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Have you ever wanted to learn Spanish but just could't find the right program, enough time, or a good place to practice? Language Guy Mark Frobose presents his new Spanish 1 for the Road. Rest assured, your language learning worries are now over forever.
With Spanish 1 for the Road, you're finally given a fast, easy, and fun way to learn authentic Spanish while you drive in the privacy of your car or from any location. Spanish 1 for the Road frees you of the necessity trying to learn Spanish while tied to a computer, shackled to a classroom desk, or paying big bucks for a private instructor. With Spanish 1 for the Road, for the first time ever, you are able to learn to speak Spanish naturally, while on the go, while driving to and from work, while waiting on a train, or even while standing in line at the supermarket. By turning your otherwise wasted down time into language learning up time, Spanish 1 for the Road becomes your constant Spanish teaching companion. You'll learn so much so fast that you'll always be looking forward to your next lesson.
Mark Frobose, the Language Guy®, talks you through the basics of the Spanish language while his trained and educated native Spanish speakers teach you to communicate, speak, and understand authentic Spanish and speak it just like the natives do. Always understand while you learn with American multilingual expert language teacher Language Guy® Mark Frobose guiding you through Spanish in plain English. A variety of educated native Spanish speakers work with Mark to teach you their language, authentic Spanish, the way it's really spoken. Learn to speak your own original Spanish sentences within the first 10 minutes of the program. Remember more of what you learn with the best built-in memory technique in the industry.
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Teaches Spanish On the Go
I've tried Learn in Your Car Spanish, Behind the Wheel Spanish, and Paul Noble, to name a few.
This is the most "on the move friendly" Spanish program that I've tried so far.
It uses a building block approach, so even just a few minutes in the car or while jogging teaches you something you can use right away with native speakers. Love the memory reviews too.
- Paul


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Hostage Situation Reported at Credit Union on Bryant Drive
A hostage situation is currently being reported at Alabama Credit Union on Bryant Drive in Tuscaloosa.
Stephanie Taylor of the Tuscaloosa News tweeted that police have confirmed a hostage situation in progress at Alabama Credit Union at 220 Paul Bryant Drive .
Police confirm hostage situation at credit union on Bryant Drive, heading there now.
— Stephanie Taylor (@TNews_Steph) January 10, 2017
The suspect in the case has taken someone hostage inside the bank, and Bryant Drive is currently closed.
Credit Union on University Blvd in Tuscaloosa AL currently being robbed! @spann pic.twitter.com/ILAvh6TbTT
— Joy Simpson (@zjpate) January 10, 2017
We'll keep you updated as this situation continues to develop.

subset: commoncrawl


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DJ Shadow will soon offer fans a peek inside his formative years, compiling a collection of previously unreleased instrumental tracks created from 1992 to 1996. Total Breakdown: Hidden Transmissions From the MPC Era, 1992-1996 focuses on his early experimentation on the Akai MPC. It includes tracks from a scrapped 1994 EP he planned with the Gift of Gab, like the demo that eventually became The Private Press single Six Days.
There's no release date scheduled yet, but the project will eventually be available on CD, double-vinyl and digitally via DJShadow.com with bonus tracks. Below you can listen to the 1992 track Dreams Of A Piece via LRG.
Hit continue reading to check the album tracklist & download link for this track.
Track List, CD/Vinyl Version:
1. Intropy (Original Version)
2. Vee In Detroit
3. Dreams Of A Piece
4. The Not-So-Quiet Storm
5. Fast Rap Fanfare
6. Intensely Hitting
7. Mystical With Solo
8. Alright
9. Don't Try It
10. From The Old School
11. Freddi's Popcorn
12. Quickstep
13. Movin' On (Gab Demo 1)
14. Falling Up
15. Mellow But Chunky
16. Total Breakdown (Gab Demo 2)
17. Heavy Mood
18. Affectations
19. Perilous Journey (Gab Demo 3)
20. Aye
21. Brittle But Magic
22. Atmospheric Disturbances
23. Swimming Upstream (Gab Demo 4)
24. Up For Grabs (Outro)
DJShadow.com bonus Tracks:
25. Give Up The Drums
26. Slow Poke
27. Vortex


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My ex wife is famous for that ! Everything is a big deal to her, especially if it comes to money and she wants some.
Well, there was maybe just this one time...
I was working for a Japanese company and stuffed up. The boss came to see me and said, "If you ever do this again I will bring a ceremonial knife into this office and watch you ritually disembowel yourself."
Seemed like a pretty big deal to me.


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difficulty: Medium
time: 35
Pomì Organic Strained Tomatoes 1 box
This dish is a staple of Roman cooking, but like many other fantastic recipes of my peninsula, this one made its way onto every Italian's kitchen and is now well represented almost everywhere you go.
Although this is an easy recipe, the quality of the ingredients used can truly make an enormous difference, especially when it comes to the right cured meat and the right tomatoes; you want to achieve a balance between the saltiness of the guanciale, the sweetness of the tomatoes and the hotness of the pepper…it is a bit of an exercise, but it won't take too much practice.
Always remember to cook your pasta about one minute less than indicated in the box to make sure it comes out truly "al dente".
Dice the Guanciale or slice it in thin strips.
In a non-stick pan large enough to finish your pasta when it's ready, sauté the meat in 2 tablespoons of hot olive oil until golden.
Add the garlic chopped roughly and the crushed red pepper, sauté for a couple of minutes then add the tomatoes. Season with salt and pepper, add the basil ripping it in pieces with your hands, lower the flame and stir well.
The sauce will have to cook uncovered for about 15 minutes to reduce at its best texture.
Bring a pot of salted water to a boil, cook the pasta for about 1 minute less than indicated on the box, drain and add to the Amatriciana sauce. Finish on a high flame stirring and jumping the pasta to make sure that the sauce is evenly distributed.
Serve garnished with a touch of finely chopped parsley, a generous sprinkle of Parmigiano or Pecorino and a drizzle of the best Extra Virgin Olive Oil.
Prep Time: 10 min - Cook Time: 20-25 min


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People's Movement Against Nuclear Energy (PMANE) president SP Udayakumar earlier had said that Tamil Nadu Chief Minister J Jayalalithaa encouraged the anti-nuclear protest and without her support, the protest could not have sustained in the state.
In an interview to Mail Today, Udayakumar said, "It is she who encouraged us. Without her support we could not have continued the struggle," adding that he demanded for the release of 18 anti-nuclear protestors. After Chidambaram's statement about "ulterior motive", speculations have been surfaced saying - was he trying to hint something regarding Jayalalithaa?
Chidambaram attended a meeting with Congress workers in Sivaganga on Sunday and also expressed his concern about heavy power crisis in Tamil Nadu. The Union Minister, while addressing load shedding issue in the state, urged Jayalalithaa's government to resolve the problem.
The minister also tried to save the Congress led UPA government as he claimed that its decision to vote against Sri Lanka to support US moved UN resolution was taken "after a serious thinking."
OneIndia News


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View other listings in Vermont, Canterbury, Box Hill, Blackburn, Hawthorn.
Online about 1 year ago
Rent budget
Preferred stay length
Move date
Hi everyone!
I'm from Germany and I will be teaching at a Secondary School in Vermont, Melbourne, VIC, from September to December 2016. Therefore I'm looking for a flat nearby.
I'm tidy, sociable and open-minded and love cooking, eating (even more than cooking) and doing sports. I love getting to know new cultures and I'm really looking forward to my stay in Melbourne.


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Richmond vs. Duquesne - College Basketball Pick, Odds, and Prediction
Duquesne Dukes (6-12) at Richmond Spiders (10-9)
College Basketball: Wednesday, January 28, 2015 at 7:00 pm (Robins Center)
The Line: Richmond Spiders -13.5 -- Over/Under: TBA See the Latest Odds
The Richmond Spiders and the Duquesne Dukes face off on Wednesday at the Robins Center in A-10 college basketball action.
The Richmond Spiders are 10-9 this season and they have lost two of their last three games. Richmond is averaging 64.2 points and they are shooting 46.6 percent from the floor while defensively they are allowing 60.1 points and their opponents are shooting 42.4 percent. Kendall Anthony leads the Spiders averaging 15.9 points per game. Terry Allen is averaging 11.8 points and 5.5 rebounds, T.J. Cline is averaging 11.5 points, and ShawnDre' Jones is averaging 8.6 points. Richmond is 9-2 at home and 3-3 in the A-10 this season.
USA TODAY Sports
The Duquesne Dukes are 6-12 this season and they have lost their last five games. As a team Duquesne is averaging 71.7 points and they are shooting 45.4 percent form the floor while defensively they are allowing 72.6 points and their opponents are shooting 43.3 percent. Derrick Colter leads the Dukes averaging 12.7 points per game. Micah Mason is averaging 10.8 points, Jordan Stevens is averaging 9.5 points, and TySean Powell is averaging 8.6 points. Duquesne is 1-5 on the road and 1-6 in A-10 play this season.
Richmond is 5-0 against the spread in their last five games, 5-0 against the spread against the A-10, and 0-4 against the spread in Wednesday games. Duquesne is 4-1 against the spread following a game that they didn't cover the spread, 1-5 against the spread in their last six road games, and 7-3 against the spread following a loss. The favorite has covered the point spread in six of the last seven meetings of these two.
Richmond is looking at a big number to cover here but they should get it done at home.


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a style of central African popular dance music with electric guitars, Caribbean rhythms, and often several vocalists.
a style of African popular music that originated in Zaïre (now the Democratic Republic of Congo), characterized by syncopated rhythms and intricate contrasting guitar melodies
Tagged: s
Read Also:
- Soul
noun 1. the principle of life, feeling, thought, and action in humans, regarded as a distinct entity separate from the body, and commonly held to be separable in existence from the body; the spiritual part of humans as distinct from the physical part. 2. the spiritual part of humans regarded in its moral aspect, or […]
- Soul-brother
noun, Informal. 1. a black male, especially a fellow black male. sprawl
- Soul-cake
noun, British. 1. a round, sweet bun or small, oval cake, traditionally made to celebrate All Souls' Day.
- Soul-destroying
adjective 1. (of an occupation, situation, etc) unremittingly monotonous


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Orange Blossom Soap
9 ounces water
1 1/2 teaspoons ground cinnamon
2 tablespoons ground orange or lemon peel
1 teaspoon ground ginger
3 drops cinnamon essential oil
Melt grated Castille soap in 9 ounces water in a saucepan over very low heat. Add the cinnamon, peels and ginger; stir well. Add the cinnamon oil, stir again and pour into small oiled molds and let the soap set. This can take from a few hours to a week, depending on the size of the mold.
When dry, unmold, wrap in tissue and put it in a dry cabinet to cure for about a month.
Print Orange Blossom Soap
We invite you to print the Orange Blossom Soap to use later or to share with a friend.
- Print recipe (blank)
- Print recipe (body)
- Print recipe (bath)


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Letter(s) from Maria to Richard (Holland)
Letter 1
Hello! I write to you the first letter! I hope that you will become interested in my letter and we learn each other. My name is Masha. To me of 25 years. I want to find the man which will be more senior than me. Because I am young and at me not enough life experience... I think, hundred at you wide experience and for this reason I want to try to build with you relations!!! I work as the seller in grocery shop. At us amicable collective. I very much like my work. I was born and have grown in city Nizhni Novgorod. It is very old and beautiful city!!! I live with mum and the younger little sister. My mum does not work, and the sister still studies, but will soon stop and Will work. And how you concern to what I the Russian girl??? How you in general concern to Russian people??? At me Russian the man once was, but soon we have left it, because it used It is a lot of alcohol, offended and humiliated me... And once even has lifted a hand on me!!! And almost all Russian men such!!! I hope you not such??? I want to try to build serious relations with you and that create happy family. I not so can frequently write to you because I do not have computer And I should write to you from Internet - cafe. If all of you are still interested in me then write to me, I shall necessarily answer!!! Tell to me about itself. How you live, where live, why there is no girl??? Now I shall finish the letter and if you really are interested in me, Then I wait for your answer. Yours Masha.
Letter 2
Hello Richard! How are you? I hope that at you all well. Thanks for
Prompt reply. I very much am interested in acquaintance to you. And consequently I want.
To learn you it is better.
Now I want to tell to you not much about my city. It to be not so far
From our capital of Moscow. Our population of city makes about
1500000 person. But in comparison with Moscow our city not such big.
Nizhni Novgorod - ancient city with fine architectural
Constructions. Not simply separate monuments, and the whole streets store
Memory of a history of our city. I hope that to you interestingly to learn about
I city has more. At us it is a lot of rivers and it is sometimes simply surprising
To leave for a wood and to have small walk. Also it will be pleasant for me if
You can tell to me about your country and about your city.
I very well am able to prepare, as me to this
Mum has learned mine. She now the pensioner, but earlier she worked in
Kindergarten also was the cook on kitchen. I hope that once you can
To try my dishes and I is sure that they will like you.
Also I hope that you want to learn about my hobbies and a hobby. I very much like
To go in for sports and always I try to hold the figure in good
Condition. As I know that men like harmonous girls.
Also I very much like to read books and to look various films about love!)
You know that I work as the seller in small shop and at
Me is to contact not always a lot of time you. But I always shall be
To try to answer you as soon as possible. I work 6 days in a week.
My day begins in 7 mornings as I should be on work already in
8.30 hours. Our shop opens in 9.00 mornings. Visitors it happens
It is a lot of, but in separate where I work them very little. But I hope that
Once them begins more. Now I need to go, but I shall look forward to hearing from you and I shall try to tell to you about myself even more that you could understand
What I the person. I shall wait for your fast reply. Yours Masha.
P.S. Send to me more than the photos, please.
Letter 3
Hello Richard! I am pleased to see your letter, I hope that at you all
Well. It was pleasant for me to receive your following letter
Today. How are you doing? In me all today it is good, I have remarkable
Mood. Probably it because I have found your letter today.
However I want to tell in this letter a little that, that like to eat
And to be prepared you already know, that I very well am able and I like
To prepare. I am able to prepare many things. They - various soups, a ragout, pies,
Salads and it is a lot of another. When I do pies which I am usual I
I prepare for them with a cherry and apples. You love pies? If yes it, with
What? I love the Russian kitchen. And you prefer what kitchen? You
Tried Russian kitchen? And if yes that what dishes have liked you? I
I want to know about you all. As with each letter I understand more and more
That our interest grows to each other. It is pleasant for me to read your letters and
To learn about you it is more and more and more. As we should know each other
To make any the decision in the future. You love children? How many
You would like to have children and whom? I am interested very much with these questions. I Any more I do not want to have something with the Russian men. I suffered
Because of them very much and more I do not want it. They think only of themselves and not Know, that it is necessary to be for the woman happy. Last mine
Attitudes have been destroyed because of that that I the man simply has thrown me
Also has left to other girl. It has hurted me and has destroyed my feelings.
After that I do not want to build more the attitude with Russian
Men. It is difficult for me should speak about it, but I think,
That we should not hide something from each other. And why you are lonely??? I am already tired from lie and Pains and consequently I want to learn you better and to understand that you for the person. I search only for happiness and it does not suffice me.
Attitudes are under construction on Trust and honesty, therefore I with you am completely frank and Sincere. On it I to finish the letter and I shall always wait your fast
The answer. It is pleasant for me to receive your letters as they do me
More happily.
Yours faithfully Masha.
Letter 4
Hello Richard! I am happy to see your letter again. We is pleasant for me that
Acquaintance every day all moves ahead. I am glad to know that you also
Be interested in me and I see your relation to me. It is pleasant for me that you
Seriously concern to ours acquaintance and probably once we can
To be together and to try to build family. But while I want to tell to you about
The childhood. When I was small I tried a lot of various
Hobby and hobbies. From the smallest age my parents
Have given me to a dancing circle, and then I went to school chorus. But
I when did not want to be the singer and consequently I have thrown these hobbies.
But soon I began to mature and have acted in university, where I only
Thought of study. I have finished university perfectly well. But mine destiny not
Has developed also to me it was necessary to go to work in shop the seller.
Today at me very bad day was. My day has not developed
Since the morning. When I went for work and the machine passed street,
Passing past has poured over me water. Now at us it is warm also a snow began
On the sly to thaw. Then, when I went for work, in the bus at me have pulled out money
From a pocket... It has very much afflicted me... (((But at me at once it was cheered up
When I have seen your letter!!!) it seems to me that we really approach each other.
If you have any questions, I always with pleasure shall answer them. Now to me
It is necessary to go and I shall write to you later and always I wait for your answer.
Please Also send me more than your photos. Yours Masha.


text:
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Offshore low cost PHP Web Development Company India
Published on January 24th 2012 | Web Design
Published on January 24th 2012 | Web Design
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ePixel Technologies is IT Consultant company in India. It is one of the top companies of PHP development India. Our PHP website design makes easy interaction with user and also easy to load the conten...
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Published on December 1st 2011 | Business
Published on December 1st 2011 | Business
ePixel Technologies is a fast growing and leading PHP web development India. Website is way to get more benefits from business. As we are living in latest technology, website is a mirror of your compa...


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I was recently diagnosed with 4 hepatic adeoma's in my liver (the largest is a hair under 5cm). I was also diagnosed with endometriosis and placed on BC when I was 13, so I've been on it for 21 years now. Mostly levonorgestrel, but also Yaz, Yasmin and ortho tricycline. They took me off two months ago because of the adenoma's. Has anyone else had this experience, and if so how did you deal with your endo? My symptoms are out of control, hair loss, pain, lethargy, joint pain and my back is killing me.
Ethinyl Estradiol/Levonorgestrel - Long term use for endometriosis and hepatic adenoma?
- Asked
- 20 Aug 2016 by Bluebuttterfly
- Topics
- endometriosis, estradiol, estradiol/levonorgestrel, ethinyl estradiol/levonorgestrel, levonorgestrel, benign liver tumor, liver, liver disease, diagnosis
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text:
Just like an account captures the audienceis fascination with memorable heroes, discord plus an engaging solution, an individual narrative gives you the opportunity to reveal a crucial second in your lifetime by participating visitors' imaginations. Aspects like depiction and conversation, in addition to exclusive attention to point of view and intent, will help an individual account that shares a piece of yourself with people is crafted by you. Pick a Persuasive Subject Your narrative will be many getting in case you come up with something of particular importance that is good. You might try making lists of remarkable life activities, such as special locations you have visited, individuals who have affected your daily life, beloved interests or occasions that you learned significant instructions to select a subject. Then, choose the topic that has one of the potential to be progressed into a tale. Over a separate little bit of paper, dissect the elements of your tale by writing down why the big event or person was important to your daily life and heroes who'll come in the account, the primary conflict. Art Your Own Style Speech could be the infusion of the writeris temperament into an article, employing word alternative, depth and pointofview to make it seem like mcdougal is really currently talking with followers. First person perspective, the pronouns " my use," "my" and " " can be an important element of style for personal narratives since it enables readers begin to see the narrative unfold during findings and your own steps.
Dactyl: a dactyl is just a foot with three syllables.
Individual stories can also be generally instructed before tense, like confirming of what happened the details. This enables mcdougal better possibility to examine the value of the event and think about lessons. Show, Don't Inform The gatherings that affected the writer's existence are n't just recited by superior particular narratives. Utilize placing descriptions, physical details and dialogue to create the narrative to life onpaper. In case your essay is approximately a teacher from senior school who prompted you to make an application as an example, for a grant, you might dramatize this minute by recreating the location of the classroom and any remarkable facts you recall, together with the talk. By using detail to aid them photograph the action, your article should put viewers right at the center of your own account. Resolve It With Representation They should be plainly conscious of why your subject is essential for you when http://unimogtr.com/exploratory-essay-topics-4/ followers walk-away from your essay. By figuring your plot with representation on what you've just told them you'll make this happen.
Reveal everything you know here.
Instead of pointing to session or a unique meaningful, try obtaining more individual by discussing how a knowledge improved you. Work as a camp counselor assisted you acquire greater command capabilities, or happening a tasks getaway on your church offered you the chance to encounter new civilizations possibly a summertime. Viewers may abandon by outlining the significance of the function your story more pleased should you conclude the tale.


text:
Majors and Programs
College of Liberal Arts and Sciences: Pre-Human Medicine
Physicians study, diagnose, and treat illness and injury. All medical schools recommend a broad pre-professional education that includes courses in biology, chemistry, physics, mathematics, English, social sciences, arts, and humanities. The Pre- Human Medicine major is designed for incoming freshman students who are unaware of what major they would like to pursue as an undergraduate student. Students who select Pre-Med, as their incoming major, will be advised in the College of Liberal Arts and Sciences to assist in helping students find a major that supports their academic interests as well as starting students in the required Pre-Med coursework needed for admittance into a Medical School. Most students earn a bachelor's degree while taking the courses required for admission to medical school. You may enroll in any major at Iowa State University to complete these courses. The coursework of the major you select should reflect your interests while meeting the academic requirements for medical school. Each medical school has specific requirements and we strongly suggest students research different medical schools, prior to entering Iowa State, to ensure they are aware of the academic requirements of each program.Questions/Concerns can be directed to our Pre-Health Adviser at email@example.com
Related Curriculums
Premedical and Health Sciences
Related Career Keys


text: I have a motorola milestone running Android 2.1 and this morning my data connection appears to have stopped working. I've tried via wifi and via 3G and neither seem to have any joy. The speed with which browsers, market etc. timeout suggests it's not even trying The last few weeks I've been seeing a lot of force-closes on my IMAP email account (to the point where I couldn't even download any emails) so perhaps that's connected? What should I do to identify the source of the problem / fix it? Am I going to have to do a factory reset and re-install everything?


text:
What's On
Guernsey Jazz Orchestra
04 November 2017 - 04 November 2017
The Guernsey Jazz Orchestra presents Big Band Fireworks. The Guernsey Jazz Orchestra takes to the stage to present an evening of music from some of the biggest names in jazz. From Gordon Goodwin to Frank Sinatra, Phil Collins' Big Band to Michael Bublé there's something for everyone. Together with some of Guernsey's most talented vocalists, GJO's exciting programme includes the classics as well as modern Big Band charts. With cabaret seating, in-hall bar and a spectacular showcase of live music, this is a night not to be missed! Tickets £12 or £120 per table of ten
Coach-House Gallery art expo
05 November 2017 - 02 December 2017
Frances Lemmon and Keith Langlois show work together until December 2nd. Wide variety of paintings on view; bold graphic musical murals, Guernsey folklore illustrations, bespoke framing, 3D lettering and horses as well as watercolour landscapes and small oil nautical studies. Opening times are 11-5pm Mon-Sat
The Café Club
10 November 2017 - 10 November 2017
Join Father Daughter duo Katie and Alan Gough (Saxophone and Piano) for an evening of light entertainment. Katie studied saxophone at the Birmingham Conservatoire and is now working locally as an instrumental teacher and freelance musician. Alan is well known as a conductor, teacher and freelance musician. An evening of east listening Jazz Standards and popular well-known melodies should prove to be popular and kind to the ears. Held in Café St James Tickets are £15 and include supper. Please tel: 711361 to learn more
Guernsey Symphonic Winds
11 November 2017 - 11 November 2017
The Guernsey Symphonic Winds, conducted by Alan Gough, return to the stage with an exciting programme crammed full of musical delights. Adding their sonorous sounds is the Guernsey Welsh Male Voice Choir, conducted by Fiona Le Tissier, who sing a variety of ontrasting works before joining forces with the full band for a Grand Finae. Tickets £10, £8 senior citizens and students, £8 restricted view. Children under 15 free. Tel: 711361 to book
St James Lecture Lunch
14 November 2017 - 14 November 2017
St James Lecture Lunch Series with Andrew Dyke - 'Guernsey Farmhouses and their Evolution' With 40 years in practice and a substantial emphasis on conservation and historic building work, Chartered Architect Andrew Dyke has worked on farmhouses, manoirs, mills, cottages, towers and many of Guernsey's iconic buildings including Castle Cornet, Town Church, Saumarez Manor, historic fortifications and the little chapel to name a few. In his capacity as Buildings Advisor to the National Trust of Guernsey for over thirty years he has had responsibility for all the buildings and structures owned by them or in their care. Andrew has also been responsible for reintroducing the use of the traditional thatch as well as clay and lime mortars on the island. In this fascinating talk, Andrew looks at our Guernsey farmhouses and how they evolved from medieval times to the 19th century along with some of their interesting characteristics Tickets £20 and must be booked in advance of the event. Call 711361 to book
Meet the Author
17 November 2017 - 17 November 2017
Meet the Authir - Peter Niven at Afternoon Tea Café St James. Join local author peter Niven for afternoon tea and a talk surrounding the writing and publication of his book 'St James the Less' Tickets £12.50 and must be booked in advance of the event. Tel: 711361 to book
Guernsey Symphony Orchestra
18 November 2017 - 18 November 2017
2017 Credit Suisse Series Guernsey Symphony Orchestra presents Brave New World. Guest Conductor: Ewa Strusinska and Guest Soloist: Rachel van der Tang (cello) In the final concert of the 2017 series the orchestra presents Leonard Bernstein's Overture to Candide, Max Bruch's Kol Nidrei (cello), Antonin Dvorak's Silent Woods (cello) and Dvorak Symphony No.9 'New World Symphony'. Tickets £15, £12.50, £10, Students half price, £6 restricted view. Tel:711361 to book
Fox Art Exhibition
22 November 2017 - 15 December 2017
An opportunity to view a selection of works following the judging of this annual competition by judge Allan Drummond. Now in its 34th year, the Fox Art Competition has helped progress the career of many leading artist in the Channel Islands and attracts a wide range of entries. On view in Café St James during building opening hours. Preview on Wednesday 22 November 5:30pm - 6:45pm. All welcome.
The Guernsey Bach Choir
25 November 2017 - 25 November 2017
The Guernsey Bach Choir & Orchestra present J.S. Bach Christmas Oratorio Parts 4- 6. Conducted by Alan Gough, Organ James Henerson and soloists Nicki Kennedy (Soprano), Alexandra Gibson (Contralto), Nicholas Hurndall Smith (Tenor) and Andrew Rupp (Bass. Tickets £14, £12 senipr citizens, £12 students, £10 restricted view Tel: 711361 to book Kindly sponsored by Polygon Group Ltd
Wire-Wrap Jewellery Workshop
30 November 2017 - 30 November 2017
I will go through some skills for wrapping sea glass and shell pieces to make pendants, using soft jewellery craft wire and jewellery pliers. Materials, and use of tools to make 2-3 pendants will be included. At Sula Framing and Gallery, Sandy Hook, L'Islet. Book at the link given, or contact Sula Gallery tel. 245396


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Trouble is brewing for the OKCoin Bitcoin exchange by the look of things. To be more precise, the exchange's parent company is forced to pay damages for facilitating money laundering. This decision was made by a Chinese civil court a few weeks, but the news only became public yesterday evening.
Given the legal grey area of Bitcoin in China, this civil court ruling does not come as a complete surprise. At the same time, it is the first time a major [civil] court ruling occurs against a Bitcoin exchange operating in the Chinese market. Whether or not this will become the new norm, remains to be seen.
Facilitating money laundering is never a good idea, and OKCoin.cn found that out the hard way. Do not confuse this entity with the Bitcoin exchange itself, as it is registered in Singapore. The parent company has been battling in court over these money laundering allegations for several years.
OKCoin Will Need To Get A Proper Business License
It all started when Huachen Commercial and Trading Co. Ltd. took OKCoin.cn to court. The commercial institution claimed they lost US$1.8m to a hacker, who then used the money to purchase Bitcoin. Once that process was completed, the hacker withdrew funds to Macau. OKCoin.cn appealed this verdict, but their efforts have been futile.
The civil court took exception to how OKCoin is conducting its ID verification. Every exchange platform – Bitcoin or otherwise – requires users to verify their identity. However, the parent company did not perform an adequate job, according to the ruling. Keeping in mind how the criminal in this case managed to set up multiple OKCoin accounts with fake information, the decision can easily be justified.
Note from the Author: OKCoin and other Chinese exchanges have stepped up their KYC and AML game ever since this court ruling in 2014.
But that is not all, as the OKCoin parent company was found guilty of operating illegally. While the company holds an official license, it doesn't allow them to [facilitate] trade Bitcoin for profit-making purposes. As a result, the judge ordered the company to get a proper business license.
With the majority of Bitcoin trading volume originating from China, this is not good news by any means.For OKCoin, obtaining the business license they need can be a difficult process. Moreover, this court case may set a precedent for future Bitcoin regulation in China.
Header image courtesy of OKCoin
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text: As per title we are pleased to offer a special price on the supply and fit of Eibach springs here at AmD Essex. The price will be £299.95 inc vat supplied and fitted for 2WD cars. This will also include four wheel laser alignment. For 4WD cars it will be £349.95 inc vat, also including four wheel laser alignment. We would also recommend replacing top mounts at the same time and these are avaliable at an extra cost. To book in for this offer or either contact me on -01708 861827 -firstname.lastname@example.com -or pm me via the private messaging system. If you require any further information, ask away.


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At the Anne and Henry Zarrow School of Social Work, we seek to provide a flexible program that meets the needs of all students from all walks of life. To this end, the school offers two MSW degree options: a 60 hour program for students with any bachelor's degree and a 33 hour advanced standing degree for students who have received a BSW degree from an accredited university within the last five years.
To find out more information about the advanced standing option, please view our advanced standing page. The 60 hour program can be completed on either a part-time or full-time basis. To find out more about the part-time option, please view our part-time page. To find out more about the full-time option, please view our full-time page.
At the Anne and Henry Zarrow School of Social Work students have the option of selecting one of two concentrations: Direct Practice (practice emphasis with individuals, families and small groups) or Administration and Community Practice (practice emphasis with larger groups, organizations and communities). For Advanced Standing students, this decision is made when applying. For part-time or full-time students, this decision is made during the last semester of the foundation curriculum. To read more about these options, please view our concentrations page.
The University of Oklahoma, 660 Parrington Oval, Norman, OK 73019-0390 (405) 325-0311.


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PHILADELPHIA -- Michael Vick left the Giants-Eagles game in the fourth quarter with a broken right hand, and his return is doubtful. Mike Kafka took over at quarterback with 8:07 left to play, and the Giants leading 22-16.
Vick's health continues to be a major concern for the Eagles, as he was unable to complete a game for the second consecutive week. Vick suffered a concussion during a Week 2 loss to the Falcons, and Kafka finished that game as well. It was not immediately known how long this injury would keep Vick sidelined. However, this season is taking an early toll on Vick's body.


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Cement production in Pakistan will be around 47Mt by 2009 with the expansion in production capacity of the existing 28 units, which will be enough to meet both domestic and export needs, it is learnt.
Sources said that three investors, from the UAE, Egypt and Qatar, are also considering setting up cement plants here. However, they said, the final decision to this effect would be taken by January next year whether to set up these plants in Sindh or Balochistan. The production from these units would be in addition to 47Mt that is anticipated from the present 28 units.
The biggest export market of Pakistan's cement is Afghanistan, where competition is growing among the Central Asian States, Iran and Pakistan. However, Pakistan is in a better position because of low freight charges. The cement sector has seen production of 6.32Mt during the last four months, bringing total production till October to 33Mt from 21Mt during January 2006.
This production is expected to rise to 38Mt by the end of December as a result of on-going capacity enhancement by existing units. Attock Cement has completed its expansion project and the expansion program of Bestway Cement would be completed soon. D G Khan Cement's new unit in Chakwal is likely to start production soon, whereas the other units have either started their capacity enhancement program or are planning.
Cement manufacturers see growing demand of the commodity at home due to planned construction of dams and increase in reconstruction activities in Afghanistan.
Cement crisis last year had forced the government to intervene. As a result, the government allowed import of unlimited quantities of cement without any customs duty and withholding tax through the private sector to meet the growing demand. The government had also earmarked an amount of Rs 720 million in the budget to arrest cement price hike.
The government has been paying substantial subsidy to cement importers to stabilise the prices.
Sources said that cement import has already been stopped because of much reduced prices in the domestic market. The government hopes that cement prices will remain under control because of the commitment of local manufacturers to raise the level of production and increase production capacity.
Published under Cement News


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I am a South African citizen with Permanent Residence status in Canada. I will be flying from Canada to South Africa for a visit. I will be flying from Montreal to Paris (CDG), and Paris (CDG) to Amsterdam, and finally from Amsterdam to Johannesburg through Air France. In Paris, I will be transferring from terminal 2E to 2F.
The layovers in Paris and Amsterdam are very short, both less than 2 hours. Do I require a visa for these transfers? I am getting conflicting answers.
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Is a Visa Required to Transfer from CDG Terminal 2E to 2F??
I am a South African citizen with Permanent Residence status in Canada. I will be flying from Canada to South Africa for a visit. I will be flying from Montreal to Paris (CDG), and Paris (CDG) to Amsterdam, and finally from Amsterdam to Johannesburg through Air France. In Paris, I will be transferring from terminal 2E to 2F.
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As long as you stay airside you do not need visas - since you are not actually entering the country. This will depend on if you are on a single ticket - with baggage checked all the way through and receiving boarding passes at your first flight.
If they are separate tickets and you need to collect and recheck baggage you will need to go landside and then may need visas (you would have to check this with the appropriate embassays.
In any case - you will have to go through security again before each flight - so be sure you have enough time between flights.
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Thanks for your reply - much appreciated! It's a single ticket, so luggage will be checked all the way through. I have a very short layover so I will be wearing running shoes for sure!
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You will go through a passport control when entering EU from a non Schengen zone.
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The official airport website http://www.aeroportsdeparis.fr/ADP/en-GB/Passagers/Home/
contains maps and useful transfer advice, which you can print out and take with yoiu to help navigation. As soon as you step off the plane, check a monitor to be sure that your next departure gate has not been changed.
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Thank you for the info. Danon, you mention passport control. Does that mean they need to see a visa of some sort?
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"As long as you stay airside you do not need visas - since you are not actually entering the country. "
...is simply untrue.
Where on earth does the person giving this dreadful advice think those terminals are?
To transfer terminals (or even walk to the gate), you have to go through French territory. By doing so, even if you don't go through passport control, international treaties give a traveller considerable claims on France (like asylum, protection and the right to medical treatment). To avoid being taken advantage of, most Western countries (such as the one the offerer of this awful advice is a citizen of) require most travellers from developing countries to have transit visas, even if they are not leaving an airport's airside.
There are a wide range of exceptions to this, and South Africans travelling to other rich countries MIGHT be among them. However at least one site (http://visados.com/en/visa-for-France )implies South African tourists are NOT an exception, and DO need a visa even for airside transfer. If that site is accurate, you won't even be allowed on the flight out of South Africa.
You need to consult your nearest French consulate URGENTLY for clarification, unless someone can demonstrate between now and tomorrow morning that the visados site is wrong. And don't rely on general websites like this for information on something so complicated and important.
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Flying between Paris and Amsterdam will require a Schengen visa because it is a "domestic" European flight. The only case where you would not require a visa is if your plane from Canada just stops in Paris and then continues to Amsterdam without you leaving the aircraft. In such a case, TWOV rules may apply.
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In any case, you will not be allowed to board the flight in Canada if your documentation is inadequate because the airlines are fined 20,000 euros in such cases.
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I took the opposite route (2F to 2E) when I transited in CDG (Prague to Toronto) & went through passport control (because I was leaving EU). Normally a person will pass passport control in the first airport if going to another Schengen country (Holland) but I would ask AF if they can provide transit pass in your case as you'll continue outside Schengen. 2E & 2F are in the same building.
Here is the map
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Have flown numerous time to switz with a transfer of planes at LHR. Stayed airside (yes, even changed terminal via "airside" buses" and never went through immigration until I got to Switz.
Have also done the same through CDG to Switz. Not sure why it was not considered a "domestic" flight - but we did go through Immigration in Switz.
Since this is a 2 stop itinerary - why not check with the embassays - I don;t know rules for South africans.
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As Kerouac says you need a Schenegen visa as you're travelling from Paris to Amsterdam
details here www.consulfrance-montreal.org/-Visas-for-Tourists-or-for-a-Simple-
If you were simply transiting CDG then you could have got away with a Transit visa
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Your safest bet is to contact either the French Embassy in Ottawa or a consulate closest to where you live.
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You will go through passport control when transferring from 2E to 2F to enter Schengen. There's no way to remain "outside" Schengen.
Were your experiences prior to Switzerland joining Schengen?
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I meant when you flew through CDG. The UK isn't part of Schengen.
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Canadian citizens do not need a visa to enter Schengen
if you have Permanent Resident status in Canada ( even if you are not a citizen ), you might be ok,
...for such a serious questions, you might wish to contact authorities - not Fodorites.
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>>Have flown numerous time to switz with a transfer of planes at LHR. Stayed airside (yes, even changed terminal via "airside" buses" and never went through immigration until I got to Switz<<
But - nyt -- first, this question has nothing to do w/ either the UK or CH and . . you don't need a visa to visit any of the countries involved. This is a wholly different situation.
I sure hope the OP doesn't take the first response as the be all/end all and doesn't come back to the thread.
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I meant to say >>you personally don't need a visa to visit any of the countries involved<<
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Sometimes one forgets that although "we" don't need visas to enter most countries ,some travelers
We were checking in for the flight to Sydney when the employee of the airline pointed out we did not have Australian visas. I just assumed , being Canadian , it was not necessary.
I was mortified, but luckily,
we were issued visas on the spot and continued the trip.
A lesson learned.
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I am a Permanent Resident of Canada so I am hoping that counts in my favor. The French Consulate in Montreal only answers Visa-related queries by email and the Embassy in Moncton advised that they no longer process visas so are unable to comment. No response from Montreal yet ... getting pretty nervous. I have contacted Air France and CIBT. Both say no visa is required but from what I have read on the CDG website, I am confused. Will likely end up delaying or cancelling trip altogether
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I have South African friends who have to wait for weeks to get a visa valid for France, including a personal interview each time although they have been to France a number of times.
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But - aren't you traveling on a SA passport? And isn't that the controlling authority? I don't know myself. Just asking.
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Yes, I have a South African passport. I would have thought all I would have needed was that, my Canadian Permanent Residence Card and my onward ticket.
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I don't think any other country cares about your residency in Canada...it's not a passport or a visa and is only valid in Canada as far as I know.
Our South African friend has a UK indefinite leave to remain (permanent residency) and he still has to apply weeks in advance for schengen. They don't care about his uk residency at all...
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As I said before the issue might be that you'll transferring to another Schengen flight. AF & KLM are one company. If this visa thing such a big issue may they'll be willing to exchange portion of your ticket (YUL-CDG-AMS) for the direct YUL-AMS flight (there is one daily) with KLM. In AMS (Schiphol) there is no need for visa as long as next flight is non-Schengen.
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Thank you, hkto. That is a good idea. I will try to contact KLM.
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From what I can see, the problems is what PP said...transferring to another flight in the zone.
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The OP could also change CDG-AMS-JNB to CDG-JNB.
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Can anyone comment on this : http://www.consulfrance-toronto.org/spip.php?article545
Consulate General of France in Toronto site says: "The visa section of the Consulate General of France in Toronto reminds all Landed immigrants of Canada, holding a valid Permanent Resident Card that they do not require a visa for an Airport Transit in the Schengen Zone."
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They're referring to an international to international transit, i.e. YUL-CDG-JNB.
The link that danon posted above explains it better:
If you are holding a valid Permanent Resident Card of Canada, or a valid Green Card of the USA AND you are not going out of the international zone of the airport AND you are holding confirmed airplane tickets (NO « stand-by ») AND your next destination after France is not a Schengen country, then, no matter what your nationality is, you do not need a Type A visa. You can transit without a visa.
If you're still ticketed as YUL-CDG-AMS-JNB, what you need is a short stay visa not a Type A transit visa.
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Thank you for all the replies. I appreciate your input. After several days of trying to contact the airline (and several hours of being on hold), I was finally able to change my ticket. Praise God!
Sign in to comment.


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"The Wahhabist Saudi Royal Family, Caliphs, Grand Muftis, Ayatollahs, Imams, chieftains and warlords who are in charge in these deeply sexist, rigidly gender-segregated, pathologically puritanical places would be surprised indeed to learn that they are not practicing "Islam." "
Actually they are. I've read the Quran. All 114 surahs. Without annotations—not an easy slog.


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charles.pe2278847wrote a review of Deeluxe Rough Diamond Snowboard Boot - Men's on November 19, 2011
These boots are sick!
I got the Deeluxe Rough Diamond Snowboard Boot a couple weeks ago. The boot was very comfortable straight out of the box. The heel locking system in the inner liner works excellent, I've never had a boot that made me feel as locked in as I am in this boot. The speed lace system in both the inner liner and outer boot works lightning fast and are very easy to use. The dual power straps around the shin give excellent support. I took the boot up onto the mountain for the first time the other day and I loved the boot as much as I did when I first tried them on.
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We have previously implicated transient receptor potential vanilloid 4 (TRPV4) channels and alveolar macrophages in initiating the permeability increase in response to high peak inflation pressure (PIP) ventilation. Alveolar macrophages were harvested from TRPV4−/− and TRPV4+/+ mice and instilled in the lungs of mice of the opposite genotype. Filtration coefficients (Kf) measured in isolated perfused lungs after ventilation with successive 30-min periods of 9, 25, and 35 cmH2O PIP did not significantly increase in lungs from TRPV4−/− mice but increased >2.2-fold in TRPV4+/+ lungs, TRPV4+/+ lungs instilled with TRPV4−/− macrophages, and TRPV4−/− lungs instilled with TRPV4+/+ macrophages after ventilation with 35 cmH2O PIP. Activation of TRPV4 with 4-α-phorbol didecanoate (4αPDD) significantly increased intracellular calcium, superoxide, and nitric oxide production in TRPV4+/+ macrophages but not TRPV4−/− macrophages. Cross-sectional areas increased nearly 3-fold in TRPV4+/+ macrophages compared with TRPV4−/− macrophages after 4αPDD. Immunohistochemistry staining of lung tissue for nitrotyrosine revealed increased amounts in high PIP ventilated TRPV4+/+ lungs compared with low PIP ventilated TRPV4+/+ or high PIP ventilated TRPV4−/− lungs. Thus TRPV4+/+ macrophages restored susceptibility of TRPV4−/− lungs to mechanical injury. A TRPV4 agonist increased intracellular calcium and reactive oxygen and nitrogen species in harvested TRPV4+/+ macrophages but not TRPV4−/− macrophages. Kf increases correlated with tissue nitrotyrosine, a marker of peroxynitrite production.
- pulmonary edema
- filtration coefficient
- superoxide
- nitric oxide
- Ca2+ channels
- peroxynitrite
- 4-α-phorbol didecanoate
previous experimental studies on ventilator-induced lung injury (VILI) indicate a rapid increase in lung vascular permeability after overdistension with high airway pressures and lung volumes (5, 6, 30). The increased permeability is calcium-dependent because it was attenuated by gadolinium, an inhibitor of stretch-activated, nonselective cation channel activity (31, 34). We (14) recently presented evidence that the transient receptor potential vanilloid-4 (TRPV4) channel was this stretch-activated cation channel. High airway pressure-induced permeability increases were abolished in lungs of TRPV4 knockout (KO) and in wild-type (WT) mice treated with inhibitors of TRPV channels, arachidonic acid production, and P-450 epoxygenases.
Recent studies also suggest that the alveolar macrophage (AM) has a critical role in the rapid initial increase in permeability after lung overdistension because macrophage depletion attenuates VILI in rats (7, 10). The AM is a sentinel cell that can initiate lung injury and inflammation because macrophage depletion attenuates lung injury after challenges with sepsis (9), live bacteria (15), or ischemia-reperfusion (48). The number of AM recovered by bronchoalveolar lavage (BAL) decrease within minutes after initiation of ventilation with high airway pressures, indicating a rapid activation and firm adhesion (7, 10). Pugin et al. (35) compared cultures of various lung cell types during cyclical strain and identified the lung macrophage as the major source of inflammatory cytokines, chemokines, and matrix metalloproteinase-9 (MMP-9) during mechanical stretch. AM are also sources of reactive oxygen and nitrogen species (28). Although TRPV4 has been identified in macrophages by immunohistochemistry (2), the role of the TRPV4 channel in macrophage function and the mechanisms whereby activation of macrophages initiates overdistension-induced lung injury have not been studied.
In the present study, we addressed the hypothesis that TRPV4 expressed in macrophages plays a central role in the initial transduction of VILI. We instilled WT (TRPV4+/+) macrophages into lungs of KO (TRPV4−/−) mice and instilled TRPV4−/− macrophages into lungs of TRPV4+/+ mice. The filtration coefficient (Kf) was measured in lungs isolated from these mice in response to ventilation with low or high peak inflation pressures (PIP). The instilled TRPV4+/+ macrophages restored the Kf increase produced by high PIP in TRPV4−/− mouse lungs. Harvested macrophages were also labeled with specific fluorescent dyes sensitive to levels of intracellular calcium, superoxide, and nitric oxide (NO) and challenged with a TRPV4 channel agonist, 4-α-phorbol didecanoate (4αPDD), and the fluorescence was measured. Cross-sectional areas of the two macrophage genotypes were also measured in response to overnight culture and the TRPV4 agonist. Lung tissue samples from six experimental groups were immunostained for nitrotyrosine, and the color intensity was evaluated.
TRPV4 KO Mice
The TRPV4 KO mice were generated by Cre-lox excision of exon 12 of the TRPV4 gene, which codes for the pore-loop region and the adjacent transmembrane domain (21). Mice were genotyped using mouse tail DNA using PCR primers described in our (2) previous online supplement. PCR products resolved on agarose gel indicate a 2.1-kb band for TRPV4+/+ mice and a 1.1-kb band for TRPV4−/− mice, the latter reflecting excision of exon 12. The TRPV4−/− mice have been backcrossed with the C57BL/6 background mice for 8 generations so that C57BL/6 could be used as controls (14). The 8-fold backcross with the C57BL/6 background would lead to retention of only 0.38% of the non-C57BL/6 background, and previous studies on Kf in mouse lungs indicated no significant differences between the α1G KO mice and littermates bred on the C57BL/6 background and littermates of these TRPV4 KO mice relative to baseline lung Kf values or Kf increases after 10 μmol of the TRPV4 agonist, 4αPDD (2, 43).
The TRPV4−/− breeding colony was housed in the University of South Alabama Vivarium and monitored by veterinary personnel. All protocols were approved by the Animal Care and Use Committee of the University of South Alabama College of Medicine and met all National Institutes of Health guidelines.
Isolated Lung Preparation
C57BL/6 WT male mice (Charles River Laboratories) and TRPV4−/− mice weighing 19.6–32.0 g (24.1 ± 0.3 g) were anesthetized with an intraperitoneal injection of 100 mg/kg pentobarbital sodium. The trachea was cannulated, and the mice were ventilated with a gas mixture of 20% O2-5% CO2-75% N2 by using a Harvard rodent ventilator (model 683; Harvard, South Natick, MA). The tidal volume was adjusted to obtain a PIP of ∼9 cmH2O at a respiratory rate of 90 breaths/min, with a positive end-expiratory pressure (PEEP) of ∼2.5 cmH2O. The chest was opened, 100 IU of heparin sodium was injected into the right ventricle, and a suture was placed around the pulmonary artery with aorta. Cannulas (0.86-mm inner diameter and 1.27-mm outer diameter) were placed in the pulmonary artery and left atrium, and lung and heart were excised en bloc and suspended from a balance beam attached to a force transducer (model FT03 C; Grass, Quincy, MA). The initial 1–2 ml of perfusate, which contained residual blood cells and plasma, were discarded and not recirculated. All lungs were perfused with 1% bovine serum albumin-3% clinical grade dextran in Earle's balanced salt solution by using a roller pump (MINIPULS 2; Gilson, Middleton, WI) at a constant flow rate of 0.75 ml/min in a recirculating system that had a system volume of 10 ml. The venous outflow was collected in a reservoir, the height of which could be adjusted to increase venous pressure. Pulmonary arterial pressure and pulmonary venous pressure (Ppv) were zeroed at the midlung level, airway pressure was measured by using Cobe pressure transducer (Lakewood, CO), and pressures and lung weight were continuously recorded on a Grass model 7D polygraph (14).
After a 30-min equilibration period and attainment of an isogravimetric state, Ppv was increased by 6 cmH2O for 20 min. The change in capillary pressure (Ppc) was determined by double occlusion before and after the Ppv increase. Kf (in ml·min−1·cmH2O−1·100 g−1) was calculated as the rate of lung weight gain between 18 and 20 min divided by the change in Ppc. All Kf values were normalized to 100-g predicted lung weight (PLW) on the basis of the ratio of lung-to-body weight (BW) according to PLW = (0.00452 ± 0.0003) BW (29).
Lung wet-to-dry weight ratios.
After the measurement of final Kf, the lung was harvested, wet weight was determined, and samples were desiccated at 80°C for 1 wk before the dry weight was determined. The wet weight divided by dry weight was calculated for the wet-to-dry lung weight ratio (W/D ratio).
High and low PIP ventilation protocols.
The lungs were ventilated for 30 min with 9 cmH2O PIP and with ∼2.5 cmH2O PEEP at 40 breaths/min followed by an increased venous pressure for 20 min for measurement of Kf. Lungs were then divided into one of two protocols as shown in Fig. 1. The low PIP protocol required two additional 30-min periods of 9 cmH2O PIP ventilation followed by Kf measurements at the end of each 30-min period. In lungs assigned to a high PIP protocol, lungs were sequentially ventilated for 30-min periods at 25 and then 35 cmH2O PIP. Each period was ended with Kf measurements as previously described (14). In all groups, perfused buffer was maintained at 37°C, and Kf was measured at 30, 80, and 130 min, i.e., the venous reservoir was raised at these times for the 20-min Kf measurement period, and the weight gain slope was measured during the last 2 min of the venous pressure increase. The temperature was maintained by direct measurement of the perfusate using a type K thermometer (Extech Instruments, Waltham, MA) inserted into the right ventricle of the isolated heart-lung preparation.
Macrophage transfer protocol.
WT (TRPV4+/+) or TRPV4 KO (TRPV4−/−) mice were anesthetized with intraperitoneal injection of 100 mg/kg pentobarbital sodium. To obtain AM, BAL was performed 3 times with 0.5 ml of PBS. Pooled BAL fluid from 2 mice of each genotype was centrifuged at 500 g for 10 min, and the cells were resuspended in 50 μl of PBS for tracheal instillation in each experimental mouse. This resulted in an average of 517,000 ± 74,000 TRPV4+/+ and 627,000 ± 113,000 TRPV4−/− cells per instillation. Differential BAL cell counts indicated 88.7 ± 0.5% macrophages, 10.9 ± 0.6% lymphocytes, and 0.3 ± 0.1% neutrophils in TRPV4+/+ BAL (n = 4) and 87.6 ± 1.3% macrophages and 11.7 ± 1.2% lymphocytes and 0.6 ± 0.2% neutrophils in TRPV4−/− BAL (n = 4). TRPV4−/− mice were anesthetized and intubated. Then, 50 μl of the macrophage suspension was injected into the lung through tracheal tube followed by 15 min of low PIP ventilation with 2 cmH2O PEEP to spread the fluid to the alveoli. The tracheal tube was removed, mice were kept in the cages for 16 h, and then Kf and W/D ratio were measured.
Experimental groups.
Isolated lung experiments were performed on groups (n = 5) ventilated with the low or high PIP protocols with or without the addition of AM from either WT or KO mice. C57BL/6 mice were used as WT controls as the KO breeders were backcrossed some 8 generations against this background. Kf measurements at baseline and after high vascular pressure injury were not statistically different between C57BL/6 mice and true littermates (unpublished data). Using the low PIP (LP) protocol, groups included: 1) LP WT group of WT mouse lungs ventilated with low PIP; 2) LP WT+KO AM group of WT lungs instilled with KO AM ventilated with low PIP; 3) LP KO group of TRPV4 KO lungs ventilated with low PIP; and 4) LP KO+WT AM group of KO mice instilled with WT AM ventilated with low PIP. Groups ventilated with the high PIP (HP) protocol included: 5) HP WT group of WT mouse lungs ventilated with high PIP; 6) HP WT+KO AM group of WT lungs instilled with KO AM ventilated with high PIP; 7) HP KO group of TRPV4 KO lungs ventilated with high PIP; and 8) HP KO+WT AM group of KO mice instilled with WT AM ventilated with high PIP.
Macrophage TRPV4 Expression
AM were harvested from either WT (n = 2) or TRPV4−/− (n = 2) mice. Total RNA was extracted from the pooled macrophages in each genotype using a Qiagen RNeasy Plus Mini Kit with on-column removal of genomic DNA. For comparison, we also isolated total RNA from cultured murine lung epithelial (MLE) and alveolar macrophage (RAW) cell lines (n = 2 in each case). After first-strand cDNA synthesis using 1.5 μg of total RNA (SuperScript II; Invitrogen, Eugene, OR), PCR was performed to amplify TRPV4 mRNA. Primers designed to amplify a sequence of the TRPV4 mRNA coding for the pore-loop region of the protein were sense 5′-TCACGAAGAAATGCCCTGGAGTGA-3′ and antisense 5′-ACTGCAACTTCCAGATGTGCTTGC-3′ (612-bp predicted product length), and those for GADPH mRNA as a control were sense 5′-TGTGTCCGTCGTGGATCTGA-3′ and antisense 5′-CCTGCTTCACCACCTTCTTGAT-3′ and were obtained from Integrated DNA Technologies (IDT). PCR reactions (50 μl) included 3-μl cDNA, 2-μl primer mix (5 μM each sense and antisense primers), and 45-μl Platinum Blue PCR SuperMix (Invitrogen). cDNA was amplified in a thermal cycler (Hybaid) using the following conditions: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, for 35 cycles, with a final extension at 72°C for 10 min. Products were separated on a 1.8% agarose gel and visualized by ethidium bromide staining.
Confocal fluorescence imaging.
Macrophage fluorescence studies were performed using a spinning-disk laser confocal microscope (RS-3 UltraVIEW; PerkinElmer). AM were cultured overnight in coverslip bottom 35-mm petri dishes. During fluorescence measurements, the dishes had a volume of ∼2.5 ml and were continuously perfused at 8 ml/min with 37°C Earle's balanced salt solution bubbled with 5% CO2-balance air. For measurement of intracellular Ca2+, cells were loaded with Ca2+ indicator solution (containing HEPES buffer, 10 μM fluo-4 AM, 0.01% DMSO, 0.02% Pluronic F-127) for 30 min at 37°C in the dark. Ca2+ fluorescence was measured by excitation at 488 nm and emission filtered at 510 nm. To measure intracellular NO production, cells were loaded for 30 min with 10 μM 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) diacetate, fluorescence was measured by excitation at 495 nm, and emission was measured at 515 nm. Superoxide production was measured by loading the cells with 500 nM MitoTracker Red dye for 30 min, fluorescence was measured by excitation at 578 nm, and emission was measured at 599 nm. Fluo-4 AM, DAF-FM, and reduced MitoTracker Red were obtained from Invitrogen. 4αPDD was obtained from Sigma (St. Louis, MO). Drugs were dissolved in DMSO before use. Macrophage cross-sectional areas and diameters were measured by drawing a region of interest around individual cells and calculating mean intensity, cross-sectional areas, and diameters using UltraVIEW software. The system was calibrated using 6-μm calibration beads. Fluorescence was measured before and after challenge with 10 μM 4αPDD, a TRPV channel agonist.
Scanning Electron Microscopy
Macrophages were placed on glass coverslips and fixed with 3% glutaraldehyde in cacodylate buffer. Specimens were rinsed in cacodylate buffer, postfixed with osmium tetroxide for 30 min, dehydrated, and chemically dried with hexamethyldisilazane (HMDS; Ted Pella, Redding, CA). The coverslips were attached to stubs, and the specimens were coated with gold-palladium in a Denton DSM-5A cold sputter module (Denton Vacuum, Moorestown, NJ). Specimens were viewed and photographed in a Philips XL20 scanning electron microscope (FEI, Hillsboro, OR).
Immunohistochemistry Staining for Nitrotyrosine
Sections of mouse lung tissue from LP KO, HP KO, LP WT, HP WT, HP KO+WT AM, and HP WT+KO AM groups were fixed with 4% paraformaldehyde, processed, and embedded in paraffin. Sections (5 μm) were incubated overnight at 4°C with 1:500 primary antibodies to nitrotyrosine (Cell Signaling Technology) followed by incubation for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody (1:100; Santa Cruz Biotechnology) as previously described (2). Sections were stained with diaminobenzidine and counterstained with hematoxylin. The presence of nitrotyrosine was indicated by the brown peroxidase reaction product. Light micrographs were obtained, and a semiquantitative analysis was performed using MetaMorph multispectral software (CRi, Woburn, MA). The software allowed selective threshold intensities of red, green, and blue to be adjusted. Blue intensity was adjusted to remove air spaces, and tissue red intensity of tissue areas above threshold were recorded. Taking the difference of these parameters, a staining coefficient was calculated. Although the threshold areas correlated with injury, a better correlation was calculated by multiplying the threshold area by average pixel intensity. The mean values (n = 4) for each group were normalized by dividing by the value of the LP KO group.
Statistical Analysis
All values are expressed as means ± SE. One- or two-way ANOVA with repeated measures followed by a Student-Newman-Keuls posttest or linear regression analysis was used. Significant differences were determined where P < 0.05.
Macrophage Genotype Effects on Kf and Edema
The permeability and edema responses of isolated perfused mouse lungs to PIP and the addition of macrophages with or without functional TRPV4 channels are shown in Fig. 2. In groups ventilated with the low PIP protocol (Fig. 2A), the Kf in the LP WT+KO AM group was significantly (35%) higher than that in the LP KO group at the 130-min time period and its baseline value. There were no other differences between other groups or time periods. Figure 2B shows Kf measured after the high PIP ventilation protocol. Kf in the HP KO group did not change significantly from baseline after ventilation with either 25 or 35 cmH2O PIP. In the HP WT, HP WT+KO AM, and HP KO+WT AM groups, Kf increased significantly after ventilation with 25 cmH2O PIP and showed a further significant increase after the 35 cmH2O PIP ventilation period. The Kf for these 3 groups increased ∼2.2-fold after 35 cmH2O PIP compared with Kf after 9 cmH2O PIP ventilation but were not significantly different from each other at any time period. However, Kf in these 3 groups were significantly greater than the Kf for the HP KO group after both the 25 and 35 cmH2O PIP ventilation periods.
The W/D weight ratios were indicative of the edema gained during the experiments. Figure 2C shows the terminal W/D ratios for the 4 low PIP and 4 high PIP groups. W/D ratios after the low PIP ventilation were 4, 15, and 24% greater in the LP WT, LP WT+KO AM, and LP KO+WT AM groups compared with those of the LP KO group. After low PIP ventilation, the W/D ratios of the LP KO+WT AM group were significantly greater than those for the LP KO group but not different from the other groups. The W/D ratios for all high PIP groups except the HP KO group were significantly greater after high PIP ventilation than the same group after low PIP ventilation. W/D ratios in the HP WT, HP WT+KO AM, and HP KO+WT AM groups were significantly greater than those of the HP KO group by 8, 19, and 21%, respectively. These data indicate that macrophage TRPV4 was required for the permeability increase and increased edema after high PIP ventilation and that the presence of TRPV4 on TRPV4+/+ macrophages restored the pressure-induced permeability increase in TRPV4−/− lungs.
Macrophage TRPV4 and GAPDH Expression
PCR product was measured in WT and KO macrophages obtained by BAL. TRPV4 has previously been identified by immunohistochemistry in mouse lung epithelium, endothelium, and macrophages, but the TRPV4 messenger RNA in AM has not previously been reported. The primers used for the PCR reaction were specific to the pore region of the TRPV4 channel, which we reasoned must be absent in the KO mice. The PCR product indicated that the TRPV4 pore region (612 bp) was intact in the macrophages from WT mice but not the TRPV4 KO mice (data not shown). PCR product for mGADPH (100 bp) used as a control was present in both TRPV4+/+ and TRPV4−/−macrophages.
Effects of TRPV4 Activation on Macrophage Function
Macrophage intracellular calcium.
AM obtained by BAL were labeled with fluo-4 dye as described. Perfusion of the cells with buffer containing 4αPDD produced a heterogeneous increase in fluorescence indicating calcium entry into the TRPV4+/+ macrophages. However, no significant increase was observed in the TRPV4−/− macrophages. Figure 3A shows fluorescence micrographs of fluo-4-labeled TRPV4−/− and TRPV4+/+ macrophages after 4αPDD, whereas Fig. 3B indicates the transient responses of individual TRPV4−/− and TRPV4+/+ macrophages over the first few minutes after adding 4αPDD. Fluorescence intensity increased heterogeneously in TRPV4+/+ macrophages but not in TRPV4−/−macrophages. Figure 3C summarizes the mean calcium responses of TRPV4−/−(n = 67) and TRPV4+/+ (n = 64) macrophages before and after challenge with 4αPDD. Mean fluorescence was not different between TRPV4−/− and TRPV4+/+ macrophages under baseline conditions, but fluorescence intensity was significantly higher in the TRPV4+/+ macrophages after challenge with 4αPDD compared with TRPV4+/+ baseline measurements or TRPV4−/− macrophages at baseline and after 4αPDD treatment. These studies indicate a significant intracellular calcium increase was induced by 4αPDD activation of TRPV4 in the TRPV4+/+ macrophages, but minimal calcium increase was observed in the TRPV4−/− macrophages.
Reactive oxygen species.
AM from TRPV4−/− and TRPV4+/+ mice were labeled with reduced MitoTracker Red dye, which makes a fluorescent product that is trapped in the mitochondria when oxidized by superoxide anions. TRPV4+/+ and TRPV4−/− macrophages stained with MitoTracker Red are shown in Fig. 4A after treatment with 4αPDD. There were no significant differences in fluorescence intensity between macrophage genotypes under baseline conditions, but the fluorescent intensity of the TRPV4+/+ (n = 38) macrophages after 4αPDD treatment was significantly increased compared with baseline values for either untreated TRPV4−/− (n = 55) or TRPV4+/+ macrophages or TRPV4−/− macrophages treated with 4αPDD (Fig. 4B). Although oxidized MitoTracker Red accumulated in the mitochondria, this may also reflect oxygen free radicals produced outside the mitochondria because superoxide and hydrogen peroxide can diffuse into the mitochondria from other cellular sources. However, TRPV4 activation significantly increased production of reactive oxygen species in TRPV4+/+ macrophages.
Reactive nitrogen species.
AM from TRPV4−/− and TRPV4+/+ mice were also labeled with DAF-FM, a NO-sensitive dye, and fluorescence intensity was measured. Figure 5A indicates TRPV4+/+ and TRPV4−/− macrophages labeled with DAF-FM dye after 4αPDD, whereas Fig. 5B summarizes the mean fluorescence of TRPV4−/− (n = 19) and TRPV4+/+ (n = 32) macrophages under baseline conditions and 30 min after treatment with 4αPDD. There was no difference in fluorescence intensity between macrophage genotypes at baseline, but the fluorescent intensity of the TRPV4+/+ macrophages after 4αPDD treatment was significantly increased compared with baseline values for TRPV4−/− and TRPV4+/+ macrophages and for 4αPDD-treated TRPV4−/− macrophages. These studies suggest that TRPV4 activation also increased NO production, likely initiated by the increased calcium entry through the TRPV4 channel.
Macrophage spreading.
After plating BAL macrophages on glass coverslips, we observed spreading and lamellipodia formation over 24 h in culture, which was greatly increased by treatment with 4αPDD. Figure 6 shows scanning electron micrographs (SEM) of TRPV4−/− (Fig. 6, A and B) and TRPV4+/+ (Fig. 6, C and D) macrophages. Figure 6, A and C, shows freshly harvested TRPV4−/− and TRPV4+/+ macrophages, respectively, obtained by BAL, whereas Fig. 6, B and D, shows TRPV4−/− and TRPV4+/+ macrophages after 24-h incubation in DMEM with 10% FBS and 30-min posttreatment with 4αPDD. The diameters of freshly harvested macrophages of both genotypes were approximately 8–10 μm. Although the diameter of the TRPV4−/− macrophages remained at ∼10 μm with or without 4αPDD challenge, many of the TRPV4+/+ macrophages formed prominent lamellipodia (Fig. 6, arrow) and spread to diameters of ∼27 μm or 3.4-fold that of the freshly harvested macrophages. Figure 7 summarizes mean diameters (Fig. 7A) and cross-sectional areas (Fig. 7B) measured from SEM of TRPV4+/+ and TRPV4−/−macrophages. Each group represents measurements from 7 to 18 cells determined using the UltraVIEW software. Although these data represent cells taken at random, all TRPV4+/+ cells did not spread in a homogeneous manner. Using a diameter threshold of 11.5 μm, none of the TRPV4−/− macrophages exceeded this diameter regardless of treatment. In contrast, 27% of TRPV4+/+ macrophages exceeded this diameter after 24-h incubation in FBS, and 42 and 65% of TRPV4+/+ macrophages exceeded the threshold diameter after incubations with 4αPDD for 30 and 60 min, respectively.
Macrophage cross-sectional areas were also measured in fluo-4-labeled macrophages using the UltraVIEW software (data not shown). Although the groups exhibited the same statistical differences as observed for SEM, the magnitude of the differences were not as great. We concluded that the thin lamellipodia could not be visualized as accurately using the fluorescent dye as in the SEM. Whereas the cross-sectional diameter of 4αPDD-treated TRPV4+/+ macrophages was 252% greater than 4αPDD-treated TRPV4−/− macrophages using analysis of SEM, the relative increase between TRPV4+/+ (n = 23) and TRPV4−/− (n = 19) macrophages was only 43% determined using the fluo-4-labeled macrophages. Thus activation of the mechanogated TRPV4 channel initiates signaling pathways for cell adhesion and spreading, but the response is heterogeneous in TRPV4+/+ macrophages.
Immunohistochemistry Staining for Nitrotyrosine
Samples of lung tissue from the isolated lung experiments were fixed and immunostained for 3-nitrotyrosine. Brown reaction product indicates the presence of reaction product due to nitrotyrosine, and the blue color is a tissue counterstain. The presence of nitrotyrosine is generally accepted as a marker of peroxynitrite activity. Figure 8 shows examples of nitrotyrosine staining in lung tissue samples from the LP KO (Fig. 8A), LP WT (Fig. 8B), HP KO (Fig. 8C), HP WT (Fig. 8D), KO+WT AM (Fig. 8E), and WT+KO AM (Fig. 8F) groups. A significantly greater amount of brown staining was apparent in the high PIP ventilated lungs (Fig. 8, D–F) compared with low PIP ventilated lungs (Fig. 8, A and B) and high PIP TRPV4−/− lungs (Fig. 8C). Thus an increase in nitrotyrosine reaction product was present in all high PIP ventilated groups except HP KO, as indicated in the HP WT, WT+KO AM, and KO+WT AM lungs.
The brown immunostaining of nitrotyrosine was quantitated using MetaMorph software to threshold for the red color spectrum. The percent area stained for nitrotyrosine was then multiplied by the average pixel density to obtain a staining coefficient. Six groups were selected that exhibited differences in Kf. Figure 9 shows the relationship of lung section nitrotyrosine immunostaining (n = 4) for each group compared by linear regression with mean Kf values for that group. A significant correlation (r2 = 0.87) was observed with the relationship, Kf = 0.133 + 0.185 nitrotyrosine. This indicates a strong correlation of tissue nitrotyrosine to lung injury.
VILI is a complex syndrome induced by tensile failure and a cellular response to mechanical stress (5, 6, 30) and significantly contributes to mortality during ventilatory support for acute respiratory distress syndrome (ARDS) (4). The rapid onset of the increase in vascular permeability following overdistension of the lungs suggests a rapid intracellular cascade of signaling events that is amplified by increases in cytokine production and neutrophil infiltration some hours later (36, 46). The acute endothelial permeability increase induced by low or high PIP ventilation was evaluated using the gravimetric Kf, the product of endothelial hydraulic conductivity and vascular surface area. Kf is a sensitive and repeatable measure of vascular permeability to fluid in the fully recruited lung (29, 32, 33). The rapid permeability response to lung overdistension has been blocked by gadolinium, an inhibitor of stretch-activated and store-activated cation channels (31, 34) indicating that an increase in endothelial intracellular calcium was a necessary event for the increase in permeability (23). Calcium entry is also essential for numerous receptor ligand-mediated vascular permeability responses (22, 23, 39). Recently, we (14) have established the molecular identity of the stretch-activated cation channel responsible for the high PIP ventilation-induced permeability increase in mouse lungs as the TRPV4 cation channel. Kf increases in isolated mouse lungs induced by high PIP ventilation were abolished in TRPV4 null mouse lungs and in WT lungs after treatment with ruthenium red, a TRPV inhibitor; methanandamide, an arachidonic acid inhibitor; and miconazole, a P-450 inhibitor. The increases in lung intracellular calcium induced by lung overdistension were also attenuated in the TRPV4 null mice and ruthenium red-treated mouse lungs.
We used C57BL/6 mice as WT controls (14) because previous studies on Kf in mouse lungs by us (2) and Wu et al. (43) indicated no significant differences between the α1G KO mice or their littermates bred on the C57BL/6 background or littermates of TRPV4 KO mice relative to either baseline lung Kf values or the Kf increases after 10 μmol of the TRPV4 agonist, 4αPDD. Further comparisons of high vascular pressure injury using 30 cmH2O venous pressure by us (16) on Kf in lungs of TRPV4 littermates with the same venous pressure injury using C57BL/6 mice (unpublished data) also indicated no statistical difference between either baseline or postinjury Kf values. Therefore, we believe there was justification for using C57BL/6 mice as WT controls for the responses studied in these experiments.
An essential role of AM in VILI has also recently been demonstrated. We (7) and Frank et al. (10) depleted macrophages in rat lungs using clodronate-filled liposomes and reported an attenuation of VILI. High volume and pressure ventilation caused rapid adhesion of macrophages after only a few minutes as indicated by a reduced macrophage recovery in lavage fluid. The increased alveolar protein leak, increased lung edema, decreased lung compliance, and reduced gas exchange were attenuated by depletion of macrophages. Macrophage depletion also decreased plasma chemokine expression and alveolar lavage neutrophil counts induced by injurious ventilation.
In the present study, we hypothesized that the TRPV4 channel in macrophages contributes to macrophage activation and the increase in vascular permeability after ventilator-induced injury. The high PIP ventilation protocol did not increase Kf in the TRPV4 null mouse lungs. In contrast, instillation of TRPV4+/+ macrophages in TRPV4 null mice 24 h before performing the isolated lung experiments restored the increased Kf response to high PIP ventilation. Ventilation with the low PIP protocol resulted in a small (30%) but significant increase in Kf compared with baseline after 130-min ventilation with 9 cmH2O PIP in TRPV4+/+ lungs supplemented with TRPV4−/− macrophages, but Kf did not change over time in TRPV4−/−lungs with added TRPV4+/+ macrophages. However, after high PIP ventilation (35 cmH2O PIP), Kf increased by 115% in the HP WT+KO AM group and by 100% in the HP KO+WT AM group compared with baseline. These increases were not significantly different from the increase in the HP WT lungs (125%) relative to baseline. The edema accumulation indicated by the W/D ratios followed the general trend of the Kf changes but were determined by multiple factors in addition to vascular permeability. Lungs in all groups accumulated some edema due to the Kf measurements as indicated by experimental W/D ratios that exceeded previously determined baseline lung W/D ratios of 4.47 ± 0.19 in TRPV4+/+ mice and 4.21 ± 0.13 in TRPV4−/− mice of unventilated lungs (47). Thus TRPV4+/+ macrophages were sufficient to increase permeability in TRPV4 null mouse lungs in response to high PIP ventilation. Although we did not deplete the resident macrophages before adding macrophages of the opposite genotype, the fact that addition of TRPV4+/+ macrophages in TRPV4−/− lungs did not produce an increase in permeability after ventilation with the low PIP protocol indicates that the macrophage transfer protocol did not activate the TRPV4+/+ macrophages sufficiently to produce significant injury. Rather, mechanical stress was necessary to activate the TRPV4+/+ macrophages sufficiently to result in injury.
The TRPV channels are cation channels with six transmembrane-spanning segments, a pore-loop region between transmembrane domains 5 and 6, and a greater permeability to calcium than sodium (27). TRPV4 was initially identified as a channel activated by membrane stretch induced by osmotic stimuli (20). Further study demonstrated that TRPV4 is also activated by heat (12, 41), mechanical stimuli (27), the synthetic phorbol ester, 4αPDD (2, 40, 41), and cytochrome P-450 epoxygenase-dependent formation of epoxyeicosatrienoic acids from arachidonic acid (2, 42, 44). Peak gating of TRPV4 with mechanical stretch occurs at core body temperature, with little response at room temperature (26). Phospholipase A2 mediates the formation of arachidonic acid, which can activate TRPV4 via cytochrome P-450 epoxygenase products (19, 40) such as the epoxyeicosatrienoic acids (8). TRPV4-mediated calcium currents are self-limiting through a negative feedback of calcium concentration and can be augmented by tyrosine phosphorylation of the channel protein (45). In previous mouse lung studies, TRPV4 protein was found in lung endothelial cells, epithelial cells, and macrophages using immunohistochemistry (16). Although and TRPV2 and TRPV4 mRNA was observed in lung tissue using PCR (17), TRPV4 mRNA in isolated macrophages has not been reported, and the function of TRPV4 in macrophages has not been previously studied (25). Although immunohistochemistry antibodies recognize the TRPV4 protein in both genotypes, primers were designed specifically for the pore-loop region of the TRPV4 channel to differentiate between TRPV4+/+ and TRPV4−/− macrophages.
AM serve as sentinel cells for phagocytosis of inhaled bacteria and can initiate and coordinate the inflammatory response (18). Activation of AM can initiate the vascular permeability response in lungs after challenge with bacteria, bacterial toxins, and ischemia-reperfusion (9, 15, 48). In addition to phagocytic activity, activated macrophages may produce cytokines IL-1, IL-6, and TNF-α, the chemokines IL-8 and keratinocyte-derived chemokine (KC), as well as MMP, NO, and superoxide (38). Macrophages can produce NO through the eNOS pathway and in greater amounts by the iNOS pathway (9, 24), whereas increased superoxide production occurs largely through the NADPH oxidase pathway (3). Equimolar combination of NO with O2− produces the powerful oxidant peroxynitrite (28). Peroxynitrite can diffuse greater distances than either NO or O2−, and excessive amounts can increase vascular permeability and produce cell injury (28). In the present study, we demonstrated an increased intracellular calcium, an increased NO production, and an increased superoxide production after challenge with 4αPDD in WT macrophages but not in TRPV4 KO macrophages. The high levels of 3-nitrotyrosine in lung tissue after high ventilation in the WT lungs, WT lungs after KO macrophage transfer, and KO lungs after WT macrophage transfer, but not in KO lungs alone, suggests that mechanical activation of the WT macrophages by lung overdistension produced peroxynitrite. Tissue nitrotyrosine has previously been correlated with peroxynitrite production, and in the present study it was correlated with the increase in Kf in the mouse lungs (28). Nitrotyrosine has been shown to be elevated in ARDS, and nitration of surfactant proteins and an increased protein nitrotyrosine were inversely correlated with survival in ARDS (13, 18).
The importance of TRPV4 channels in mechanical activation of macrophages was also demonstrated by the increased cell spreading of WT as opposed to KO macrophages on contact with the glass surface of the coverslip culture dishes. Increased spreading of TRPV4+/+ macrophages occurred after overnight culture in media with 10% FBS, which suggests that firm attachment to the dish surface initiated a signal cascade for cell spreading that was mediated by cell membrane distortion and activation of TRPV4 channels. Further activation of the TRPV4 channels with 4αPDD caused a significant additional spreading of TRPV4+/+ macrophages compared with TRPV4 KO macrophages. Although firm adhesion of macrophages to the culture dish surface implies an adhesion process that does not normally occur in uninjured lungs, the rapid disappearance of lavage macrophages in lungs ventilated with high airway pressures suggests that mechanical activation of macrophages induces a similar firm adhesion in vivo that could be associated with macrophage activation and lung injury (1, 10). ICAM-1 has been shown to be upregulated in various types of lung injury and is an important adhesion molecule for lung epithelial cells and macrophages that may induce this firm adhesion in vivo (11, 37).
In summary, we present for the first time evidence for a functional role of TRPV4 channels in mechanical activation of macrophages and subsequent lung injury. The increased cell spreading of WT macrophages compared with TRPV4 KO macrophages even in the absence of the TRPV4 agonist indicates that the mechanogated TRPV4 channels are important for macrophage spreading and possibly other mechanical functions such as phagocytosis, adhesion, and motility. The increase in calcium entry, NO, and O2− production in WT but not TRPV4−/−macrophages after the TRPV4 agonist indicates that TRPV4 channels are capable of macrophage activation. The presence of increased nitrotyrosine in lungs after high PIP ventilation also suggests a mechanism for the increases in Kf observed. A correlation of the nitrotyrosine staining and Kf suggests that TRPV4 activation of the reactive nitrogen and oxygen pathways forms peroxynitrite, which could account for the rapid increase in vascular permeability in lungs after high pressure and volume ventilation. Restoration of high PIP-induced injury to TRPV4 null lungs by instillation of WT macrophages supports the critical role of mechanical activation of macrophage TRPV4 in initiating this injury.
This research was supported by National Heart, Lung, and Blood Institute Grants P01-HL-66299, HL-084159, and HL-092992.
No conflicts of interest, financial or otherwise, are declared by the author(s).
We acknowledge Sue Barnes for technical assistance.
- Copyright © 2010 the American Physiological Society