostly involving overexpressing systems, which may not truly model the in vivo situation. Indeed, experiments in TLR2 knockout mice and several infection models often failed to show a pronounced anti-inflammatory phenotype, suggesting redundancy in microbial pattern recognition (5, 6, 7, 8, 9).
Lipoproteins are present in the cell wall of all bacteria and may represent a general principle for how bacteria induce an immune response and tissue damage. Other than the complex macromolecular structure of the cell wall backbone, the basic structure of lipopeptides, an unusual S-propyl-substituted cysteine carrying three lipid residues plus a variable amino acid chain, has been chemically synthesized. Synthetic lipopeptides allow us to study TLR2-driven inflammation without the confounding effects of contaminants such as bacterial toxins, metabolic products, or other cell wall components. In previous studies, the synthetic bacterial lipopeptide Pam3CysSK4 (N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2R,S)-propyl)-(R)-cysteinyl-seryl-(lysyl)3- lysine) has been identified as a specific TLR2 agonist (10) acting mainly through TLR2/1 heterodimeric receptors (11). In the present study, we used Pam3CysSK4 to study TLR2-driven inflammation and neuronal damage in experimental meningitis as an important example of invasive bacterial infection. For comparison with a viable pathogen, meningitis was induced in further animals by intrathecal inoculation with live encapsulated Streptococcus pneumoniae, the most common microorganism in bacterial meningitis.
Materials and Methods
Bacterial culture
D39, an encapsulated strain of S. pneumoniae serotype 2 originally derived from a clinical isolate, was grown in standard casein plus yeast medium with 5% CO2 at 37°C. During log phase growth, bacteria were pelleted by centrifugation and resuspended in pyrogen-free 0.1 M PBS. CFU per milliliter were then determined photometrically (absorption at 620 nm) using a standard curve. Adequate dilutions in pyrogen-free PBS were used to produce defined inocula. CFU calculations were verified by plating of serial dilutions.
Rat experiments
All animal experimental procedures were reviewed by institutional and state authorities. The general experimental procedure was as described earlier (12). The experiments were performed on male Wistar rats (280–330 g) anesthetized with i.p. thiopental sodium (Byk Gulden). A dose of 100 mg/kg was used for induction of anesthesia, while 20 mg/kg was given for maintenance at ∼2-h intervals (total dose per animal, 160–180 mg/kg). Adequate anesthesia was verified by absence of response to a painful stimulus. Animals were tracheotomized and mechanically ventilated (model AP-10; K. Effenberger). End-tidal CO2 was monitored continuously (Heyer Artema MM204). Body temperature was measured by a rectal probe and maintained at 37.8 ± 0.4°C using a heating pad. Mean arterial blood pressure was measured continuously by a transducer (Statham P109-EZ; Spectramed) connected to a catheter placed in the left femoral artery. From this catheter, arterial blood samples were analyzed for PaO2, PaCO2, and pH at 0, 2, 4, and 6 h. The left femoral vein was cannulated for infusion of saline as a volume replacement. A 3×3-mm area of the parietal bone lateral to the sagittal suture was thinned to allow laser Doppler flow (LDF) measurements (Periflux 4001 Master; Perimed) in cortical blood vessels. A catheter was placed into the cisterna magna through an occipital burr hole and connected to a pressure transducer (Statham P109-EZ; Spectramed) for continuous intracranial pressure (ICP) measurement. The 100 μl of cerebrospinal fluid (CSF) removed from this catheter was replaced with 100 μl of Pam3CysSK4 (1 mg/ml; EMC Microcollections) or with 100 μl of bacterial suspension containing 107 CFU. Controls received 100 μl of endotoxin-free PBS. At the end of the experiment, CSF samples were obtained to determine the CSF leukocyte count. In experiments involving live bacteria, CFU in the CSF were determined by the plating of serial dilutions on blood agar plates. Animals were then killed by i.v. injection of 3 M potassium chloride to induce cardiac arrest.
Mouse model of meningitis
Mouse experiments were conducted using a previously published model (12, 13). In brief, male C57BL/6 mice (∼20 g) or TLR2−/− mice (14) were anesthetized with i.p. ketamine (100 mg/kg; DeltaSelect) and xylazine (20 mg/kg; Bayer). A skin incision was made to expose the lumbar spine. Using a 30-gauge needle and a microliter syringe, 40 μl of PBS, Pam3CysSK4 solution or bacterial suspension containing 5 × 105 CFU were slowly injected into the spinal canal at vertebrae L2 or L3. The skin incision was closed using dermal clips. Animals were then allowed to wake up and given free access to food and water. Absence of paresis and adequate waking were verified. After 24 h, the animals were again deeply anesthetized. To obtain a CSF sample, a skin incision was made over the head and neck. After dissection of the suboccipital muscles under a preparation microscope, the cisterna magna was punctured and CSF withdrawn using a 27-gauge butterfly cannula connected to a microliter syringe. White blood cells were then counted in the CSF using a Fuchs-Rosenthal chamber at ×400 magnification, and CFU per milliliter were determined as discussed when appropriate. Brains were removed, snap frozen in methylbutane on dry ice, and stored at −80°C until further processing.
Cryostat sections (20 μM) were thaw-mounted on coated glass slides, including the hippocampal structures at several levels. For the evaluation of neuronal damage, we performed TdT-TUNEL using a commercially available kit (Qbiogene). Labeled sites were visualized using a FITC-conjugated Ab. Sections were counterstained using Hoechst 33258 (1/10,000; Invitrogen Life Technologies). For quantification, the TUNEL-positive nuclei in the dentate gyrus were enumerated on all sections using a fluorescent microscope at ×1000 magnification. Cell count was divided by the area of the dentate gyrus as determined using Stereo Investigator software (version 4.0; MicroBrightField).
Generation of primary neuronal and glial cell cultures
Primary rat neuronal cultures were prepared from the cerebral cortex of embryonic day 18 rat embryos according to a previously published protocol (15). Following dissociation, 150,000 cells/cm2 were seeded into 24-well plates pretreated with poly-l-lysine (0.5% w/v in PBS, 1 h at room temperature) and coating medium (dissociation medium with 0.03‰ w/v collagen G, 1 h at 37°C). The cultures were maintained at 36.5°C with 5% CO2 in neurobasal medium with B27 supplement (Invitrogen Life Technologies) and fed every 4 days by replacing one-third of the medium. Cultures typically consist of >90% neurons, <10% astrocytes, and <1% microglia until 14 days in vitro (16). Experiments on rat neuronal cultures were conducted after 5–8 days in vitro.
Primary cultures of purified murine cortical neurons were generated from forebrains of embryonic day 17 mice, as previously described (17). Briefly, cortices were dissociated by trituration with papain (Cell Systems) in Earle's balanced salt solution (Invitrogen Life Technologies) for 5 min at 37°C. Subsequently, cells were resuspended in 0.25% trypsin inhibitor and 0.25% BSA (both from Sigma-Aldrich) in Earle's balanced salt solution, and incubated at 37°C for 5 min. Cells were pelleted by centrifugation at 1000 × g for 5 min and were plated onto poly-d-lysine-coated glass slides. Neurons were maintained in DMEM with GlutaMAX medium (Invitrogen Life Technologies) supplemented with 10% FBS and penicillin/streptomycin in humidified 5% CO2/95% air at 37°C. Glial cell cultures were prepared from the brains of newborn mice (postnatal days 0–3) following previously described methods (18). After digestion with trypsin and mechanical dissociation, cells were seeded in 75 cm2 flasks in DMEM with 10% FBS, 1% penicillin/streptomycin, 2 mM l-glutamine, and 0.1% glucose. After 8–10 days, microglia were detached by shaking for 2 h at 200 rpm, retrieved from the supernatant, and reseeded into 24-well plates in neurobasal medium with B27. Experiments on microglia were then conducted after 24 h in culture. The remaining astrocytes (>99% glial fibrillary acidic protein GFAP positive cells) were maintained by subcultivation in DMEM with supplements as described.
TLR2 mRNA expression.
Total RNA was isolated from ∼106 cells according to standard methods (19) using TRIzol reagent (Invitrogen Life Technologies). Contaminating DNA was removed by incubation with DNase (Promega), followed by phenol/chloroform extraction and ethanol precipitation. RNA was quantified by measuring the OD. Reverse transcription of 2 μg of extracted RNA was performed with random hexamer primers (Roche Applied Science) and Moloney murine leukemia virus reverse transcriptase (Promega) following the manufacturer's instructions. Real-time PCR was performed using FastStartDNA SYBR Green I and a light cycler instrument (Roche). PCR conditions were 10 min at 95°C followed by 45 cycles at 95°C for 15 s, 64°C for 10 s, and 72°C for 15 s (amplification product data acquisition at 81°C). All reactions were performed in duplicate, and the mean threshold cycle was used for analysis. The TLR2 expression of each sample was normalized on the basis of its β-actin and GAPDH mRNA content. The following sequence-specific primers were used: TLR2 forward, 5′-GATATTCTGAGTTCCGTGAG-3′ and reverse, 5′-TGTTACCGTTTCTATTTTACC-3′; β-actin forward, 5′-ACCCACACTGTGCCCATCTA-3′ and reverse, 5′-GCCACAGGATTCCATACCCA-3′; and GAPDH forward, 5′-AGATTGTCAGCAATGCATCCTGC-3′ and reverse, 5′-CCT TCTTGATGTCATCATACTTGG-3′.
Immunocytochemistry for TLR2
Cells were fixed and immunostained as previously described (17). To identify neurons and microglia, cells were stained with the Ab microtubule-associated protein-2 (Chemicon International) or with IB4-Alexa (Invitrogen Life Technologies), respectively. The mouse TLR2 Ab 6C2 was obtained from eBioscience. Specificity of the TLR2 Ab was confirmed by the absence of signal in TLR2−/− microglia.
Effects of Pam3CysSK4 in cell culture
Primary rat neuronal cultures were challenged by addition of Pam3CysSK4 to the medium at a final concentration of 0.1 μg/ml. At 24, 48, and 72 h, cell death was assessed by the measurement of lactate dehydrogenase (LDH) in the supernatant, an indicator of disintegration of the cell membrane (20). Moreover, cells were stained with the intercalating dyes acridine orange (final concentration, 2 μg/ml) and ethidium bromide (2 μg/ml), both from Sigma-Aldrich, to allow microscopic differentiation of live, apoptotic, and necrotic cells (21). Acridine orange stains all nuclei green, whereas ethidium bromide is excluded by cells with an intact cell membrane. Live cells are identified by green, regular-sized nuclei. Nuclear condensation or fragmentation identifies apoptotic cells, whereas red staining identifies late apoptotic or necrotic cells. In additional experiments, neurons were not directly challenged with Pam3CysSK4 but exposed to preconditioned medium from Pam3CysSK4-stimulated microglia instead. For this purpose, microglia was exposed to 0.1 μg/ml Pam3CysSK4 in neurobasal medium with B27 for 6, 24, or 48 h. Following centrifugation, the supernatant was filtered and then used to replace the medium of primary neuronal cultures as described. For controls, supernatant was collected from unstimulated microglia at identical times. Cell damage in the neuronal cultures was then determined by measuring the LDH concentration in the supernatant after 48 h of exposure to the preconditioned medium.
Data are presented as mean ± SD. Two group comparisons were performed with Student's t tests after ensuring normal distribution; otherwise, Mann-Whitney U tests were used. Multiple group comparisons on samples with normal distribution were performed with ANOVA followed by Student-Newman-Keuls post hoc testing. Otherwise, nonparametric ANOVA (Kruskal-Wallis) and Dunn's post hoc analysis were used. The impact of TLR2 genotype on the survival rate of D39-challenged mice was examined using Fisher's exact test.
Intrathecal administration of Pam3CysSK4 induces the pathophysiological hallmarks of bacterial meningitis
In anesthetized rats (n = 3), intracisternal application of pyrogen-free PBS did not induce relevant changes of regional cerebral blood flow as measured by LDF or ICP during the experimental period of 6 h (Fig. 1⇓, A–C). At the end of the experiment, LDF was 103 ± 13% of the individual baseline (Fig. 1⇓A), and ICP was increased by 0.5 ± 1.5 cm H2O (Fig. 1⇓B). Leukocyte count in the CSF at 6 h was 25 ± 15 leukocytes/μl (Fig. 1⇓C). In contrast, instillation of Pam3CysSK4 into the cisterna magna of anesthetized rats (n = 3) was followed by a gradual increase of ICP and regional cerebral blood flow. At 6 h after application, LDF was 155 ± 17% of the individual baseline (p < 0.05; Fig. 1⇓A), whereas ICP was 11.9 ± 0.7 cm H2O above baseline (p < 0.01; Fig. 1⇓B). By comparison, inoculation with viable pneumococci resulted in a more pronounced increase of LDF (at the end of the experiment: 216 ± 23% vs 155 ± 17% of the individual baseline; p < 0.05 vs Pam3CysSK4 and p < 0.05 vs control), whereas the increase of ICP was not different between the two challenges (at 6 h: 12.3 ± 0.3 cm H2O with D39 vs 11.9 ± 0.7 cm H2O with Pam3CysSK4, p was not significant vs Pam3CysSK4 and p < 0.01 vs control). At the end of the experiment, the CSF contained 3278 ± 355 leukocytes/μl in Pam3CysSK4-challenged rats, whereas controls had 25 ± 15 leukocytes/μl (p < 0.01; Fig. 1⇓C). In D39 infected rats, the CSF revealed 3984 ± 1656 leukocytes/μl (p < 0.01 vs control and p is not significant vs Pam3CysSK4) and a bacterial concentration of 5.4 ± 1.6 × 108 CFU/ml. In all experimental groups, arterial blood gas analysis and invasive blood pressure monitoring revealed normal values throughout the entire experimental period (data not shown).
Pam3CysSK4 causes leukocyte influx into the CSF in wild-type but not in TLR2-deficient mice
Wild-type mice (n = 4) receiving intrathecal pyrogen-free PBS had 11 ± 2 leukocytes/μl CSF at 24 h after surgery. Intrathecal challenge with Pam3CysSK4 caused a dose-dependent influx of leukocytes into the CSF. At 24 h after application of 2 μg of Pam3CysSK4 per animal (n = 3), 2607 ± 349 leukocytes/μl were counted in the CSF, while a higher dose of 20 μg of Pam3CysSK4 per animal (n = 6) increased pleocytosis to 6741 ± 1969 leukocytes/μl (p < 0.05; Fig. 1⇑D). The leukocyte influx was absent in TLR2−/− mice (n = 8) 24 h after application of 20 μg of Pam3CysSK4 (24 ± 35 leukocytes/μl, p < 0.05 vs wild-type mice) (Fig. 1⇑E). Compared with the 24-h interval, a higher number of leukocytes (20913 ± 5667/μl) were detected at 12 h after application of 20 μg of Pam3CysSK4 in wild-type mice (n = 5; p < 0.05), whereas TLR2-deficient mice (n = 4) again showed no invasion of leukocytes (15 ± 7/μl; Fig. 1⇑E). In meningitis induced by live D39 pneumococci, two of seven wild-type mice died within the experimental period compared with no deaths in six TLR2−/− mice (p was not significant using Fisher's exact test). CSF was not available from the two wild-type mice that died. In the remaining wild-type mice, challenge with live pneumococci resulted in a similar CSF pleocytosis at 24 h as was observed with challenge by 20 μg of Pam3CysSK4 (5103 ± 2451 leukocytes/μl, n = 5; p was not significant vs Pam3CysSK4 and p < 0.01 vs control). Unlike Pam3CysSK4, D39 also caused leukocyte influx (2428 ± 830 leukocytes/μl) in TLR2-deficient mice (n = 6; p < 0.01). D39-induced pleocytosis was less pronounced in TLR2−/− than in wild-type mice (p = 0.05).
Pam3CysSK4 induces neuronal damage in a TLR2-dependent fashion in vivo
In the dentate gyrus, TUNEL-positive cells were found in all animals 24 h after challenge (Fig. 2⇓). In wild-type control mice (n = 3), 73 ± 18 apoptotic cells/mm2 were present following intrathecal application of pyrogen-free PBS. Meningitis induction with 20 μg of Pam3CysSK4 increased apoptotic cell number to 120 ± 18/mm2 (n = 7; p < 0.05), whereas D39-induced meningitis resulted in 146 ± 40 apoptotic cells/mm2 (n = 5; p < 0.05 vs control and p was not significant vs Pam3CysSK4). In TLR2−/− control mice (n = 3) receiving intrathecal PBS, 81 ± 3 apoptotic cells/mm2 were present (p was not significant vs wild-type control mice). No significant increase of this number was observed following challenge in TLR2−/− control mice (n = 8) with Pam3CysSK4 (74 ± 11 apoptotic cell/mm2) or with live pneumococci (92 ± 15 apoptotic cell/mm2; n = 5).
Expression of TLR2 is cell type-specific
In purified primary cultures from rat cortex, real-time PCR demonstrated minor levels of TLR2 mRNA in neurons or astrocytes with a 50- to 600-fold higher presence in microglia (Fig. 3⇓A). Accordingly, immunocytochemistry revealed the presence of TLR2 Ag on microglial cells, but not on cortical neurons (Fig. 3⇓, B–J).
Pam3CysSK4 does not induce neuronal cell death in vitro
In unchallenged cortical rat neurons, the proportion of apoptotic cells did not increase during the experimental period. We observed 32.3 ± 8.7% apoptotic neurons at 24 h, 31.0 ± 7.4% at 48 h, and 30.7 ± 6.8% at 72 h (Fig. 4⇓A). Accordingly, the concentration of LDH in the supernatant remained essentially unchanged with 10.1 ± 3.0 U/ml at 24 h, 9.1 ± 2.6 U/ml at 48 h, and 12.3 ± 5.0 U/ml after 72 h (Fig. 4⇓B). Challenge with 0.1 μg/ml Pam3CysSK4 did not result in a significant increase of apoptotic neurons (35.5 ± 7.7% at 72 h; p = 0.12 vs control at 72 h; Fig. 4⇓A) or of LDH release into the supernatant (12.0 ± 4.4 U/ml at 72 h; p = 0.86 vs control at 72 h; Fig. 4⇓B). Conversely, exposure to 0.5 μM staurosporine resulted in apoptosis of 97.6 ± 2.1% of the neurons (p < 0.01 vs control at 72 h; Fig. 4⇓A) and a strong increase of LDH in the supernatant (38.3 ± 10.9 U/ml; p < 0.01 vs control at 72 h; Fig. 4⇓B). Cells exhibiting a necrotic staining pattern were not observed in any of the groups.
Next, primary mouse microglia was stimulated with 0.1 μg/ml Pam3CysSK4 (Fig. 4⇑, C and D). Supernatant was collected at 6, 24, or 48 h and used to challenge cortical neurons as described (Fig. 4⇑C). Supernatant from unstimulated microglia was collected at identical times to be used as a control. Supernatant from microglia after 6 h of incubation with Pam3CysSK4 did not induce an increase in LDH release from neurons compared with unstimulated microglia. LDH concentrations after incubation with "stimulated" vs "unstimulated" supernatant were 53.2 ± 17.9 U/ml vs 52.3 ± 16.2 U/ml at 24 h, p = 0.89; 63.8 ± 37.3 U/ml vs 64.1 ± 33.7 U/ml at 48 h, p = 0.98; and 80.5 ± 37.5 U/ml vs 69.1 ± 37.0 U/ml at 72 h, p = 0.46. Supernatant from microglia stimulated for 24 h with Pam3CysSK4 induced a time-dependent LDH release into the neuronal culture medium (incubation of neurons for 24 h: 64.1 ± 13.7 U/ml; 48 h: 78.4 ± 31.1 U/ml, 72 h: 89.8 ± 37.1 U/ml) that was significantly different from the effect of the supernatant collected from unstimulated microglia after 24 h (incubation of neurons for 24 h: 48.0 ± 14.1 U/ml, p < 0.01; 48 h: 53.5 ± 25.8 U/ml, p = 0.04; 72 h: 61.3 ± 30.6 U/ml, p = 0.05). Similar results were observed with the supernatant from stimulated microglia after 48 h (incubation of neurons for 24 h: 68.1 ± 19.1 U/ml vs 41.3 ± 6.4 U/ml, p < 0.01; 48 h: 85.0 ± 40.7 U/ml vs 49.5 ± 19.5 U/ml, p < 0.01; 72 h: 84.3 ± 35.8 U/ml vs 55.2 ± 23.0 U/ml, p = 0.03). Fig. 4⇑C shows LDH release from neurons after 48 h of supernatant exposure. Pam3CysSK4 did not induce neurotoxic properties in the supernatant from TLR2−/− microglia (Fig. 4⇑D).
The major finding of our study is that the selective activation of TLR2 by a synthetic bacterial lipopeptide in vivo causes inflammatory changes typically associated with experimental bacterial meningitis. Lipopeptide-induced intracranial inflammation has all the hallmarks of bacterial meningitis, i.e., influx of leukocytes, increase of blood flow, development of intracranial hypertension as well as neuronal cell death (21, 22, 23, 24, 25, 26). In particular, the time course of the inflammatory response to the synthetic bacterial lipopeptide in the mouse experiments resembles an earlier study with purified pneumococcal cell wall components (22). In this study, challenge also resulted in a self-limiting acute influx of leukocytes into the CSF with high leukocyte concentrations at 6 h and receding numbers at 24 h. In our rat model, the maximum increases of LDF and ICP after challenge with Pam3CysSK4 were similar to previous findings in meningitis induced by purified pneumococcal cell wall components (12, 23). Although the effects of Pam3CysSK4 on leukocyte influx and ICP increase were not different from those of viable pneumococci, we observed a significantly stronger LDF increase with live D39. As the likely basis of this effect, we have previously identified release of hydrogen peroxide by S. pneumoniae as an independent vasodilator during early experimental meningitis (27). Studies of neuronal apoptosis induction in vivo by bacterial cell wall components have not previously been published. Earlier studies using viable pneumococci have argued that neuronal damage during meningitis is the combined effect of bacterial toxins and the inflammatory host response (13, 28, 29). Our present findings show that stimulation of the TLR2 pathway alone induces neuronal apoptosis in vivo.
Our data present clear evidence that TLR2 signaling is highly sufficient to trigger meningitis in vivo, including the induction of neuronal apoptosis in the dentate gyrus. The clinical relevance of this finding is underlined by the fact that pneumococcal cell wall components, a known TLR2 agonist (30, 31), are excessively released after antibiotic-induced lysis of bacteria and that patient outcome is correlated to their concentration in the CSF (32). The ubiquitous presence of lipopeptides on bacterial surfaces, including other meningitis-inducing pathogens, further broadens the clinical importance.
Conversely, the relative impact of TLR2 on inflammation and host damage in bacterial meningitis caused by a viable pathogen has not been clearly delineated. In vitro, ectopical expression of TLR2 confers responsiveness to PG and LTA of Gram-positive S. pneumoniae in Chinese hamster ovary fibroblasts (30, 33) and human embryonic kidney HEK 293 cells (8, 33, 34). In vivo, several studies report a reduced ability of TLR2−/− mice to limit the growth of Gram-positive bacteria (5, 6, 7, 35, 36). In a previous study (8), higher titers of S. pneumoniae in CSF and blood were found in TLR2−/− mice compared with wild-type mice 24 h after meningitis induction. Typical CNS complications, i.e., ICP increase and formation of brain edema, were more pronounced and clinical scores were worse in the knockout mice. However, CSF leukocyte count and tissue levels of proinflammatory cytokines were not significantly changed, and aggravation of disease in TLR2−/− mice may have resulted from pronounced cytotoxic effects of the pneumococcal metabolism. Reduced CSF pleocytosis was later observed in pneumococcal meningitis of mice lacking the adaptor protein MyD88, which is situated downstream of both TLR2 and TLR4 (37). Similarly, aggravation of the clinical course, higher bacterial titers in brain tissue, enhanced brain edema, and increased mortality were reported in TLR2−/− mice in meningitis due to S. pneumoniae or Listeria monocytogenes (7). These studies suggest that TLR2 plays an important role in the host defense during Gram-positive meningitis. However, these reports do not allow differentiation between effects of an inadequate suppression of bacterial growth and consequences of the absence of TLR2 on the inflammatory host response, nor can meningitis-associated phenomena be traced back to specific receptors. Indeed, our own findings in D39-induced meningitis do not support a more severe clinical course in TLR2-deficient mice in the presence of equivalent bacterial concentrations in the CSF. Rather, reduced CSF pleocytosis and prevention of excess neuronal damage in the knockout mice despite an identical bacterial load suggest a prominent contribution of TLR2 signaling to inflammation and host damage during meningitis.
Neuronal loss, predominantly in hippocampal structures, is a hallmark of bacterial meningitis (21, 24, 38, 39, 40). In pneumococcal meningitis, at least two distinct pathways leading to neuronal loss have been demonstrated: live pneumococci induce an early caspase-3-independent, AIF-dependent wave of apoptosis, while later, the release of bacterial cell wall components leads to a different type of cell death involving release of cytochrome c, formation of the apoptosome, and ultimately activation of caspase-3 (21, 24, 28, 29, 41). It has been argued that induction of apoptosis by bacterial cell wall components occurs secondary to the activation of immune competent host cells (28). In vitro, bacterial cell wall components induce cell death in organotypical slices and in neuronal/glial coculture, but not in primary neurons cultured alone, and NO release from activated glia has been suggested as the relevant cytotoxic principle (42). We did not observe an induction of cell death in neuronal monocultures exposed to Pam3CysSK4. These findings are conclusive and are supported by the absence of TLR2 in neurons. As demonstrated in another model, our experiments confirm that TLR2-dependent release of neurotoxins by microglia causes neuronal cell death (43). Additionally, initiation of TLR2-mediated and caspase-dependent cell death by pneumococcal cell wall has only been reported in TLR2-expressing cells such as endothelial cells or dendritic cells (41, 44). Our finding of low TLR2-mRNA expression in resting astrocytes does not argue against an additional contribution of these cells in vivo because astrocytes have been shown to up-regulate TLR2 upon activation by bacterial compounds (45).
Taken together, we have established a model of meningitis induced by a synthetic TLR2 ligand. Synthetic bacterial lipopeptides offer an opportunity to study TLR2-dependent events independently of bacterial metabolism and other bacterial cell wall components and thus enable further dissection of the complex mechanisms of intracranial immune response.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This work was supported by Grant SFB 507 B6 from the Deutsche Forschungsgemeinschaft (to O.H., J.S.B., and J.R.W.) and a Rahel Hirsch grant from the Charité-Universitätsmedizin Berlin (to S.L.).
↵2 O.H. and J.S.B. contributed equally to this work.
↵3 Address correspondence and reprint requests to Dr. Joerg R. Weber, Department of Cell Biology and Neurobiology, Center for Anatomy, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. E-mail address: joerg.weber{at}charite.de
↵4 Abbreviations used in this paper: PG, peptidoglycan; ICP, intracranial pressure; CSF, cerebrospinal fluid; LDF, laser Doppler flow; LDH, lactate dehydrogenase.
- Received September 7, 2006.
- Accepted February 27, 2007.
- Copyright © 2007 by The American Association of Immunologists


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Posts Tagged 'White Wizard Games'
Game Designer Interview: Rob Dougherty on Star Realms and White Wizard Games
If you've gotten the impression that Star Realms is my favorite game, you're right. So when a new big Kickstarter is happening for the game, you better believe I get excited. I had the chance to talk to Rob Dougherty, CEO of White Wizard Games and co-designer of Star Realms, about what's different this time. Read on…Read More
Review: Hero Realms
Designers: Darwin Kastle, Rob Dougherty Publisher: White Wizard Games Category: Deckbuilding Game Player Count: 2 (more with variants) Price: $19.99 MSRP I gotta be honest with you all. I've been putting this review off. Star Realms is my favorite game of all time. I play 10-20 games of it a day online, and I'm even going to be featured on a future…Read More
The Complete Star Realms Buyer's Guide
(This guide was first posted in January 2017. Three years later, it really, really needed updated! Star Realms is still cranking out new content, and I'm here to help! This post is now current as of January 2020.) You may have heard that I like Star Realms. Like, a lot. It's fast, exciting, and inexpensive… sort…Read More
Review: Star Realms
Designers: Rob Dougherty and Darwin Kastle Publisher: White Wizard Games Category: Deckbuilding/Strategy Game Player Count: 2 BoardGameGeek Rating: 7.65 (16,264 votes) Price: $14.99 When I was around 10-years-old, the Magic: the Gathering craze began full swing in America. I have fond memories of building deck after deck, reading Pro Tour Qualifier reports (.txt files!) on The Dojo, and…Read More


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Diablo Immortal and How Hype Culture Can Negatively Impact Game Reveals
The negative reaction to titles like Diablo Immortal and Metroid Prime: Federation Force both show how toxic hype culture can ruin game reveals.
The negative reaction to titles like Diablo Immortal and Metroid Prime: Federation Force both show how toxic hype culture can ruin game reveals.
Nintendo announced through a message on the Nintendo eShop that the online features of the Metroid Prime: Blast Ball demo will be shut down in North America on December 31 at 23:59PT.
Nintendo has gone live with a new trailer for the Nintendo 3DS exclusive shooter Metroid Prime: Federation Force, which is available today.
Nintendo has been fighting an uphill battle with Metroid Prime: Federation Force for a little over a year. Originally revealed at E3 2015, the title has been under constant bombardment across-the-board by both Nintendo and Metroid fans.
Nintendo has released a new trailer for the upcoming Nintendo 3DS exclusive Metroid Prime: Federation Force.
Metroid Prime: Federation Force is a little more than a week from launch, and Nintendo is once again trying to turn the tide of popular opinion with a trailer. Dubbed the "Story Trailer," the quick clip shows
Have you been dying to get your hands on Metroid Prime: Blast Ball? Probably not, but even so -- the three-on-three Metroid-like shooting game will be free-to-play from now until early September. With many online still critical
Nintendo has gone live with a new trailer for the upcoming Nintendo 3DS exclusive Metroid Prime: Federation Force.
Nintendo recently revealed amiibo support for Metroid Prime: Federation Force on Twitter.
Using either Samus or Zero Suit Samus amiibos will unlock both a custom paint job in the style of the character and special upgrades for
Though the reveal of the game at last year's E3 showing was controversial to say the least, Nintendo has remained adamant that fans will find something to love in Metroid Prime: Federation Force. The company took to
The gaming community still isn't too hot on Metroid Prime: Federation Force, however some brand new screenshots have surfaced via this weeks Weekly Famitsu. The screens showcase a fair amount of gameplay for the mission-based game
Metroid Prime: Federation Force may get a lot of flack on the internet for not being the next big Metroid game, but today's latest gameplay footage (brought to you by GameXplain) shows off an otherwise solid cooperative
Metroid Prime Federation Force also received its time in the spotlight during today's Nintendo Direct, with some new gameplay footage and information on the lore.
Heading into E3 2015, many were expecting big things from Nintendo's Digital Event broadcast and while many saw great new titles like Star Fox Zero and Yoshi's Woolly World, fans were also hoping for the long-awaited return


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Danfoss TP9000
Hi, I have a Danfoss TP9000 programmer installed 'Downstairs' in the kitchen with a TS2 temperature sensor wired into the Hall.
From what I've read and understand, I need to :-
- Install an Extension Kit Add-on to replace the TP9000.
- Install a Wired Smart Thermostat (add on) to manage the temperature downstairs.
Is this correct?
Am I right in saying that the Wired Smart Thermostat will actually Just be battery powered and won't need to connect to any wires because it will control the downstairs temperature via the Extension kit?
BTW - I already have Tado V3+ installed - That manages my 'Upstairs" Heating Zone. This is why I'm buying Add-ons and not the full-kit. I already have the Wifi dongle and upstairs thermostat.
Hope anyone can help?
Best Answer
GrilledCheese ✭✭✭
Yes, the extension kit will replace the TP9000. The wires connected to pins 5 and 6 of the Danfoss backplate are for the remote sensor and will not be used when you switch to the Tado backplate. You will need to replace the TS2 with a Tado wall thermostat and it will connect wirelessly to the extension kit. This thermostat will run on batteries, so you can change its location if you prefer.
Your Tado thermostat upstairs is wired and should not need any local changes. Tado support will obviously have to make changes at their end.
One of your thermostats can be used to manually control the HW. Decide which one you prefer and ask the support team to configure it for you.5
Many thanks for your help Mr Cheese!
I'll buy the items and give it a go. Will report back when installed and fully working as expected.
Much appreciated.0


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Music Room is an online store aiming at making music education much easier for students – it offers sheet music, classy books about music, tuition books and, of course, musical instruments of all kinds.
FreeShippingWorldwide.org is a directory of online shops offering free shipping worldwide, complete with ratings, user reviews and descriptions. We also did our best to complement it with user-friendly interface and shop search feature.
Thanks for visiting our site! Don't hesitate to bookmark it and spread the word about it among your friends. We are sure they will love it.
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Clinton Temporary Employment Agencies brought to you by the Internet Chamber of Commerce. Choose a company to see comprehensive business information.
Er Management Inc
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More information about Er Management Inc
Note: If you are aware of any errors or ommissions on this page, there is a link on each business record's page where it can be reported.


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"1945 willys mb. Real deal real piece of history. Has always been a military jeep. Not a Cj painted like one..
I have restored her to parade ready condition. Rebuilt engine, rear end, transmission, brakes, winter of 12. 4wd works great. No problems starts right up. $22kobo"


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As we always strive to cater to our customers' specific needs and requirements, we currently offer sanitizing service follow. Please note pricing will be based on the total Sq Ft of area serviced. In order to get your customized quote, feel free to give us a call, we're happy to oblige.
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We don't have any photos of this beach yet. Why don't you be the first to upload one?Upload Photo
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Sand dunes run along the coastline to the north of the bathing water. There is an active sand quarry to the north of St Annes North bathing water.
|Chance of rain
|Min temp.
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|Wind 7mph
|Chance of rain
|Max temp.
|(feels like
|Wind 13mph
|Max UV Index
The tide at St Annes North Beach is currently rising. High tide will be at approximately 11:55 PM, and the next low tide will be at approximately 6:40 AM tomorrow.
Today's air pollution levels are forecast to be Low
Approximate sea surface temperature: 14.8°CDetailed weather and tides »
Pay car parks and roadside parking available.
Dogs not allowed on the stretch between the old pier and the cinema, May 1st to September 30th.
No lifeguard cover at this beach
Good water quality
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Island Cinemas (1.6 miles)
The Big One (1.8 miles)
Ken Webster Hypnotist Show (1.9 miles)
Pasaje Del Terror (1.9 miles)
The Evolution Of Magic (1.9 miles)
Adrenalin Zone (2 miles)
St. Annes Beach Huts (2 miles)
Escape Blackpool (2.3 miles)
All distances are approximate
around 2 miles south-southeast as the seagull flies, around 2 miles by road.
around 2 miles north as the seagull flies, around 2 miles by road.
around 3 miles north as the seagull flies, around 3 miles by road.
Location: St Anne's,
Location type: Urban
OS Grid: SD305304
County: Lancashire
Country: England
Local Authority: Fylde
Parish: Saint Anne's on the Sea
Nearest postcode: FY8 2RN
(for sat-nav purposes only, a beach does not have a postal address!)
Important! Beach information is compiled from a variety of different sources and is correct to the best of our knowledge at the time of compilation. However, information may be incorrectly supplied by our sources or go out of date after publication. BritishBeaches.uk makes no warranty as to the accuracy of any information on this page.
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In case you have a mid-to-large scale business, the mobile app could be a very crucial portion of your small business growth strategy. Therefore, you will need to pick a mobile app development company which has an innovative approach together with futuristic thinking. But before you judge the pros and cons from a company, you'll want to start by assessing your organizational needs and appearance whether the chosen company can fulfill those. Let's consider 7 proven strategies for seeking the mobile app company that you pick:
1. Understand what Your App Requirements Are
It could seem to be a no-brainer but using a thorough understanding of your app development requirements can certainly help you in filtering the mobile app development companies. This process should focus on the close research into the specific business demands and objectives, which you need to convert into meaningful app features and functionalities. We percieve that many our clients struggle while determining what they need from other business app. To enable them to progress, we offer a questionnaire which enables bring them toward their goal:
What sorts of products/services are you currently trying to sell?
Where are you wanting your app to get: on Android, iOS, Windows-based Mobile Database integration Platforms?
Who will make your member list?
What kind of mobile app do you want- native, hybrid or web-based apps?
When you are convinced as to what you want in the app, you are ready to go to a higher step.
2. Question their App Delivery Time-to-Market
Faster 'time-to-market' is the new demand in today's' ages of digital disruption. If your chosen mobile app development firm is slow at rolling out apps to the market, it comes with an increasing possibility that you'll go into default your competition. The quicker your organization app hits the marketplace, higher could be your ROI. Therefore, give full attention to choosing mobile app development companies which deliver high-quality apps quickly, whilst keeping planned the changing trends inside the app development market.
3. Require a Critical look at their Portfolios
When you open your website from a mobile app development company, begin by checking their portfolios. Portfolios give you a good introduction to the company domains and project themes the organization has been involved with, and the type of technical expertise they hold. You can also get in touch with the company to know when they've labored on the sort of app you need before and have the opportunity to deliver it.
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In terms of choosing the right mobile app development company, you'll always find the development cost and app quality to stay conflict. A great deal of organizations go for start-ups to maintain the event cost low and get a end product which has little market relevancy. On the other hand, it can be wrong to assume that best mobile app development companies offering quality app solutions is going to be always high-priced. What you must bear in mind here is that just strong market players can afford to provide quality apps at efficient costs. If you choose to go for long-established companies with good market visibility, you will recognize that their charges are comparatively lesser compared to start-ups while others.
5. See How Transparent Their Process Is
The product or service development cycle and transparency policy followed by the mobile app development company also play a very important role within the process. Low code methods of mobile application development have gained immense popularity for faster app delivery, but few leading mobile app development companies in US use it nowadays. Moreover, search for companies that provide transparent communication and feedback loops from the development process for holding better mutual understanding, and promptly respond to errors and iteration requests.
6. Check Their Experience and Reputation Online
Market reputation and experience speak volumes concerning the credibility of your mobile app development company. Apart from examining the portfolios, keep an eye on the number of positive reviews posted about a particular company, years of experience in the app development domain, as well as the different kinds of mobile application development services which they provide. Bare in mind that you could achieve good Roi (ROI) from your app only when the company chosen by you can deliver solutions with innovative, out-of-the-box ideation. Also, if a company projects in-depth familiarity with multiple smart phone requirements and many types of popular Mobile Database integration Platforms including Android and iOS, rest assured that they're able to deliver what you look for from their website.
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Paraquat Induced Changes in Reserve Carbohydrates, Fatty Acids and Oleoresin Content of Young Slash PinesAuthor(s): Claud L. Brown; Terry R. Clason; Jerry L. Michael
Date: 1976
Source: Proceedings of the Lightwood Research Coordinating Council. 20-21
Publication Series: Miscellaneous Publication
PDF: Download Publication (136 KB)
DescriptionParaquat was fed into the terminal leaders of five-year-old slash pine trees and collected at weekly intervals for 4 weeks.Cytological observations showed a decrease in starch levels and a corresponding increase in content of oleoresin. Quantitative analysis indicated a decrease in starch accompanying increases in fatty acids, monoterpenes, and resin acids.
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CitationBrown, Claud L.; Clason, Terry R.; Michael, Jerry L. 1976. Paraquat Induced Changes in Reserve Carbohydrates, Fatty Acids and Oleoresin Content of Young Slash Pines. Proceedings of the Lightwood Research Coordinating Council. 20-21
Keywordsresin soaking, terpene biosynthesis, starch content, monoterpenes, resin acids
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Treatment of squeezed tunnel overChameliya Hydropower Project has reached a major milestone, with civil contractor China Gezhouba Group Corporation (CGGC) completing the treatment of the squeezed tunnel section.
Bibek Subedi
Chameliya Hydropower Project has reached a major milestone, with civil contractor China Gezhouba Group Corporation (CGGC) completing the treatment of the squeezed tunnel section.
Nepal Electricity Authority (NEA), the owner of the 30MW project located in Darchula in the Far West, said the breakthrough in the treatment of the 232m squeezed section was achieved on Friday night.
The contractor, which had resumed civil work in October 2016 after more than two years of hiatus, has increased work pace and has expressed commitment to complete the task well before the deadline, according to NEA.
The contractor has also completed first layer shotcrete—a process of spraying concrete throughout the inner layer of the tunnel for its durability—of the treated tunnel, and is planning to complete the second layer within a couple of days.
"Now, the contractor will spend a week cleaning the 4.06km tunnel before starting the construction of a 47m rock-trap," said Ajay Kumar Dahal, project chief. "Rock-trap is a pit built in the tunnel to sediment pebbles and stones so turbines and other equipment of the hydropower plant are not damaged."
It will take around three months for the contractor to complete the rock-trap, Dahal added. After the completion of the rock trap, CGGC has to block three audit tunnels that were opened while digging the main tunnel.
As per the revised work schedule prepared by the civil contractor and approved by NEA at the end of September 2016, CGGC has to complete remaining civil work by September 2017. "However, we have requested the contractor to complete its job a month earlier," said Dahal. "It has not given a written commitment, but has said it will try to complete the work before the deadline."
Similarly, as per the revised work schedule, the Korean contractor assigned to do electro-mechanical and hydro-mechanical works will take additional three months to bring the project online after the civil works are completed.
"They have also been requested to complete the job a month earlier and they have also made same promises as the civil contractor did," Dahal told the Post. "If both the contractors complete their job before the deadline, we can bring the project online by October 2017, two months before the revised deadline."
The construction of the project had been stalled since May 2014 after the government refused to make an additional payment of Rs1.09 billion which the contractor had asked for due to cost variance resulting from the squeezing of the tunnel.
The contractor agreed to resume work after being summoned to the Energy Ministry and told to do so immediately by Minister Janardan Sharma. NEA also agreed to release a provisional payment of the disputed bill before the issue was resolved in order to maintain a regular cash flow for the project.
Once the dispute is settled, the contractor has to return the excess if it has been paid too much and it will receive additional payment in case of a deficit.


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It was too small to turn around a wheelchair in, and my unconventional futon/couch would be impossible to drag out. house.
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Home Equity Loan With No Appraisal Buying A House For Your Child To Rent The American Dream Is Fading, a Third of Those Surveyed Believe – Think of it this way: If it's Monday and you have $1,000 — the exact amount of your rent, which is due Tuesday. It may also mean some tough choices like buying a smaller house than you want or.Here's How the latest home equity-tapping tools Stack Up – When it comes to utilizing a wealth of built-up equity. little-to-no mortgage balance. Safe Stay fees and costs: Upfront fee of 3%; potential various costs if buyback occurs or occupant leaves..
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EUR/CAD to 1.46?
EUR/CAD to 1.46?
The euro also came under selling pressure today as investors realized that Deutsche Bank's lower settlement deal with the U.S. Department of Justice is not real. There was a media report on Friday that said they reached an agreement to lower the penalty to $5.4 billion from $14 billion and while talks are underway investors there's been no official deal. The problem now is that a lower number has now been thrown out there so if the actual settlement is higher and closer to $10 billion, the euro could still be punished and for this reason along with the ongoing banking crisis in Europe, we believe the euro will underperform. In contrast, the Canadian dollar extended its gains as oil prices pressed higher. Fundamentally, we believe that oil has bottomed and the CAD following the OPEC deal. It's a busy week in Canada but we don't have any major economic reports until Friday so fundamentally we could see further pressure on EUR/CAD.
Technically today's high in EUR/CAD coincided with the 200-day SMA – an important indicator that has previously served as support and resistance for the pair. We believe this level should hold and EUR/CAD should find its way back down to the 50-day SMA at 1.4610. If EUR/CAD breaks above the 200-day SMA near 1.4750, the 50% Fibonacci retracement of the 2008 to 2012 sell-off should cap gains.Chart Of The Day