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21 Parks near Port Orchard, Washington
Bethel, Washington 0.63 miles SW
East Port Orchard, Washington 1.7 miles N
Parkwood, Washington 2.41 miles NE
Navy Yard City, Washington 4.3 miles NW
Manchester, Washington 5.28 miles NE
Port Orchard, Washington (map) | (360) 871-4065
Sites: 50 | Latest Rate: $30.00 | 7 Reviews
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Sep 2014 - $30.00
Only a few 50amp sites so check if you need it. Pretty much in the trees with total shade. Makes it nice and mostly private. No sewer at site but good spot in campground. A 1/4 mile hike to the water or drive to day use area. Great spot to stay while exploring Bremerton Navy exhibits. Close enough to drive to town for good food. - Kirt
Sep 2013 - $30.00
From the pictures on the website, we thought we'd have a waterfront site, but we had to walk or drive down to the day use area to see the water. It was really nice down by the water, and I enjoyed seeing the ferries along with the historical sites. Our site was nice, but very dark, whether the day was sunny or not. We had some nice fires, but didn't expect such darkness. It is a nice place, and the facilities are great. The rangers are very helpful, but now only work part time because of budgets. Good, yes, but not what we expected from the website. - LeeL
Sep 2013 - $27.00
They are listed as having 50 amp but it is only in one or two sites. The 30 worked fine for the time of year we were here. It was cool and rainy so we didn't need AC. Park roads were paved and they only had one loop open. Roads in this loop were a bit tight in a couple of places but not a big problem. Sites are gravel and pretty level for the most part. The sites we had had water and electric and there's an on site dump station that has easy access in and out. The showers will cost you 1 token (50 cents) for 3 minutes. You will spend the first minute an a half waiting for the water to warm up. They didn't spend any time cleaning the shower for the 5 days we were here. The sites were quiet and you could hear the ferries going into Bremerton but it was not loud. I'd stay here again. - pumper9x9
Bremerton, Washington (map) | (360) 478-6460
Sites: 23 | Latest Rate: $32.00 | 2 Reviews
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May 2014 - $32.00
We heard a lot about this place from other campers that post on RV Park Reviews. This camp ground was packed to the brim, dogs-kids every where and boy was it noisy--fun as hell to be around. The park offered a lake access below where we were parked and there was a lot of games going on on the beach. We will for sure be coming back again next year. - kara9
Jul 2007 - $17.00
There are only 2 full hook up sites available. One of which is used by the camp host. Mosquitoes were very bad. Some nice hiking trails. Only got about 4 min worth of hot water in shower and it cost me 50 cents for this. Dump station was closed. - retnavrib
Bremerton, Washington (map) | (360) 479-1181
Sites: 23 | Latest Rate: $20.00 | 3 Reviews
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Aug 2015 - $20.00
Nice location, gravel surface, sites not too spacious but we had no one on either side of us for the 3 nights we stayed there. A couple of non-Elks groups used the picnic facilities adjacent to the RV parking but they were gone by dark. Friendly staff in lodge. Water and electric at site but no dump. Couldn't get level on Site #9 so chose #8 instead. - RVtravelers2
Jul 2014 - $20.00
Beautiful location and views. Campsites adequate with nice park area. Restrooms were clean. - hbcamper
Jun 2012 - $20.00
You must be an Elks Lodge Member. The sites are fairly large and easy to access. There is a large grassy area where we ran our two Labs. beautiful views and the lodge is very nice. - User2627
Gig Harbor, Washington (map) | (253) 858-8138
Sites: 95 | Latest Rate: $50.00 | 70 Reviews
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Jul 2016 - $50.00
Pretty RV park (not a resort) nestled under well-trimmed evergreen trees. The roads are paved and the sites are gravel. It is on a hillside so sites are terraced and roads are steep in a couple of places. Office staff was friendly and helpful. Our site was a long, fairly level pull-through with 50 amp. The back-in sites were of various sizes. The bathrooms, showers, and laundry were modern and clean. WiFi worked some of the time. The park is less than a mile from downtown Gig Harbor and close to a bike trail.
Larger RVs should use the Burnham Drive exit when arriving from the east.
We enjoyed our stay here and would gladly come back again. - JoyceM
Oct 2015 - $40.00
This campground is in a beautiful setting, large mature trees, hilly terrain with sites terraced into the hills. Many of the sites are hard to get into, we were lucky and our pull through was easy, but was right on top of our neighbor, 4 feet between slides. Road and sites are gravel and not real level. Wi-Fi was weak and the cable had fair reception. - Traveler II
Sep 2015 - $58.00
We called to make reservations nearly a month ahead of time. We were told we couldn't reserve a specific site, but we would get a site meeting our requirements. When we arrived we were told that no pull-through sites were available. When we mentioned that several sites were empty, she said they were reserved. Total disconnect here--we were told we couldn't reserve and yet...
We finally gave up arguing and backed into the site they gave us. Too many trees for satellite, WiFi was very slow, cable was good. Site had enough room to set up chairs outside and put out the awning.
Park personnel did a very good job of cleaning up after guests left, and maintaining the grounds.
We stayed for 10 days, as we had our son flying in, and we wanted to see all the sites in Seattle. We parked in Bremerton, and took the ferry across to Seattle where we picked up transportation to the sites.
Gig Harbor is not a resort by any means, but a decent park.
We found the add-ons for pets and an extra person excessive, and we will not use this park again. - The Sauntering Sampsons
Belfair, Washington (map) | (360) 275-0668
Sites: 184 | Latest Rate: $45.00 | 9 Reviews
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Jun 2015 - $45.00
We had space T24 on the beach loop - 30 amp, water, and sewer. The temperature soared to 92 degrees the first day, but our AC worked fine on 30 amps (we're a 50 amp unit). The pull-through sites could be a little longer for today's rigs, but if everyone parks properly, things seem to fit okay. Friendly staff. - NotSoBadBob
Sep 2013 - $37.00
Nice scenery. Some of the pull-throughs are a little sharp for the bigger rigs, but most are okay. We arrived about 1/2 hour before check-in time and had no trouble getting right in. Ranger and staff were pleasant. We stayed in the Beach Loop at a pull-through site with 50 amp, water, and sewer. - NotSoBadBob
Jul 2011 - $35.00
The location of Belfair is great for visiting the Seattle area. The park itself is a wonderful park, and is well maintained. Very laid back, and I was impressed to see the volunteer workers cleaning campsites as soon as a camper left, prior to new arrivals. They do enforce the no check in before 2:30p, which was not a big deal. We are in a 40' MH /c 3 slides, and the spaces are limited for a rig our size, but there are several to choose from. If you don't need services, there are great sites without hookups that would easily fit our rig. The ferry terminal was a 30 minute drive, and we got to Seattle in 1 hr, shopped, and got back without any traffic hassle. Don't attempt to take your MH to the ferry terminal, parking is very limited. We left ours after checkout in the day use area and went on the ferry for the day. - red baron
Poulsbo, Washington (map) | (360) 779-4305
Sites: 35 | Latest Rate: $25.00 | 2 Reviews
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May 2012 - $25.00
Small park, but not much available in the area. We spent two months. Backed up to Hwy 3 and had some road noise. Not bad at night. Clean sites and medium in size. We would stay again. - RETE8
Aug 2011 - $31.00
This is a nice, clean park. The full time residents (95%) are all friendly. The negatives are lots of road noise, day and night. The park sits on the road to the Seattle ferry. Also, there is a 4 page, double sided, fine print set of rules and a two page registration form. They are trying to keep the undesirables out, but have overdone it. This park is not designed for an overnight stay. If you are looking for a long term stay in this area, I would recommend this park. - retiredfields
Poulsbo, Washington (map) | (360) 598-5988
Sites: 88 | Latest Rate: $30.00 | 33 Reviews
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Jun 2016 - $30.00
There are few RV parks in the general area for competition, and frankly it shows. This is one of the most cramped parks we ever stayed in during our two years on the road, particularly if you are in one of the 50-amp pull-thru spots. Putting out our awning partially covered the adjacent unit's bedroom slide, if that will give you any idea. There are also quite a few long termers. Having said that, it is close to Bainbridge Island, Poulsbo and Silverdale - all big plusses. It is also very quiet (except that you can hear when adjacent units' furnaces kick in and out throughout the night). - coffeetime
Aug 2015 - $30.00
Online photos showed shaded spacious sites but those were only being utilized by permanent RV residents. The pull thru sites were long and narrow with no room to sit outside or even open our awning. Slides almost touched the neighboring RV on both sides. The back end of our fifth wheel was so close to the road coming into the park it felt like we would get hit. So glad we only stayed 4 nights & it was cool while we were there so we didn't need to use ac. Laundromat was clean. Nothing much to do in Poulsbo either. - RendezVous
Jul 2015 - $37.00
Site was gravel, very narrow, width of a parking space at WalMart. Minimal room for slide-outs, no room for awning. No trees or shade, no privacy. Could not sit outside, no room to grill. Several full-time residents with their accumulated possessions all around. Not a pleasant camping experience, probably one of the two worst we have experienced. - RDV
9565 Scenic Beach Road N.W., Seabeck, WA 98380 Seabeck, Washington 98380 (map) | 360-830-5079
Sites: 52 | Latest Rate: $28.00 | 1 Reviews
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May 2015 - $28.00
This is an outstanding campground. There are no utilities in the campsites, but there is potable water and a RV dump station. The camp is located on the north end of the beautiful Hood Canal. During clear weather, the rocky beach has a spectacular view of the Olympic Mountains. The campsites vary from private to very private. Our site was totally private and deep in the lush dappled sunlight of cedar and fir trees. There are two good playgrounds for kids. Many sites are small and somewhat uneven, but they work for small and moderate RVs. Pull-through sites would fit all but the largest bus type RVs. We were there on Memorial Day weekend, and the campground was full. Fellow campers were generally quiet, and all noise was done by 10 PM. I would happily return. - SeaTeacher
Federal Way, Washington (map) | (888) 226-7688
Sites: 118 | Latest Rate: $45.00 | 15 Reviews
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Jul 2016 - $45.00
Large site and with fifth wheel parked, it made a very private area with rig parked between site and road. Quiet, full hookup but very popular area. Reservations suggested. I made mine in March for July and 6 days was the longest "open" time. - Edd505
Jun 2016 - $45.00
We arrived at 3:30 in the afternoon. Wait at gate 12 minutes. Gate attendant was very professional and friendly. Drive through the park was very tight with trees leaning over the roadway. We drive a 40 ft DP. The site we were in was the only site that our rig would fit in, it was a drive through, but we had to drive in and back out.
Campsites are medium distance apart and paved. Hookups were well placed. One note - there were no trash cans, just recycle.
Beach access is very nice with a very large parking lot. The beach is typical salt water with a very low slope.
We will not be returning to camp. - Half_Timer
Sep 2015 - $43.00
Difficult to drive through park, winding, low branches and pull-thru unlevel, hard to level out. Tight parking. Only one campground open, Self Registration, ranger on site part time. Watch speed limit. - billandmemakes3
Des Moines, Washington (map) | (253) 661-4956
Sites: 50 | Latest Rate: $25.00 | 7 Reviews
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Jun 2014 - $25.00
I rated this low. For $25/night it had to be the worst WA state park we have ever stayed in. Sites 1-4 are the ones where supposedly the large RVs should be. They are as close together as any commercial campground, so no privacy, noise from your neighbor's generator, etc. There were other pull through sites that would have worked. The website just didn't show they were available for larger rigs, but we saw a RV larger than ours on one of them, That would have been a much more pleasant spot with a little more privacy. $25/night for no hooks and no working dump station felt like it was priced too high. You also get charged extra if you are not a WA state resident and want to make a reservation. Unlike most Western WA state parks, there is little or no vegetation between any of the RV sites in this park. Only sites with any privacy are in the tent or very small class B RV area. You are in the flight path of SeaTac so you do get that noise. On the plus side, nice hiking trail in the park and easy walk to the beach area. Probably not a park we'll stay in again. - DRFink
Jul 2013 - $23.00
Nice woodsy park between Seattle and Tacoma. We were in a 45' motorhome and except for one tight turn because of a parked camper sticking out, I was able to navigate thru the park and to the campsite with care, but no problem. The showers do not work (was told by camp host). The place is a bit overgrown. I had to trim a tree as not to scrape going into my site. Site 1 - 4 are best for big rigs. Easy to back in to and easy to pull out. No hookups (as advertised) and a good price. The beach is great. A nice paved path along the waterfront is nice. A good restaurant called Wally's Chowder House a couple of miles away was wonderful. The park is great for camping. Just not very well maintained with the lack of budget in the WA State Park System. - JoeVinnie
Jul 2011 - $22.00
The park was extremely well maintained. There were many trails to explore, and the beach access had excellent low-tide pooling for investigation. Our site right next to the creek was wonderful. Detractions included the airline traffic heading to/from Seatac (pretty minor, but noticeable) and the dry camping (offset by running a generator to recharge batteries). - bradrf


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Cornelius Clarke was born on January 26, 1968 in Drogheda, County Louth, Ireland. He is known for his work on Hard Times (2009), Night Wolf (2010) and Belly (2011). See full bio »
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Hotels in Newtownabbey, United Kingdom
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"Rooms are lovely and modern but too dark and no air conditioning,which was very much needed"
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"Amazing gem in the middle of the city for a perfect romantic getaway. Cocktail bar and restaurant were excellent and service was first class for our entire stay. Would definitely visit again."
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"Lovely place to stay in Belfast. Nice, Clean and Customer Service is 2nd to none. Breakfast is 5 star. All the staff members pay personal attention to you and your needs. I had a very important meeting next day and I asked for a Laundry service at 10'o clock in the night and I was hoping answer ..."
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A fond farewell, and changes ahead for Gimme Noise
Photo by Stacy Schwartz
Today marks my last day at the helm of this blog, and the end of a journey that I began with City Pages nearly four years ago. Naturally, I have quite a few thoughts about closing this chapter, and you can read all about the history of Gimme Noise and my thoughts on the past four years in this farewell column, which is also running in print today.
Over the next few weeks you'll be hearing a lot from frequent Gimme Noise contributor Erik Thompson, who has graciously agreed to step in and run the blog until the arrival of your new music editor,Reed Fischer
, on January 18.
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Save Big On Open-Box & Pre-owned: Buy "The Lords of Salem [Blu-ray]" from Amazon Warehouse Deals and save 46% off the $14.99 list price. Product is eligible for Amazon's 30-day returns policy and Prime or FREE Shipping. See all Open-Box & Pre-owned offers from Amazon Warehouse Deals.
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Of course this isn't a film for everyone, someone looking for another gore fest or cookie cutter teeny bopper horror film should look else where as "The Lords of Salem" is a horror film that stands out from the norm in every way possible and delivers itself as a true horror experience.
All these websites, blogs, and reviewers can trash the film but its obvious to me they just don't know a true piece of horror when they see it, one site or actually person in particular is Brad Miska from bloody-disgusting.com, I think he is a horrible reviewer for their website and has bad taste in movie choices. You want to read a review, than check out zombiehamster.com review for the lords, its dead on - zombiehamster.com/reviews/theatrical-release/the-lords-of-salem-2012/.
So now let the hate begin, I know all you angry reviewers/movie watchers out there can't wait to trash my review and give it a bunch of "Unhelpful" votes which is fine, I knew what I was getting myself into. Regardless "The Lords of Salem" is something the horror genre needed weather you liked it or not.
If "Lords of Salem" was made in the 1970's (perhaps even as late as 1981) then it would have been hailed as an iconic horror film, much in the same way as such other greats of the genre of that time, as Dario Argento's "Suspiria", "Rosemary's Baby" or even that of Stanley Kubrick's "The Shining". Sadly, however, the film finds life in 2013 where most of the audiences that will go and see it will neither understand or have the patience for Zombie's latest creation.
We find ourselves following along the life of DJ Heidi Hawthorn (aka Heidi LaRox), played by Sheri Moon Zombie, living life in Salem, Massachusetts, as night time radio's hottest DJs. When One night after a show with her cohorts and hosts Herman `Whitey' Salvador (Jeff Daniel Phillips) and Herman Jackson (Ken Foree), Heidi receives an old wooden box containing a vinyl record and a note saying it is from the band The Lords. Heidi takes the box home, and while she and Whitey are hanging around Heidi's apartment, Whitey plays the record. The music is mostly made up of string instruments being played in some strange rhythmic beat.Read more ›
the story is basically about immolated witches using what looked like a 78 rpm record in the present time to crack the doorway open for their return via the descendants of their executors. the vehicle for their musical transmission is local salem deejay Heidi hawthorne, played by sheri moon zombie, who really does a nice job, i thought. not perfect, but alternately energetic and morose, and always working hard. in her alone moments, i thought she was most effective, conveying simultaneously what recovering from addiction must be like, as well as what demonic entities entering her head must be like. she's lost, and nothing helps you get found like a coven of modern-day practicing witches. this film, less than zombie's others, is filled with more seemingly normal people, though many turn out to be not so. i really thought judy geeson, patricia quinn, and (especially) dee wallace were awesome as the coven of witches lurking under heidi's nose. they worked well together, were funny and dark, and, by the end, were very much who I was rooting for. bruce davison in full jeff bridges regalia was awesome as always, and ken foree and jeff Daniel Phillips were their usual, hardworking, effective selves. special mention must be made of meg foster, who is sublimely uncomfortable as the crone witch leader of yore.Read more ›
Most Recent Customer Reviews
Solid Rob Zombie horror flick. Better than most other horror films I see. It has a really great underlying satanic vibe the whole film that keeps it unsettling; something I crave... Read morePublished 7 days ago by Sluttering0504
Just what to expect from the best. One of the best of Zombie flicks !Published 13 days ago by Amazon Customer
I am a fan of Rob Zombie's movies. Having said that, this particular flick left me wishing I hadn't spent my money or my time on it. Read morePublished 14 days ago by Shawna Albus
Lords of Salem was a good story by an incredible writer/director (Rob Zombie) and a very worthy cast (led by his wife, Sherry Moon Zombie). Read morePublished 22 days ago by Amazon Customer
I was beyond unimpressed with this movie. From his early work Rob has come a long way, but this one is a waste of time and money. Read morePublished 28 days ago by Richard S. Barber


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28 March 2014 at 3:00 PM
Taylor Swift & Lily Aldridge Are the Chicest Ladies Who Lunch!
About this photo set: Taylor Swift heads out for lunch with her friend Lily Aldridge on Friday afternoon (March 28) in New York City. The 24-year-old singer and the 28-year-old supermodel… Read More Here
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Ramblas Building - Feel at Home Barcelona, Barcelona
- Free WiFi
Main amenities
- 12 smoke-free apartments
- Breakfast available
- Airport shuttle
- Childcare
- Air conditioning
- Daily housekeeping
- Free grocery shopping service
- Library
- Front desk safe
- Self-serve laundry
- Multilingual staff
- Concierge services
What's around
- In El Raval
- Palau Guell (0.1 mi / 0.1 km)
- Gran Teatre del Liceu (0.1 mi / 0.2 km)
- Las Ramblas (0.2 mi / 0.4 km)
- Barcelona Cathedral (0.5 mi / 0.9 km)
- Palau de la Musica Catalana (0.8 mi / 1.2 km)
For families
- Free cribs/infant beds
- Babysitting or childcare
- Babysitting or childcare (surcharge)
- Sofa bed
- Kitchen
- Cookware, dishware, and utensils
An amazing experience especially with the helpful and friendly staff!
Great spot. Nice, clean, secure building. Just 1 block from Las Rampas. Felt safe and…
Ramblas Building - Feel at Home Barcelona, Barcelona
Hotel Info: 01 65 69 441from €223
- Standard Apartment, 2 Bedrooms
- Standard Apartment, 2 Bedrooms, Balcony
- Penthouse, 1 Bedroom, Terrace
Recommended hotels
At a glance
Key facts
Hotel size
- This hotel has 12 rooms
- This hotel is arranged over 6 floors
- Check-in time 2 PM-8 PM
- Check-out time is 11 AM
- Express check-in/out
Airport shuttle service is available on request. Contact the property in advance to make arrangements.
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
- Minimum check-in age is 18
Travelling with others
- Up to 4 children (12 years old and younger) stay free when occupying the parent or guardian's room, using existing bedding
- Babysitting/childcare *
- Pets allowed (dogs and cats only) *
- 1 per room (up to 5 kg)
- Free WiFi in public areas
- Free WiFi in rooms
- Airport shuttle (available on request) *
- Train station pickup *
- Cruise terminal shuttle *
- No parking
- Discounted offsite parking
Other information
- Smoke-free property
Nearby Four Star Barcelona hotels
In the hotel
- Continental breakfast (surcharge)
- Bicycle rentals on site
- Sailing nearby
- Scooter/moped rentals nearby
- Scuba diving nearby
- Front desk (limited hours)
- Concierge services
- Tours/ticket assistance
- Limo or Town Car service available
- Laundry facilities
- Free grocery shopping service
- Luggage storage
- Multilingual staff
- Number of buildings/towers - 1
- Year Built 2007
- Elevator/lift
- Safe-deposit box at front desk
- Library
- In-room accessibility
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Espresso maker
- Coffee/tea maker
- Iron/ironing board
- Sofa bed
- Sofa bed size double
- Furnished lanai
- Separate dining area
- Separate living room
- Private bathroom
- Shower only
- Free toiletries
- Hair dryer
- Satellite TV channels
- IPod docking station
- DVD player
- Desk
- Free WiFi
- Full-sized refrigerator/freezer
- Microwave
- Kitchen
- Stovetop
- Cookware, dishware, and utensils
- Dishwasher
- Daily housekeeping
- In-room safe (laptop compatible)
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Ramblas Building - Feel at Home Barcelona, Barcelona's small print
Also known as
- Barcelona Feel
- Ramblas Feel Home Barcelona
- Ramblas Feel Home
- Feel Barcelona
- Feel Home Apartment
- Feel Home Apartment Barcelona
- Feel Home Barcelona
- Feel At Home Barcelona - Ramblas Building Catalonia
- Feel At Home Barcelona Hotel Barcelona
- Ramblas Feel Home Barcelona Apartment
- Ramblas Feel Home Apartment
Cash transactions at this property cannot exceed EUR 2500, due to national regulations. For further details, please contact the property using information in the booking confirmation.
For transfers, guests must contact the property 72 hours prior to arrival at the number on the booking confirmation.
Mandatory fees
You'll be asked to pay the following charges at check in or check out:
- A tourist tax will be collected at the property upon arrival. The tax applies up to a maximum of 7 nights per person, per stay. Children under 17 years of age are exempt from this tax.
Deposit: EUR 150.00 per stay
Optional extras
Late check-in after 8 PM can be arranged for an extra charge of EUR 35
Continental breakfast is offered for an extra charge of EUR 3,50 for adults and EUR 3,50 for children (approximately)
Babysitting/childcare is available for an extra charge
Pets are allowed for an extra charge of EUR 50 per accommodation, per stay (maximum EUR 50 per stay)
Airport shuttle service is offered for an extra charge of EUR 50.00 per vehicle (one way)
Cruise ship terminal shuttle and train station pick-up service is offered for an extra charge
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
Hotel registry number: HUTB-002459-68
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Upcoming events
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|Thursday August 01, 2013
Start: 5:00 pm
End: 7:00 pm
Come and join us for a Geocaching Meet and Greet at the Dauphin Street Fair. We will be meeting up and getting together between 5pm-7pm. More details to come closer to the event.
Dauphin & District Chamber of Commerce hosts it's annual Street Fair, 8:00 am until midnight in the downtown core! Start the day off with a pancake breakfast and bring the family down for fun activities like face painting, bouncy castle, dunk tank, and of course shopping.


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Oct. 16, 2010
By Kaitlyn Comiskey
Athletic Media Relations
FORT COLLINS, Colo. – The CSU football team wasn't the only team to get a homecoming weekend win.
The CSU volleyball team improved to 17-2 overall, 7-0 in the Mountain West, and pushed its winning streak to eight matches, sweeping New Mexico - 25-16, 25-21, 25-19. With the loss, the Lobos fall back to 11-8 overall and 3-2 in the Mountain West.
CSU came out strong to start, swinging hard and ending the first set hitting an impressive .400 as a team. Danielle Minch notched a deep cross-court kill early in the set to give the Rams a lead, and teammates Katelyn Steffan and Brieon Paige followed with a solid block-kill on the outside, giving the Rams a two-point lead.
Steffan ended the first set hitting .667, contributing two block assists as well. Jacque Davisson also helped the Rams to their first set victory, notching three kills and three digs.
"I knew we were playing pretty well, but you don't get the real scope of it until you get to look at these numbers," commented Head Coach Tom Hilbert. "You will win every match you play if your outside hitters hit that well."
Setter Evan Sanders won a one-handed joust in the beginning of the second set, putting CSU ahead, 7-5. Paige came out swinging hard in the second set, but had her two hard-hit quicks out of the middle dug in a rally. Yet Paige had the final say, going up for a solo-block to end the rally for the Rams. Paige finished the night with two block-assists as well as two solo-blocks.
The Lobos served themselves into trouble in Set 3, racking up three service errors, with eight total on the night. Davisson cranked a big kill to middle back, pushing the Rams to a 20-16 lead. Sophomore Dana Cranston served up an ace the next point, giving CSU a comfortable lead, and allowing them to go on and take Set 3.
Minch tied Davisson for the team lead in kills for the night with 10, hitting an impressive .375. Davisson also recorded a team-high seven digs. Steffan contributed to the Rams .372 hitting percentage on the night as well, finishing with five kills and hitting .714.
The night was a special one for the Rams not only because of homecoming, but it was also alumnae night. And a crowd of 3,200 packed Moby Arena to welcome back 35 former CSU volleyball players who were recognized on the court during the intermission between Sets 2 and 3.
"It was an exciting weekend with everyone here," said Minch. "It's good to see all the people that have played here and have been through all this before that we're going through right now, and just having the crowd there was awesome."
Colorado State continues a four-match homestand, facing in-state foe Denver on Wednesday, Oct. 20, before hosting the "Border War" against rival Wyoming next Friday.
Wednesday's match vs. DU marks CSU's final non-conference match of the regular season. The match is scheduled to start at 7 p.m. The Rams will be wearing special pink uniforms and the match has been designated CSU's annual "Dig Pink" event to help raise awareness for breast cancer.
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Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients.
Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot.
Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression.
XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury.
Citation: Lu P, Struijs M-C, Mei J, Witte-Bouma J, Korteland-van Male AM, de Bruijn ACJM, et al. (2013) Endoplasmic Reticulum Stress, Unfolded Protein Response and Altered T Cell Differentiation in Necrotizing Enterocolitis. PLoS ONE 8(10): e78491. doi:10.1371/journal.pone.0078491
Editor: Derya Unutmaz, New York University, United States of America
Received: July 8, 2013; Accepted: September 12, 2013; Published: October 23, 2013
Copyright: © 2013 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
Endoplasmic reticulum (ER) stress-related inflammation is involved in the pathogenesis of various chronic inflammatory diseases, including inflammatory bowel disease [1,2]. In the ER, secretory and transmembrane proteins are folded into their native conformation, and proper protein conformation needs the assistance of molecular chaperones such as 78 kDa glucose-regulated protein (GRP78). As such, highly secretory cells, like Paneth cells, have high basal levels of the molecular chaperone GRP78 to maintain homeostasis of protein folding in the ER [1,3]. When misfolded or unfolded proteins accumulate in the ER, ER stress occurs. To restore ER homeostasis, mammalian cells activate a process called unfolded protein response (UPR), which is marked by induction of a number of UPR-related genes including GRP78 and C/EBP homologous protein (CHOP). There are at least three ER stress sensors on the ER membrane, which are inositol-requiring transmembrane kinase-endoribonuclease-1 (IRE1), pancreatic ER kinase (PERK), and activated transcription factor 6 (ATF6) (Figure S1).
Growing evidence shows that ER stress and the UPR play crucial roles in intestinal homeostasis and inflammation. In the colon and small intestine of patients with inflammatory bowel disease, ER stress and the UPR go hand in hand with increased GRP78 expression [4] and spliced X-box binding protein 1 (XBP1s) mRNA [5,6]. Furthermore, multiple single nucleotide polymorphisms of X-box binding protein 1 (XBP1) were found to be associated with inflammatory bowel disease in independent whole genome analysis studies [7–9]. However, ER stress and the UPR have not been studied in necrotizing enterocolitis (NEC), an acute intestinal inflammatory disease in premature infants.
NEC is the most common gastrointestinal emergency in the neonatal intensive care unit, with high morbidity and mortality rates in very low birth weight infants [10]. NEC is most common in the ileum, and is characterized by inflammation and coagulation necrosis [11]. Current treatment strategies for NEC are antibiotic administration and, if required, eventually intestinal surgery to remove the affected part of the intestine. Infants, who underwent such surgery and survive, often experience severe long-term complications, such as intestinal strictures, recurrent sepsis, short bowel syndrome, growth impairment, and poor neurodevelopmental outcome [10]. These adverse sequelae underscore the importance of defining the pathophysiology of NEC to develop new strategies for treatment and prevention.
In this study, we aimed to study ER stress and the UPR in the ileum of the NEC patients. Our data show that ER stress and the UPR occurred only in a subset of acute NEC patients but not in patients who recovered from NEC. Moreover, ER stress and the UPR correlated with poor surgical outcome, increased interleukin 6 (IL6) and interleukin 6 (IL8) expression, and altered T cell differentiation.
Study population and sample collection
This study was conducted with the approval of the 'Central Committee on Research involving Human Subjects', Hague, the Netherlands, and the written informed consents were obtained from the guardians on the behalf of the infant participants involved in the study. Infants who underwent bowel resection at the neonatal intensive care unit at Sophia Children's Hospital between November 2003 and September 2010 were recruited. Fresh ileal tissue samples were collected during surgery, and fixed in 4 % paraformaldehyde in phosphate buffered saline or snap-frozen in liquid nitrogen immediately. Diagnosis and staging of NEC were based on Bell's criteria [12], and diagnosis was confirmed during surgery and by histopathological evaluation of resected intestinal tissue. Preterm patients who underwent bowel resection for stage III acute NEC were referred to as A-NEC, and NEC patients who underwent reanastomosis (stoma closure) were referred to as R-NEC. Patients who underwent resection for diseases other than NEC were included as control patients. Control patients for A-NEC (A-CTRL) only contained preterm patients, and control patients for R-NEC (R-CTRL) contained patients who were born prematurely or at term. The corrected gestational ages during surgery of A-CTRL and A-NEC patients and R-CTRL and R-NEC patients were matched. The patient demographics and reasons for surgery on control patients are shown in Table 1 and Table 2, respectively.
|A-CTRL n=5
|A-NEC n=12
|R-CTRL n=10
|R-NEC n=13
|3 (60.0%)
|10 (83.3%)
|6 (60.0%)
|10 (76.9%)
|Birth weight (gram)
|905 (760, 1935)
|1175 (785, 1526)
|2323 (1400, 3411)$
|950 (858, 1548)$
|Gestational age at birth (weeks)
|26.9 (26.8, 33.9)
|29.5 (27.4, 31.1)
|34.8 (28.5, 39.1)
|29.4 (26.9, 31.2)
|Postnatal age at surgery (days)
|8.0 (1.0, 24.5)
|10.5 (5.5, 19.8)#
|43.5 (0.8, 83.0)$
|78.0 (52.0, 120.5)$,#
|Gestational age at surgery (weeks)
|31.0 (28.8, 34.1)
|31.0 (28.6, 32.9)#
|40.6 (38.6, 42.1)
|42.1 (36.0, 45.2)#
|Survival (1 year after surgery)
|5 (100.0%)
|6 (50.0%)#
|10 (100.0%)
|12 (92.3%)#
|Reason for surgery
|Ileum perforation after incarcerated hernia
|Isolated perforation
|Milk curd syndrome
|Meconium ileus and Meckels' diverticulum
|Ileum perforation after incarcerated hernia
|Perforation with gastroschizis
|Milk curd syndrome
|Meckels' diverticulum
|Meconium ileus and perforation
|Midgut volvulus
Cell Culture and Treatment
The human monocytic cell line THP-1 (derived from the peripheral blood of a 1 year old human male) was purchased from ATCC, and the cells were cultured in Dulbecco's modified Eagle's minimal essential medium supplemented with 10 % fetal bovine serum, 50 μg/ml streptomycin and 50 U/ml penicillin. The fetal human intestinal epithelial cell line HIEC (a kind gift from Prof. Jean-François Beaulieu) was cultured in Opti-MEM I GlutaMAX medium supplemented with 5 % fetal bovine serum, 0.01 M HEPES and 5 ng/ml epidermal growth factor [13]. Both cell lines were cultured in a 37 °C incubator with 5 % CO2. To induce ER stress and the UPR, cells were treated with 5 μg/ml of tunicamycin (TM, purchased from Sigma-Aldrich) for 6 hours, and cells treated with dimethyl sulfoxide were used as control.
RNA isolation and complementary DNA synthesis
Total RNA from snap-frozen ileal tissue was isolated using RNeasy midi kit (Qiagen, Venlo, the Netherlands), and total RNA from THP-1 cells and HIEC cells was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Düren, Germany). The quality of the RNA samples was analyzed by Bioanalyzer (Agilent Technologies), and only RNA samples with an RNA integrity number greater than 7 were used for further analysis. Complementary DNA was synthesized from 1.5 μg RNA using M-MLV reverse transcriptase (Promega, Leiden, the Netherlands).
XBP1s detection
Complementary DNA was amplified using XBP1 forward primer (5'-GGA GTT AAG ACA GCG CTT GGG GA-3') and XBP1 reverse primer (5'-TGT TCT GGA GGG GTG ACA ACT GGG-3') to generate cDNA products encompassing the IRE1 cleavage sites [8]. The unspliced X-box binding protein 1 (XBP1u) and XBP1s mRNAs generate 164-bp and 138-bp PCR products, respectively. These fragments were resolved on 2 % agarose gel to distinguish the PCR products of XBP1u and XBP1s.
Quantitative PCR (qPCR)
Relative mRNA expression was determined using DyNAmo HS SYBR Green qPCR Kit (Finnzymes, Vantaa, Finland) on the ABI prism 7900HT Fast Real-Time PCR system (PE Applied Biosystems, Foster City, CA, USA), and was normalized against the mRNA expression levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or cluster of differentiation 4 (CD4) as described previously [14]. All qPCR primer sequences are given in Table 3.
|Forward Primer (5' to 3')
|Reverse Primer (5' to 3')
Western blot
Briefly, 20 µg of homogenates from snap-frozen tissue samples were separated on SDS-PAGE and subjected to Western blot. The blots were probed with antibodies against GRP78 (Santa Cruz Biotechnology, CA, USA), CHOP (Abnova, Heidelberg, Germany) and beta ACTIN (Abcam, Cambridge, UK). Signal was detected using IRDye 800CW conjugated secondary antibodies on the Odyssey Imager (Li-cor, Westburg, Leusden, the Netherlands).
Histology, immunohistochemistry (IHC) and immunofluorescence (IF)
Paraformaldehyde fixed ileal tissues were embedded in paraffin, and 4-μm-thick sections were stained with hematoxylin and eosin to study the ileal morphology. For IHC and IF, antigen unmasking was carried out by heating the sections for 20 minutes in 0.01 M sodium citrate (pH 6.0) at 100 °C [15]. Antibodies against GRP78 (Santa Cruz Biotechnology, CA, USA), human alpha defensin 5 (HD5, HyCult Biotechnology, Uden, the Netherlands), and Forkhead box P3 (FOXP3, eBioscience, San Diego, CA, USA) were used in combination with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) and 3,3'-diaminobenzidine as staining reagent for IHC, or in combination with DyLight 594 conjugated secondary antibody and DyLight 488 conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for IF.
Statistical analysis
Data were presented as the total numbers (percentage), medians (interquartile range), or mean ± SEM. The comparisons were made by Fisher's exact test, Mann-Whitney test, or Kruskal-Wallis test followed by Dunns comparison. The data were considered statistically significant at P < .05.
ER stress and the UPR in NEC patients
Firstly, we analyzed splicing of XBP1 mRNA to determine the activation of the IRE1 pathway, 1 of the 3 ER stress sensors and a hallmark of the UPR. There was no XBP1s detected in R-CTRL (n=10) and R-NEC (n=13) patients, and XBP1s was only detected in 1 out of 5 A-CTRL patients (20%) and in 4 out of 12 A-NEC (33%) patients (Figure 1A and Table 2). Based on the occurrence of XBP1s, A-NEC patients were further divided into two subsets, A-NEC patients in whom XBP1s was not detected, i.e., unspliced, (A-NEC-XBP1u) and A-NEC patients in whom the XBP1s was detected (A-NEC-XBP1s). The patient demographics of the two subsets of A-NEC patients are shown in Table 4.
(A) Splicing of XBP1 mRNA was checked in all patients, and representative PCR products of XBP1u and XBP1s are shown using DNA electrophoresis. Dimethyl sulfoxide (DMSO)-treated HIEC cells were used as negative control for XBP1s, and TM-treated HIEC cells were used as positive control for XBP1s.
Mucosal mRNA expression levels of GRP78 (B) and CHOP (C) in the ileum of patients were quantified using qPCR and normalized to GAPDH mRNA levels. Asterisks indicate statistical significant differences between indicated groups.
(D) The Western blot result shows the representative protein expression of GRP78 and CHOP in A-NEC-XBP1u and A-NEC-XBP1s patients, and beta ACTIN was used as loading control.
|A-NEC-XBP1u n=8
|A-NEC-XBP1s n=4
|p Value
|6 (75.0%)
|4 (100.0%)
|Birth weight (gram)
|1001 (781, 1190)
|1501 (926, 1909)
|Gestational age at birth (weeks)
|27.8 (26.0, 29.7)
|30.8 (29.9, 32.2)
|Postnatal age at surgery (days)
|11.5 (7.8, 19.8)
|7.0 (4.3, 30.8)
|Gestational age at surgery (weeks)
|29.4 (27.8, 32.1)
|32.6 (31.4, 34.9)
|Survival (1 year after surgery)
|5 (62.5%)
|1 (25.0%)
Secondly, mRNA expression levels of activated transcription factor 4 (ATF4) [16] and growth arrest and DNA damage-inducible protein 34 (GADD34) [17] were analyzed to determine whether the PERK pathway, the second pathway sensing ER stress and the UPR, was activated. Neither increased ATF4 nor GADD34 mRNA expression was observed in any of the patient groups, and no significant differences in ATF4 and GADD34 mRNA expression levels were detected between A-NEC-XBP1s patients and A-NEC-XBP1u patients (Figure S2A and S2B).
Thirdly, to investigate the activation of the ATF6, the third sensor of ER stress and the UPR pathway, mRNA expression of protein disulfide isomerase family A member 4 (PDIA4) was quantified [6]. Similar to ATF4 and GADD34, no significant differences in PDIA4 mRNA expression levels were detected among the patient groups, and the PDIA4 mRNA expression in A-NEC-XBP1s patients was comparable to those in A-NEC-XBP1u patients (Figure S2C).
Finally, expression levels of GRP78 and CHOP, general markers of ER stress and the UPR, were determined in the ileum of patients by qPCR and/or Western blot. The GRP78 expression in R-NEC patients was higher than that in A-NEC-XBP1u patients. Interestingly, A-NEC-XBP1s patients expressed more GRP78 than A-NEC-XBP1u patients (Figure 1B). Similarly, CHOP mRNA expression levels in A-NEC-XBP1s patients were higher compared with A-NEC-XBP1u patients (Figure 1C). Western blot analysis also revealed increased GRP78 and CHOP protein expression in A-NEC-XBP1s patients compared with A-NEC-XBP1u patients (Figure 1D).
Intestinal morphology and localization of GRP78
In line with our previous study [18], most (7 out of 12) of A-NEC patients showed severe morphological damage characterized by crypt-villus loss and/or almost complete villus atrophy, i.e., crypt-villus ratio 1:1 (Figure 2A). Only 5 out of 12 A-NEC patients showed mild damage with relatively normal crypts and villi (Figure 2B). Focusing on A-NEC patients, 2 out of 4 A-NEC-XBP1s patients showed complete crypt-villus loss but a remaining surface epithelium (Figure 2C), and 1 A-NEC-XBP1s patient hardly had epithelium left (Figure 2D). In R-NEC patients, epithelial morphology was restored compared with A-NEC patients, and only mild mucosal damage was observed (Figure 2E).
Ileal tissues from NEC patients were stained with hematoxylin and eosin and representative sections are shown.
(A) Ileum of an A-NEC-XBP1u patient with severe morphological damage.
(B) Ileum of an A-NEC-XBP1u patient with mild morphological damage.
(C) Ileum of an A-NEC-XBP1s patient with complete crypt-villus loss but remaining surface epithelium.
(D) Ileum of an A-NEC-XBP1s patient who hardly had epithelium left.
(E) Ileum of an R-NEC patient with mild mucosal damage.
GRP78 positive (GRP78+) cells were observed in the submucosa, lamina propria, and epithelium of the ileum of all patients (Figure S3A and S3B). GRP78+ cells were also found at the base of crypts in most of R-CTRL and R-NEC patients (Figure S3B). In contrast, most of A-CTRL and A-NEC patients did not have GRP78+ cells at the base of crypts (data not shown). The position and morphology of these GRP78+ cells suggest that these cells are most likely Paneth cells. Therefore, we performed double-stainings for HD5, a specific Paneth cell marker, and GRP78. The GRP78+ cells at the base of crypts were also positive for HD5 (Figure S3C), indicating that these cells are indeed Paneth cells.
IL6 and IL8 expression levels in NEC patients
Gene expression levels of IL6 and IL8 were evaluated using qPCR. Mucosal IL6 mRNA expression was increased in A-NEC-XBP1s patients when compared with A-NEC-XBP1u patients, and both A-NEC-XBP1s and A-NEC-XBP1u patients showed higher IL6 mRNA expression than R-NEC patients (Figure 3A). There was a trend towards increased IL8 mRNA expression in A-NEC-XBP1s patients compared with A-NEC-XBP1u patients, and both A-NEC-XBP1s and A-NEC-XBP1u patients showed higher IL8 mRNA expression than R-NEC patients (Figure 3B).
The mRNA expression levels of IL6 (A,C & E) and IL8 (B, D & F) in the ileum of patients (A&B), THP-1 cells (C & D) and HIEC cells (E & F) were quantified using qPCR and normalized to the mRNA expression levels of GAPDH. Asterisks indicate statistical significant differences between indicated groups.
To analyze whether ER stress and the UPR can increase IL6 and IL8 expression directly, THP-1 cells and HIEC cells were treated with TM, an inducer of ER stress and the UPR. In both cell lines, TM-treatment induced ER stress and the UPR characterized by the induction of XBP1s (Figure 1A and Figure S4A) and up-regulation of GRP78 (Figure S4B to S4D). Importantly, TM-treatment indeed significantly up-regulated the mRNA expression levels of IL6 and IL8 in both cell lines (Figure 3C to 3F), which is similar to what was observed in A-NEC-XBP1s patients.
Altered cluster of differentiation 4 positive (CD4+) T cells differentiation in NEC patients
We determined the gene expression of hematopoietic cells (except erythrocytes and plasma/B-cells) and CD4+ T cells in A-NEC patients using cluster of differentiation 45 (CD45) and CD4 as markers, respectively. Mucosal mRNA expression levels of CD45 and CD4 were significantly decreased in A-NEC-XBP1s patients compared with A-NEC-XBP1u patients when normalized to GAPDH (Figure 4A and 4B), suggesting that overall there were less hematopoietic cells and CD4+ T cells in A-NEC-XBP1s patients. However, there were no significant differences in CD4 expression levels when normalized to CD45 (Figure S5A), implying that the numbers of CD4+ T cells relative to hematopoietic cells were not altered between A-NEC-XBP1s and A-NEC-XBP1u patients.
Mucosal mRNA expression levels of CD45 (A), CD4 (B), RORC (C), IL17A (D), FOXP3 (E) and CTLA4 (F) in the ileum of patients were quantified using qPCR and normalized to GAPDH mRNA levels. Asterisks indicate statistical significant differences between indicated groups.
Since there was an overall decrease in CD4 expression levels in A-NEC-XBP1s patients compared with A-NEC-XBP1u patients, we analyzed the expression of helper T (Th1, Th2 and Th17) cell markers and regulatory T (Treg) cell markers using GAPDH as internal control. Mucosal mRNA expression levels of T-box 21 (TBX21) and GATA binding protein 3 (GATA3) did not differ between A-NEC-XBP1s and A-NEC-XBP1u patients (Figure S5B and S5C), indicating that the total abundances of Th1 and Th2 cells were not changed between A-NEC-XBP1s and A-NEC-XBP1u patients. The mRNA expression levels of RAR-related orphan receptor C (RORC) and interleukin 17A (IL17A) were down-regulated in A-NEC-XBP1s patients compared with A-NEC-XBP1u patients (Figure 4C and 4D), demonstrating that there were less Th17 cells in A-NEC-XBP1s patients. Similarly, mRNA expression levels of FOXP3, a hallmark of immune-suppressive Treg cells, were down-regulated in A-NEC-XBP1s patients compared with A-NEC-XBP1u patients (Figure 4E), suggesting reduced Treg cell numbers in A-NEC-XBP1s patients. Interestingly, the mRNA expression levels of FOXP3 and cytotoxic T-lymphocyte antigen 4 (CTLA4) were up-regulated in R-NEC patients compared with A-NEC-XBP1u patients (Figure 4E and 4F), implying an enhanced immunosuppressive function in R-NEC patients.
We also identified FOXP3 positive (FOXP3+) cells by immunohistochemistry. FOXP3+ cells were detected in 6 out of 12 A-NEC patients and all R-NEC patients, and there was a trend towards increased abundance of FOXP3+ cells in R-NEC patients compared with A-NEC patients (data not shown). In A-NEC patients, FOXP3+ cells were confined to the lymph nodes, submucosa and lamina propria in the crypt region (Figure S6A). In R-NEC patients, besides the lymph nodes, submucosa and lamina propria in the crypt region, FOXP3+ cells also localized to lamina propria within the villi, which correlated with areas containing lymphocytic infiltrates. Remarkably, in some R-NEC patients FOXP3+ cells were associated with, and interspersed between, epithelial cells (Figure S6B).
Finally we normalized the expression of Th cell markers and Treg cells to CD4 mRNA to determine the relative amount of these cells compared with CD4+ cells. In A-NEC-XPB1s patients compared with A-NEC-XBP1u patients, the relative expression levels of TBX21 and GATA3 were up-regulated (Figure 5A and 5B), while the relative expression of RORC, IL17A and FOXP3 remained unaltered (Figure S5D to S5F). These data suggest that more CD4+ T cells had differentiated into a Th1 or Th2 cell than into a Th17 or Treg cell in the A-NEC-XPB1s patients.
Mucosal mRNA expression levels of TBX21 (G) and GATA3 (H) in the ileum of patients were quantified using qPCR and normalized to the mRNA expression levels of CD4. Asterisks indicate statistical significant differences between indicated groups.
ER stress and the UPR are involved in various chronic inflammatory diseases, including inflammatory bowel disease. However, the role of ER stress and the UPR in NEC, an acute inflammatory disease in premature infants, was unknown. In the present study, we first analyzed ER stress and the UPR in NEC patients, and we determined IL6 and IL8 expression and the differentiation of CD4+ T cells. Our data indicate that acute NEC patients with ER stress and activation of the UPR show increased IL6 and IL8 expression with altered T cell differentiation.
There are three main pathways (IRE1, PERK and ATF6) sensing ER stress. Splicing of XBP1 mRNA, which is activated by ER stress via IRE1 pathway, is associated with intestinal inflammation [5]. The spliced form XBP1s is a transcriptional activator of UPR target genes; whereas the unspliced form XBP1u is a transcriptional suppressor of UPR target genes [19]. XBP1s was detected in 1 out of 5 A-CTRL patients and in 4 out of 12 A-NEC patients, but not in R-CTRL and in R-NEC patients. Two general ER stress and UPR markers, GRP78 and CHOP, were both up-regulated in A-NEC-XBP1s patients compared with A-NEC-XBP1u and R-NEC patients, which confirmed the occurrence of ER stress and the UPR in A-NEC-XBP1s patients. Analysis of PERK and ATF6 pathways revealed that the expression levels of ATF4, GADD34 and PDIA4 were similar between the patient groups studied. With respect to these data, we suggest that PERK and ATF6 pathways were not activated, likely suggesting that only the IRE1 pathway was activated in A-NEC-XBP1s patients. These data indicate that ER stress and activation of the UPR plays a role in NEC, as ER stress and the UPR was observed in a subset of A-NEC patients, but might not occur in all NEC patients. A reason for this might be the complexity of this devastating disease. NEC can be caused by one or a combination of risk factors, i.e., bacterial colonization, formula nutrition and ischemia [20,21]. Because of this, one can consider NEC as a multi-factorial disease, most likely leading to differences in the pathogenesis. The occurrence of ER stress and the UPR in only a subset of A-NEC patients supports the multifactoriality of NEC.
We observed increased GRP78 expression levels and increased GRP78+ Paneth cell abundances in R-NEC patients compared with A-NEC-XBP1u patients. Paneth cells are among the most highly secretory cell types in mammals, and these cells synthesize large amount of secretory proteins including anti-microbial peptides [22]. To cope with the high basal protein synthesis level, Paneth cells express high basal levels of ER chaperones including GRP78. Studies report that the Paneth cell number is lower in the preterm infants compared with term infants [23,24]. This is in line with our results showing reduced HD5 positive and GRP78+ Paneth cells in A-CTRL and A-NEC patients. Paneth cells play a pivotal role in the innate immune response and host defense by producing anti-microbial peptides that shape the gut microbiota [25], and Paneth cell dysfunction has been suggested in NEC [26]. In a previous study we already demonstrated Paneth cell hyperplasia after recovery from NEC [27]. Therefore, the increased GRP78 expression in R-NEC patients might be due to up-regulated Paneth cell activity, i.e., increased protein synthesis, but this aspect needs more investigation.
There is growing evidence showing that IL6 and IL8 are involved in the pathogenesis of NEC [28]. In the present study, we detected increased mucosal IL6 and IL8 expression in A-NEC patients compared with R-NEC patients. Several studies suggest that plasma IL6 concentrations reflect the clinical severity of NEC [29–32]. Plasma IL8 levels were elevated in infants with NEC compared with those with sepsis [33], and it was also higher in infants with stage 3 NEC compared with those with less severe NEC [34]. Our results are in line with the previous studies analyzing IL6 and IL8 levels in plasma of NEC patients and imply an essential role for mucosal IL6 and IL8 in the pathogenesis of NEC. Interestingly, A-NEC-XBP1s patients showed increased IL6 expression and a trend towards increased IL8 expression compared with A-NEC-XBP1u patients. It has been reported that ER stress activated NF-κB [35], and XBP1s induces the production of IL6 and IL8 in various cell types [36–38]. Moreover, a positive correlation between GRP78 and IL8 expression in the intestine of IBD patients has also been observed [6]. Using the human monocytic cell line THP-1 and the fetal human intestine epithelial cell line HIEC, we confirmed the correlation between ER stress and increased IL6 and IL8 expression in vitro. Specifically, TM induced GRP78 expression, splicing of XBP1, and up-regulated the expression of IL6 and IL8 mRNAs in both cell lines.
Naive CD4+ T cells may differentiate into Th cells and Treg cells with their distinct cytokine profiles and immune functions [39]. Treg cells can suppress the effector functions of other types of T cells, and therefore are essential for the immune homeostasis [40]. Recently, reduced numbers of Treg cells were reported in NEC ileum compared with infants without NEC [41]. We did not find differences in FOXP3 and CTLA4 gene expression levels between age-matched A-CTRL and A-NEC patients or between R-CTRL and R-NEC patients, but we noticed that R-NEC patients expressed higher FOXP3 and CTLA4 levels than both A-NEC-XBP1u and A-NEC-XBP1s patients. Accordingly, compared with A-NEC patients, there was a trend towards to increased FOXP3+ cell in the ileum of R-NEC patients, and FOXP3+ cells appeared to be associated with epithelial cells of R-NEC patients exclusively, suggesting altered Treg cell differentiation and decreased immunosuppressive function in A-NEC patients. However, detailed functional studies are needed to further confirm the important role of Treg in NEC recovery since FOXP3 can also be induced in activated T effector cells [42].
Detailed analysis reveals that there might be a link between ER stress and altered CD4+ cells differentiation as seen in A-NEC-XBP1s patients. Total CD4 gene expression levels were lower in A-NEC-XBP1s patients than in A-NEC-XBP1u patients. Using the key transcription factors as T cell markers, we demonstrate that the total abundance of Th1 and Th2 cells remained unaltered, while the total abundance of Th17 cells and Treg cells were decreased in A-NEC-XBP1s patients compared with A-NEC-XBP1u patients. These data imply that decreased abundances in Th17 and Treg cells are the cause of the decrease in total abundance of CD4+ T cells. Accordingly, the relative amounts of Th1 and Th2 cells were higher in A-NEC-XBP1s patients when TBX21 and GATA3 were normalized to CD4. We also confirmed the altered Th17 cell differentiation using the signature cytokine IL17A. ER stress and the UPR can regulate Th17 cells, as overexpression of CHOP in developing Th17 cells suppresses IL17A production [43]. Th17 cells and IL17A can directly inhibit Th1 cells, and thereby suppress the development of intestinal inflammation [44]. Interestingly, it has been shown that the Th17-cell capacity is inversely related to developmental age and that preterm infants show a significant Th17-cell bias [45]. Therefore, it can be hypothesized that ER stress inhibits the differentiation of Th17 cells in A-NEC-XBP1s patients, whom were born prematurely, resulting in more severe inflammation and necrosis. Yet, further studies are necessary to confirm this hypothesis. Nevertheless, all data in conjunction point to an imbalance in CD4+ T cells differentiation in A-NEC-XBP1s patients. Additionally, changes in
RORC, IL17A and FOXP3 gene expression levels might also reflect a dysregulation of a group of innate immune cells called innate lymphoid cells. However, the exact role of these innate lymphoid cells in NEC needs further investigation.
XBP1s correlated with a poor epithelial morphology and surgical outcome. Two of 4 A-NEC-XBP1s patients showed complete crypt and villus loss, and 1 A-NEC-XBP1s patient only had limited epithelium left. Three of the 4 A-NEC-XBP1s patients died within 1 year after surgery. In contrast, A-NEC-XBP1u patients had relatively better epithelial morphology than A-NEC-XBP1s patients and surgical outcome was also better in A-NEC-XBP1u patients as 3 out of 8 A-NEC-XBP1u patients died within 1 year after surgery compared with 3 out of 4 A-NEC-XBP1s patients. However, there was no significant difference between the survival rates of A-NEC-XBP1s and A-NEC-XBP1u patients (25.0% versus 62.5%), probably due to the small patient numbers. Finally, in most R-NEC patients, the epithelial morphology was restored, and only 1 out of 13 R-NEC patients died within 1 year after surgery.
In summary, ER stress and the UPR correlate with the disease severity in a subset of NEC patients. Compared with A-NEC-XBP1u patients, A-NEC-XBP1s patients suffer from more severe inflammation, have increased ER stress and activated UPR, as demonstrated by XBP1s and increased GRP78 and CHOP expression, and have a worse surgical outcome. Accordingly, A-NEC-XBP1s patients express higher levels of IL6, and show a trend towards increased IL8 expression compared with A-NEC-XBP1u patients. The total abundance of CD4+ T cells is reduced in A-NEC-XBP1s patients compared with A-NEC-XBP1u patients, and T cells differentiation is also altered. Specifically, in A-NEC-XBP1s patients, Th17 and Treg functions seem suppressed and the relative amounts of Th1 and Th2 cells seem up-regulated in A-NEC-XBP1s patients. In line with this, the mucosal damage is more severe in A-NEC-XBP1s patients compared with A-NEC-XBP1u patients. Furthermore, R-NEC patients did not show splicing of XBP1 mRNA. In R-NEC patients, IL6 and IL8 are suppressed, Treg cells are up-regulated and epithelial morphology is restored. Together, these data imply that that prevention/avoidance of ER stress and the UPR in NEC patients may contribute to improved disease outcome.
Supporting Information
Figure S1.
Three pathways of ER stress and the UPR. IRE1 functions as an endoribonuclease and protein kinase. Upon activation, IRE1 removes a 26-bp nucleotide fragment from cytosolic unspliced XBP1 mRNA to generate spliced XBP1 which encodes a transcription factor inducing UPR target genes.
During ER stress, PERK is autophosphorylated, resulting in phosphorylation of EIF2A. This in turn leads to an arrest in protein translation and accumulation of ATF4. GADD34 is also induced by the PERK pathway and dephosphorylates EIF2A to restores protein translation.
Upon ER stress, ATF6 is cleaved and the active ATF6 p50 subunit moves to the nucleus to modulate gene expression such as PDIA4.
Figure S2.
Expression of genes of PERK and ATF6 pathways in the ileum of patients. The mucosal mRNA expression levels of ATF4 (A), GADD34 (B) and PDIA4 (C) in the ileum of patients were quantified using qPCR and normalized to the mRNA expression levels of GAPDH.
Figure S3.
Localization of GRP78 in the ileum of NEC patients. The localization of GRP78 in the ileum of NEC patients was investigated using IHC (A & B) and IF (C).
(A) Representative staining of GRP78 in A-NEC patients is shown. Black arrows indicate the GRP78+ cells in submucosa (SM), lamina propria (LP) and surface epithelium (SE).
(B) Representative staining of GRP78 in R-NEC patients is shown. Black arrows indicate the GRP78+ cells in SM, LP, SE and base of crypts (BC).
(C) Representative IF staining shows the co-localization of GRP78 and HD5 in the Paneth cells in R-NEC patients. Green signals in the top-left panel show the GRP78 staining, red signals in the top-right panel show the HD5 staining, and the bottom-left panel shows the merged signals. White arrows indicate the GRP78 and HD5 co-localized Paneth cells.
Figure S4.
TM-induced ER stress and the UPR in vitro. (A) The splicing of XBP1 induced by TM was detected, and the representative PCR products of XBP1u and XBP1s in THP-1 cells are shown using DNA electrophoresis.
(B) The induction of GRP78 protein expression by TM in HIEC and THP-1 cells was demonstrated using Western blot.
The mRNA expression levels of GRP78 in the DMSO-treated and TM-treated THP-1 (C) and HIEC (D) cells were quantified using qPCR and normalized to the mRNA expression levels of GAPDH. Asterisks indicate statistical significant differences between DMSO and TM groups.
Figure S5.
Expression of CD4+ T cells in the ileum of patients. Mucosal mRNA expression levels of CD4 in the ileum of patients were quantified using qPCR and normalized to the mRNA expression levels of CD45 to demonstrate the amount of CD4+ cells in all hematopoietic cells (A). Mucosal mRNA expression levels of TBX21 (B) and GATA3 (C) in the ileum of patients were quantified using qPCR and normalized to the mRNA expression levels of GAPDH. Mucosal mRNA expression levels of RORC (D), IL17A (E) and FOXP3 (F) in the ileum of patients were quantified using qPCR and normalized to the mRNA expression levels of CD4.
Figure S6.
Localization of FOXP3+ cells in A-NEC and R-NEC patients. (A) Representative FOXP3 stainings in A-NEC patients is shown. Black arrows indicate the FOXP3+ cells in the lymph node (LN), submucosa (SM), and lamina propria in the crypt region (LPC).
(B) Representative FOXP3 stainings in R-NEC patients is shown. Black arrows indicate the FOXP3+ cells in the LN, SM, LPC and lamina propria within the villi (LPV). Black triangles indicate the FOXP3+ cells which interspersed between epithelial cells (IBE). The bottom-right panel with an asterisk represents a higher magnification of IBE.
We are grateful for the help in tissue acquisition of Dr. Nanda Burger-van Paassen, Dr. Patrycja Puiman and our surgical colleagues. We also thank Prof. Jean-François Beaulieu for providing HIEC cells.
Author Contributions
Conceived and designed the experiments: PL MCS JBG IBR. Performed the experiments: PL JM JWB AMKM ACJMB. Analyzed the data: PL MCS. Wrote the manuscript: PL IBR. Final approval of manuscript: JBG IBR.
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Fresh Fruit Delivered for Any Occasion
Pittman and Davis offers a variety of fresh citrus gifts. Choose from sweet navel oranges, big ruby red grapefruit and Texas Grapefruit, to other seasonal fruit selections, such as Pears, Clementines, Tangelos and other gourmet fruit gift ideas. Texas Grapefruit and other Texas Citrus are our specialities! Send citrus gifts for all occasions, from an anniversary, birthday or corporate gift idea, to even a wedding. Pittman & Davis' gifts taste as delicious as they look!
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Our guarantee is simple: You'll be absolutely delighted with every citrus fruit gift and gourmet food item you send or we'll either replace your gift, or refund your purchase price. Start shopping today for your citrus gifts. Order from Pittman and Davis, directly at PittmanDavis.com


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The Foundation helmet from Sector 9 was made to protect your head while you ride hard.
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There's been a thread about it here already, it might have some info for you in it already. From what I see, it is legitimate, and should be automatic if you're linked to your PowerUp account here, but apparently there's been some trouble with it for some people.
Forums → Kongregate →
Power Up Rewards Kong Plus (locked)
1 post


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Wholistic Beauty Connected™
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Wholistic Beauty Connected is a bath, body and wellness store. We carry our own line of products formulated with wholesome natural ingredients. We also sponsor wellness workshops as a catalyst to beginning the process of self-nurturing and healing. Simply put, we are here to help you remember how to be kind to yourself.
We are located in the Russell Trade Center SUITE 401, come visit us, you will be pleasantly enlightened. Contact Phone: 313.923.7119 (enter the Trade Center through the "DOCK" doors
visit us at: wholisticbeautyconnected.com


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4 Comments
Realtime comments disabled
TheNetworkGeek 1858 days ago
Wow, that's some heavy Photoshopping by AdWeek!
tylerwillis 1858 days ago
Big day for Barsha Ray (how I think it happened: they did a google image search, this photo came up because Barsha Ray is a comedian who's name showed up next to Baratunde's on a comedy event website). Fact checking, for the win.
Google Search: http://b
cc_chapman 1858 days ago
Wow, someone at AdWeek needs a slapping *laugh*
OriginalNajeema 1858 days ago
How did they make this mistake? I don't understand how something like this happens.


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[Watch] Here's What Happens When Your Side Chick Crashes Your Wedding
It's a scene out of a Tyler Perry movie.
Though critics have been skeptical about Lauryn Hill's comeback, she is showing no signs of being fazed. The Grammy Award-winning star announced 12 additional stops on her intimate music showcase and, this weekend, made her debut at the Coachella Music Festival in Southern California.
Though there were sound issues, L-Boogie kept the energy high and ploughed through some of the hit tracks off her classic album, The Miseducation of Lauryn Hill. Head to Rap Radar and watch the video from Coachella. Judging from the clip, it seems safe to say Hill is on her way back to the top.
It's a scene out of a Tyler Perry movie.
A few fellow students have taken to Instagram displaying an appalling lack of remorse


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Glee Cast - I Won't Give Up Soundtrack Lyrics
Glee Cast – I Won't Give Up Soundtrack Lyrics [ from "Glee: The Music, The Graduation Album" TV (2012) ]
When I look into your eyes
It's like watching the night sky
Or a beautiful sunrise
There's so much they hold
And just like them old stars
I see that you've come so far
To be right where you are
How old is your soul?
I won't give up on us
Even if the skies get rough
I'm giving you all my love
I'm still looking up
And when you're needing your space
To do some navigating
I'll be here patiently waiting
To see what you find
'Cause even the stars they burn
Some even fall to the earth
We've got a lot to learn
God knows we're worth it
No, I won't give up
I don't wanna be someone who walks away so easily
I'm here to stay and make the difference that I can make
Our differences they do a lot to teach us how to use
The tools and gifts we got yeah, we got a lot at stake
And in the end, you're still my friend at least we did intend
For us to work we didn't break, we didn't burn
We had to learn how to bend without the world caving in
I had to learn what I've got, and what I'm not
And who I am
I won't give up on us
Even if the skies get rough
I'm giving you all my love
I'm still looking up
Still looking up.
I won't give up on us (no I'm not giving up)
God knows I'm tough enough (I am tough, I am loved)
We've got a lot to learn (we're alive, we are loved)
God knows we're worth it (and we're worth it)
I won't give up on us
Even if the skies get rough
I'm giving you all my love
I'm still looking up
Share "Glee Cast I Won't Give Up" Lyrics
Soundtrack Information
|Glee: The Music, The Graduation Album TV (2012)
|"Glee: The Music, The Graduation Album" TV (2012)
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Watch The Video of these Lyrics
For this Video look below Lyrics
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