text:
Monday, May 17, 2010
The Things You Learn on the Public School Bus
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Sunday, May 16, 2010
Why I Chose to Have Kids
A few of my lesser reasons for choosing to have kids include some of the more fun ones. I wanted to see how I would do at being a parent. I hope my kids will be like me--I'm one of my favorite people! I wanted a chance to raise kids who might think and support the same ideas I do. They choose what to believe, but I'm hopeful. I enjoy being able to see them develop, change, and grow. My husband is good with kids and I really enjoy seeing that fun side of him with our own kids. And I think there are a couple of other reasons I cannot recall at the moment.
My most important reason for having children has to do with the intimacy that makes children possible. There is no more concrete representation of my marriage and my commitment to my husband than to create an entire other human being with him; a child that is partly me and partly him. Along with that purely physical joining, we also commit to dealing with each other for at least 18 years. Making decisions together regarding parenting issues for the duration of their minority. Though there is no guarentee that we will always be together, at the time we decided to have children, we had every intention of devoting ourselves to each other and our family for 20 or more years.
There are things I did not think were important. I did not think they would provide me with company. I can do that quite well by myself. I do not expect them to take care of me, though I hope they like me well enough to consider it. I did not worry I might screw the kids up irretrievably. I was pretty sure I could do better than that. I was not trying to compensate for any lack in my own upbringing. My own childhood was not perfect, some of which was out of my parents control, and some of which was. There are aspects of my childhood that my kids will not have to deal with and decisions to prevent those were made long before my decision to actually have children.
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Friday, May 14, 2010
Being a Parent
The sad fact is that people didn't need to decide why to have kids. Children came as a natural result of living. There were few reliable methods of contraception. Preventing children is against the teaching of Catholicism (yes, yes--they've okayed the rhythm method, but I'll bet someone could easily argue that having sex knowing you couldn't conceive is right up there with withdrawal). Fertile women could do nothing more than raise kids. Many families may have considered a child 'just another mouth to feed.' Children were a common commodity and not necessarily well-received.
I'm fortunate to live at a time and in a political environment when reliable contraception is available. My reproduction is almost completely within my control without sacrificing an essential part of what it means to be human and in love (sex--hopefully good). I am glad that I've been able to decide to become a mother.
By most accounts kids are difficult, inconvenient, costly, time-consuming, and resource-consuming. When they're infants, they wake you up, need to be fed frequently and you have to change their diapers. When they're toddlers they have nightmares, have a hard time going to sleep, still need to be fed frequently, and the diaper situation may be less frequent--but the job is necessarily bigger. As they become kids there's education, toys, play dates, and toilet training. All along you are constantly watching out for personal hygiene, removing trash more frequently, doing extra laundry, cooking, shopping, putting away toys, asking them to put away toys, trying to teach chores. It is easy to understand why people choose to not have children.
I'm not exactly sure why I knew I was going to have kids at even a young age. Perhaps it had to do with people around me talking as though it was an inevitability. In fact, even though I knew children could be rewarding, I was completely surprised by the sheer joy I felt after having them.
Thursday, May 13, 2010
Today: Just Nice
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Tuesday, May 11, 2010
A New Rule: Trying to Tame the Laundry
Usually I find their clothes on the floor of their room, on their desks or dressers, on the floor of their closet, hanging over their stuffed hampers, under beds, and even on chairs. Not only does this cause a big issue for my work of the laundry, but it also keeps their rooms well beyond navigable, not to mention how much dirtier the rooms look--and they are always at some level of disaster.
I have tried reminders and explanations to no avail. I have sat with them while they cleaned up. Since I have no job left other than toddler-parenting and housekeeping with my two oldest now attending public school, I have been very attentive of the laundry recently. Once I finished what was in the laundry room, I was quite satisfied with myself. Until I realized that I had almost as much laundry still in the kids' rooms.
Now we have a nightly inspection of each of their rooms. All clothing needs to be on hangars, in the hamper, or in their drawers. Not on the floor. Not on chairs (unless it's laid out for the next day). Not on top of dressers, desks, or chairs. Not in the bottom of the closet. For each item left out they pay me a dollar. It had to be enough to make them notice and it had to represent how much it means to me. This is no small change for them since they only get $4 and $5 a week. Just one item each night would have them in the red.
It worked well tonight, but it's a matter of discipline at this point. Discipline on my part! I have to remember to check each night (warning given first to help them plan their time, but only for a while). I have to remember to remove the amount from their allowance (there's an app for that), if there is anything to remove. Reminder added into my calendar for each night. Done!
Perhaps the money will not give them internal motivation. Maybe when they move out of the house they will keep their houses like pig sties. I could not care less. That will be their problem. This, right now, is a problem I am having with them. I am willing to try this idea for the mindless laundry pick up that needs to be done regularly.
Monday, May 10, 2010
My View on Co-Sleeping: Oh No, You Can't Sleep Here
Sunday, May 09, 2010
Happy Mother's Day
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Saturday, May 08, 2010
Book Club: Anna Karenina
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Friday, May 07, 2010
In Which I Am a Bad Mom: "Mom, Why Don't You Make My Lunch Like All the Other Kids' Moms?"
Because I know you can do it yourself, just like you've been doing it since you were 7.
Because I want you to know you don't have to rely on other people for things you can do yourself.
Because you know best what you want.
Maybe the other kids don't know how to do it themselves.
Because I don't want to.
And Flurpee, at 8, is making her own lunches, too.
Thursday, May 06, 2010
The short version: I was going to wait until next year. I couldn't give any warning because I didn't know I was going to do it myself. Attending school for a couple of months is better than being at home and getting very little accomplished.
The long version: I actually had a 'revelation' while recording the podcast. I said something off the cuff that I later realized was an accurate, succint statement of why Flurpee was so hard to motivate at home. Once she knew she was going to public school eventually, she was waiting for "real" school to start. I made the statement on Saturday. I realized how true it was on Sunday. I wondered about whether the school would take such a late enrollment on Monday. Called on Monday. Had her evaluated on Monday. And had her in school on Wednesday.
Even my husband did not realize that I was planning to put her in school. Normally, this is one of those decision about our family that I would consult with him first and make sure he was on board. Since he was on-board with the plan to send Flurpee next year and we had already decided which grade to put her in, I figured the timing would not change things drastically. He was quite shocked however when I told him I scheduled an evaluation for Flurpee and asked him to pick up the health forms on his way home from work. A short talk brought him around to my way of thinking.
Enrolling Flurpee at the end of the year would mean a chance for her to learn the ropes with her big sister still around. She would have her first school experience while things were exciting and not particularly demanding. If there were any issues, I would know about them to help over the summer.
Now I am home with just Omega.
Tuesday, May 04, 2010
Podcast Links
Tobin's Labs I'm glad to know about another homeschool science supplier
Home Science Tools
Rainbow Resource
Institute for Excellence in Writing
The Book Peddler
Teaching Textbooks
Slide stains from Home Science Tools and Tobin Labs--in case you are literally bleeding for your homeschool and want to make a teaching moment out of it.
It's a Podcast! A Podcast for Active-Minded Parents and Homeschoolers #1: Introductions and Mass Hope
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Monday, May 03, 2010
I decided to do this as a personal challenge. I only like to blog when the mood strikes me. I was curious about whether I could come up with any ideas when I needed them instead of waiting for inspiration.
Obviously, this first post was a given!


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Celebration Trailer (2014)
01 January 2014 7 mins
Imagine waking up in the most perfect American suburban landscape. Carefully manicured lawns, pristine white picket fences and not a spec of litter in sight. Welcome to Celebration, Florida, the town that Disney built. It pushes the classic Americana tropes to the nth degree. CELEBRATION questions this perfection, can it all be real? Through colored filters and handheld camera it provides and interpretive study of place.
Watch the official Celebration 2014 trailer in HD below or find more Celebration videos on Vidimovie.

subset: commoncrawl


text:
Viola Davis says being the favourite to win an Oscar and then losing is "devastating".
The 51-year-old actress is the odds on favourite to take home the gong for Best Supporting Actress for her role as Rose Lee Maxson in 'Fences', but isn't getting her hopes up as she says she's been in this position before, and says the pressure of thinking she's going to win "makes it worse" when someone else's name is called.
Speaking during an appearance on 'Jimmy Kimmel Live!' on Thursday (16.02.17), Viola said: "You know, I've been the odds on favourite to win before, and not. And it's devastating. It makes it worse.
"You're sitting there and you're like, 'I'm the odds on favourite, okay' and then your name is not called, it's like falling from a 100 storey building.
"As opposed to if you know you're losing, it's like falling from a one storey building."
Meanwhile, Viola admitted she's "always surprised" to be invited to award ceremonies at all.
The 'Suicide Squad' star – who recently picked up a Golden Globe and a BAFTA both for Best Supporting Actress for 'Fences' – said recently: "I'm the girl on the carpet that's tense. [I'm] with the good looking husband. I'm great … I always feel when I come to London, though, that I feel like I've been invited to the best party. I'm always surprised when I'm invited to this party. This is something I never thought I would be invited to and I am."
And the actress hasn't been able to ask her 'Fences' co-star Denzel Washington – who also directed the drama – for advice as he always takes a very chilled approach to these events.
She added: "Denzel never has any messages. He'll just say, 'Where are you going?' And I'll say, 'I'm going to the BAFTAs,' and he'll say, 'Have fun!' "


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Economie du Développement Durable et Economie Sociale et Solidaire: des relations complexes pour des enjeux majeurs
[Social Economy and the Economics of Sustainable Development: complex relationships, major stakes]
Social Economy and Sustainable economics are two independent fields that sometimes refer one to another. The former is primarily concerned with "social capital" issues, while the latter deal with "natural capital" stakes. When referring to the other field, there is no systematic analysis of the interrelations between their respective goals, tools, methods and concepts. As a matter for fact, it happens that goals of social economy actors are opposite to those of sustainable economy actors; conversely, they sometimes lead to the same conclusions and policies. In this paper we explore the positive and negative relations between the two fields in order to evaluate in a systematic way how they could cooperate. We show that social economy concepts were developed much earlier and, thus, without reference to sustainable development issues. Nowadays, social economy studies remain primarily focused on social capital development. However, sustainable economics might find very useful tools and concepts in the field of social economy, which should encourage cooperation between scientists and actors of the two fields.
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|Date of creation:
|15 Sep 2011
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Phone: +49-(0)89-2180-2459
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Web page: https://mpra.ub.uni-muenchen.de
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- Jean Gadrey & Florence Jany-Catrice, 2005.
"Les nouveaux indicateurs de richesse,"
halshs-00198357, HAL.
- Florence Jany-Catrice, 2006. "Les nouveaux indicateurs de richesse," Post-Print halshs-00282336, HAL.
- Susan Rose-Ackerman, 1996. "Altruism, Nonprofits, and Economic Theory," Journal of Economic Literature, American Economic Association, vol. 34(2), pages 701-728, June.
- Jérôme Blanc, 2008. "Responsabilité sociale des entreprises et économie sociale et solidaire : des relations complexes," Post-Print halshs-00345374, HAL. Full references (including those not matched with items on IDEAS)
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text:
|12 Nov 2011
Windows 7 Ultimate x64
If you have tried different LAN drivers, BIOS firmware and cables then I would try and return it then. If it cant do 1Gbps as advertised then you can say its not adiquate for your needs.
|My System Specs
|13 Nov 2011
Windows 7 x64 (Ultimate)
Yeah, I had thought of that and eventually I will have to do that... its just a pisser really since everything else is working perfectly fine, it is the first time everything goes well in the build, cables are tucked in pretty neat, clean install, everything and the thought of disassembling and putting another board back together is just a pita... but it must be done I guess.
Thanks everyone for the help and the time.
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The Network of Youth Groups across Scotland
Youth Scotland is Scotland's leading charity for the support and delivery of community based youth work. Our vision is to have in Scotland young people who are confident, resilient, aspirational and ready to reach their potential.
Through our network of over 1,000 youth groups and projects, we deliver services which help over 64,000 young people to learn, succeed and make positive contributions to their communities.
We are passionate about young people and believe that youth work changes lives.
With over 80 years of experience, we are innovative and are ambitious for the young people we support.
We champion all those who support young people particularly the role of volunteers in youth work.
Our current Treasurer, and Chair of the Finance & Resource Group, Jan Roxburgh, is stepping down after four years on the Board of Trustees of Youth Scotland. She is leaving behind a financially strong and resilient organisation which is highly respected and recognised for the difference it makes in young people's lives.
The essential role of Treasurer is to bring financial experience to the Board of Trustees. Any experience of the youth work or third sector, while desirable, is not essential.
The remit of the Treasurer is to:
Chair the Finance and Resource Group
Meet with the Chief Executive on a regular basis (planning and reporting)
Carry out finance functions as required
Ensure compliance with the Auditors and oversee the Investments and Risk Management Policies
We have a committed Board of Trustees ably led by the new Chair, Dona Milne, a consultant in public health and steeped in youth work practice and policy with many years' experience. The Board is drawn from across the public, private and voluntary sectors and is passionate about the work we do.
We are seeking applications from individuals who are interested in serving as Treasurer for an initial three year period from November 2017.
Over the next three years, incorporating the Year of Young People in 2018, we will:
Provide more non-formal learning opportunities for young people to contribute both to their personal self-development and prospects for employment;
Continue to support our paid and voluntary work force throughout Scotland;
Promote the added value of community based youth work directly and in collaboration with numerous partners
Ensure that Youth Scotland continues to be an effective and sustainable organisation within the charitable and youth work sector in Scotland.
This is an exciting voluntary post for someone who is committed to helping support the financial management and sustainability of Youth Scotland. Significant accountancy experience at a senior level in the public, private or voluntary sector would be an advantage as would active involvement in the governance of a charity or voluntary organisation.
For an informal discussion with our Chair, Dona Milne, or with our Chief Executive, please initially contact our Chief Executive, Ian McLaughlan at firstname.lastname@example.com or on 0131 554 2561.
An application pack will be available on the Youth Scotland website to interested applicants which will provide a comprehensive set of publications on all relevant aspects of our organisation.
The Treasurer and Chair of the Finance & Resource Group of Youth Scotland needs to possess a range of skills, attributes and personal characteristics to enable them to effectively lead the Board and organisation. Above all, the Treasurer must demonstrate a commitment to the vision, mission and values of Youth Scotland.
Along with the Chair of the Board of Trustees (other colleagues on the Board, the Chief Executive, Finance Officer and the Executive) the Treasurer provides a key leadership of the organisation helping to maintain proper financial controls, systems and accountability.
The Treasurer along with the Chair and Chief Executive ensures strong and transparent corporate governance on all aspects of finance management of the organisation, upholding the values of Youth Scotland and develops trust, respect and honesty in all its activities.
Ideally, a
The Treasurer leads by example in being objective in finance decision-making and accountable for such decisions taken by the Board.
The Treasurer is also the Chair of the Finance & Resource Group, and in that role provides support and encouragement for Members of the Finance & Resource Group drawing out their particular expertise and knowledge to inform recommendations to, and decisions taken by, the Board.
As part of the Office – Bearers Group, the Treasurer promotes the essential relationships between the Treasurer, the Chair and the Chief Executive always respecting their different roles in the management and leadership of the Charity.
Along with the Chair and the Chief Executive, the Treasurer will represent Youth Scotland at strategic finance and investment meetings.
Ideally, applicants will be able to demonstrate knowledge and experience of the third sector in Scotland and be able to represent Youth Scotland in finance related meetings with its members, Area Associations, funders, government and the media.
The Treasurer needs to have a passion and clear commitment to the organisation and to the young people it supports. This often means balancing strong corporate governance with lateral thinking in developing and managing effective services and projects.
The Board of Youth Scotland meets 5 times per year, mostly early Thursday evenings and an annual development day on a Saturday in which the Treasurer plays a key part.
The Treasurer is also the Chair of the Finance & Resource Group which meets two or three weeks prior to the Board. It is expected that the Treasurer and the Chief Executive will discuss and agree the agenda prior to the Board meeting to identify priorities and ensure continuity between meetings.
The Treasurer is also responsible for arranging, in conjunction with the Auditors, the Annual Financial Report at the Youth Scotland AGM in November each year.
The Treasurer and Chair of the Finance & Resource Group has the governance oversight for Finance, Investments, Risk, Audit, Property and HR within Youth Scotland.
Youth Scotland
Balfour House
19 Bonnington Grove
E: firstname.lastname@example.com
©2017 Youth Scotland.


text:
Jacob Rees-Mogg praises 'magnificent' press after row over High Court's Brexit judgment
JACOB REES-MOGG has moved to defend sections of the media who were criticised for their coverage of the High Court's Article 50 ruling.
The Brexiteer said the "free press" was "entitled" to disagree and voice their disapproval of judges if it disagrees with them as he rebuked fellow Conservative MP Dominic Grieve for describing the coverage of the Brexit decision as "chilling".
Last week's controversial High Court ruling means Theresa May not start the official process of exiting the EU without first consulting MPs in the House of Commons.
The Lord Chief Justice – sitting with Master of the Rolls Sir Terence Etherton and Lord Justice Sales – said they made their ruling on a "pure question of law" and have been heavily criticised by many over the decision.
Jacob Rees-Mogg lauded the British press for standing up to High Court judges
We have a free press and it is entitled to attack judges if it disagrees with them
Mr Grieve, a former Attorney General told BBC Newsnight he was "horrified" by the way the media had covered the ruling, saying: "The judges did exactly what was asked of them."
He also described the attacks on the judiciary as "chilling and outrageous" and "smacking of the fascist state".
Speaking to The Times: "Some of the things which have been said about the court's judgment by politicians has been utterly disgraceful.
His Conservative colleague Dominic Grieve criticised the media's coverage of the decision
"Some members of Parliament do not appear to understand this judgment had nothing to do with subverting the will of the people."
Speaking on BBC's Daily Politics, Mr Rees-Mogg was asked if he agrees with the views of his Conservative colleague.
He replied: "No, I don't. Dominic is perhaps the cleverest man in the House of Commons and a brilliant lawyer but on this I think he's wrong.
"We have a free press, we have an outspoken press and it is entitled to attack judges if it disagrees with them.
"Ministers are not, and had ministers [attacked judges], the Lord Chancellor would have been right to tell them not to, and members of Parliament in the House of Commons may not attack judges except on a specific motion.
"So, there are controls on the legislature and the executive on how we relate to the judiciary but a free press is a free press and have been magnificent in pushing forward the interests of the British people."
The Government has announced its intentions in appealing the decision at the Supreme Court.
A pro-Brexit group has claimed 100,000 will march alongside Nigel Farage against the decision to give Parliament a say on triggering Article 50 on December 5.
Leave.EU said the protesters will march from Trafalgar Square before gathering in Parliament Square, within sight of the Supreme Court, as the challenge is being heard.
A Leave.EU spokesman told the Telegraph: "We will also be launching with all Leave campaigns including members of all political parties a march on the Supreme Court to make a point that 'Brexit means Brexit'
The Supreme Court has scheduled up to four days for the hearing and proceedings will be streamed live via its website.


text:
Pramitol 25E
Buy Now
Dan from Howard, Ohio writes,
I just purchased 1 gal. of Pramitol 25E for our fence line in a horse pasture, my understanding is to use 10oz...
per gal. of water is this correct, and how long before the horses can be let back into the field.
Pramitol 25E mixing ratios vary depending on many factors. We recommend to contact Control Solutions 281-892-2500 for the best and effective application in your area.
Answer last updated on: 07/30/2014
3 of 16 people found this answer helpful. Was it helpful to you? Yes No


text:
|SalesVantage.com >> Article Archives >> Selling Tactics >> Succeeding in Sales
Not every one is cut out for a career, or even an assignment in sales. Additionally, it is often difficult for people to make the switch from "Customer Service Officer," a role with an emphasis on serving the customer, to that of "Customer Sales Representative" and a need to be a professional sales person.
However, in today's world, where customers have numerous choices of service and product providers, an organization without a sales culture featuring a core of well-trained, highly motivated sales professionals, is not going to be as successful as it would with these two ingredients.
Selling is a people business. So what does it take to be successful in selling?
A partial, and admittedly by no means complete, list of personal criteria for succeeding in selling includes:
1. Customer Focus and Concern -- a successful sales person will build relationships based on trust, honesty, integrity, and concern for his customers. You have to be able to understand, from your customer's perspective, the needs, wants and desires of each individual customer. What are their key needs, wants, desires and how can you and your organization satisfy these either cost efficiently or through adding value (or both)?
2. Loyalty to the Needs of the Customer -- having the ability to be an internal advocate and fighter for the customer, and being able to lead (directly or indirectly) internal teams and processes towards the absolute satisfaction of your customers.
3. Accepting and Learning from Rejection -- everyone in sales experiences rejection. A sales person cannot take rejection personally, but must use each instance as a learning experience. Those who allow sales rejections to upset them personally and emotionally, are likely to carry these emotions into their personal lives. A unhappy or emotionally distraught person is unlikely to find success in a sales career.
4. Understanding the Value of Selling -- customers today cannot be expected to know all there is about our products. When they need more information, they turn to our sales forces to help educate them. This is why consultative selling approaches, rather than the old fashioned hard sell approaches, are working best in so many industries. Selling is a value-added process, when it is done right. Each sales person needs to be a critical component in this value adding job function.
5. Being a Constant Student -- successful sales people are not born, they are well trained and tend to be constant learners. A desire to constantly upgrade skills is a key criteria for success, resulting in a self-propelled drive to read, listen to audio recordings, or watch video tapes, from other successful sales people and about things that impact their customers and their customers' products.
6. Believing in yourself, your products and your services -- customers can easily tell when a sales person does not fully believe in the products and services they are selling. Success requires a complete belief in what you are selling, including full confidence and belief in one's own consultative selling ability.
7. Commitment -- at a minimum, a three-level commitment is required: a) a commitment to continuing trying, no matter what the odds or what your recent experiences have been, b) a commitment to focus on the needs of the customer, not the needs of one's own organization, and c) a commitment to one's self to constantly upgrade skills and to constantly monitor one's own motivation requirements.
8. Goal Setter -- the old adage that "what gets measured gets accomplished" is very true in sales. A successful sales person will set his/her own stretch goals, ones that focus on the selling process (number of attempts/calls, hours spent upgrading skills, etc.) as well as on outcomes (sales, success ratios, etc.).
9. Honesty and Trustworthiness -- one cannot build a long-term career in sales without being fully honest and trusted. As in criteria number one above, client relationships must be built on honesty, integrity, trust, and a true concern for one's customers. After all, customers prefer to purchase from those they can trust.
10. Keeping Outgoing Personality Under Control -- many people think they will be good at selling because they have an outgoing personality and they enjoy interacting with people. While it's true that an extrovert has many tendencies and qualities of a good sales person, it is also important to remember that one of the most critical selling skills is that of listening. An outgoing personality that asks interesting questions is far more likely to be successful in sales than a person who likes to talk about themselves and/or their products and services.
11. Enthusiasm -- last, but certainly not least, is to have positive enthusiasm for one's job, products, company, and even life in general. Positive and enthusiastic people are so much more pleasant to deal with that we all find ourselves buying from them just because the sales/buying experience has been so enjoyable.
There are many more personal criteria required for being successful in sales, but this list is a good start. And without these 11 criteria as one's core personal competencies, all other personal attributes will not lead to the kind of success one is capable of achieving.
Steven Howard is Asia's leading marketing consultant and positioning specialist, with over 22 years experience. The author of two books and numerous articles, you may reach him at www.howard-marketing.com or firstname.lastname@example.com.
More articles by Steven Howard
More articles on Selling Tactics


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Talk:Russia and the Caucasus
Moved from Wikitravel:votes for deletion by Evan
Article Hierarchy[edit]
I do not really like these this and that names. It means we are doing a region join. I think this is 2 regions and mashing them together is like trying to make babyfood. Its interesting to eat but normally its a mishmash of a meal. There are two destinations here Russia, or more specifically European Russia, which needs to be written about a lot more, and the countries of the Caucasus, which are a perfectly valid region around the Caucus Mountains, of which more articles should be written too. Perhaps its because we have not developed a regional name for the Commonwealth of Independent States of the Former Soviet Union or the Region of the World formerly ruled by Soviet Socialist Regimes that we have this problem. To me this name implies East (Europe) and North (Asia) from Black Sea to Arctic. -- Huttite 14:33, 1 Jan 2006 (EST)


text:
M-AUDIO Axiom Axiom 25 Drivers Download
|Hardware Type:
|Axiom 25
|Windows XP, Vista, 7, 8, 10
|Download Size:
|Database Update:
|Available Using DriverDoc:
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text: US M&A 2014–15: Full steam ahead 1 Cover title, initial cap only, goes here Cover: Stand first. Omnihill oribusapis erum ad mo dolore de expere pro od quidign ihilicit moditatur? Cabore nissint ibustiis ma natur, nias anis et latium quo omnis a quunturibus aut auditis 1 US M&A 2014–15: Full steam ahead A review of the drivers behind 2014's banner M&A year and a look ahead to 20152 White & Case Contents US M&A hits a home run Page 1 The US M&A market takes center stage Page 2 Three sectors driving deal value Page 4 US M&A in figures Page 5 PE exits reach five-year high Page 6 Looking ahead: All eyes on 2015 Page 7 yoshi0511/ShutterstockUS M&A 2014–15: Full steam ahead On the back of headline-grabbing megadeals and a more stable economy, US M&A staged a dramatic comeback in 2014 US M&A hits a home run John Reiss Partner, White & Case The final figures are in, and it is safe to say that 2014 was a home run for the US M&A market. Deal activity was at its highest level in five years. A number of factors have increased confidence among corporates, bringing them back to the dealmaking table with a fervor. Stock markets are strong in the United States, and many companies have substantial cash piles to invest as well as the backing of shareholders pressing for more deals. Importantly, the US economy is stable and growing—particularly compared to markets in Europe and Asia—and this is attracting an increasing number of non-US buyers to the United States. Megadeals, such as the proposed US$66 billion acquisition of Allergan by Actavis, have been at the forefront of the revival and helped to drive the value increase, as sectors, such as pharma and TMT, looked to consolidate. The number of deals also increased significantly, although not as much as value. We expect a broader, deeper and more active M&A market in 2015, with the prospect of dealmaking activity spreading to the under-US$5 billion market, which will drive volumes higher. In the private equity arena, buyers have stepped back somewhat due to extremely high seller expectations. The value of acquisitions by buyout firms was only 11 percent of total deal value, the lowest in three years and a significant drop from 33 percent in 2007. However, increased volatility in the market, which we have seen in the first weeks of 2015, may create greater opportunities if prices decline. On the other hand, there is no certainty that sellers' expectations will quickly reset. Overall, deal value fell in January compared to December, but that is consistent with patterns in recent years, with value remaining static or falling from December to January in the previous two years. The momentum built in the last year looks set to continue into 2015, but there is no room for complacency. Dealmakers will be very aware of recent stock market volatility, falling oil prices and a potential pullback in the leverage markets. Although buyers should proceed with caution, there is good reason to be optimistic about the M&A markets in 2015. Gregory Pryor Partner, White & Case2 White & Case US$1 billion). The spike in these highvalue transactions helped to boost overall deal value for the year. "Something has changed for buyers and sellers," Reiss says. "The buyers are through their restructurings. They have done what they can on the cost side. Now they have lean, growing business operations, and they are ready to transform through acquisitions. The thing that has changed for sellers is that buyers are now willing to meet their price expectations—and when you have enthusiastic buyers (even at high prices), you get willing sellers and a highly charged M&A market." Stock is an option Another indicator of the confidence that has flowed through the US M&A After a long hiatus, US M&A activity sparked back to life in 2014 to record its best performance in five years. Companies announced 4,795 deals worth US$1.4 trillion last year, a 22 percent increase in volume and a 57 percent rise in value on 2013 figures. US dealmaking has finally recovered since the beginning of the downturn in 2009, when there were just 2,771 deals worth US$688 billion. "M&A lawyers have lived through a difficult period since 2008, but in 2014, the M&A market has come roaring back," says John Reiss, the Global Head of White & Case's M&A practice. The revival has been underpinned by positive fundamentals. The US economy is expected to show growth of around 3 percent for 2014, the S&P 500 gained 11.24 percent through the year and, in December 2014, unemployment fell to 5.6 percent from 5.8 percent. This compares extremely favorably with both Europe and Asia. For example, the European Commission recently cut the Eurozone growth forecast from 1.2 percent to 0.8 percent, and the unemployment rate stands at 11.5 percent (where it has been since June). Meanwhile, 2014 witnessed China's slowest growth in nearly a quarter of a century. Deal drivers The M&A renaissance in the United States has taken place against a backdrop of soaring values and record performance in the global M&A market as a whole, with deals totaling US$3.2 trillion. At 16,554 deals, global transaction volume in 2014 was 14 percent higher than in 2013. HEADLINES n US deal values reached a five-year high in 2014 n Deal volumes were up by almost a quarter compared with 2013 n Stable economy, record cash balances and backing from shareholders have set the foundations for a rise in M&A n Megadeals are up 51 percent in volume and 67 percent in value n Deals under US$1 billion are up 18 percent in volume and 28 percent in value The US M&A market takes center stage In the United States, the stable economy, shareholder enthusiasm for deals and record corporate cash (standing at around US$1.6 trillion, according to Moody's Investor Service) have laid the foundations for recovery. The return of the megadeal in the United States has served as a key pillar of the M&A rebound and suggests that US dealmakers have the conviction to pursue deals and make large, long-term strategic plays once again. Megadeal mania There were 243 megadeals in 2014 worth a total value of more than US$1 trillion, which marks an increase of 51 percent and 67 percent compared to the 161 deals worth US$652 billion in 2013 (megadeals are deals worth over Total US M&A, 2009 – 2014 Volume Value 2009 2010 2011 2012 2013 2014 Volume Value (US$ billion) 0 200 400 600 800 1,000 1,200 1,400 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 0 50 100 150 200 250 300 350 400 450 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 51% The percentage increase in megadeals from 2013 to 2014US M&A 2014–15: Full steam ahead 3 market in 2014 was the willingness of sellers to accept stock consideration. The use of equity to fund deals has been on the rise with the number of deals in which sellers accepted equity as part of the consideration rising to 18 percent in 2014, the highest percentage recorded in the last six years. "A substantial amount of deal consideration is paid not in the form of cash but in the form of equity, evidencing confidence in the equity of the acquirer," says White & Case partner Gregory Pryor. Inbound on the up The growth supporting the rise in US deal activity did not go unnoticed abroad, and interest from international dealmakers in the United States was strong. US inbound M&A – top bidders by volume 2013 2014 0 40 80 120 160 United Kingdom Canada Japan France Germany China Switzerland Ireland Sweden Netherlands Volume The number of deals in the United States by the 10 foreign countries most active in M&A climbed from 432 in 2013 to 623 in 2014, with deal values more than trebling from US$99.1 billion to US$342.8 billion. Recovering global M&A markets and steady US growth spurred inbound investment into the United States such as German group Bayer's US$14.2 billion takeover of Merck & Co.'s consumer care branch and Chinese multinational Lenovo's US$2.1 billion acquisition of IBM's x86 server business. US inbound M&A – top bidders by value (US$ billion) 2013 2014 0 20 40 60 80 100 120 Ireland Germany United Kingdom Japan Canada Switzerland Singapore France China Italy Value (US$ billion) The thing that has changed for sellers is that buyers are now willing to meet their price expectations—and when you have enthusiastic buyers (even at high prices), you get willing sellers and a highly charged M&A market. John Reiss, Global Head of M&A, White & Case4 White & Case Three sectors driving deal value Three sectors— technology, media and telecommunications (TMT), energy, mining and utilities (EMU) and pharma, medical and biotech (PMB)—accounted for the lion's share of US deal value in 2014, with activity driven by consolidation, strong foreign appetite for assets and tax inversions. There were 1,029 deals in the TMT sector in 2014, with a total deal value of US$302 billion, up six percent from 2013. Megadeals dominated the sector. The increase in the use of wireless devices caused a blurring of the lines between television, mobile telephony and broadband. In response, cable providers and mobile operator companies have moved to consolidate and bundle these services together. Megadeals were also the driver in the EMU sector, which recorded HEADLINES n TMT was the most active sector with 1,029 deals in 2014 n M&A was driven by consolidation and bundling in the TMT market n Energy, mining and utilities recorded the highest value in 2014 with US$318 billion worth of deals, more than double that of 2013 n In the pharma, medical and biotech sector, deal value rose 134 percent between 2013 and 2014, from US$100 billion to US$232 billion 495 deals in 2014 and in which deal value more than doubled from US$139.6 billion in 2013 to US$318.1 billion. Strong appetite for midstream assets was a major contributor to the sharp rise in value, as was keen interest from foreign investors in shale gas plays. A fall in the price of oil in the final quarter also brought out buyers seeking to acquire assets at lower valuations. The 475 deals in the PMB industry last year ranked the sector as the third-largest contributor to overall deal values in the United States. PMB deal values more than doubled from US$99.5 billion in 2013 to US$232 billion. Activity was partly prompted by the need for PMB firms to replenish drug pipelines and find synergies to reduce R&D costs, but the main driver of activity in the sector was tax inversion deals, where US businesses acquire foreign companies and transfer their tax domicile to secure lower tax rates. Medtronic's US$45.95 billion purchase of Ireland's Covidien and Actavis's takeover of Warner-Chilcott for US$8.4 billion were two of the high-profile tax inversion deals in PMB last year. The US Treasury did respond by introducing regulations restricting some of the advantages associated with tax inversions. The new rules have blocked companies from using cash trapped offshore to fund overseas deals without incurring tax, although it is still possible to leverage US operations and deduct the interest against US taxable income post-acquisition. "While the government has moved to restrict inversions, we believe that these will continue to get done in certain circumstances," says White & Case partner William Dantzler. 134% The percentage increase in deal value in the PMB sector from 2013 to 2014 27% High yield bond issues in Europe featuring portability in 2014 Total US M&A – top ten sectors 2014 Volume Value (US$ billion) 0 200 400 600 800 1,000 1,200 Transportation Construction Leisure Financial Services Consumer Pharma, Medical & Biotech Energy, Mining & Utilities Business Services Industrials & Chemicals TMTUS M&A 2014–15: Full steam ahead 5 US M&A in figures * The Intelligence Forecaster is based on "companies for sale" tracked by Mergermarket in the US between 1 August 2014 and 31 January 2015. Mergermarket's Intelligence Forecaster of predicted deal flow is based on intelligence relating to companies rumored to be for sale, or officially up for sale. It is therefore indicative of sectors that are likely to be most active during 2015. Total US M&A 2009 – 2014 Volume 0 500 1,000 1,500 2,000 2,500 3,000 H1 2009 H2 2009 H1 2010 H2 2010 H1 2011 H2 2011 H1 2012 H2 2012 H1 2013 H2 2013 H1 2014 H2 2014 Value† Proportion of US M&A accounted for by private equity 0 5 10 15 20 25 30 35 2009 2010 2011 2012 2013 2014 Buyout Exit Secondary buyout 7% 7% 14% 13% 14% 18% 11% 13% 6% 13% 14% 4% 5% 14% 3% 16% 4% Inbound M&A by country, 2014 52 % Percentage of inbound deals with European buyer UK 148 deals US$42.2 billion Canada 138 deals US$33.7 billion Japan 70 deals US$36.1 billion France 57 deals US$11.8 billion Germany 50 deals US$65.2 billion China 47 deals US$11.3 billion Switzerland 35 deals US$21.2 billion Ireland 32 deals US$97.8 billion † Indicated by size, where the smallest is US$303 billion, and the largest is US$747 billion US M&A 2014–15: Full steam ahead 5 TMT, pharma and industrials poised to be most active in 2015 Lowest deal flow Highest deal flow 346 Pharma 449 TMT 180 Consumer 249 Business Services 146 Financial Services 39 Real Estate 59 Leisure 32 Transportation 281 Industrials and Chemicals 11 Defense 203 Energy, Mining and Utilities 11 Construction The M&A Forecaster* predicts sector deal flow by volume in the US M&A market for 2015.6 White & Case Private equity firms took full advantage of the US M&A rebound in 2014, seizing the opportunity to exit more portfolio companies than at any time since the bottom of the market in 2009. Private equity investors sold 962 companies worth US$261.8 billion last year, an increase of 31 percent in volume and 70 percent in value compared to the 735 exits worth US$153.6 billion secured in 2013. Indeed, exit values were five times higher in 2014 than they were in 2009. A number of factors have spurred this run of private equity exits. Stronger portfolio company performance; the return of wellfunded strategic buyers to M&A markets; a robust auction market; and, most importantly, very high sale prices have all contributed to the strong run of exits. The downside of the high valuations that private equity firms have been able to secure in a seller-friendly market is that it has been more difficult for buyout investors to purchase companies at attractive prices. Exit volumes and values have been soaring, but new deal activity has been far more sedate. There were 871 buyouts closed in 2014, an increase of only 14 percent from 2013. The value of buyouts was US$153.8 billion in 2014, lower than the US$159.3 billion recorded in 2013. While buyout activity is well up from 2009 figures, when there were only 463 buyouts worth US$48 billion, the recovery has been modest when compared to the market for exits. Now that firms have, in some respects, cleared out their portfolios, attention will have to turn to new deals in 2015, which could finally prompt an increase in buyout volumes. HEADLINES n Private equity exit values reached a five-year high n Exit volumes increased 31 percent from 2013 to 2014 n Buyout volume was up 14 percent from 2013 PE exits reach five-year high US private equity buyouts, 2009 –2014 Volume Value 0 50 100 150 200 250 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Value (US$ billion) 0 10 20 30 40 50 60 70 80 2009 2010 2011 2012 2013 2014 Volume US private equity exits 2009 – 2014 US private equity buyouts – top ten sectors 2014 Volume Volume Value (US$ billion) 0 50 100 150 200 250 Transportation Financial Services Construction Leisure Energy, Mining & Utilities Pharma, Medical & Biotech Consumer Business Services TMT Industrials & Chemicals Volume Value 0 50 100 150 200 250 300 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Value (US$ billion) Volume 0 20 40 60 80 100 2009 2010 2011 2012 2013 2014US M&A 2014–15: Full steam ahead 7 Looking ahead US M&A activity should continue thriving into 2015. However, despite an upbeat outlook, dealmakers cannot afford complacency. The beginning of 2015 has seen stock market volatility and a continued slide in oil prices— at the time of writing, Brent Crude Oil prices were at a six-year low and have seen a drop of 60 percent since the middle of last year. One major theme of 2014 was the discrepancy between the increases in value and volume. "We note that while aggregate deal value is up substantially, which is creating a lot of the excitement around the M&A market, the fact is that aggregate deal volume has not increased as much. A fall in the number of megadeals in 2015 could affect the momentum built up through the last year," says White & Case partner Morton Pierce. As dealmakers move into 2015, they would, therefore, do well to remember the following lessons from the year past: Watch the regulators Antitrust scrutiny has impacted a number of deals and the US Treasury has tightened the rules around tax inversions, a development that prompted AbbVie to withdraw its bid for Shire Pharmaceuticals. Dealmakers should be following these developments closely. Stick to strategy Shareholders have, for the most part, rewarded boards that have pursued transformative deals. This should not, however, be read as a green light to pour cash that has been sitting on balance sheets into a slew of opportunistic deals. Investors will back transactions, but only if they are underpinned by solid deal rationales. Diversify your acquisition capital When prices are high, it is worth mitigating risk by using a variety of acquisition capital to fund Looking ahead: All eyes on 2015 a transaction. Companies may want to pay with stock to share some of the risk with their targets. And even cash rich corporates may want to use debt to finance at least some portion of their deals, given that debt is widely available and attractively priced. Do your diligence The competition for prized assets is likely to be just as fierce in 2015 as it was in 2014. However, the desire to prevail over rivals and get the deal done as quickly as possible should not preclude bidders from doing thorough due diligence on their targets. While there has been something of a slowdown in the first weeks of 2015, we believe the prospects for dealmaking in 2015 remain positive. However, the real acid test of the US M&A recovery will be whether there is a deepening of the market beyond the megadeals that dominated the headlines in 2014. "We continue to have a strong US economy and a deal market that is attractive to both domestic acquirers and foreign acquirers. That bodes well for a strong 2015," says White & Case partner Daniel Dufner. "There is an entire well of untapped M&A activity. We hope that in 2015, we will not only see megadeals, but also see activity floating down-market." © George Diebold/Blend Images/Corbis8 White & Case Contacts John M. Reiss Partner, New York T +1 212 819 8247 E email@example.com Private equity Oliver C. Brahmst Partner, New York T +1 212 819 8219 E email@example.com Bank Finance Eric Leicht Partner, New York T +1 212 819 8796 E email@example.com Tax J. William Dantzler Partner, New York T +1 212 819 8543 E firstname.lastname@example.com Benefits Henrik P. Patel Partner, New York T +1 212 819 8205 E email@example.com Gregory Pryor Partner, New York T +1 212 819 8389 E email@example.com Antitrust Rebecca H. Farrington Partner, Washington, DC T +1 202 626 3599 E firstname.lastname@example.com CFIUS Richard J. Burke Partner, Washington, DC T +1 202 626 3687 E firstname.lastname@example.com United Kingdom Ian Bagshaw Partner, London T +44 20 7532 1575 E email@example.com France François Leloup Partner, Paris T +33 1 55 04 15 17 E email@example.com Germany Dr. Joerg Kraffel Partner, Berlin T +49 30 880911 400 E firstname.lastname@example.com Asia Eric Berg Partner, Hong Kong T +852 2822 8718 E email@example.com Barrye L. Wall Partner, Singapore T +65 6347 1388 E firstname.lastname@example.com Central and Eastern Europe Michal Smrek Partner, Prague T +420 255 771 224 E firstname.lastname@example.com Scandinavia Darragh Byrne Partner, Stockholm T +46 8 506 32 360 E email@example.com NY0215|TL|B|09858v1 © Orjan F. Ellingvag/CorbisDisclaimer This publication contains general information and is not intended to be comprehensive nor to provide financial, investment, legal, tax or other professional advice or services. This publication is not a substitute for such professional advice or services, and it should not be acted on or relied upon or used as a basis for any investment or other decision or action that may affect you or your business. Before taking any such decision, you should consult a suitably qualified professional advisor. While reasonable effort has been made to ensure the accuracy of the information contained in this publication, this cannot be guaranteed and neither Mergermarket nor any of its subsidiaries or any affiliate thereof or other related entity shall have any liability to any person or entity which relies on the information contained in this publication, including incidental or consequential damages arising from errors or omissions. Any such reliance is solely at the user's risk. Published in association with Mergermarket Part of the Mergermarket Group www.mergermarketgroup.com For more information, please contact: Robert Imonikhe Publisher, Remark, part of the Mergermarket Group Tel: +44 207 010 6100 whitecase.com In this publication, White & Case means the international legal practice comprising White & Case llp, a New York State registered limited liability partnership, White & Case llp, a limited liability partnership incorporated under English law and all other affiliated partnerships, companies and entities. This publication is prepared for the general information of our clients and other interested persons. It is not, and does not attempt to be, comprehensive in nature. Due to the general nature of its content, it should not be regarded as legal advice. © 2015 White & Case LLP


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Endothelial activation and surface expression of cell adhesion molecules (CAMs) is critical for binding and recruitment of circulating leukocytes in tissues during the inflammatory response. Endothelial CAM expression plays a critical role in the intestinal microvasculature in inflammatory bowel disease (IBD), as blockade of leukocyte α4-integrin binding by gut endothelial CAM ligands has therapeutic benefit in IBD. Mechanisms underlying expression of vascular cell adhesion molecule (VCAM)-1, a ligand for α4-integrin in primary cultures of human intestinal microvascular endothelial cells (HIMEC) has not been defined. We investigated the effect of curcumin, phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt), and mitogen-activated protein kinase (MAPK) inhibitors on VCAM-1 expression and function in HIMEC. CAM expression was assessed and HIMEC-leukocyte adhesion was visualized under static and flow conditions. Western blotting and in vitro kinase assays were used to assess Akt and MAPK activation. Nuclear factor-κB (NF-κB) activation and nuclear translocation of its p65 subunit were determined. Tumor necrosis factor (TNF)-α/lipopolysaccharide (LPS)-induced VCAM-1 expression in HIMEC was suppressed by Akt small-interfering RNA, curcumin, and inhibitors of NF-κB (SN-50), p38 MAPK (SB-203580) and PI 3-kinase/Akt (LY-294002). VCAM-1 induction was partially suppressed by p44/42 MAPK (PD-098059) but unaffected by c-Jun NH2-terminal kinase (SP-600125) inhibition. Curcumin inhibited Akt/MAPK/NF-κB activity and prevented nuclear translocation of the p65 NF-κB subunit following TNF-α/LPS. At physiological shear stress, curcumin attenuated leukocyte adhesion to TNF-α/LPS-activated HIMEC monolayers. In conclusion, curcumin inhibited the expression of VCAM-1 in HIMECs through blockade of Akt, p38 MAPK, and NF-κB. Curcumin may represent a novel therapeutic agent targeting endothelial activation in IBD.
- phosphatidylinositol 3-kinase
- protein kinase B
- mitogen-activated protein kinase
- nuclear factor-κB
in the dysregulated, chronic inflammation that characterizes human inflammatory bowel disease [IBD; Crohn's disease (CD) and ulcerative colitis (UC)], alterations in intestinal microvascular endothelium have been identified at the morphological and functional levels, including increased expression of cell adhesion molecules (CAM) and enhanced leukocyte recruitment (4). To further characterize microvascular endothelial changes in gut inflammation, human intestinal microvascular endothelial cells (HIMEC) cultures have been generated, demonstrating inducible expression of the molecules E-selectin, intercellular adhesion molecule (ICAM)-1, MAdCAM-1, and vascular cell adhesion molecule (VCAM)-1 following activation with inflammatory cytokines [i.e., tumor necrosis factor (TNF)-α, interleukin-1β] and lipopolysaccharide (LPS) (4). Characterizing mechanisms underlying gut microvascular endothelial activation and CAM expression represents an essential area for defining mechanisms of IBD pathogenesis as well as developing novel targets for therapy.
VCAM-1 is involved in firm adhesion and transmigration of leukocytes expressing the α4-integrin ligand. α4-Integrin-expressing leukocytes play a critical role in the pathogenesis of rodent models of IBD and a spontaneous form of chronic colitis that occurs in primates (i.e., cotton-top tamarin). Recent clinical trials have confirmed the importance of α4-leukocyte trafficking in the intestine in human IBD, as natalizumab and MLN02 (10, 30) have demonstrated benefit in CD and UC respectively. The clinical efficacy of α4-integrin inhibition is believed to result from interruption of interaction with MAdCAM-1, but at present it is not known whether an alternative ligand, VCAM-1, may also be playing a role in IBD chronic inflammation.
VCAM-1 upregulation by proinflammatory cytokines has been reported in various cells (6, 7, 35). Induction of VCAM-1 in endothelial cells by TNF-α requires nuclear factor (NF)-κB, activator protein-1, phospholipase C, and phosphatidylinositol 3-kinase (PI 3-kinase) activation (16, 23). Downregulation of VCAM-1 expression with subsequent impairment of endothelial-leukocyte adhesion in rodents was linked to inhibition of PI 3-kinase/protein kinase B (Akt) signaling (19–21, 25, 32). Endothelial expression of VCAM-1 in both human and animal models of IBD has been difficult to confirm at a histological level. Smooth muscle and mesenchymal expression of VCAM-1 has been readily demonstrated, but endothelial expression in tissue histology has been less definitive. However, specific targeting of VCAM-1 in rodent models of IBD has demonstrated clear evidence of benefit, suggesting that this molecule is playing an important role in IBD chronic gut inflammation.
Curcuma longa Linn, Zingiberaceae (curcumin), the major yellow coloring pigment found in the household spice turmeric, has been used for centuries in food preparation and Ayurvedic traditional medicine to treat inflammatory disorders (34). Curcumin has low toxicity, has treated chronic gut inflammation in animal models, and has shown benefit in a randomized, multicenter crossover trial in the treatment of UC (12). Mechanistically, curcumin inhibits: 1) production of reactive oxygen species; 2) nitric oxide synthase activity; 3) lipoxygenase activity; and 4) cyclooxygenase-2 activity during inflammation (9, 5, 17, 29).
Prior work by our group has shown that expression of the α4-integrin ligand MAdCAM-1 in HIMECs following activation with TNF-α/LPS is regulated through the PI 3-kinase/Akt pathway but neither E-selectin nor ICAM-1 expression was affected by this signaling mechanism (24). In this study, we demonstrate that, in addition to activation of p38 mitogen-activated protein kinase (MAPK) and NF-κB, VCAM-1 expression in TNF-α/LPS-stimulated HIMEC also requires signaling through PI 3-kinase/Akt. Moreover, VCAM-1 expression and leukocyte adhesion in TNF-α/LPS-activated HIMEC were inhibited by curcumin, LY-294002 (PI 3-kinase/Akt), SN-50 (NF-κB), and SB-203580 (p38 MAPK) pretreatment. The present data further define the intracellular mechanisms of PI 3-kinase/Akt-mediated activation in gut microvascular endothelium and leukocyte recruitment through α4-integrin binding, identifying potential endothelial targets for IBD therapy.
TNF-α and antibodies to ICAM-1, VCAM-1, E-selectin, and the Tissue-Cell-Staining kit were purchased from R&D Systems (Minneapolis, MN). Nonradioactive In vitro MAPK Kinase Assay Kits and MAPK antibodies were from Cell Signaling Technology (Danvers, MA). The nonradioactive in vitro Akt Kinase Assay Kit was obtained from Upstate Cell Signaling (Lake Placid, NY). PD-098059, SB-203580, SP-600125, SN-50, and LY-294002 inhibitors were obtained from Calbiochem (La Jolla, CA). RNA Cell Protect reagent and RNeasy Plus Mini Kits were obtained from Qiagen (Valencia, CA). Akt primers catalog no. PPH0008A were obtained from SA Biosciences (Frederick, MD). Akt small-interfering RNA (siRNA) (target siRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA (positive control), and nontargeting siRNA (negative control) were obtained from Dharmacon (Chicago, IL). The iScript cDNA synthesis kit, Sybr Green Master Mix, iQ5 software, and all other electrophoresis reagents were obtained from Bio-Rad (Hercules, CA). Unless otherwise indicated, all other chemicals used in this study were purchased from Sigma Chemical (St. Louis, MO).
HIMEC isolation and culture.
All experiments were approved by the Human Research Review Committee of the Medical College of Wisconsin. HIMECs were isolated and cultured as previously described (4). Experiments were performed on three independent HIMEC lines unless otherwise specified. All images displayed were a representative result of one of the three independent experiments.
Activation and pharmacological modulation of HIMEC.
HIMEC activation was achieved following TNF-α (100 U/ml)/LPS (1 μg/ml) stimulation for specified time periods. Curcumin (10 μM), SN-50 (18 μM), LY-294002 (10 μM), SB-203580 (10 μM), PD-098059 (10 μM), SP-600125 (10 μM), and vitamin E (25 μM) were used to determine the effect of these inhibitors on VCAM-1 expression in HIMEC. Of note, we have performed dose-response experiments for all inhibitors stated, and the most potent but nontoxic dose for our experimental analysis was chosen (data not shown).
Assessment of CAM surface expression and leukocyte-HIMEC adhesion assay.
CAM surface expression in HIMEC and U-937-HIMEC interaction were assessed as described previously (4, 26, 28).
Semiquantitative RT-PCR.
VCAM-1 mRNA expression was determined as described previously (24), using VCAM-1 (forward: 5′-GGA ACC TTG CAG CTT ACA GTG ACA GAG CTC CC-3′, reverse: 5′-CAA GTC TAC ATA TCA CCC AAG-3′)- and β-actin (forward: 5′-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3′, reverse: 5′-CTA GAA GCA TTG CGG TGG ACG ATG GAG GG-3′)-specific primers. RNA without reverse transcription was used as negative control (no RT), and expression of β-actin served as an internal control.
MAdCAM-1 mRNA expression was determined using real-time PCR as described previously (11, 27), using MAdCAM (forward: 5′-GAA GTG ATC CCA ACA GGC TC-3′, reverse: 5′-TTC CAG AGG TGA TAG GTG GG-3′) and GAPDH (forward: 5′-TGC ACC ACC AAC TGC TTA GC-3′, reverse: 5′-GGC ATG GAC TGT GGT CAT GAG-3′) specific primers. Normalized gene expression was analyzed using iQ5 software.
Western blot analysis.
SDS-PAGE and Western blot analysis were performed using antibodies to VCAM-1, Akt, and MAPK as described previously (28).
Akt in vitro kinase activity assay.
Active Akt was immunoprecipitate from cell extracts and then an in vitro kinase assay was performed using glycogen synthase kinase (GSK)-3 Fusion Protein as a substrate according to the manufacturer's instructions. Phosphorylation of GSK-3 was measured using phospho-GSK-3α/β (Ser21/9) antibody.
Akt siRNA transfection and VCAM-1 expression.
Transfection was done using Amaxa's primary endothelial transfection kit and Nucleofector device according to the manufacturer's instructions. HIMEC were transfected with 100 nM of either Akt siRNA (target gene), GAPDH siRNA (positive control), or nontargeting siRNA (negative control). Pooled siRNAs for each gene were obtained from Dharmacon. After transfection (24 h), cells were either unstimulated or stimulated with 100 U/ml TNF-α and 1 μg/ml LPS for 6 h. RNA was then isolated using Qiagen's RNeasy Plus Mini Kit according to the manufacturer's instructions. Reverse transcription was done with 1 μg of RNA using Bio-Rad's iScript cDNA synthesis kit. Akt gene knockdown and VCAM gene expression were analyzed via real-time PCR using Bio-Rad's Sybr Green Master Mix, 2 μl of cDNA, and 250 nM primer in 25-μl reactions. Cycling parameters were 95°C for 3 min and then 45 cycles of 95°C for 10 s and 60°C for 30 s. Generation of a single product was confirmed with a melt cycle. Real-time data were analyzed using Bio-Rad's iQ5 software. Primer sequences were as follows: α-actin forward CTG GAA CGG TGA AGG TGA CA and reverse AAG GGA CTT CCT GTA ACA ATG CA, VCAM forward TGT TGA GAT CTC CCC TGG AC and reverse CGC TCA GAG GGC TGT CTA TC, and GAPDH forward TGC ACC ACC AAC TGC TTA GC and reverse GGC ATG GAC TGT GGT CAT GAG. Akt primers were obtained from SA Biosciences.
c-Jun NH2-terminal kinase and p38 MAPK in vitro kinase activity.
c-Jun NH2-terminal kinase (JNK) and p38 MAPK activity were determined as previously described (28).
p44/42 MAPK in vitro kinase activity.
Active p44/42 MAPK was immunoprecipitated from cell extracts, using an immobilized phospho-p44/42 MAPK (Thr202/Tyr204) antibody. Next, an in vitro kinase assay is performed using Elk-1 protein as a substrate. Phosphorylation of Elk-1 was detected by Western blotting using phospho-Elk-1 (Ser383) Antibody.
Assay of transcription factor NF-κB.
Using TransAM NF-κB and Nuclear Protein Extraction Kits, NF-κB activity was measured according to the manufacturer's protocol (Active Motif, Carlsbad, CA). The samples were analyzed in a 96-well plate containing the immobilized NF-κB consensus site (5′-GGG ACT TTCC-3′) oligonucleotide. The activated form of NF-κB in nuclear extract binds to this oligonucleotide. Use of an antibody against NF-κB p65 subunit and a horseradish peroxidase (HRP)-conjugated secondary antibody results in a colorimetric readout, which was quantified at 450 nm using a Beckman DU-650 spectrophotometer. Data from triplicate wells were expressed as a means ± SD.
Immunofluorescence staining.
Immunofluorescence staining was performed using NF-κB p65 subunit antibody and a FITC-conjugated secondary antibody as described previously (24).
TNF-α/LPS induces expression of VCAM-1 gene and protein in HIMEC.
We have previously demonstrated that TNF-α/LPS activation of HIMEC significantly increased ICAM-1, E-selectin, and VCAM-1 expression (3, 26). In this study, we characterized the signaling pathways underlying VCAM-1 gene and protein expression in HIMEC following TNF-α/LPS activation.
VCAM-1 mRNA expression was determined by semiquantitative RT-PCR. VCAM-1 mRNA expression in HIMEC following TNF-α/LPS stimulation was time-dependent, and the maximum response was achieved at 4 h, lasted for 12 h, and then returned to basal levels by 24 h (Fig. 1A, left). Corresponding to the VCAM-1 mRNA expression, Western blot analysis demonstrated that TNF-α/LPS activation of HIMEC induced increased VCAM-1 protein significantly by 12 h, which was sustained up to 24 h, and then decreased by 36 h and returned to basal levels by 48 h (Fig. 1B, left). Time-matched controls for VCAM-1 mRNA and protein expression are shown in Fig. 1, A and B, right. Individual blots were stripped and reprobed with an anti-actin antibody to ensure equal protein loading.
Effect of curcumin on Akt signaling in HIMEC.
TNF-α/LPS stimulation of HIMEC resulted in phosphorylation of Akt in a time-dependent manner, which was initially evident at 15 min, lasted up to 60 min, and declined by 120 min (Fig. 2A). No Akt phosphorylation was detected in unstimulated control HIMEC at any given time point (data not shown). Pretreatment of HIMEC for 30 min with either curcumin (10 μM) or the PI 3-kinase inhibitor LY-294002 (10 μM) before TNF-α/LPS stimulation completely abolished the Akt phosphorylation (Fig. 2B), whereas SN-50, an NF-κB inhibitor, did not affect the Akt phosphorylation. Total nonphosphorylated Akt was probed to demonstrate equal protein loading.
Akt in vitro kinase activity assay.
The active Akt was immunoprecipitated from TNF-α/LPS-activated HIMEC by immobilized Akt antibody. The Akt in vitro kinase assay was performed using GSK-3 fusion protein as a substrate. GSK-3 underwent phosphorylation in TNF-α/LPS-activated HIMEC but not in control samples using phospho-GSK-3α/β (Ser21/9) antibody and Western blotting. Curcumin and LY-294002 pretreatment of HIMEC resulted in inhibition of GSK-3 phosphorylation and Akt activity (Fig. 3).
Effect of curcumin on MAPK signaling pathways in HIMEC.
The effect of curcumin on the kinase activity of the individual MAPK was investigated using an in vitro kinase activity system. As shown in Fig. 4A, TNF-α/LPS activation of HIMEC increased the JNK activity, which was evident by c-Jun phosphorylation using phospho (Ser63)-c-Jun antibody. Pretreatment of HIMEC with curcumin, SP-600125, and LY-294002 before TNF-α/LPS activation abolished the JNK activity. Figure 4B demonstrates that TNF-α/LPS activation of HIMEC increased the p38 MAPK activity, which was evident by ATF-2 phosphorylation. Phosphorylation of ATF-2 at Thr71 was measured by Western blotting using phospho-ATF-2 (Thr71) antibody. Pretreatment of HIMEC with SB-203580, LY-294002, and curcumin before TNF-α/LPS activation inhibited the p38 MAPK activity. As shown in Fig. 4C, TNF-α/LPS activation of HIMEC increased the p44/42 MAPK activity, which was evident by Elk-1 phosphorylation detected by Western blotting using phospho-Elk-1 Antibody. Pretreatment of HIMEC with curcumin, PD-098059, and LY-294002 before TNF-α/LPS activation inhibited the p44/42 MAPK activity.
VCAM-1 expression in HIMEC is dependent on Akt/p38 MAPK/NF-κB activation.
The contribution of the PI 3-kinase pathway to VCAM-1 and ICAM-1 expression in human monocytes has been previously demonstrated (1). Downregulation of VCAM-1 expression and impairment of monocyte-endothelial cell adhesion in rodents through inhibition of the PI 3-kinase/Akt pathway with lovastatin has been previously reported (25). However, the role of the PI 3-kinase/Akt pathway, and its potential contribution to TNF-α/LPS-induced VCAM-1 expression in HIMEC had not been specifically defined. The next series of experiments focused on the PI 3-kinase/Akt signaling pathway in HIMEC, since this signaling mechanism is intimately involved in cellular differentiation and was previously demonstrated to play a critical role in α4-integrin-expressing leukocyte binding through MAdCAM-1 expression in HIMEC. Interestingly, HIMEC expression of E-selectin and ICAM-1 did not involve PI 3-kinase/Akt, suggesting that specific signaling pathways underlie specific CAM expression during inflammation (24).
As demonstrated in Fig. 5, A–C, LY-294002 (PI 3-kinase/Akt), SB-203580 (p38 MAPK), SN-50 (NF-κB) inhibitors, vitamin E, and curcumin effectively inhibited VCAM-1 mRNA, protein, cell surface expression, and leukocyte binding, whereas SP-600125 (JNK) inhibitor had no effect on VCAM-1 expression and PD-098059 (p44/42 MAPK) inhibitor only partially decreased VCAM-1 mRNA, protein, and cell surface expression (Fig. 5, A–C). Curcumin inhibited VCAM-1 expression in a dose-dependent fashion (Fig. 5D). These findings suggest that activation of Akt/p38 MAPK/NF-κB and possibly p44/42 MAPK may play a role in TNF-α/LPS-induced VCAM-1 expression in HIMEC, and curcumin was a potent inhibitor of all of these inflammatory pathways.
Gene silencing of Akt in HIMEC.
To confirm whether a functional effect of VCAM-1 expression was linked to enhanced Akt expression in HIMEC, we performed gene-silencing experiments using transfection with siRNA specific for Akt. With the use of appropriate controls (GAPDH siRNA as a positive control and nontargeted siRNA as a negative control), specific knockdown of Akt by Akt siRNA was demonstrated by real-time PCR (Fig. 5E, right). After confirming significant silencing of Akt expression, the effect on VCAM-1 expression after TNF-α/LPS stimulation was measured by real-time PCR (Fig. 5E, left).
Effect of curcumin on MAdCAM-1 gene expression in HIMEC.
Involvement of PI 3-kinase/Akt activation in MAdCAM-1 expression following TNF-α/LPS stimulation of HIMEC was previously demonstrated by our group (24). Our data suggest that VCAM-1 and MAdCAM-1 expression in HIMEC share similar signaling pathways following TNF-α/LPS activation. Given the potent inhibitory effect of curcumin on VCAM-1 expression, we determined its effect on MAdCAM-1 expression in HIMEC. As demonstrated in Fig. 6, curcumin significantly inhibited MAdCAM-1 mRNA expression.
NF-κB inhibitor attenuates TNF-α/LPS-induced VCAM-1 expression.
Involvement of NF-κB activation in E-selectin and ICAM-1 expression following TNF-α/LPS stimulation of HIMEC has been previously demonstrated (24). We investigated whether NF-κB activation also plays a role in VCAM-1 expression in TNF-α/LPS-activated HIMEC. As shown above in Fig. 5, A–C, pretreatment of HIMEC with the NF-κB inhibitor (SN-50) attenuated VCAM-1 gene, cell surface expression, and leukocyte binding. Figure 7A demonstrates that NF-κB-DNA binding activity was completely inhibited by SN-50 and curcumin pretreatment of HIMEC before TNF-α/LPS activation, using a cell-based ELISA-NF-κB assay. Western blot analysis from nuclear protein fractions of TNF-α/LPS-activated HIMEC show the immunoreactivity of NF-κB subunit p65, which was also inhibited by both SN-50 and curcumin (Fig. 7B). Furthermore, Western blotting showed that inhibitory factor κB-α is rapidly degraded in TNF-α/LPS-activated HIMEC in <30 min and then recovers by 60 min, resulting in NF-κB activation (Fig. 7C). Translocation of NF-κB subunit p65 in the nucleus was effectively blocked with both SN-50 pretreatment and curcumin (Fig. 7D).
Together these results suggest that PI 3-kinase/Akt, MAPK, and NF-κB are the key regulatory pathways for VCAM-1 expression in HIMEC following TNF-α/LPS activation.
Immunohistochemical localization of VCAM-1 in colonic microvessels.
In frozen sections from non-IBD resected human colon (i.e., diverticular disease, colon cancer resection margins), mucosal microvascular endothelial VCAM-1 expression was assessed by immunohistochemistry using a diaminobenzidine-HRP-based substrate system. VCAM-1 immunoreactivity (shown by dark brown precipitate) is evident in select mucosal and submucosal microvessels (Fig. 8). Of note, not all microvessels showed positive immunoreactivity in these colonic specimens.
Schematic of Akt activation leading to VCAM-1 expression.
We hypothesize that TNF-α/LPS activation of HIMEC results in PI 3-kinase activation and subsequent Akt phosphorylation, as demonstrated in the summary figure (Fig. 9). Activated Akt will in turn activate MAPK cascades and NF-κB pathways, which will ultimately result in increased gene and protein expression of MAdCAM-1 and VCAM-1, the two major endothelial ligands for α4-expressing leukocytes, which preferentially home to the mucosal immune compartment in the intestine.
The present study has confirmed that TNF-α/LPS stimulation of HIMEC resulted in activation of MAPKs and NF-κB and increased expression of VCAM-1 along with MAdCAM-1, E-selectin, and ICAM-1 (24, 26). We defined the mechanisms and signaling pathways that underlie VCAM-1 expression in TNF-α/LPS-activated HIMECs. Our findings indicate that, in addition to activation of the MAPK and NF-κB, the PI 3-kinase/Akt signaling pathway is integral to VCAM-1 expression in TNF-α/LPS-activated HIMEC.
VCAM-1 expression in HIMEC required p38 MAPK, NF-κB, and PI 3-kinase/Akt activation, which is in marked contrast to the signaling cascades required for E-selectin and ICAM-1 expression, which only required NF-κB but not PI 3-kinase/Akt activation (24). Our findings are paralleled by studies that have shown that the PI 3-kinase/Akt inhibitor LY-294002 did not exert inhibitory effects on either E-selectin or ICAM-1 expression in human umbilical vein endothelial cells (22). These results raise the question of selectively blocking PI 3-kinase/Akt as a therapeutic target that would interrupt α4-mediated leukocyte transmigration to the gut endothelium in the treatment of IBD, while not affecting other cell adhesion pathways involved in cellular homeostasis and leukocyte recruitment.
PI 3-kinase consists of two subunits (p85 and p110), and, upon activation, p110 catalyzes the phosphorylation of membrane phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate, leading to Thr308 and Ser473 Akt phosphorylation and activation (31). The PI 3-kinase/Akt pathway plays a role in multiple aspects of cell physiology and homeostasis, since Akt activation will result in cell proliferation, differentiation, chemotaxis, survival, enhanced glucose metabolism, and nitric oxide production (18). In the present study, we demonstrate that VCAM-1 expression in TNF-α/LPS-activated HIMEC is also regulated by the PI 3-kinase/Akt pathway. The importance of PI 3-kinase/Akt signaling in expression of endothelial ligands for leukocyte α4-integrin, a critical molecular homing mechanism that is specific to the gut, suggests that this signaling cascade plays an essential role in gut-specific differentiation of these endothelial cells. However, PI 3-kinase/Akt activation in large-vessel endothelial populations has also been reported (22), which implies that additional signaling pathways will likely be required to achieve the complete gut-specific differentiation exhibited by HIMEC.
Previous attempts to target the signaling cascades involved in inflammatory activation in IBD have met with mixed success. The initial success of a p38 MAPK inhibitor in a pilot study (14) has yet to demonstrate success in a pivotal multicenter trial. Two other inhibitors of inflammatory signal transduction have failed to demonstrated therapeutic benefit (8). The reasons underlying the failure of these inhibitors of inflammatory signaling cascades are unclear. The inhibitors may have too broad of an effect on diverse cell physiology. Another factor could be the pharmacokinetic issues involving drug delivery to areas of inflammation. An additional concern regarding the therapeutic potential of signal transduction inhibitors in inflammation centers on the timing of their use in complex disorders such as IBD. Selective inhibitors of signal transduction may not be effective for both induction and maintenance of remission in a chronic inflammatory disease but may be more efficacious for one of these two phases in the disease process. Given that most IBD clinical trials will initially focus on induction of remission, agents that may preferentially function in the maintenance of remission may not be assessed with the best approach. A prime example of the potential selective efficacy of an agent that will function in maintenance of remission is highlighted by the compound curcumin. Although curcumin has been shown to be effective in the treatment of animal models of IBD where it prevented development of gut inflammation and decreased morbidity and mortality in both dextran sulfate sodium and trinitrobenzene sulfonic acid colitic mice (33), its major benefit in human trials has been demonstrated in maintenance of remission. Curcumin has been shown in a multicenter crossover trial to offer therapeutic benefit for maintenance of remission in UC patients who were concomitantly on 5ASA therapy (12). Because this agent has been historically used as a component of the spice turmeric in food preparation, one can hypothesize that its therapeutic effect may be more subtle, and this may underlie its demonstrated success in maintaining remission over time. Therefore, curcumin may not be successful in inducing remission in severe active inflammation but offer benefit as a long-term agent for the maintenance of colitis remission.
At present, there are emerging therapeutic agents that specifically target the endothelium for IBD treatment. Work over the past 15 years has demonstrated the central importance of the α4-integrin pathway in leukocyte recruitment to the gut in IBD inflammation. Initial therapeutic observations in animal models of IBD have been translated into human trials with several selective and nonselective α4-integrin inhibitors. Natalizumab, the first anti-α4-integrin inhibitor, has demonstrated significant therapeutic benefit for the treatment of CD and multiple sclerosis. This agent is believed to block the interaction of α4-expressing circulating leukocytes with their endothelial integrin ligands MAdCAM-1 and VCAM-1. Although this agent demonstrated a weaker induction effect, responding patients demonstrated a strong and durable remission extending beyond one year of treatment. At present, there are ongoing trials with multiple anti-α4-integrin inhibitors (i.e., natalizumab, MLN02) for the treatment of both CD and UC (2).
In summary, we have demonstrated that HIMEC express mRNA and protein for VCAM-1 in response to TNF-α/LPS. The regulation of VCAM-1 gene expression in HIMEC is dependent on NF-κB activation, which is similar to ICAM-1 and E-selectin expression. However, VCAM-1 expression is also dependent on PI 3-kinase/Akt signaling, which resembles MAdCAM-1 signaling patterns in HIMEC. VCAM-1 functions as a ligand for the α4-integrin, which is a shared function with MAdCAM-1, the molecule that is felt to play the central role in leukocyte recruitment to the gut. Both VCAM-1 and MAdCAM-1 expression is linked to the PI 3-kinase/Akt activation, suggesting that α4-leukocyte recruitment to the gut is linked to endothelial activation of this signaling cascade, and curcumin inhibited the expression of both molecules. These data suggest that inhibitors of endothelial PI 3-kinase/Akt activation may function to inhibit enhanced CAM expression and warrant evaluation for the treatment of IBD.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-065948 and by the Digestive Disease Center of the Medical College of Wisconsin.
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