After dedicating her life to children with disabilities, she entered this world from the opposite side when her daughter was born with lissencephaly, a rare genetic brain development disorder that results in significant physical and cognitive disabilities.
" I love seizure tracker. I think this is an awesome tool to help keep track of seizure activity and medications."
Tracking Daily Variables - Record and track daily variables that may effect your seizure activity on Seizure Tracker. Create reports that graphically illustrate what time and day these variables occurred.
Seizure Tracker Data Share Tool - Seizure Tracker is providing tools to researchers that will help improve your life!
NIH/NINDS Clinical Research Program - Check out the NIH/NINDS Clinical Research Program and the exciting research happening on their Bethesda, MD campus.
Seizure Tracker Clinical Trial Monitoring Tool - Based on the classic Seizure Tracker interface, the CTMT provides a simple electronic logging system for multi-center clinical trial enrollment. The development of this tool was funded through a grant from the Tuberous Sclerosis Alliance.
Track Epilepsy-Related Medication Blood Levels - Record and track your epilepsy-related medication blood levels on Seizure Tracker. Create reports that graphically illustrate when blood draws were done and what the levels were at a the time of draw.


Darlene Barriere: Author. Speaker. Coach.: What Stands in Your Way?
What keeps you from realizing your dreams? From moving forward in a difficult situation? From getting past something you endured as a child?
We all face some form of adversity at some time in our lives. Whether it was as a child, a youth or as an adult. And some of us seem to have a greater load than others. Possibly experiencing heavy challenges at every stage of life.
Perhaps you endured physical, sexual and/or emotional abuse as a child. Perhaps you were raised to feel as though you aren't enough and that you'll never measure up. Or you were rejected, told that you shouldn't have been born.
Maybe you lost a loved one, someone you were very close to. Someone who was a mentor. Someone you deeply respected. Perhaps you feel guilt or shame for what you perceive was a role you played in harming someone, either when you were a child or as a youth.
No matter what has happened in your life, no matter what your choices were, be it present or past, you can get beyond it. The questions are: What stands in your way? What exactly is holding you back? What is it that you fear?
Success? The fear of success stands in the way of more people than you might think. Perhaps you're one of those who worries about what will happen to your life if your dream comes to fruition. How it will impact your family time, leisure time or time for yourself.
Let's face it though. If we're being honest, it isn't usually success that we fear the most. At least not when we're initially imagining our dreams. Most of us fear failure.
I have news for you. You will fail. Failure isn't only an option, it's an opportunity. It was Thomas Edison who said: I have not failed. I've just found 10,000 ways that won't work. Use failure as an opportunity to determine what doesn't work, then find what does work.
The fear of failure can hit us in so many ways. Failure in the sense that we look inadequate or that we won't measure up. Fear that we'll prove all the negative messages we received as a child are actually true.
But they aren't true. They're lies. They were lies when they were being relayed to you. They're lies now that you've adopted them in your psyche.
Notice what I said in the last line. You've adopted them in your own psyche.
No matter what others have told you, either directly or indirectly, it's what you tell yourself that determines where you will go in your life.
So how exactly do the thrivers keep thriving? What is their secret to not getting stuck. Or better, how do they not stay stuck when triggers are all around them?
Thrivers first shed the old tapes. They shed the lies. They shed stuff that prevents them from doing the things they dream of doing. The stuff that stops them moving from dream potential to dream production.
Thrivers replace negative self-talk with an I-can mindset. They tell themselves the opposite of what was told to them. They tell themselves that they are worthy. That they are capable. That they do measure up. And they believe every word of those truths.
Thrivers don't allow others to dictate their success. They don't allow others to interfere with their success. And lastly, they don't allow others to determine their success.
It's not how successful you are; it's how you measure success.
Thrivers have a strong sense of right and wrong. They measure success, not solely in monetary terms, but also in intrinsic terms. How they make a difference in the world. How they contribute to the greater good.
So what stands in your way of being a thriver?
As per your article the only thing standing in the way of my success is me. However, I am one of the fortunate ones who was never demeaned by my parents, so most of my ``stories`` came from other sources. Thankfully I was able to work through and in my view have been very successful,especially if you measure your success by your happiness.


text:
Census Tract 401, Appomattox, Virginia
Census Tract 401 is a Low-Income CommunityOpportunity Zone located in Appomattox, Virginia.
This 62 square milecensus tract has a population of approximately 7,100and is the only Opportunity Zone in Appomattox County.
The adjacent map shows the location of this Opportunity Zone in Virginia.
Opportunity Zone Demographics
List of Similar Opportunity Zones
Census Tract 401 Demographics
The charts below illustrate how this Opportunity Zone compares to the broader city, county, and state on various socioeconomic indicators:
This Opportunity Zone has a median household incomeof approximately $60,000, which is 21% lowerthan the median household income for the state of Virginia of $76,000.
Poverty Rate
The percentage of households below the poverty line in this Opportunity Zone is 17%, which is 7% higherthan the rate for the state of Virginia of 9.9%.
This Opportunity Zone has a median home valueof approximately $160,000, which is 45% lowerthan the median home value for the state of Virginia of $290,000.
This Opportunity Zone has a percentage of population with high school diplomaof approximately 86%, which is 4% lowerthan the rate for the state of Virginia of 90%.
This Opportunity Zone has a median ageof approximately 40, which is 3% higherthan the median age for the state of Virginia of 39.
Similar Opportunity Zones
See all OZs in Virginia
See all OZs in Appomattox County
See all OZs in Appomattox

redpajama_set_name: RedPajamaCommonCrawl


Loans for house purchase were at their highest since 2009 last year, according to new figures published by the Central Bank.
The data show net mortgage lending rose by €964 million or 1.3 per cent in the year to November with net growth of €131 million in that month alone.
Commenting on the latest figures, Brokers Ireland, which represents 1,250 firms, said the data does not tally with the reality experienced by consumers.
Rachel McGovern, director of financial services at the organisation, said the Banking and Payments Federation Ireland (BPFI) figures for the third quarter showed actual mortgage drawdowns, as opposed to approvals, at 8,727 for the quarter, to a value of €1.985 million, bringing lending for house purchase to €5.093 million for the first nine months of 2018.
"This would indicate that while the situation with mortgages is steadily improving it is still a long way off what could be considered the norm," said Ms McGovern.
"On that basis we can expect that mortgage lending for house purchase will come in way under the €10 billion figure for the year," she added.
The rise in mortgage loans comes as total loans to households fell 1.1 per cent in the year to the end of November, according to the Central Bank figures.
Net consumer lending totalled €22 million during the month with new lending exceeding repayments by €233 million on an annual basis.
Net lending for other purposes, such as education, was negative during the month under review.
Deposits from households were €4.6 billion higher than withdrawals over the year at 4.7 per cent, the largest annual growth rate seen since 2008.
Irish households continued to be net funders of the banking system with banks holding €12.3 billion more in household deposits than loans at the end of November, with the loan-to-deposit ratio standing at 0.88.
Annual growth in total bank lending remained negative, with a decline in lending of 0.2 per cent in the year, the data shows.


The Nifty finished Monday's session at 10,800, down by 18 points or 0.17 per cent.
Sensex witnessed 11 stocks advancing and 20 declining at the end of today's trade.
NEW DELHI: Jaiprakash Associates (number of shares traded: 11.40 crore) emerged as the most traded stock on NSE on Monday.
It was followed by Reliance Communications (5.67 crore), ICICI Bank (2.78 crore), Vakrangee (2.48 crore), GMR Infra (2.23 crore) and Idea Cellular (1.71 crore) in that order.
Headline indices Nifty and Sensex ended the day in the negative territory after a choppy trade owing to losses in a majority of sectors, led by metals, bank, IT, capital goods, consumer durables and telecom. However, gains in oil and auto stocks kept the fall restricted.
The BSE Sensex fell for 74 points, or 0.21 per cent, settling at 35,548, while the Nifty finished Monday's session at 10,800, down by 18 points or 0.17 per cent.
Overall, 20 stocks remained in the green in the Nifty pack, compared with 30 stocks that went home with losses.
Among the sectors, Nifty Auto and private bank index closed the day in the green, while Nifty Pharma settled almost flat. All other indices closed the day in the red, with Nifty Metal as the top loser, falling 1.74 per cent.


A prolapsed gland of the eyelid (or, "cherry eye") refers to a pink mass that protrudes from an animal's eyelid. Usually, the gland development is anchored by an attachment that is made up of fibrous material. This condition occurs in both cats and dogs—however it mostly affects younger pets.
How Can I Identify Cherry Eye in Pets?
The most common sign of cherry eye is an oval mass that protrudes from your pet's third eyelid. This can occur in both eyes or just one eye and can be accompanied by irritation and swelling.
This condition is often associated with a congenital weakness of the gland's attachment to the pet's eye. It is unknown however, if this condition is inherited. For cats, cherry eye can happen to any breed, however it is more commonly seen in Burmese or Persian cats.
How is Cherry Eye Diagnosed?
Your veterinarian will review the mass in your pet's third eyelid to see if there are any underlying causes for the condition. The diagnosis for the prolapsed gland could be everted or scrolled cartilage in the third eyelid, a prolapse of fat in the pet's eye or abnormal cells in the third eye.
What is the Treatment for Cherry Eye?
A treatment option for cherry eye includes a surgical replacement of the gland in the pet's eye, or even removing the entire gland if the condition is that severe. If medications are recommended, they are typically topical, anti-inflammatory treatments that are effective in reducing swelling.


Fight-or-Flight vs. Rest and Digest-Which Mode Are You In?
One thing I check all of my patients for at every visit is to see if they are in a "Sympathetic Dominant mode". If you are a patient, you may have heard me say this before during a treatment! I check for this mode in all my patients using our amazing tool of muscle testing.
If a patient is in a sympathetic dominant, or "fight or flight" mode, his/her body will be very stressed, and every muscle will be overly strong (hypertonic). When I pinch a muscle, it should weaken. If if doesn't weaken upon pinching, you better bet that we will first be working on shifting that patient out of sympathetic dominant mode, and into a more balanced nervous system, so that we can continue treatment with accurate testing and good results.
If you are in any way stressed, overly busy, have suffered an injury or are ill, sleep deprived, malnourished, dehydrated, or under any other body stresses, you may very well be in a sympathetic dominant mode. When in this mode, healing is difficult, sleep is difficult, thinking is difficult, etc.. The body will likely eventually break down under this stress, possibly in more pathological ways.
One of the most significant benefits of chiropractic kinesiology is that it activates the body's parasympathetic nervous system! Yes, adjustments can actually shift a patient out of the fight-or-flight mode, and into a more relaxed state. Oftentimes, just one adjustment can cause a total parasympathetic shift. Chiropractic can do this by down regulating (reducing) sympathetic nerve firing such as pain signals, while consequentially increasing parasympathetic activity. This is why oftentimes a patient will feel a feeling of calm relaxation, and sometimes even euphoria, after a treatment. I know that many of my patients, in fact a large majority, have always told me that they feel more calm, relaxed, and de-stressed after a visit!
As we start a new work week, I look forward to helping all of my patients maintain balance in their health and lives this week!


I have been offshore charter fishing in excess of 38 years. My current vessel is the Seahawk III, a 35 foot Harris based in Holden Beach, NC. The price of fuel has gone so far through the roof, the goverment has slowly chipped away at our fishing seasons and I am just flat getting a little long in the tooth for the offshore game. So i decieded to charter for the other things I love to do. Hunting and inshore fishing. I have been hunting/inshore/freshwater fishing here in Bruinwick County just about my whole life.
For me this is what it is all about. I enjoy seeing people, especially kids, learn to hunt and fish and be able to bring them home for dinner or shoot game for the table. When I was 6 years old I was taught how to hunt and fish and I was able to teach my son these things when he was about the same age. I feel I have taught alot of kids to enjoy hunting and fishing and hope that they will do the same for others, both adults and children. It is so worthwhile for me to see someone do something that they thought hey probably couldn't do. Their faces shows it all when they do it, if it's catching a nice fish or hunting a squirrel or deer and coming back satisfied and happy with their selves. This is what it is all about!! Even if you don't get a fish or deer you are out in nature and can see and hear all God has created!!


Great apartment right next to Assiniboine Park! Find out how convenient it is to live in the desirable Bruce Park neighbourhood. Step out your door and step into the nicest park in Winnipeg. Lovely walking and cycling trails abound. Plenty of great restaurants, shops and grocery stores nearby. Easy access to downtown. Excellent bus service. Solid, safe and modern building. Heat and water included. Parking available.


Home›Latest›Tip Jar: For Art or Money?
I believe the success I've had as a composer is a direct result of me composing for money instead of art. Let me explain. Some years ago I got the opportunity to compose production music for a large music library. But instead of sending them a sampling of the bluesy acoustic music I had been writing, I asked them to send me the top 10 most-used tracks in their library from the last 12 months. Then I spent a week transcribing all of them.
There were amazing similarities between all the songs. Each one was heavy rock with big attention-grabbing intros and outros. They all had a first verse and chorus section with limited instrumentation and no melody, followed by a similar verse and chorus with more instrumentation and melodies, and almost all of them had a cut time bridge section. I did some research and learned why these songs (Most of them were done by different composers) were used more than the rest. These songs were selected primarily because it made the editor's (who selects which music is cut into a TV show) life easier.
Each one of these songs had sections that could be moved around and looped. They had sparse sections so they could be in the background and big attention grabbing sections for when there is no dialogue.
Bottom line is… I didn't compose a thing until I knew exactly who my audience was. In this case it was an audience of one person… The editor. I didn't submit music that was important to me. I found out what was important to my audience and created something specifically for them. This resulted in my 34 tracks being played millions of times.
We musicians face this issue all the time: Do I create art and hope there is an audience for it or do I find an audience and create for them?
In my experience there is more monetary success when creating for a specific audience. I think this concept can be applied to many different aspects in the music business.
Here are a few steps I recommend.
1. Define who your audience is… Who is hiring you? Are you a producer being hired by bands or labels? Are you a private music teacher being hired by parents to teach a child? Are you a gigging musician being hired by nightclubs or promoters?
2. Define who the decision maker is. If you are a producer, you need to learn who in the band or label is calling the shots… Who are you really working for? If you are a private music teacher teaching children it's most likely the mother who is making the decisions. If you are a gigging musician you should find out who the primary booking agent is.
3. Find out what is important to the decision maker. Ask questions like: What's the most important part of this project to you? Do you have any specific goals or deadlines I should know about? What was the best experience you had with a similar project? What was so great about it? What is the best way to communicate with you? Phone? Email? Text?
4. Put yourself in the decision maker's shoes. Imagine what would be important to you if you had their job. What kind of actions would earn your trust. What would someone do or say that would make you want to brag to your friends or colleagues about the awesome experience you had. Basically, be the person you would want to hire.
5. Make a personal connection. Try to meet your buyer/decision maker face to face. The best connections happen when you meet someone face to face, smile, shake their hand and look them in the eye. Go out of your way to make this happen with your buyer/audience if you can. It makes a world of difference. Smile when you are on the phone with the decision maker… People want to work with positive people and they can hear your smile. Connect with them on social media, LinkedIn, Facebook, etc. If you have a monthly newsletter, include them to your email list.
• Confirm the expectations of the job with your client prior to showing up, and give them the opportunity to communicate back to you so you can be sure to deliver a great experience.
• Show up early. A simple way of proving you are a professional and value your clients is to be very prompt.
• Follow up after the job has been completed and thank the client. This could be a simple email or phone call.
• Find out what their mailing address is and send them a gift. It could be a simple handwritten card or a bottle of their favorite alcohol. High-class touches like this will put you at the front of the line the next time there is the opportunity to work together.
Clearly you will need to adjust these suggestions to your specific situation. The point I'm making here is to find out how to make your audience happy and confident before you spend the time, energy and money creating something for an audience who might not exist. And once you have defined an audience, work very hard to make their life better. Money is the natural consequence of delivering value. Focus on delivering great value and the money will follow.
TIM GUILLE is an entrepreneur in the music industry. His compositions have been used in hundreds of television shows and networks ranging from NFL to Pawn Stars. He owns All Play Music (allplaymusic.com) a successful private lesson company in Los Angeles and is the Co-Founder of Music Money Formula (musicmoneyformula.com) where he provides business coaching and support for music educators around the world.


text:
Sign In Download
October 11, 2015 Gaming, Media
Unraid Featured On linustechtips
Linus Sebastian provides content to his audience (nearly 2 million youtube subscribers) in various forms: hardware reviews/unboxings, news analysis, build guides, and much more. After the release of Unraid 6, we reached out to Linus to see if he had any interest in demonstrating some of our capabilities in one of his shows. Just recently, CoolerMaster had asked Linus to do a video showcasing their Mastercase 5 in a build guide. Linus thought this would be a good opportunity to showcase the adaptability of the case to a variety of build needs, including two gamers running off the same CPU. In the video, Linus goes over component selection, BIOS configuration, and Unraid OS configuration for his build. If anyone wants to see what high-performance virtualization-capable hardware can do, this is a must-see!
On a personal note, we were very happy to see that this particular motherboard provided individual IOMMU groups for each of the PCI device slots on the Asus motherboard (no ACS Override required). The motherboard also featured two distinct USB controllers (an Intel and an ASMedia). One could be passed through to a VM completely while the other can be used for the Unraid USB flash boot device. The only thing that we were missing was an on-board graphics device so we could have avoided the need for taking up a PCI slot for host-based graphics (the Unraid console). That said, the performance results really speak for themselves. This is a great demonstration of the power of high-performance personal computing. I could easily imagine college roommates going in 50/50 on a gaming rig with overpowered hardware like this. When they are both there, they split the CPU 50/50 and still get great performance. When only one is there, they could use ALL the CPU cores and get even higher performance! Also consume less power, generate less heat, and act as a redundant storage system for their most important data.
Discuss this in the Unraid Community Forum
Registration required to post.
Unraid
Replace Registration Key
Copyright © 2005-2020 Lime Technology, Inc.
Unraid® is a registered trademark of Lime Technology, Inc.


text:
B&G Family
Our Growth
We are very proud of what we've built.
In 1982, Brenda and Gregory J. Hamer, Sr. created B&G Food Enterprises, LLC when they received a franchise from Taco Bell to operate a restaurant in Morgan City, Louisiana. Today, B&G operates over 150 restaurants in Louisiana, Mississippi, and Texas. B&G is the largest Taco Bell Franchisee in Louisiana and maintain our Corporate Headquarters in Morgan City, Louisiana.
In 1997, Brenda and Greg received their first KFC franchise. We acquired our first Long John Silver's in 2011. Today, B&G is a franchise of Taco Bell, KFC, and Long John Silver's.
Brenda and Greg began their partnership as juniors at Morgan City High School, and they were married in 1967, while Greg was still attending LSU.
Brenda is not only the Corporate Secretary of B&G, but she is also the center point of the family. Nothing delights her more than spending time with her three children and her nine grandchildren.
Greg Sr. is the CEO of B&G and prior to going full time into B&G was active in the oilfield service industry for over 20 years prior to going full time into B&G.
His public service included 12 years in elected office at both the city and parish (county) level; he also served on a public hospital board and the Louisiana Coastal Commission and the University of Louisiana Board of Trustees.
His civic activities have been numerous, and his restaurant activities include being Emeritus Director of the National Restaurant Association and a past Chairman of the National Restaurant Association Education Foundation and a current trustee. His long tenure with the Louisiana Restaurant Association has seen him honored as President in 2000, and as a member of the LRA Hall of Fame. He was also honored as Active Member of the Year and Chapter President of the Year. The St. Mary Chamber of Commerce recognized B&G as company of the year in 1998, and Greg was Citizen of the Year in 2017. The Community Foundation of Acadiana honored Greg as Philanthropist of the year in 2011.
He is a supporter of several organizations related to Louisiana State University and a rabid Tiger fan. He is a current member of the Dean's Advisory Council of the Ourso College of Business at LSU and on the Board of the Tiger Athletic Foundation (TAF). He is also on the Board of the Community Foundation of Acadiana.
B&G has grown to provide a livelihood for well over 3000 employees, without whom our success could not have been achieved. Our biggest blessing has been that the success of B&G has enabled all of our children and their spouses to be active in the business which has kept our family close.
B&G is a family business, and we strive every day to treat all of our employees as we would want to be treated.
Meet Mr. Greg Hamer
2017 NRAEF Board Chairman
Operating over 150 restaurants throughout Louisiana, Texas and Mississippi.
HomeAboutB&G FamilyOur GrowthLocationsOur Pride
Copyright © 2022 Bgfood.com
closearrow-circle-o-downfacebook-squarebarsyoutube-playinstagram


Parafin Ltd. ("We") are committed to protecting and respecting your privacy.
For the purpose of the Data Protection Act 1998 (the Act), the data controller is Parafin Ltd. of 26 Chelsham Road, London SW4 6NP (registration number 08536891).
— Information that you provide by filling in forms on our site www.parafin.co.uk (our site). This includes information provided at the time of registering to use our site, subscribing to our service or requesting further services. We may also ask you for information when you report a problem with our site.
— If you contact us, we may keep a record of that correspondence.
— We may also ask you to complete surveys that we use for research purposes, although you do not have to respond to them.
— Details of your visits to our site including, but not limited to, traffic data, location data, weblogs, operating system, browser usage and other communication data, whether this is required for our own billing purposes or otherwise and the resources that you access.
— To estimate our audience size and usage pattern.
— To store information about your preferences, and so allow us to customise our site according to your individual interests.
— To speed up your searches.
— To recognise you when you return to our site.
— To ensure that content from our site is presented in the most effective manner for you and for your computer.
— To provide you with information, products or services that you request from us or which we feel may interest you, where you have consented to be contacted for such purposes.
— To carry out our obligations arising from any contracts entered into between you and us.
— To allow you to participate in interactive features of our service, when you choose to do so.
— To notify you about changes to our service.
— If we are under a duty to disclose or share your personal data in order to comply with any legal obligation, our customers, or others. This includes exchanging information with other companies and organisations for the purposes of fraud protection and credit risk reduction.
You have the right to ask us not to process your personal data for marketing purposes. We will usually inform you (before collecting your data) if we intend to use your data for such purposes or if we intend to disclose your information to any third party for such purposes. You can exercise your right to prevent such processing by checking certain boxes on the forms we use to collect your data. You can also exercise the right at any time by contacting us at info@parafin.co.uk.


text:
Tag Archive: U.S. Unemployment Rate=
FX Daily, October 4: The US Jobs Data to Close a Sobering Week
by Marc Chandler
Overview: The recovery of US shares yesterday signaled today's fragile stability. Gains in Japan, Australia, and Taiwan blunted the losses elsewhere in the region, including a 1% slide in Hong Kong. The MSCI Asia Pacific Index fell for the third week. China's markets have been closed since Monday and will re-open Monday and may play some catch-up.
FX Daily, August 2: End of Tariff Truce Trumps Jobs
Overview: The market was finding its sea legs after being hit with wave and counter-wave following the FOMC decision, and more importantly, Powell's attempt to give insight into the Fed's thinking. Trump's tweet than signaled an end to the tariff truce with a 10% levy on the $300 bln of imports from China that have not been subject to action previously.
FX Daily, July 05: Dollar is Bid Ahead of Jobs Report
Overview: The dovish response to news that Lagarde was nominated to replace Draghi was extended by the dismal German factory order report that has pushed the euro to new two-week lows and kept bond yields near record lows. The focus ahead of the weekend is squarely on the US employment data, where a second consecutive poor report will fan expectations for a large Fed cut to initiate an easing cycle.
FX Daily, May 03: Ahead of US Jobs Report, the Greenback Remains Firm
Overview: The US April jobs data stand before the weekend, and the greenback is holding on to most of yesterday's gains as participants wait for the report. Equities in the Asia Pacific region were mixed without leadership from China and Japan, where the markets remain closed for the extended holiday. On the week, Australia's ASX was the worst performing.
FX Daily, August 03: Greenback Remains Firm Ahead of Jobs, JGBs Stabilize, Italian Debt Moves into Spotlight
The US dollar is trading at the upper end of its recent ranges against the euro and sterling. The euro finished below $1.16 yesterday for the first time since the end of June and has not been able to resurface that level so far today.
Look Past Disappointing Jobs Data, Luke
The US jobs report was broadly disappointing. However, the Federal Reserve will look through it and investors should too. A June hike is still by far the most likely scenario. The US created 164k net new jobs in April, and when coupled with the 32k upward revision in March, it was near expectations.
US Jobs Data Optics Disappoint, but Signal Unchanged
The US jobs growth slowed in March more than expected, but the details of the report suggest investors and policymakers will look through it. The poor weather seemed to have played a role. Construction jobs fell (15k) for the first time since last July, and the hours worked by production employees and non-supervisory worker slipped.
FX Daily, March 09: Today is about Jobs, but Not Really
The US Administration has softened its initial hardline position of no exemptions for the new steel and aluminum tariffs. There is little doubt that the actions will be challenged at the World Trade Organization and the idea that national security includes the protection of jobs for trade purposes will be tested. At the same time, US President Trump has agreed to meet North Korea's Kim Jong Un.
FX Daily, February 02: A Note Ahead of US Jobs Report
The US dollar is sporting a firmer profile against all the major currencies after weakening yesterday. Frequently, it seems the Australian dollar leads the other currencies, and we note that it is making a new low for the week today. Briefly, in Europe, it slipped below its 20-day moving (~$0.7985) average for the first time since December 13.
FX Daily, January 05: Dollar Given Reprieve Ahead of Employment Report
As the US dollar finished last year, so too did it begin the New Year, and after extending its losses, the bears have paused. Technical factors had been stretched, but it appears to have been old-fashioned macroeconomic considerations to have helped the dollar to move off the mat. Quickly summarized, these considerations are a larger than expected Australian trade deficit, slippage in Japan's service sector PMI, a larger than expected drop in the...
Headline US Jobs Disappoint, but Earnings as Expected
The headline US non-farm payrolls disappointed, rising by 148k instead of the consensus of 180k-200k. However, the other details were largely as expected and are unlikely to change views about the trajectory of Fed policy or the general direction of markets. It is a very much steady as she goes story.
What Central Banks Have Done Is What They're Actually Good At
by Jeffrey P. Snider
As a natural progression from the analysis of one historical bond "bubble" to the latest, it's statements like the one below that ironically help it continue. One primary manifestation of low Treasury rates is the deepening mistrust constantly fomented in markets by the media equivalent of the boy who cries recovery.
Aligning Politics To economics
There is no argument that the New Deal of the 1930's completely changed the political situation in America, including the fundamental relationship of the government to its people. The way it came about was entirely familiar, a sense from among a large (enough) portion of the general population that the paradigm of the time no longer worked. It was only for whichever political party that spoke honestly to that predicament to obtain long-term...
Four Point One
The payroll report for October 2017 was still affected by the summer storms in Texas and Florida. That was expected. The Establishment Survey estimates for August and September were revised higher, the latter from a -33k to +18k. Most economists were expecting a huge gain in October to snapback from that hurricane number, but the latest headline was just +261k.
FX Daily, November 03: Dollar Firms Ahead of What is Expected to Be Strong US Jobs Data
The US dollar is firm but is not going anywhere quickly. The lack of fresh interest rate support and uncertainty over the US tax proposals, which the Brady, the Chair of the House Ways and Means Committee hopes to have a revised version out after the weekend so the committee work can begin on Monday.
US Storm-Skewed Report Means Nothing about Anything
US interest rates and the dollar rose in response to the data. It was firm before the report. The US Dollar Index is up for a fourth consecutive week. It is the longest streak since Q1. US 10-year yields are near 2.40%, an area that has blocked stronger gains for nearly six months.
FX Daily, October 6: Look Through the US Jobs Report
Traders are putting the final touches on another strong weekly performance for the US dollar. Strong economic data, including the PMIs, auto sales, and factory orders have surprised to the market. The ADP report warns that the storms that flattered some high frequency data will likely skew today's employment report (both headline and details) to the downside. Of course, investors will quickly look for the number of people who could get to work due...
FX Daily, September 01: Manufacturing PMIs, US Jobs, and Implications of Harvey
As the markets head into the weekend, global equities are firmer, benchmark 10-year yields are mostly lower, and the dollar is consolidating after North American pared the greenback's gains yesterday. Manufacturing PMIs from China, EMU, and the UK have been reported, while in the US, the August jobs data stand in the way of the long holiday weekend for Americans.
Great Graphic: Unemployment by Education Level
The US reports the monthly jobs data tomorrow. The unemployment rate stood at 4.4% in June, after finishing last year at 4.7%. At the end of 2015 was 5.0%. Some economists expect the unemployment rate to have slipped to 4.3% in July. Recall that this measure (U-3) of unemployment counts those who do not have a job but are looking for one.
Constructive US Jobs, but Where Do the Euro Bulls make a Stand?
The US created 209k jobs in July and jobs growth in June was revised higher (+9k) to 231k. The underemployment rate was unchanged at 8.6%. The unemployment rate ticked down to 4.3%, matching the cyclical low set in May. This is all the more impressive because the participation rate also ticked up (62.9% from 62.8%).
Page 1123Next »


text:
Harry J. Zeldenrust (1934) is een Nederlands theoloog, predikant en vredesactivist.

Levensloop
Zeldenrust studeerde theologie aan de Vrije Universiteit Amsterdam en aan de Katholieke Universiteit Nijmegen. Hij was enige jaren gereformeerd predikant te Schiermonnikoog, later in Venlo en Uden. Van 1974 tot 1999 was hij als studiesecretaris verbonden aan de pacifistische kerkelijke vredesbeweging Kerk en Vrede, waar hij in de voetsporen trad van Krijn Strijd en Hannes de Graaf. Zeldenrust vertolkte een genuanceerd pacifistisch standpunt, waarbij hij zich veelal liet inspireren door de Duitse theoloog Dietrich Bonhoeffer. Hij was medewerker aan en redacteur van diverse publicaties van de Theologische Werkgroep van Kerk en Vrede, nam deel aan diverse gremia van het IKV en was vele jaren een vaste medewerker aan de jaarlijkse Liturgiekrant voor de Vredesweek, een uitgave van Pax Christi Nederland.

Publicaties
Ervaring en Keuze. Een theologische verkenning op het spoor van Dietrich Bonhoeffer (1976)
Geweldloze weerbaarheid. Een nieuw model in de strijd tegen onrecht (1979)
Een revolutie zonder geweld. André Trocmé en zijn kijk op de geweldloze revolutie (1981)
De weerbaarheid van een kind. Verhalen over geestelijke weerbaarheid (1988)
Voettocht naar vrede. Van rechtvaardige oorlog naar een vrede door gerechtigheid (1994)

Literatuur
P. van Dijk, H. Noordegraaf en G. Witte-Rang, Gidsen en Getuigen op de Pelgrimage naar Vrede, een bundel opstellen ter gelegenheid van het afscheid van Harry Zeldenrust als studiesecretaris van Kerk en Vrede (1999)

Nederlands pacifist
Nederlands vredesactivist
Predikant van de Gereformeerde Kerken in Nederland

redpajama_set_name: RedPajamaWikipedia


Founded in 1977, Pafilia Property Developers is the largest privately owned residential developer in Cyprus, dedicated to delivering the best in concept, design, quality and service. The company offers a diverse and innovative portfolio of property types and styles, each an exemplar of superior living, all built to the highest standards. Owner-managed by Mr. Elias Eliades and family, the Pafilia brand is synonymous with quality and has gained a reputation for excellence. Pafilia's dynamic team, family values and customer centric approach are key attributes to the company's success. Integrity, commitment to excellence, market knowledge and wealth of experience has earned Pafilia its place at the forefront of the property industry and gained it over 10,000 satisfied homeowners. Our commitment to provide absolute customer satisfaction is constant. At every stage of the process, from designing and planning to the final finishing touches, we consider the emotional and practical priorities of the residents.


text:
The Journal of Immunology
ImmunoHorizons
AAI.ORG
filter your search All ContentThe Journal of Immunology
Next in The JI
Translating Immunology
Pillars of Immunology
COVID-19, SARS, and MERS Articles
Monkeypox and Other Poxvirus Articles
Annual Meeting Abstracts
About the JI
The JI Editorial Board
Reagents and Abs
Conditioned medium analysis
Measurement of cytokines
Real-time RT-PCR
Luciferase assay
Small interfering RNA (siRNA)
Measurement of intracellular pH
Purification of tumor-infiltrating immune cells
Tumor cell-conditioned medium enhances TLR ligand-induced IL-23p19 expression in monocytes/macrophages
The conditioned medium enhances IL-23p19 promoter activity
Characterization of the small molecule in the conditioned medium responsible for the increase in TLR-stimulated IL-23p19 promoter activity
Lactic acid in the conditioned medium enhances the IL-23p19 gene expression induced by TLR ligands
Pathway involved in the enhancing activity of lactic acid
Lactic acid enhances secretion of proinflammatory cytokines by human monocytes/macrophages
Lactic acid enhances the IL-23/IL-17 pathway
Lactic acid enhances the expression of IL-23p19 mRNA in tumor-infiltrating immune cells
Tumor-secreted lactic acid enhances TLR ligand-dependent IL-23 expression in monocytes/macrophages
Predicted lactic acid signaling pathway
Lactic acid is not only a terminal metabolite of glycolysis but also a proinflammatory mediator
Research Article| June 01 2008
Tumor-Secreted Lactic Acid Promotes IL-23/IL-17 Proinflammatory Pathway1
Hiroaki Shime;
Hiroaki Shime
*Department of Molecular Genetics and
Masahiko Yabu;
Masahiko Yabu
Takashi Akazawa;
Takashi Akazawa
Ken Kodama;
Ken Kodama
†Department of Surgery, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka, Japan; and
Misako Matsumoto;
Misako Matsumoto
‡Department of Microbiology and Immunology, Hokkaido University Graduate School of Medicine, Sapporo, Japan
Tsukasa Seya;
Tsukasa Seya
Norimitsu Inoue
Address correspondence and reprint requests to Dr. Norimitsu Inoue, Department of Molecular Genetics, Osaka Medical Center for Cancer and Cardiovascular Diseases, 1-3-3 Nakamichi, Higashinari-ku, Osaka 537-8511, Japan. E-mail address: inoue-no@mc.pref.osaka.jp
Received: May 23 2007
Accepted: March 23 2008
Published: June 01 2008
Copyright © 2008 by The American Association of Immunologists
J Immunol (2008) 180 (11): 7175–7183.
https://doi.org/10.4049/jimmunol.180.11.7175
Hiroaki Shime, Masahiko Yabu, Takashi Akazawa, Ken Kodama, Misako Matsumoto, Tsukasa Seya, Norimitsu Inoue; Tumor-Secreted Lactic Acid Promotes IL-23/IL-17 Proinflammatory Pathway1. J Immunol 1 June 2008; 180 (11): 7175–7183. https://doi.org/10.4049/jimmunol.180.11.7175
IL-23 is a proinflammatory cytokine consisting of a p19 subunit and a p40 subunit that is shared with IL-12. IL-23 is overexpressed in and around tumor tissues, where it induces local inflammation and promotes tumor development. Many tumor cells produce large amounts of lactic acid by altering their glucose metabolism. In this study, we show that lactic acid secreted by tumor cells enhances the transcription of IL-23p19 and IL-23 production in monocytes/macrophages and in tumor-infiltrating immune cells that are stimulated with TLR2 and 4 ligands. DNA elements responsible for this enhancing activity of lactic acid were detected in a 2.7-kb 5′-flanking region of the human IL-23p19 gene. The effect of lactic acid was strictly regulated by extracellular pH. Furthermore, by inducing IL-23 overproduction, lactic acid facilitated the Ag-dependent secretion of proinflammatory cytokine IL-17 but not IFN-γ by TLR ligand-stimulated mouse splenocytes. Interestingly, this effect was observed even in the absence of TLR ligand stimulation. These results suggest that rather than just being a terminal metabolite, lactic acid is a proinflammatory mediator that is secreted by tumor cells to activate the IL-23/IL-17 proinflammatory pathway but not the Th1 pathway. Targeting the lactic acid-induced proinflammatory response may be a useful approach for treating cancer.
Immune cells often infiltrate in and around many kinds of tumors. Initially, the immune system protects the host from cancer development and, indeed, the infiltration of NK cells in cancers is associated with a favorable prognosis (1). However, the infiltration of innate-immune cells such as macrophages correlates with a poor prognosis, which suggests that these cells may be directly involved in tumor development and metastasis by inducing angiogenesis and tissue remodeling. Moreover, many cancers often arise at sites of chronic inflammation caused by infections with microbes like Helicobacter pylori and hepatitis viruses. Noninfectious chronic inflammation such as that caused by asbestos is also associated with tumor development (1, 2, 3).
Infectious inflammation is associated with the secretion of several cytokines by innate immune cells in response to pathogen-associated molecular pattern stimuli (4). One of these cytokines is IL-23. IL-23 is a member of the proinflammatory heterodimeric cytokine family and consists of a p19 subunit and a p40 subunit that is shared with IL-12 (5, 6). Whereas IL-12 mainly induces the development of IFN-γ-producing Th1 cells, IL-23 is involved in maintaining the Th17 cells that are generated in response to IL-6 and TGF-β (7, 8, 9, 10) and activates memory T cells (CD44high and CD62Llow) (11). IL-23 also induces the production of the proinflammatory cytokines IL-17 and IL-22 (11, 12). IL-23 is mainly produced by APCs, such as monocytes/macrophages and dendritic cells (DCs)3 in response to stimulation with TLR2 and 4 ligands, such as peptidoglycan (PGN), LPS, and bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) (13, 14, 15).
Recently, the Oft group (16) showed that IL-23 but not IL-12 is overexpressed by macrophages and DCs in human and mouse tumor tissues. They also showed that IL-23 is an important molecule that leads to the up-regulation of IL-17 and the matrix metalloprotease 9, to an increase in angiogenesis, and to a reduction in CD8+ T cell infiltration in the tumor microenvironment (16). Significantly, IL-23p19-deficient mice but not IL-12p35-deficient mice developed chemically induced tumors less frequently than wild-type mice, and tumors transplanted into IL-23 receptor-deficient mice showed reduced growth (16). Furthermore, it was shown that Th17 cells are gradually increased in the tumor microenvironment during tumor development (17) and that IL-17 up-regulates the production of a variety of proinflammatory cytokines (18) and proangiogenic factors (19) to promote tumor development (20). Therefore, the activation of the IL-23/IL-17 pathway promotes the incidence and growth of tumors by inducing local inflammatory responses. However, it remains unclear what induces inflammation and IL-23 overproduction in the tumor microenvironment. Notably, peripheral blood cells from patients with lung cancer that had been cultured with TLR stimuli overproduced IL-12/23p40; this response was eliminated in most patients after tumor resection (21). On the basis of these studies, we speculated that the tumor itself produces a factor(s) that promotes IL-23 overproduction. To test our hypothesis, we searched for tumor-secreted factors that might modulate the production of IL-23 by monocytes/macrophages stimulated with TLR ligands. In this study, we show that lactic acid secreted from tumor cells up-regulates TLR signal-dependent transcription of the IL-23p19 subunit in human and mouse monocytes/macrophages to enhance IL-23 secretion. Therefore, we predict that the lactic acid that is secreted by many tumor cells is a proinflammatory mediator that promotes tumor development.
The CADO-LC10 cell line, which was established from a human lung adenocarcinoma (22), was cultured in high glucose DMEM (4.5 mg/ml glucose; Sigma-Aldrich). Human PBMC, human monocytes, mouse splenocytes, the mouse macrophage-like cell line J774.1 (RIKEN cell bank), and the mouse melanoma cell line B16 were cultured in RPMI 1640 (Sigma-Aldrich). All media were supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured at 37°C under a 5% CO2 atmosphere.
We purchased the TLR ligands: PGN of Staphylococcus aureus and LPS from Sigma-Aldrich and a synthetic tripalmitoylated lipopeptide, Pam3CSK4, from InvivoGen. BCG-CWS was prepared as described previously (23, 24). l-Lactic acid was purchased from Sigma-Aldrich and Wako Pure Chemical and sodium lactate was purchased from WAKO. We used anti-mouse IL-23p19 Ab (G23-8; eBioscience) to neutralize IL-23 activity and rat IgG1 Ab (eBRG1; eBioscience) as an isotype control; both Abs were used at a concentration of 10 μg/ml. We purchased recombinant GM-CSF from PeproTech and anti-GM-CSF receptor α-chain Ab (S-20) from Santa Cruz Biotechnology.
Conditioned medium was prepared from CADO-LC10 cells that had been cultured for 3 days. The medium was passed through a 0.22-μm pore size filter (Millipore) and stored at −80°C. For some experiments, the conditioned medium was subjected to molecular size fractionation by using Microcon YM-10 centrifugal filter devices, which separates molecules at the nominal 10-kDa molecular mass cutoff (Millipore). The flow-through fraction (<10 kDa) was supplemented with 10% FCS and the retentate (>10 kDa) was diluted with serum-free culture medium to obtain the original volume. In other experiments, the conditioned medium was treated with 50 μg/ml proteinase K at 37°C for 1 h. To remove proteinase K, the medium was passed through a Microcon YM-10 and the flow-through fraction was used for further experimentation. The control media were subjected to the same treatments as the conditioned media.
To examine how glucose concentrations in the culture medium of CADO-LC10 affects the subsequent enhancing activity of the conditioned medium, we cultured confluent CADO-LC10 cells for 3 days in fresh glucose-free DMEM (Invitrogen) supplemented with 1 or 4.5 mg/ml glucose and 10% FCS. In separate experiments, we inhibited lactic acid production by culturing the CADO-LC10 cells in the presence or absence of 20 mM oxamic acid (Sigma-Aldrich) for 2 days. The pH of the conditioned media and the lactic acid-containing media neutralized with NaOH was measured with a pH meter (Beckman Coulter) at 37°C under a 5% CO2 atmosphere. l-Lactic acid concentrations in the conditioned media were measured by using a Determiner LA Kit (Kyowamedics). In the enhancing activity analysis, the conditioned media of CADO-LC10 cells described above were added to cells with an equal volume of cell culture media (i.e., 50% of the medium consisted of conditioned medium).
Human PBMC were isolated from healthy volunteers by using Ficoll-Paque Plus (GE Healthcare Bio-Sciences). Human monocytes were purified from the PBMC by using the MACS system (Miltenyi Biotec) and monocyte isolation kit II (Miltenyi Biotec). To measure human IL-23 production, 1.5 × 105 monocytes were cultured in 96-well tissue culture plates in the presence or absence of lactic acid for 24 h and then treated with 10 μg/ml PGN for 24 h. The IL-23, IL-12/23p40, and IL-6 levels in the culture supernatants were measured by using human IL-23 (Bender MedSystems), human IL-12p40, and human IL-6 (BioSource International) ELISA kits, respectively. To measure mouse IL-23 production, 1.0 × 105 J744.1 cells were stimulated with lactic acid and 10 μg/ml PGN. The measurements of mouse IL-23 production were performed by using a mouse IL-23 ELISA kit (eBioscience).
Mouse splenocytes isolated from an OVA-specific, MHC class II-restricted αβ TCR-transgenic mouse, OT-II (25), were cultured at 5 × 105 cells/well in 96-well tissue culture plates with 0.2 μg/ml OVA323–339 peptides (Biosynth International) in the presence or absence of TLR ligands and lactic acid. After 4 days of incubation, the cytokines in the culture supernatants were measured by using IL-17A (R&D Systems) and IFN-γ (BioSource International) ELISA kits. These experiments using animals were conducted according to our institutional guidelines.
Total RNA was isolated from cells by using the SV96 Total RNA Isolation System (Promega) according to the manufacturer's instructions, after which it was treated with RNase-free DNase I. cDNA was synthesized at 42°C for 50 min by using oligo(dT)12–18 primers and SuperScript III reverse transcriptase in the presence of RNase inhibitor (Invitrogen). Diluted cDNA samples were mixed with a pair of primers derived from human IL-23p19 or β-actin cDNA sequences and PCR was performed by using SYBR Green PCR master mix (Applied Biosystems) and an Applied Biosystems 7500HT sequence detection system. The following PCR primers were designed: for human IL-23p19, forward primer, 5′-AGTGTGGAGATGGCTGTGACC-3′ and reverse primer, 5′-GCTGGGACTGAGGCTTGGAATCTG-3′; for human IL-12/23p40, forward primer, 5′-ATGCCGTTCACAAGCTCAAGTATG-3′ and reverse primer, 5′-GAACGCAGAATGTCAGGGAGAAGT-3′; and for human β-actin, forward primer, 5′-TCACCCACACTGTGCCCATCTACGA-3′ and reverse primer, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′. Copy numbers were calculated from the amount of cDNA cloned into the pGEM-T easy vector (Promega) and normalized to β-actin. PCR for mouse IL-23p19 and β-actin was performed by using the TaqMan PCR Core reagent kit, TaqMan probes, and primer sets of the TaqMan Gene Expression assay system (for IL-23p19, Mm00518984_m1 and for β-actin, Mm00607939_s1; Applied Biosystems). The relative expression of IL-23p19 was normalized to that of β-actin and calculated by using the ΔΔCt method (16, 26).
A luciferase reporter plasmid for monitoring IL-23p19 transcription was constructed as follows. A 6.7-kb fragment of the human IL-23p19 gene from the SacI site (−6654 bp) to the ATG initiation site that contained the 5′-flanking region and 5′-untranslated region of the gene was amplified by PCR using the RP11-348M3 clone (Research Genetics) as the template. The fragment was then subcloned between the SacI and NcoI sites of the pGL3 promoter vector (Promega) to generate the p19–5′ luc vector. We also constructed a reporter gene plasmid containing the 2.7-kb 5′-flanking region of the human IL-23p19 gene as follows. The p19–5′ luc plasmid was digested with SacI and XmaI, treated with T4 DNA polymerase (Takara Bio), and self-ligated. We then inserted the XhoI and BamH I fragments of the PGK promoter-driven neomycin resistance gene from pGKEM7-neoW into the SalI and BamH I sites of the plasmid to generate the p19–5′ 2.7k luc neo vector. The luciferase reporter plasmid for monitoring NF-κB activity was constructed as follows. The following synthetic oligonucleotides were annealed: 5′-TCGAGAAATGGGGACTTTCCGCTGGGGACTTTCCGCAAACCGC-3′ and 5′-GGTTTGCGGAAAGTCCCCAGCGGAAAGTCCCCATTTC-3′ (the underlined sequences indicate canonical NF-κB binding sites). The annealed oligonucleotides were then subcloned together with a SacII/NcoI fragment of the minimal promoter sequence of the pNF-κB-Luc vector (Stratagene) between XhoI and NcoI sites of the pGL3 promoter vector containing the PGK promoter-driven neomycin resistance gene to generate the pGL3–2κB luc neo vector. J774.1 cells were then transfected with the p19-5′ luc vector and pGKEM7-neoW, the p19-5′ 2.7k luc neo vector, or pGL3–2κB luc neo vector by using FuGENE 6 (Roche) according to the manufacturer's instructions. The transfected cells were selected with 200 μg/ml G418. The cells were seeded in 96-well tissue culture plates at 1 × 105 cells/well and incubated with stimulants for 24 h as described above. After incubation, the cells were lysed with GloLysis buffer (Promega) and the luciferase activity was measured by using the Bright-Glo luciferase assay system (Promega) and a Mithras LB940 multimode reader (Berthold Technologies).
We purchased SMARTpool siRNA reagents for the human LDHA gene from Dharmacon and the Allstars negative control siRNA rhodamine from Qiagen. CADO-LC10 cells were transfected with siRNA (73 nM) by using XtremeGENE siRNA Transfection Reagent (Roche) according to the manufacturer's instructions. This transfection procedure was repeated on the second day to increase the RNA interference efficiency. On the fourth day, the cells were collected and plated at 1.4 × 106 cells/well in 12-well plates. After incubation for 24 h, we collected the conditioned medium and incubated the cells further for 24 h. The collected media were combined and used for further analysis. The expression of LDHA in siRNA-transfected CADO-LC10 cells was evaluated by performing semiquantitative RT-PCR and Western blotting. The following PCR primers were used: for human LDHA, forward primer, 5′-GCACGTCAGCAAGAGGGAGAAAG-3′ and reverse primer, 5′-AGGTAACGGAATCGGGCTGAATC-3′ and for human β-actin, forward primer, 5′-GCGGGAAATCGTGCGTGACATT-3′ and reverse primer, 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′. Anti-LDHA (N-14; Santa Cruz Biotechnology) and anti-β-tubulin (TUB 2.1; Sigma-Aldrich) Abs were used for Western blotting. The expression levels of proteins were calculated by using Image J software (http://rsb.info.nih.gov/ij/) and were normalized to those of β-tubulin expression.
Intracellular pH was measured by using BCECF/AM (Invitrogen) (27). Fluorescence at 535 nm with excitation at 485 and 388 nm was measured for 0.1 s every 30 s after adding lactic acid or hydrochloric acid (WAKO) by using an ARVO MX 1420 multilabel counter (PerkinElmer).
Tumor-infiltrating immune cells were purified from tumors formed by B16 melanoma cells in C57BL/6 mice by using CD45 MACS MicroBeads (Miltenyi Biotec) as described previously (28). The purified cells were stained with FITC-conjugated anti-CD45.2 (104; eBioscience), FITC-conjugated anti-CD11b (M1/70; eBioscience), and PE-conjugated anti-CD11c (HL3; BD Biosciences) and examined by using the FACSCalibur system (BD Biosciences).
In measurement of cytokines, real-time PCR, and luciferase assay, data represent mean values ± SD of triplicate stimulations. Differences between groups were analyzed for statistical significance by the Student t test. Representative data from at least two independent experiments are shown in the figures.
To determine whether tumor-secreted factors might modulate the TLR ligand-stimulated production of IL-23 by monocytes/macrophages, we first generated medium conditioned by the lung adenocarcinoma cell line CADO-LC10. Monocytes isolated from normal human PBMC were then stimulated with PGN in the presence (LC10) or absence (medium) of the conditioned medium. PGN induced IL-23p19 transcription in human monocytes/macrophages and the transcription was significantly increased by the presence of the conditioned medium (Fig. 1,A, left). The conditioned medium alone did not induce IL-23p19 transcription in unstimulated monocytes/macrophages (Fig. 1A, left). Similar results were obtained for PBMC stimulated with BCG-CWS (Fig. 1 A, middle).
Tumor cell-conditioned medium enhances IL-23 expression in TLR ligand-stimulated monocytes/macrophages. A, Conditioned medium of the lung adenocarcinoma cell line CADO-LC10 enhances the TLR ligand-induced IL-23p19 mRNA expression in human monocytes/macrophages (left). Human monocytes were stimulated with 10 μg/ml PGN (▪) or PBS (□) in the presence (LC10) or absence (Medium) of 50% of the conditioned medium for 4 h. The conditioned medium was also fractionated according to molecular size, with the cutoff being at the nominal 10-kDa molecular mass (left). Human PBMC were stimulated with 5 μg/ml BCG-CWS in the presence or absence of the conditioned medium (middle and right). The relative expression of IL-23p19 (left and middle) and IL-12/23p40 (right) mRNA was measured by real-time RT-PCR and normalized to β-actin expression. N.D., Not detected. B, J774.1 cells were stimulated with 10 μg/ml PGN for 24 h in the presence or absence of the conditioned medium and the IL-23 that was secreted was measured by ELISA. C, Schematic representation of the human IL23A gene locus and two luciferase reporter genes carrying the 5′-flanking regions of the IL-23p19 gene. The position from the translation start site (+1) is indicated in each construct. Shown are the untranslated regions (□), the coding region of IL-23p19 and the USP52 exon (▪), the luciferase gene (▦), the SV40 late poly(A) signal (○), and the TATA box sequence (arrow). The arrowheads indicate putative NF-κB binding sites (–2311 to −2302, −1261 to −1252, −1049 to −1040, and −256 to −245) in the 2.7-kb 5′-flanking region. D, J774-p19-5′ luc cells were stimulated with 10 μg/ml PGN in the presence of the whole conditioned medium or the size-fractionated conditioned medium (left) or conditioned medium that had been treated with 50 μg/ml proteinase K (Pro K) for 1 h (right). Luciferase activity was then measured. E, Conditioned media were prepared from CADO-LC10 cells cultured in medium containing 1 or 4.5 mg/ml glucose (Glc). The control media (Medium) were subjected to the same treatments as the conditioned media (LC10). These media were added at 50% to determine the enhancing activity. The data represent mean values ± SD (n = 3). **, p < 0.01.
We then size-fractionated the conditioned medium and the control medium into two fractions bearing the >10-kDa or <10-kDa molecules and performed the same experiment described above. The PGN-stimulated expression of IL-23p19 in the monocytes/macrophages was more strongly enhanced by the lower molecular mass fraction (5.9-fold) than by the higher molecular mass fraction (1.7-fold) (Fig. 1 A, left). Thus, it appears that the tumor cells secrete a small molecule that augments TLR ligand-induced IL-23p19 expression in monocytes/macrophages.
We also examined the effect of the conditioned medium on the transcriptional expression of IL-12/23p40 in human PBMC. Although the unfractionated medium and the higher molecular mass fraction clearly enhanced the transcription of IL-12/23p40, the lower molecular mass fraction did not (Fig. 1 A, right, and data not shown). The enhancement of IL-12/23p40 expression was significantly inhibited by the anti-GM-CSF receptor α-chain Ab, suggesting that GM-CSF in the conditioned medium mainly enhanced IL-12/23p40 expression (data not shown).
The conditioned medium also enhanced the PGN-induced secretion of IL-23 by the mouse macrophage-like cell line J774.1 (Fig. 1 B).
To examine the effect of the conditioned medium on IL-23p19 gene promoter activity, we performed a luciferase reporter assay using p19-5′ luc, which is a luciferase reporter plasmid containing the 6.7-kb 5′-flanking DNA region of the human IL-23p19 gene (Fig. 1,C). We first established several stable J774.1 cell lines that contained the reporter plasmid p19-5′ luc (J774-p19–5′ luc cells). When these cell lines were stimulated with PGN, IL-23p19 promoter activity was increased (Fig. 1,D, left). A further increase was observed when the cells were treated with PGN in the presence of the conditioned medium (Fig. 1,D, left). Here again, the lower molecular mass fraction of the conditioned medium was proficient in stimulating IL-23p19 promoter activity (Fig. 1,D, left). We also generated additional luciferase reporter cells (J774-p19-5′ 2.7k luc) from p19-5′ 2.7k luc that contained only the 2.7-kb 5′-flanking region of the human IL-23p19 gene (Fig. 1 C). The reporter activity of this plasmid in J774.1 cells was increased by TLR stimuli (BCG-CWS and PGN) and this effect was further increased by the conditioned medium (data not shown).
Thus, the conditioned medium augments the stimulatory effect of TLR ligands on IL-23p19 promoter activity.
To identify the small molecule in the conditioned medium, we subjected the conditioned medium to further molecular size fractionation that separated the molecules at the nominal molecular mass of 500 Da. The <500-Da fraction, but not the >500-Da fraction, increased the IL-23p19 promoter activity in TLR ligand-stimulated J774-p19-5′ luc (data not shown). The enhancing activity of the entire conditioned medium was not diminished by treatment with proteinase K (Fig. 1 D, right) or heat treatment at 90°C for 10 min (data not shown). Thus, it appeared that the tumor-secreted small molecule we were interested in would not be a protein or peptide.
Interestingly, we found that the enhancing activity of the conditioned medium varied depending on whether the medium was obtained from tumor cell cultures in DMEM (2.4 ± 0.21), RPMI 1640 (1.6 ± 0.06), or MEM (1.0 ± 0.07). DMEM was better than RPMI 1640 and MEM had no enhancing effect at all. DMEM, RPMI 1640, and MEM contain different concentrations of glucose, namely, 4.5, 2, and 1 mg/ml, respectively. To investigate whether the glucose concentration does indeed affect the subsequent enhancing activity of the conditioned medium, we generated conditioned media from CADO-LC10 cells cultured for 3 days in DMEM supplemented with low (1 mg/ml) or high (4.5 mg/ml) concentrations of glucose. The conditioned medium prepared in high glucose DMEM enhanced the TLR ligand-stimulated IL-23p19 promoter activity in J774-p19-5′ luc cells, unlike the conditioned medium prepared in low glucose DMEM (Fig. 1,E). A high concentration of glucose alone had no effect (Fig. 1 E). Glucose was metabolized to pyruvic acid, which is catalyzed by lactate dehydrogenase (LDH) to generate lactic acid. Because tumor cells show up-regulated glycolysis, even under aerobic conditions, they generally secrete large amounts of lactic acid into the culture medium (29, 30, 31). Indeed, the conditioned media of CADO-LC10 cells cultured in 4.5 or 1 mg/ml glucose were found to contain 26.4 and 7.8 mM lactic acid, respectively (final concentrations of 13.2 and 3.9 mM in our assay). Therefore, we hypothesized that lactic acid may be the most likely candidate tumor cell-secreted factor that enhances TLR ligand-stimulated IL-23p19 transcription.
To examine whether lactic acid indeed enhances the IL-23p19 gene expression induced by TLR ligands, J774-p19-5′ luc cells were stimulated with PGN in the presence of lactic acid. The luciferase activity in J774-p19-5′ luc cells was increased by lactic acid in a dose-dependent manner (Fig. 2,A, left) and this enhancing effect was observed at all concentrations of PGN (Fig. 2,A, right). However, lactic acid alone had no detectable effect in this assay (Fig. 2,A, left, □). Lactic acid at 10 mM was as effective as the conditioned medium, which contained 27.3 ± 1.09 mM lactic acid (final concentration of 13.7 ± 0.55 mM in our assay; Fig. 2,A, left). The enhancing activity of lactic acid was also observed with cells stimulated with not only PGN but also other TLR ligands, namely, BCG-CWS, LPS, and Pam3CSK4 (Fig. 2 B).
Lactic acid enhances TLR ligand-induced human IL-23p19 promoter activity. A, J774-p19-5′ luc cells were stimulated with 10 μg/ml PGN in the presence of 0 (Medium), 5, 10, 15, or 20 mM lactic acid (left) or with 0, 2.5, 5, 10, or 30 μg/ml PGN in the presence of 15 mM lactic acid (right). B, J774-p19-5′ luc cells were stimulated with 10 μg/ml BCG-CWS (left), 100 ng/ml LPS (middle), or 100 ng/ml Pam3CSK4 (right) in the presence or absence of 15 mM lactic acid. C, J774-p19-5′ 2.7k luc cells were stimulated with 10 μg/ml PGN in the presence or absence of 15 mM lactic acid. D, J774-p19-5′ luc cells (left) and J774-2κB luc cells (right) were stimulated with 10 μg/ml PGN in the presence or absence of 15 mM lactic acid. After incubation for 4, 8, and 24 h, the cells were lysed and their luciferase activities were measured. The data represent mean values ± SD (n = 3).
We next examined the region in the IL-23p19 promoter that was responsive to lactic acid by using J774-p19-5′ 2.7k luc cells. DNA elements responsible for this enhancing activity of lactic acid like the conditioned medium were detected in a 2.7-kb 5′-flanking region of the human IL-23p19 gene (Fig. 2,C). Searching of a TRANSFAC database (32) with the TFSEARCH program version 1.3 (http://www.rwcp.or.jp/papia/) revealed four predicted NF-κB binding sites in a 2.7-kb 5′-flanking region (Fig. 1,B, arrowheads). However, when we constructed J774.1 cells transfected with a luciferase reporter construct carrying canonical NF-κB binding sites and tested their responses to TLR ligands in the presence or absence of lactic acid, we did not observe any enhancing activity (Fig. 2 D).
To test whether the lactic acid secreted from the tumor cells is indeed responsible for augmenting the TLR ligand-induced IL-23p19 promoter activity, we inhibited the production of lactic acid from CADO-LC10 cells with LDHA-specific siRNA. The expression of LDHA mRNA (Fig. 3,A, left) and protein (Fig. 3,A, right) in LDHA siRNA-transfected cells was reduced to <10 and 50% of that of control siRNA-transfected cells, respectively. We examined enhancing activity using these conditioned media (Fig. 3,B). Alternatively, we inhibited the LDH activity by adding oxamic acid, an inhibitor of LDH (Fig. 3,C). Both treatments significantly reduced the concentration of lactic acid in the conditioned medium and this was matched with a decreased ability of the conditioned medium to enhance TLR ligand-stimulated IL-23p19 promoter activity (Fig. 3, B and C). Thus, the lactic acid secreted by the tumor cells was largely responsible for the enhancing effect of the conditioned medium.
Inhibiting the production of lactic acid blocks the enhancing activity of the conditioned medium. A, CADO-LC10 cells were transfected with siRNA for the human LDHA gene (LDHA) or negative control siRNA (Control). The inhibitory effect of LDHA siRNA was assessed by semiquantitative RT-PCR (left) and Western blotting (WB; right). cDNA templates prepared from siRNA-transfected CADO-LC10 cells were serially diluted (1, 0.1, 0.01, and 0.001) and amplified with LDHA and β-actin (ACTB) primers. Cell lysates of siRNA-transfected CADO-LC10 cells were immunoblotted with anti-LDHA or anti-β-tubulin Abs. The normalized protein expression levels of LDHA are shown below the picture. B, J774-p19-5′ luc cells were cultured with the conditioned media prepared from siRNA-transfected CADO-LC10 cells in the presence of 10 μg/ml PGN (left) or 10 μg/ml BCG-CWS (right) for 24 h. C, Alternatively, CADO-LC10 cells were incubated in low or high glucose DMEM with 20 mM oxamic acid (Oxa), an inhibitor of LDH. J774-p19-5′ luc cells were cultured with these conditioned media in the presence of 10 μg/ml PGN for 24 h. The data indicate the relative enhancing activity of CADO-LC10 conditioned medium over that of the average of control medium. The concentration of lactic acid (La) in each conditioned medium was measured in B and C. The data represent mean values ± SD (n = 3). **, p < 0.01.
Lactate anions are transported together with protons into cells by monocarboxylate transporters (MCTs) in a pH-dependent manner (33). Therefore, to examine whether the enhancing activity of lactic acid depends on the pH of the medium, we incubated J774-p19-5′ luc cells with 15 mM lactic acid in the presence of NaOH, which altered the pH of the medium (Fig. 4,A). The enhancing activity of lactic acid was decreased in a NaOH dose-dependent manner. Furthermore, sodium lactate did not show any enhancing activity (Fig. 4,B). The intracellular pH of the cells decreased rapidly upon incubation with lactic acid (Fig. 4,C, left), suggesting that protons were transported into the cells along with lactic acid. However, in contrast to lactic acid, hydrochloric acid, which also decreased the intracellular pH, had no enhancing effect (Fig. 4, C, right, and D). These results suggest that only the lactate anion in its transportable state, but not the neutralized lactate anion or proton, was responsible for the enhancing activity.
The enhancing activity of lactic acid is dependent on the pH of the medium. A, pH of the medium containing 15 mM lactic acid (pH 7.28) was adjusted to 7.36 and 7.45 with 2.5 and 5 mM NaOH, respectively. J774-p19-5′ luc cells were stimulated with 10 μg/ml PGN in the presence of these media. B, J774-p19-5′ luc cells were stimulated with 10 μg/ml BCG-CWS in the presence of 0, 5, 10, 15, or 20 mM lactic acid (La) or sodium lactate (NaLa). C, BCECF-loaded J774-p19-5′ luc cells were incubated with 0, 10, 15, or 20 mM lactic acid (left) or 0, 5, 10, or 15 mM hydrochloric acid (right). Intracellular pH (pHi) was detected by examining the fluorescent pH dye BCECF every 30 s as described in Materials and Methods. The ratio of fluorescence at 535 nm after excitation at 485 nm/388 nm was calculated. D, J774-p19-5′ luc cells were stimulated with 10 μg/ml PGN in the presence of various concentrations of lactic acid (left) or hydrochloric acid (right). After a 24-h incubation, the cells were lysed and the luciferase activity was measured. The data represent mean values ± SD (n = 3).
The IL-23p19 subunit is covalently linked to the IL-12/23p40 subunit to form an IL-23 heterodimer. The heterodimer is secreted by monocytes/macrophages/DCs stimulated with TLR ligands (5, 34). To investigate whether lactic acid enhances IL-23 secretion, human monocytes were stimulated with PGN in the presence of lactic acid for 24 h (Fig. 5,A, left). The secretion of IL-23 was enhanced 1.8-fold by lactic acid. When GM-CSF was only present, IL-23 secretion was elevated 4.7-fold. When lactic acid and GM-CSF were both present, IL-23 secretion was enhanced 10.6-fold. Similarly, the PGN-induced secretion of IL-12/23p40 was slightly increased by lactic acid but synergistically enhanced by the further addition of GM-CSF (Fig. 5,A, right). These results indicate that lactic acid and GM-CSF cooperate to stimulate TLR ligand-induced IL-23 and IL-12/23p40 production by human monocytes/macrophages. Furthermore, lactic acid also enhanced the secretion of another proinflammatory cytokine, IL-6, from human monocytes/macrophages stimulated with the TLR ligand (Fig. 5 B).
Lactic acid enhances proinflammatory cytokine production by TLR ligand-stimulated human primary monocytes/macrophages. A, Human monocytes were cultured for 24 h in the presence or absence of 15 mM lactic acid with or without 5 unit/ml GM-CSF. The cells were then stimulated with 10 μg/ml PGN for 24 h and the supernatants were collected. The IL-23 heterodimer and IL-12/23p40 concentrations were measured by ELISA. B, Human monocytes were cultured for 24 h in the presence or absence of 15 mM lactic acid. The cells were then stimulated with 10 μg/ml PGN for 24 h and the IL-6 concentrations of the supernatants were measured by ELISA. C, Human PBMC were stimulated with 5 μg/ml BCG-CWS in the presence of 0, 10, 15, or 20 mM lactic acid. After 4 h, IL-23p19 (left) and IL-12/23p40 (right) transcripts were measured by real-time RT-PCR. Relative expression of these transcripts was normalized to β-actin expression. N.D., Not detected. The data represent mean values ± SD (n = 3). **, p < 0.01.
We also observed that BCG-CWS increased IL-23p19 transcription in PBMC, and like the conditioned medium (Fig. 1,A, middle), lactic acid significantly enhanced this response in a dose-dependent manner (Fig. 5,C, left). Conversely, lactic acid did not increase IL-12/23p40 transcription (Fig. 5 C, right), suggesting that lactic acid specifically acts on IL-23p19 transcription.
Because lactic acid further enhanced TLR-stimulated IL-23 production, we predicted that lactic acid should enhance IL-23 production from APCs stimulated with TLR ligand, leading to increased IL-17 production by IL-17-producing T cells. We then used OT-II-transgenic mice, which have OVA323–339 peptide-specific T cells (25). In the presence of the OVA peptide, we examined the effect of lactic acid on the IL-17 production of OT-II mouse splenocytes that had been stimulated with the TLR ligand (PGN or BCG-CWS) for 4 days. Lactic acid remarkably enhanced the secretion of IL-17 induced by PGN and BCG-CWS (Fig. 6,A, left, □ and ▪). Anti-IL-23p19 Ab significantly inhibited the lactic acid-induced enhancement of IL-17 production (Fig. 6,A, left, ▪ and ▨). This result indicates that lactic acid indeed stimulates APCs to produce IL-23, which then drives peptide-activated T cells to produce IL-17. Interestingly, in the presence of the peptide, lactic acid induced IL-17 production even without the TLR ligand and this effect was inhibited by the anti-IL-23p19 Ab (Fig. 6, A, left, and B). The peptide or lactic acid alone rarely induced IL-17 production by splenocytes (Fig. 6 B).
The IL-17 production by Ag-stimulated mouse splenocytes is augmented by lactic acid. A, Splenocytes from OT-II mice were stimulated with (▪ and ▨) or without (□ and ▦) 15 mM lactic acid and PBS, 10 μg/ml PGN, or 1 μg/ml BCG-CWS. Anti-IL-23p19 Ab (▦ and ▨) or isotype control rat IgG1 (□ and ▪) was also present, along with 0.2 μg/ml OVA323–339 peptide. B, Splenocytes from OT-II mice were stimulated with or without 0.2 μg/ml OVA peptide and 15 mM lactic acid. C, Splenocytes from OT-II mice were stimulated with 100 ng/ml LPS and 0.2 μg/ml OVA peptide in the presence (▪) or absence (□) of 15 mM lactic acid. The supernatants were collected after 4 days and the IL-17 and IFN-γ concentrations were measured by ELISA. N.D., Not detected. The data represent mean values ± SD (n = 3). *, p < 0.05 and **, p < 0.01.
We also examined the effect of lactic acid on the secretion of IFN-γ (Fig. 6,A, right). When the TLR ligand was present along with the peptide, the splenocytes produced IFN-γ. However, lactic acid did not potentiate IFN-γ production under these conditions (Fig. 6,A, right, □ and ▪). In contrast to IL-17 production, lactic acid did not induce IFN-γ production when the TLR ligand was absent (Fig. 6,A, right). Notably, the effect of lactic acid on IFN-γ production was different when the TLR ligand was replaced by LPS. Although lactic acid potentiated IL-17 production by splenocytes irrespective of the ligand used for stimulation (Fig. 6,C, left), it severely inhibited IFN-γ production (Fig. 6 C, right).
Many immune cells infiltrate the tumor microenvironment and induce local inflammation in and around the tumor. We examined whether tumor-infiltrating immune cells actually have the ability to express a high amount of IL-23 in response to lactic acid. We purified CD45-positive cells (CD11b- and/or CD11c-positive cells, 64%) that had infiltrated the tumors formed by B16 melanoma cells (Fig. 7,A). These cells were stimulated with BCG-CWS and lactic acid for 4 h. The level of IL-23p19 transcripts significantly increased in the presence of lactic acid (Fig. 7 B). This result suggests that lactic acid also enhances the production of IL-23 in tumor-infiltrating immune cells, probably in macrophages and/or DCs.
Lactic acid enhances the expression of IL-23p19 mRNA in tumor-infiltrating immune cells. A, Tumor-infiltrating CD45-positive cells were purified from tumors formed by B16 mouse melanoma cells and stained for CD11b and CD11c. The stained cells were analyzed by FACS. B, One hundred thousand tumor-infiltrating immune cells (CD45-positive cells, 86%) were stimulated with 10 μg/ml BCG-CWS in the presence or absence of 15 mM lactic acid. After 4 h, the expression of IL-23p19 and β-actin transcripts was measured by TaqMan RT-PCR. Relative expression was normalized to that of β-actin. The data represent mean values ± SD (n = 3). **, p < 0.01.
In this study, we sought to elucidate the mechanisms that induce immune cells in the tumor microenvironment to produce proinflammatory cytokines such as IL-23. We also asked, how does the tumor induce chronic inflammation without being attacked by the immune cells? We found that many tumor cell lines secrete factors that enhance TLR ligand-stimulated IL-23p19 transcription and IL-23 secretion by human and mouse monocytes/macrophages (Fig. 1,A and data not shown). We clarified that the main factor responsible for this effect is a molecule smaller than 500 Da that is protease resistant, heat stable, and only secreted from tumor cell lines when they are cultured in high glucose medium (Fig. 1, D and E, and data not shown). We then discovered that this molecule is lactic acid (Figs. 2–5). Lactic acid increased the production of proinflammatory cytokines, IL-23, and IL-6 from human monocytes (Fig. 5).
GM-CSF also enhanced TLR ligand-induced IL-23 and IL-12/23p40 production (Fig. 5). GM-CSF is secreted by many tumor cells as well as CADO-LC10 cells (35) and increases the expression of TLR2 (36). We suggest that tumor-secreted GM-CSF and lactic acid cooperate to increase IL-23 production following TLR2 ligand stimulation.
Although lactic acid is completely ionized, even under neutralized conditions (37), the neutralization of lactic acid suppressed its ability to enhance IL-23p19 promoter activity in a pH-dependent manner (Fig. 4,A). Moreover, neither sodium lactate nor acidification with hydrochloric acid enhanced IL-23p19 transcription (Fig. 4, B and D). Lactate anions are transported together with protons through MCTs and a pH gradient is necessary for effective transport of lactic acid into cells (33). Therefore, we predict that lactic acid transported into monocytes/macrophages by MCTs may be recognized by an intracellular sensor that, along with the TLR signal, activates the IL-23p19 promoter.
We found that DNA elements responsible for the enhancing activity of lactic acid are localized in the 2.7-kb 5′-flanking region of the human IL-23p19 gene (Fig. 2,C). We found four predicted NF-κB binding sites in this region (Fig. 1,C). Although NF-κB signaling plays an important role in the TLR signaling pathway (4), lactic acid did not enhance the luciferase activity of a reporter vector carrying canonical NF-κB-binding elements (Fig. 2 D). Therefore, the lactic acid signal pathway may be independent of the NF-κB pathway.
In most normal mammalian cells, the metabolism from glucose to lactate is inhibited by the presence of oxygen, which leads to the oxidization of pyruvate to CO2 and H2O in the mitochondria. However, in cancer cells, glycolysis is up-regulated, even in aerobic conditions, a phenomenon known as the "Warburg effect" or "aerobic glycolysis" (29, 30). This results in the production of large amounts of lactic acid and the accumulation of lactic acid in the microenvironment of many cancer cell types (31). High concentrations of lactate in solid tumors such as cervical carcinoma (10.0 ± 2.9 μmol/g or >8.3 μmol/g) and head and neck cancer (>7.1 μmol/g) are associated with higher frequencies of distant metastasis (31, 38, 39, 40). Moreover, low lactate tumors in primary lesions are associated with longer disease-free survival than high lactate tumors. In addition, when the LDHA activity that induces the metabolism of pyruvate to lactate is suppressed, the tumorigenicity is severely diminished (41).
In this study, we showed that lactic acid enhances the expression of IL-23p19 in tumor-infiltrating immune cells activated by TLR stimuli (Fig. 7) and in splenocytes in the presence of Ag stimulus (data not shown) and induces the Ag- and IL-23-dependent secretion of IL-17 from splenocytes (Fig. 6). Significantly, we detected this IL-23-dependent enhancing activity even when TLR ligands were absent. Since lactic acid alone did not induce IL-23 production in the absence of Ag stimulation, we predicted that an interaction between APCs and T cells mediated by Ag and lactic acid causes IL-23-dependent IL-17 production in the absence of the TLR ligand. We observed that lactic acid severely inhibited IFN-γ production by Ag-stimulated splenocytes treated with LPS (Fig. 6,C, right). The Kreutz group (42) reported that lactic acid represses the secretion of IL-12p70 by LPS-stimulated DCs. Therefore, lactic acid may inhibit IFN-γ production by suppressing the IL-12p70 production of monocytes/macrophages. In contrast, lactic acid did not affect IFN-γ production of Ag-stimulated splenocytes treated with PGN or BCG-CWS (Fig. 6 A, right), because it is assumed that PGN and BCG-CWS do not stimulate IL-12p70 production by monocytes/macrophages (14). These results suggest that upon antigenic stimulation of T cells, lactic acid mediates the activation of the IL-23/IL-17 pathway rather than the induction of IFN-γ-producing Th1 cells. We predict that lactic acid derived from tumor cells may induce inflammation but not the infiltration of CTLs in the tumor microenvironment, even in the absence of TLR ligand stimuli from microbial infections, and that inflammation promotes angiogenesis and tumor development.
The Kreutz group (42, 43) also reported that lactic acid down-regulates the cytokine production and proliferation of CTLs. Furthermore, the Murray group (44) showed that MCT1 inhibitors, which inhibit the transport of lactic acid, suppress T lymphocyte proliferation. These groups suggested that a high concentration of extracellular lactic acid or inhibition of lactic acid excretion might cause intracellular accumulation of lactic acid with consequent disturbance of T cell metabolism and function. In contrast, our results indicate that lactic acid up-regulates IL-23p19 transcription in monocytes/macrophages.
In conclusion, our results show that lactic acid acts as a novel tumor-derived factor that strongly induces the IL-23/IL-17 proinflammatory pathway without inducing Th1 responses. Thus, the production and excretion of lactic acid appears to be not only essential for the proliferation of tumor cells with up-regulated glycolysis, but also for the induction of inflammation in the tumor microenvironment, which promotes tumor progression. Therefore, lactic acid and the lactic acid/IL-23 signal pathway may be an attractive target for treating tumors.
We thank K. Kawata, T. Yasuda, and C. Kozai in our laboratory for technical assistance and Dr. M. Saio (Gifu University, Gifu, Japan) for technical support. We also thank Drs. K. Toyoshima (RIKEN, Yokohama, Japan) and I. Azuma (Muroran Institute of Technology, Muroran, Japan) for providing BCG-CWS, Dr. W. R. Heath (The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia) for providing OT-II mice, and Drs. J. Takeda and K. Yusa (Osaka University, Suita, Japan) for providing pGKEM7-neoW.
The authors have no financial conflict of interest.
This work was supported by a grant from the Cooperative Link of Unique Science and Technology for Economy Revitalization promoted by the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science, and Technology.
Abbreviations used in this paper: DC, dendritic cell; PGN, peptidoglycan; BCG, bacillus Calmette-Guérin; CWS, cell wall skeleton; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; siRNA, small interfering RNA; Pam3CSK4, N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysl-[S]-lysl-[S]-lysl-[S]-lysine/3HCl; BCECF, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein/acetoxymethyl ester.
de Visser, K. E., A. Eichten, L. M. Coussens.
. Paradoxical roles of the immune system during cancer development.
Nat. Rev. Cancer
-37.
Karin, M., T. Lawrence, V. Nizet.
. Innate immunity gone awry: linking microbial infections to chronic inflammation and cancer.
-835.
Balkwill, F., K. A. Charles, A. Mantovani.
. Smoldering and polarized inflammation in the initiation and promotion of malignant disease.
Akira, S., S. Uematsu, O. Takeuchi.
. Pathogen recognition and innate immunity.
Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, et al
. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.
Hunter, C. A..
. New IL-12-family members: IL-23 and IL-27, cytokines with divergent functions.
Nat. Rev. Immunol.
Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, B. Stockinger.
. TGF-β in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.
Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, V. K. Kuchroo.
. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.
Mangan, P. R., L. E. Harrington, D. B. O'Quinn, W. S. Helms, D. C. Bullard, C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, C. T. Weaver.
. Transforming growth factor-β induces development of the TH17 lineage.
Weaver, C. T., L. E. Harrington, P. R. Mangan, M. Gavrieli, K. M. Murphy.
. Th17: an effector CD4 T cell lineage with regulatory T cell ties.
Aggarwal, S., N. Ghilardi, M. H. Xie, F. J. de Sauvage, A. L. Gurney.
. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17.
-1914.
Zheng, Y., D. M. Danilenko, P. Valdez, I. Kasman, J. Eastham-Anderson, J. Wu, W. Ouyang.
. Interleukin-22, a TH17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis.
Begum, N. A., K. Ishii, M. Kurita-Taniguchi, M. Tanabe, M. Kobayashi, Y. Moriwaki, M. Matsumoto, Y. Fukumori, I. Azuma, K. Toyoshima, T. Seya.
. Mycobacterium bovis BCG cell wall-specific differentially expressed genes identified by differential display and cDNA subtraction in human macrophages.
Infect. Immun.
McKenzie, B. S., R. A. Kastelein, D. J. Cua.
. Understanding the IL-23-IL-17 immune pathway.
Trends Immunol.
Tsuji, S., M. Matsumoto, O. Takeuchi, S. Akira, I. Azuma, A. Hayashi, K. Toyoshima, T. Seya.
. Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors.
Langowski, J. L., X. Zhang, L. Wu, J. D. Mattson, T. Chen, K. Smith, B. Basham, T. McClanahan, R. A. Kastelein, M. Oft.
. IL-23 promotes tumour incidence and growth.
Kryczek, I., S. Wei, L. Zou, S. Altuwaijri, W. Szeliga, J. Kolls, A. Chang, W. Zou.
. Cutting edge: Th17 and regulatory T cell dynamics and the regulation by IL-2 in the tumor microenvironment.
J. Immunol.
Kolls, J. K., A. Linden.
. Interleukin-17 family members and inflammation.
Takahashi, H., M. Numasaki, M. T. Lotze, H. Sasaki.
. Interleukin-17 enhances bFGF-, HGF- and VEGF-induced growth of vascular endothelial cells.
Immunol. Lett.
Numasaki, M., J. Fukushi, M. Ono, S. K. Narula, P. J. Zavodny, T. Kudo, P. D. Robbins, H. Tahara, M. T. Lotze.
. Interleukin-17 promotes angiogenesis and tumor growth.
Matsumoto, M., T. Seya, S. Kikkawa, S. Tsuji, K. Shida, M. Nomura, M. Kurita-Taniguchi, H. Ohigashi, H. Yokouchi, K. Takami, et al
. Interferon γ-producing ability in blood lymphocytes of patients with lung cancer through activation of the innate immune system by BCG cell wall skeleton.
Int. Immunopharmacol.
Sakuma, T., K. Kodama, T. Hara, Y. Eshita, N. Shibata, M. Matsumoto, T. Seya, Y. Mori.
. Levels of complement regulatory molecules in lung cancer: disappearance of the D17 epitope of CD55 in small-cell carcinoma.
Jpn. J. Cancer Res.
Akazawa, T., H. Masuda, Y. Saeki, M. Matsumoto, K. Takeda, K. Tsujimura, K. Kuzushima, T. Takahashi, I. Azuma, S. Akira, et al
. Adjuvant-mediated tumor regression and tumor-specific cytotoxic response are impaired in MyD88-deficient mice.
Azuma, I., E. E. Ribi, T. J. Meyer, B. Zbar.
. Biologically active components from mycobacterial cell walls: I. Isolation and composition of cell wall skeleton and component P3.
J. Natl. Cancer Inst.
Barnden, M. J., J. Allison, W. R. Heath, F. R. Carbone.
. Defective TCR expression in transgenic mice constructed using cDNA-based α- and β-chain genes under the control of heterologous regulatory elements.
Immunol. Cell Biol.
Livak, K. J., T. D. Schmittgen.
. Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔCT method.
Wilson, M. C., V. N. Jackson, C. Heddle, N. T. Price, H. Pilegaard, C. Juel, A. Bonen, I. Montgomery, O. F. Hutter, A. P. Halestrap.
. Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3.
-15926.
Saio, M., S. Radoja, M. Marino, A. B. Frey.
. Tumor-infiltrating macrophages induce apoptosis in activated CD8+ T cells by a mechanism requiring cell contact and mediated by both the cell-associated form of TNF and nitric oxide.
Kim, J. W., C. V. Dang.
. Cancer's molecular sweet tooth and the Warburg effect.
Gatenby, R. A., R. J. Gillies.
. Why do cancers have high aerobic glycolysis?.
Walenta, S., W. F. Mueller-Klieser.
. Lactate: mirror and motor of tumor malignancy.
Semin. Radiat. Oncol.
Heinemeyer, T., E. Wingender, I. Reuter, H. Hermjakob, A. E. Kel, O. V. Kel, E. V. Ignatieva, E. A. Ananko, O. A. Podkolodnaya, F. A. Kolpakov, et al
. Databases on transcriptional regulation: TRANSFAC, TRRD, and COMPEL.
Nucleic Acids Res.
Halestrap, A. P., N. T. Price.
. The proton-linked monocarboxylate transporter (MCT) family: structure, function, and regulation.
Biochem. J.
Trinchieri, G..
. Interleukin-12 and the regulation of innate resistance and adaptive immunity.
Bronte, V., D. B. Chappell, E. Apolloni, A. Cabrelle, M. Wang, P. Hwu, N. P. Restifo.
. Unopposed production of granulocyte-macrophage colony-stimulating factor by tumors inhibits CD8+ T cell responses by dysregulating antigen-presenting cell maturation.
Flo, T. H., O. Halaas, S. Torp, L. Ryan, E. Lien, B. Dybdahl, A. Sundan, T. Espevik.
. Differential expression of Toll-like receptor 2 in human cells.
J. Leukocyte Biol.
Gladden, L. B..
. Lactate metabolism: a new paradigm for the third millennium.
J. Physiol.
Walenta, S., M. Wetterling, M. Lehrke, G. Schwickert, K. Sundfor, E. K. Rofstad, W. Mueller-Klieser.
. High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers.
Schwickert, G., S. Walenta, K. Sundfor, E. K. Rofstad, W. Mueller-Klieser.
. Correlation of high lactate levels in human cervical cancer with incidence of metastasis.
Walenta, S., A. Salameh, H. Lyng, J. F. Evensen, M. Mitze, E. K. Rofstad, W. Mueller-Klieser.
. Correlation of high lactate levels in head and neck tumors with incidence of metastasis.
Fantin, V. R., J. St.-Pierre, P. Leder.
. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance.
Gottfried, E., L. A. Kunz-Schughart, S. Ebner, W. Mueller-Klieser, S. Hoves, R. Andreesen, A. Mackensen, M. Kreutz.
. Tumor-derived lactic acid modulates dendritic cell activation and antigen expression.
Fischer, K., P. Hoffmann, S. Voelkl, N. Meidenbauer, J. Ammer, M. Edinger, E. Gottfried, S. Schwarz, G. Rothe, S. Hoves, et al
. Inhibitory effect of tumor cell-derived lactic acid on human T cells.
Murray, C. M., R. Hutchinson, J. R. Bantick, G. P. Belfield, A. D. Benjamin, D. Brazma, R. V. Bundick, I. D. Cook, R. I. Craggs, S. Edwards, et al
. Monocarboxylate transporter MCT1 is a target for immunosuppression.
Nat. Chem. Biol.
Follow The JI on Twitter
AAI Newsletter
Additional AAI Publications
FAR 889
PillarTalk
ImmunoCasts
Twitter JI
Twitter IH
© 2023 The American Association of Immunologists, Inc.