Tuesday, January 29, 2013

Nutrition & Crohn's Disease: Vitamins & Supplement Information From WebMD

Vitamins for Crohn's Disease: Supplement Information From WebMD:

Vitamins for Crohn's Disease

If you have Crohn's disease, good nutrition is crucial so you can stay as healthy as possible. Unfortunately, the disease -- as well as treatments for it -- can make it much harder to get enough of the vitamins and minerals you need.

Doctors often recommend vitamins for Crohn's disease to work around this problem. Whether you need Crohn's disease vitamins -- and which ones -- depends on your case and the your medical treatments.

Here's a rundown of the minerals and vitamins for Crohn's disease that your body might not be getting -- and advice on how to get more of them.

Crohn's Disease Nutrition

Poor nutrition has real risks if you have Crohn's disease. You may feel run-down and sick. Medications may not work as well. In children and teens, poor nutrition related to Crohn's disease can stunt growth.

Why does Crohn's disease affect nutrition? There are several reasons.

Inflammation and damage to the small intestine fromCrohn's diseasecan make it hard for the body to absorb substances from food, such as carbs, fats, water, and many vitamins and minerals. Surgery for Crohn’s may also make it more difficult to absorb nutrients.
Reduced appetite -- from pain, diarrhea, anxiety, and changes in taste -- makes it hard to eat enough.
Some medications for Crohn's disease make it harder to absorb nutrients.
Internal bleeding in the digestive tract can lead to anemia, which can cause low levels of iron.

Crohn's Disease Nutrition: What's Missing?

What vitamins and minerals are missing in your diet? People with Crohn's disease are likely to have lower levels of:

Vitamin B12. After surgery in the ileum -- the lower section of the small intestine -- it may not be possible to absorb enough vitamin B12. Dietary changes and oral vitamins can help. Some people with Crohn's disease need injections of vitamin B12 or a B12 nasal spray.
Folic acid. Some drugs for Crohn's disease, such as sulfasalazine or methotrexate, can lower levels of folic acid. A daily 1 mg dose of a folate supplement could help.
Vitamin D. Studies have shown the people with Crohn's disease often have low levels of vitamin D, which helps your body absorb calcium for strong bones. Many people with Crohn's disease take an 800 IU supplement of vitamin D daily.
Vitamin A, vitamin E, and vitamin K can be low in people who have trouble absorbing fats because of surgery for Crohn's disease.
Calcium. Steroids for Crohn's disease can weaken bones and affect your body’s ability to absorb calcium. On top of that, some people with Crohn's disease avoid milk because they're also lactose intolerant, further reducing calcium. Up to 50% of people with Crohn's have osteopenia, or thinning of the bones. Taking additional supplements -- often 1,500 mg of calcium a day -- can help keep bones strong and prevent other problems.
Iron. People with active Crohn's disease may develop anemia from blood loss in the intestines. The best treatment for anemia is with iron. Most people take iron tablets or liquid, but some get infusions instead.
Potassium, magnesium, and zinc may be lower in people with Crohn's disease. Taking a daily supplement can help.
Vitamins for Crohn's Disease: Diet vs. Supplements
In general, any dietitian or nutritionist would tell you it's better to get your nutrition from whole foods instead of supplements.
For some people with Crohn's disease, that's just not possible. Because of absorption problems, pain, and nausea, it may be hard to eat enough of the healthy foods that would give you the nutrition you need.
What's more, Crohn's disease -- especially when it's active -- can make your body work harder. You may need more calories and nutrients than normal -- precisely at the time when it's hard to eat. Some healthy foods, such as high-fiber nuts and seeds, can also trigger flares in some people with Crohn's disease.
In these cases, vitamin supplements for Crohn's disease can help fill any gaps in your nutrition.
Crohn's Disease Nutrition: Working With Your Doctor
While supplements can be a good idea for some people with Crohn's disease, don't start treating yourself with handfuls of vitamins every day. That's risky.
Talk to your doctor first. Some supplements could interact with your medication or make your Crohn's symptoms worse.
Before suggesting supplements, your doctor may want to check you for nutritional deficiencies. The doctor may test your levels of iron, vitamin D, vitamin B12, and other vitamins and minerals. The vitamins you need may also depend on where the damage is in your small intestine.
Schedule an appointment with a doctor. Getting good nutrition -- whether it's through dietary changes or supplements -- is essential to protect your health and help you feel better with Crohn's disease.

WebMD Medical Reference
View Article Source
Reviewed by Louise Chang, MD on April 02, 2012
© 2012 WebMD, LLC. All rights reserved.

Follow my blog with Bloglovin

Friday, January 25, 2013

Donor Feces Cure Recurrent Diarrheal Infection, Study Shows - Bloomberg

Ummmm lol I still don't know about this. There's just something that seems wrong about receiving an infusion..... of feces!

Donor Feces Cure Recurrent Diarrheal Infection, Study Shows - Bloomberg:

An infusion of feces through the nose beat the standard antibiotic as a treatment for a recurrent diarrhea-causing infection that kills about 14,000 Americans a year, a study showed.

In a trial among 43 people with recurrent diarrhea caused by a persistent bug called Clostridium difficile, 81 percent of those who received an infusion of feces from a healthy donor after treatment with vancomycin were cured, compared with only 31 percent of those treated with the drug alone, researchers from the University of Amsterdam wrote in an article published yesterday by the New England Journal of Medicine. The study was stopped early because the treatment was clearly working.

The results validate an approach called fecal microbiota transplantation, or FMT, pioneered in 1958 by doctors in Denver, and provides an alternative for the approximately one-in-four patients whose infection isn’t cleared by initial antibiotic treatment. C. difficile is the most common cause of hospital- acquired infectious diarrhea in the U.S. and costs about $1 billion a year to treat, according to the Centers for Disease Control and Prevention.

“Only patients with the most recalcitrant cases of C. difficile infection are likely to undergo FMT, usually out of desperation after multiple treatment approaches have failed,” Ciaran Kelly, a professor of medicine at Harvard Medical School, wrote in an editorial accompanying the study. The use of feces may be eliminated by the use of cultured bacteria that make the recipient resistant to C. difficile, Kelly said.

Restoring Bacteria

The procedure probably works by restoring normal gut bacteria, the researchers, led by Els van Nood, wrote in the study, which was funded by the Netherlands Organization for Health Research and Development and the Netherlands Organization for Scientific Research.

Vancomycin is sold as Vancocin by ViroPharma Inc. (VPHM) Vancocin accounted for $289 million in sales in 2011, more than half of Exton, Pennsylvania-based ViroPharma’s revenue.

A federal judge last week dismissed the company’s lawsuit against the U.S. Food and Drug Administration over generic forms of the antibiotic developed by Akorn Inc. (AKRX) and Watson Pharmaceuticals Inc. (WPI)

Optimer Pharmaceuticals Inc. (OPTR)’s Dificid, approved for sale in the U.S. in May 2011, was the first drug cleared for the infection in 25 years.

By Simeon Bennett - Jan 17, 2013 6:28 AM ET

Wednesday, January 23, 2013

Probiotics Made w/ Genetically Modified Fillers... Are they organic??

This article was on www.naturalnews.com and to tell you the truth, I don't know what to believe. Almost sounds like the writer is pushing the Sun Biotics brand.. I however, don't put much past the companies that manufacturer & produce the stuff we put in our body. I try to have good faith in whatever it is I buy that the company is not corrupt. Hard to tell though. I guess it's a good idea to find a certified organic probiotic from now on. I

Shock finding: Most probiotic supplements made with genetically modified flow agents, fillers:

(NaturalNews) A Natural News investigation into the probiotics industry has turned up alarming information about how probiotics are formulated and labeled. We've found that nearly all probiotics available in the U.S. market today are secretly formulated with genetically modified ingredients that are intentionally not listed on the label. The most common such ingredient is maltodextrin, a corn-derived flow agent.

Look for yourself at the probiotics available today, and you'll find something curious: Virtually none of them are certified USDA organic. Why is that? If you read the ingredients on the label, you might scratch your head and wonder why they can't make those ingredients organic. But the real story is found in the "dirty little secret" that probiotics supplement manufacturers are not listing on the labels: Maltodextrin and other fillers and flow agents that are derived from GM corn.

You are being lied to about probiotics ingredients

Here's the rock-bottom truth about probiotics: You are being LIED to about what they're made from because the FDA does not require manufacturers to list fillers and flow agents as ingredients.

Yep, you read that right: A probiotics manufacturer can add genetically modified corn maltodextrin to their formula and they are not required to disclose this on the label. While the maltodextrin will disqualify them from receiving organic certification, it doesn't stop them from using the word "natural" on their bottles.

Our Natural News investigation found that some probiotics contain as much as 30% maltodextrin in their formulas. Virtually all maltodextrin available today comes from genetically modified corn.

This means that consumers who think they're buying a healthy product to support digestion are actually buying products made from corn plants that are engineered to grow insecticide toxins right inside each and every kernel. Processing this into refined maltodextrin does not necessarily remove this toxin, meaning maltodextrin itself may be contaminated with the bt toxin that functions as an insecticide.

Check your probiotics label right now

If you take probiotics, check your label right now: Is it certified organic?

If not, it may contain maltodextrin derived from genetically modified corn, even if maltodextrin is not listed on the label!

You might be eating genetically modified corn byproducts and not even know it. Check with the manufacturer of your supplement and find out if they use maltodextrin or other corn-derived flow agents.

For the record, not all probiotics we investigated were made using maltodextrin, but the vast majority were. I'm not naming brands here because I don't care to be sued by a dozen manufacturers for blowing the whistle on their product formulation schemes that expose their customers to GMOs. But it's easy to see this for yourself: If it's not certified organic, it likely contains genetically modified ingredients.

How to really avoid GMOs in probiotics

The answer is simple: Buy certified USDA organic probiotics.

GMO-derived ingredients and flow agents like maltodextrin are NOT allowed to be used in certified organic formulations. And the USDA organic program is very stringent. I know this because I personally oversee production of certified organic products, and it's very detailed and very strict. Every batch of raw ingredients must be meticulously tracked, and manufacturers are audited once a year, on site, by USDA-approved inspectors who go through all your books and records to verify you're not cutting corners.

If you try to locate certified organic probiotics, you'll find there are virtually none in the marketplace. This should be your first clue that something's up. Red flag time. Why are there virtually no certified organic probiotics available today? Because the vast majority of probiotics supplements use genetically modified corn maltodextrin! Therefore, they are disqualified from USDA organic certification.

I take probiotics, and in my own search for certified USDA organic probiotics, I finally found the Sunbiotics brand. This, my friends, is the solution we've all been looking for.

Sunbiotics certified USDA organic chewable probiotics

This is the absolute best probiotic supplement available anywhere in the world today. Nothing compares to it, in my view, and I've been investigating the nutritional supplements industry for over a decade.

The Sunbiotics certified USDA organic chewable probiotics tablets are made with friendly bacteria strains carried on organic yacon root powder. (Yacon is a superfood from Peru.) The other ingredients are organic coconut sugar (delicious and low glycemic), organic vanilla flavor and silica. The silica is plant-derived. It's the same silica you get from the horsetail herb (a plant, not a horse's tail, obviously).

Each tablet delivers 18 billion CFUs of four probiotic strains:

• Lactobacillus acidophilus
• Bifidobacterium bifidum
• Bifidobacterium lactis
• Bifidobacterium longum

These Sunbiotics chewable tablets taste like yacon root candy! They're really delicious. Plus, there's no refrigeration required.

Click here to order Sunbiotics USDA organic probiotics from the Natural News store.

No soy, no dairy, no GMO, no wheat, no refined sugars, no corn, no nonsense!

What's especially remarkable about these Sunbiotics chewable supplements is what they do NOT contain. This is the whole point, of course: They do NOT contain soy, corn, wheat, refined sugars and so on.

No hidden ingredients whatsoever. No secret GMO maltodextrin like we found in other brands.

If you're looking for a trusted source of USDA organic probiotics that are delicious and chewable -- and even made with a superfood! -- look no further. This is it. This is now my top recommended source for probiotics, and it's what I'm taking.

The only problem with these is that they taste so good you actually want to eat them more than once a day.

Learn more: http://www.naturalnews.com/038783_probiotics_maltodextrin_GMOs.html#ixzz2IoS7PIlS

'via Blog this'

Friday, January 18, 2013

Fecal transplants cure diarrhea!! Wow what will they think of next

Interesting!!! Nuff said about this one.

Fecal transplants cure diarrhea, modulate testosterone levels

Nutella was not used as a placebo control.

by John Timmer - Jan 18 2013, 1:35pm EST

From some perspectives, we humans aren't really so much individuals as we are walking ecosystems—our bodies carry more bacterial cells—with their own genomes and agendas—than the total count of human cells we're composed of. Bacteria cover our skin, get to our food before we have the chance to absorb it, and in many cases stay helpfully out of the way of the immune system.

Given all that, it shouldn't be a surprise that we're finding that bacteria can have significant effects on the human body in ways that go well beyond causing an infection. Two articles that appeared this week drive that home. In one, doctors cured a recurring, diarrhea-causing infection simply by transplanting gut bacteria from a healthy individual. And in the second, the bacterial transplants altered the progression of type 1 (autoimmune) diabetes in mice—by altering the animal's testosterone levels.

Fighting bacteria with bacteria

Clostridium difficile, or C. diff, is a bacteria that tends to cause extended bouts of diarrhea. In about 20 percent of the cases that end up under a doctor's care, it will get into the digestive system and refuse to come back out, creating recurring bouts of illness that don't respond to most antibiotics. At that point, the standard of care is an intensive course of vancomycin, which only works in about 60 percent of patients. With each further recurrence, the rate of success goes down even further.

Anecdotal evidence and case reports had suggested that the problem wasn't so much the presence of C. diff as the fact that it had grown out of balance with the rest of the gut's bacterial ecosystem. To get the gut back into balance, fecal transplants had been tried and, anecdotally at least, they worked. So some doctors in the Netherlands decided to do a clinical trial, comparing a fecal transplant to standard care with vancomycin. The trial was what the researchers called "open label," meaning that people were aware they were having a feeding tube stuffed down their nose to deliver someone else's poop into their body. (A Twitter pundit suggested a Nutella infusion might make for a good placebo control.)

A few dozen healthy volunteers were screened for a huge panel of infectious diseases, and those who came through clean were asked to rush their bodies' first deliveries of the day to the hospital, where it was mixed with saline and had the particulates removed. The results then went into the gastro-nasal tube, given to patients after their original gut flora had been cleared out by a "bowel lavage."

The authors had originally planned to get 40 patients for each group, but the fecal transplants were so successful, they stopped the trial after only 16 patients had received a transplant. Of these, 13 (80 percent) were cured after a single transplant. Two of the remaining three were cured after their second, bringing the success rate up to over 90 percent. In contrast, the success rate of vancomycin treatments was down around 30 percent.

The biggest problem? Enrolling patients. Most people who agreed to participate in the trial only did so after conventional treatments failed several times, "reflecting the reluctance of patients and physicians to choose donor-feces infusion at an early stage."

Bacteria, sex, and immunity

It's easy to view this as a demonstration of the hygiene hypothesis, which posits that all sorts of health issues are linked to exposure to a variety of infectious agents, which the immune system then learns to live in harmony with. But a second paper appeared this week that cautioned against viewing things as being quite that simple.

The paper focused on the progression of type 1 diabetes, which is the product of an autoimmune attack on the insulin-producing cells. There's a special strain of mice, called NOD (for non-obese diabetic) that are prone to developing this disorder. The mice show properties that are very much like the human version of the disease: it's genetically complex, the progression is influenced by environmental factors, and it strikes females more severely than males.

And, unexpectedly, it's influenced by gut bacteria. There was some hint of this, given that other researchers had shown that a systematic exposure to bacterial proteins was able to suppress the development of the disease. But the authors found an unexpected effect when they tested how the disorder progressed in mice raised in germ-free conditions. Rather than accelerating the development of symptoms in all mice, the germ-free conditions accelerated the progression in males, making them (at least in this assay) indistinguishable from females.

So the researchers tried an experiment: they took germ-free NOD mice and did a fecal transplant from adult male animals (mice are naturally coprophagic, so the mice took a lot less convincing than did the humans). When female NOD mice received gut bacteria from males, it actually slowed the disease progression down. The apparent sex difference in autoimmune function was mediated in part by gut bacteria.

Your first thought might be that testosterone in males could create a different environment in the gut, causing it to host a different diversity of species. It's a reasonable guess, but it's wrong. Instead, the researchers found that the transplant of gut bacteria caused a surge in testosterone production by females that lasted for up to 14 weeks. This had no effect on the female's fertility, but it did clearly alter immune function. If the authors injected these mice with a testosterone inhibitor, the diabetes protection went away.

All of which indicates that the other intuitive idea—that gut bacteria influence immune function by interacting directly with immune cells—also can't be right, or at least can't be everything. Clearly, the production of testosterone, by whatever cells may be producing it, plays a key role.

Although the papers argue against some of the simpler views of human health—bacteria are all bad, or not having exposure to pathogens means an overactive immune system—they both argue that viewing our bodies as a complex ecosystem can help provide insight into human health.

New England Journal of medicine, 2013. DOI: 10.1056/NEJMoa1205037 and Science, 2013. DOI:10.1126/science.1233521 (About DOIs).

Fecal transplants cure diarrhea, modulate testosterone levels | Ars Technica:

'via Blog this'

Uceris Approved for Active Ulcerative Colitis #UC #IBD

Uceris sounds just like a drug that has been around for a while, Entercort (spelling?). It works the same exact way Uceris works; it's a form of a steroid and releases in the colon. Wonder what the difference is?/

January 15, 2013

Uceris Approved for Active Ulcerative Colitis

Santarus announced that the FDA has approved Uceris (budesonide) extended-release tablets for the induction of remission in patients with active, mild to moderate ulcerative colitis.

Uceris contains budesonide in a novel oral tablet formulation that utilizes proprietary MMX multi-matrix system technology, which is designed to result in the controlled release and distribution of budesonide throughout the length of the colon. Budesonide is a corticosteroid known to have localized anti-inflammatory effect.

Uceris will be available in 9mg extended-release tablets in 30-count bottles. Uceris is expected be available in March 2013.

For more information call (858) 314-5700 or visit www.santarus.com

Uceris Approved for Active Ulcerative Colitis - MPR:

http://www.drugs.com/newdrugs/santarus-receives-fda-approval-uceris-budesonide-induction-remission-patients-active-mild-moderate-3638.html

FDA Approves Uceris

Santarus Receives FDA Approval of Uceris (budesonide) for the Induction of Remission in Patients with Active, Mild to Moderate Ulcerative Colitis

SAN DIEGO--(BUSINESS WIRE)--Jan 15, 2013 - Santarus, Inc. announced today that the U.S. Food and Drug Administration (FDA) has approved Uceris (budesonide) extended release tablets for the induction of remission in patients with active, mild to moderate ulcerative colitis. The company expects to commence the commercial launch of Uceris in March 2013.

“The FDA approval of Uceris provides an important new therapeutic option to patients and physicians for the treatment of active, mild to moderate ulcerative colitis,” said William J. Sandborn, M.D., chief, division of Gastroenterology, director, University of California San Diego (UCSD) IBD Center and professor of clinical medicine, UCSD Health System. “Specifically, Uceris is indicated for use in the induction of remission of active disease, an acute phase of the disease often characterized by cramping, bloating, diarrhea, bleeding, fatigue, weight loss and frequent bowel movements.”Uceris contains budesonide, a corticosteroid, in a novel oral tablet formulation that utilizes proprietary MMX^® multi-matrix system colonic delivery technology. The approved dosing regimen for adult patients is one 9 mg tablet taken orally once daily in the morning for up to 8 weeks. Uceris was developed in collaboration with Cosmo Technologies Limited, a subsidiary of Cosmo Pharmaceuticals S.p.A.

Gerald T. Proehl, president and CEO of Santarus, said, “We are pleased to provide a new option for patients to treat the active phase of mild to moderate ulcerative colitis. With the incremental revenue from Uceris and continued growth in our marketed products, we anticipate another robust year of solid financial results for Santarus in 2013. We expect total revenues of $320 million to $325 million, representing significant double-digit growth over our 2012 revenue guidance."

Important Safety Information About Uceris

Uceris is a prescription corticosteroid medicine used to help get mild to moderate ulcerative colitis under control. Uceris is taken once daily in the morning with or without food for up to 8 weeks.

Do not take Uceris if you are allergic to budesonide or any of the ingredients in Uceris.
Before you take Uceris, tell your doctor if you have liver problems, are planning to have surgery, have chickenpox or measles or have recently been near someone with chickenpox or measles, have or had a family history of diabetes, cataracts or glaucoma, have high blood pressure (hypertension), decreased bone mineral density (osteoporosis), stomach ulcers, any other medical condition, are pregnant or plan to become pregnant, or breastfeed or plan to breastfeed.
Tell your doctor about all the medications you take, including prescription and over-the-counter vitamins and herbal supplements. Uceris and other medicines may affect each other, causing side effects.
Do not eat grapefruit or drink grapefruit juice while taking Uceris because these can increase the level of Uceris in your blood.
Long-time use of Uceris can cause you to have too much glucocorticosteroid medicine in your blood (hypercorticism). Tell your doctor if you have any of the following signs and symptoms of hypercorticism: acne, bruise easily, rounding of your face (moon face), ankle swelling, thicker or more hair on your body and face, a fatty pad or hump between your shoulders (buffalo hump), or pink or purple stretch marks on the skin of your abdomen, thighs, breasts, and arms.
When Uceris is taken for a long period of time, the adrenal glands do not make enough steroid hormones. Tell your doctor if you are under stress or have any symptoms of adrenal suppression during treatment with Uceris, including tiredness, weakness, nausea and vomiting, and low blood pressure.
Uceris weakens your immune system. Taking medicines that weaken your immune system makes you more likely to get infections. Avoid contact with people who have contagious diseases such as chickenpox or measles while taking Uceris. Tell your doctor about any signs or symptoms of infection, including fever, pain, aches, chills, feeling tired, or nausea and vomiting.
If you take certain other corticosteroid medicines to treat allergies (e.g., eczema, rhinitis), switching to Uceris may cause your allergies to come back. Tell your doctor if any of your allergies become worse while taking Uceris.
The most common side effects with Uceris are headache, nausea, decreased blood cortisol levels, stomach-area pain, tiredness, stomach or intestinal gas, bloating, acne, urinary tract infection, joint pain, and constipation.

Uceris is available by prescription only. For additional information, talk to your healthcare provider and see the Full Prescribing Information including Patient Labeling.

Wednesday, January 9, 2013

LDN to treat Autoimmune Diseases - Clinical Trials

LDN Clinical Trials:
Logo LDNscience.org

'via Blog this'

Clinical Trials

Am J Gastroenterol. 2007 Apr;102(4):820-8. Epub 2007 Jan 11.

Low-dose naltrexone therapy improves active Crohn's disease.

Smith JP, Stock H, Bingaman S, Mauger D, Rogosnitzky M, Zagon IS.

Department of Medicine, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.

OBJECTIVES: Endogenous opioids and opioid antagonists have been shown to play a role in healing and repair of tissues. In an open-labeled pilot prospective trial, the safety and efficacy of low-dose naltrexone (LDN), an opioid antagonist, were tested in patients with active Crohn's disease.

METHODS: Eligible subjects with histologically and endoscopically confirmed active Crohn's disease activity index (CDAI) score of 220-450 were enrolled in a study using 4.5mg naltrexone/day. Infliximab was not allowed for a minimum of 8 wk prior to study initiation. Other therapy for Crohn's disease that was at a stable dose for 4 wk prior to enrollment was continued at the same doses. Patients completed the inflammatory bowel disease questionnaire (IBDQ) and the short-form (SF-36) quality of life surveys and CDAI scores were assessed pretreatment, every 4 wk on therapy and 4 wk after completion of the study drug. Drug was administered by mouth each evening for a 12-wk period.

RESULTS: Seventeen patients with a mean CDAI score of 356 +/- 27 were enrolled. CDAI scores decreased significantly (P=0.01) with LDN, and remained lower than baseline 4 wk after completing therapy. Eighty-nine percent of patients exhibited a response to therapy and 67% achieved a remission (P < 0.001). Improvement was recorded in both quality of life surveys with LDN compared with baseline. No laboratory abnormalities were noted. The most common side effect was sleep disturbances, occurring in seven patients.

CONCLUSIONS: LDN therapy appears effective and safe in subjects with active Crohn's disease. Further studies are needed to explore the use of this compound.

PMID: 17222320

Integr Cancer Ther. 2007 Sep;6(3):293-6.

Reversal of signs and symptoms of a B-cell lymphoma in a patient using only low-dose naltrexone.

Berkson BM, Rubin DM, Berkson AJ.

Integrative Medical Center of New Mexico, Las Cruces, USA.

PMID: 17761642

Mult Scler. 2008 Sep;14(8):1076-83.

A pilot trial of low-dose naltrexone in primary progressive multiple sclerosis.

Gironi M, Martinelli-Boneschi F, Sacerdote P, Solaro C, Zaffaroni M, Cavarretta R, Moiola L, Bucello S, Radaelli M, Pilato V, Rodegher M, Cursi M, Franchi S, Martinelli V, Nemni R, Comi G, Martino G.

Institute of Experimental Neurology (INSPE) and Department of Neurology, San Raffaele Scientific Institute, Via Olgettina 58, Milan, Italy.

A sixth month phase II multicenter-pilot trial with a low dose of the opiate antagonist Naltrexone (LDN) has been carried out in 40 patients with primary progressive multiple sclerosis (PPMS). The primary end points were safety and tolerability. Secondary outcomes were efficacy on spasticity, pain, fatigue, depression, and quality of life. Clinical and biochemical evaluations were serially performed. Protein concentration of beta-endorphins (BE) and mRNA levels and allelic variants of the mu-opiod receptor gene (OPRM1) were analyzed. Five dropouts and two major adverse events occurred. The remaining adverse events did not interfere with daily living. Neurological disability progressed in only one patient. A significant reduction of spasticity was measured at the end of the trial. BE concentration increased during the trial, but no association was found between OPRM1 variants and improvement of spasticity. Our data clearly indicate that LDN is safe and well tolerated in patients with PPMS.

PMID: 18728058

Pain Med. 2009 May-Jun;10(4):663-72. Epub 2009 Apr 22.

Fibromyalgia symptoms are reduced by low-dose naltrexone: a pilot study.

Younger J, Mackey S.

School of Medicine, Department of Anesthesia, Division of Pain Management, Stanford University, 780 Welch Road, Suite 208, Palo Alto, CA 94304-1573, USA.

jarred.younger@stanford.edu

OBJECTIVE: Fibromyalgia is a chronic pain disorder that is characterized by diffuse musculoskeletal pain and sensitivity to mechanical stimulation. In this pilot clinical trial, we tested the effectiveness of low-dose naltrexone in treating the symptoms of fibromyalgia.

DESIGN: Participants completed a single-blind, crossover trial with the following time line: baseline (2 weeks), placebo (2 weeks), drug (8 weeks), and washout (2 weeks).

PATIENTS: Ten women meeting criteria for fibromyalgia and not taking an opioid medication.

INTERVENTIONS: Naltrexone, in addition to antagonizing opioid receptors on neurons, also inhibits microglia activity in the central nervous system. At low doses (4.5 mg), naltrexone may inhibit the activity of microglia and reverse central and peripheral inflammation.

OUTCOME MEASURES: Participants completed reports of symptom severity everyday, using a handheld computer. In addition, participants visited the lab every 2 weeks for tests of mechanical, heat, and cold pain sensitivity.

RESULTS: Low-dose naltrexone reduced fibromyalgia symptoms in the entire cohort, with a greater than 30% reduction of symptoms over placebo. In addition, laboratory visits showed that mechanical and heat pain thresholds were improved by the drug. Side effects (including insomnia and vivid dreams) were rare, and described as minor and transient. Baseline erythrocyte sedimentation rate predicted over 80% of the variance in drug response. Individuals with higher sedimentation rates (indicating general inflammatory processes) had the greatest reduction of symptoms in response to low-dose naltrexone.

CONCLUSIONS: We conclude that low-dose naltrexone may be an effective, highly tolerable, and inexpensive treatment for fibromyalgia.

PMID: 19453963

Inflamm Bowel Dis.. [Epub ahead of print]

Low-dose naltrexone for treatment of duodenal Crohn's disease in a pediatric patient.

Shannon A, Alkhouri N, Mayacy S, Kaplan B, Mahajan L.

Department of Pediatric Gastroenterology, Cleveland Clinic Pediatric Institute, Cleveland, Ohio.

PMID: 20014017

Annals of Neurology, Volume 9999, Issue 999A, Feb 2010

Pilot trial of low dose naltrexone and quality of life in MS

Bruce A.C. Cree, Elena Kornyeyeva, Douglas S. Goodin

Multiple Sclerosis Center at Univ. of Calif. in San Francisco 350 Parnassus Ave., Suite 908, San Francisco, CA 94117 USA

bruce.cree@ucsf.edu

Objective: To evaluate the efficacy of 4.5 mg nightly naltrexone on the quality of life of multiple sclerosis patients.

Methods: This single center, double-masked, placebo-controlled, crossover studied evaluated the efficacy of eight weeks of treatment with 4.5 mg nightly naltrexone (Low dose naltrexone or LDN) on self reported quality of life of MS patients.

Results: 80 subjects with clinically definite multiple sclerosis were enrolled and 60 subjects completed the trial. 10 withdrew before completing the first trial period: 8 for personal reasons, 1 for a non-MS related adverse event and 1 for perceived benefit. Database management errors occurred in 4 other subjects and quality of life surveys were incomplete in 6 subjects for unknown reasons. The high rate of subject dropout and data management errors substantially reduced the trial's statistical power. LDN was well tolerated and serious adverse events did not occur. LDN was associated with significant improvement on the following mental health quality of life measures: a 3.3 point improvement on the Mental Component Summary score of the SF-36 (P=.04), a 6 point improvement on the Mental Health Inventory (P<.01), a 1.6 point improvement on the Pain Effects Scale (P=.04) and a 2.4 point improvement on the Perceived Deficits Questionnaire (P=.05).

Interpretation: LDN significantly improved mental health quality of life indices. Further studies with LDN in MS are warranted.

_______________________________________________________________________________

Who is LDNscience? This is what they say......

LDNscience™ is a public information project of theMedInsight^® Research Institute.

The MedInsight^® Research Institute is a U.S.-based 501(c)(3) nonprofit working to bridge the ever-widening gap between medical research and actual medical practice. We are dedicated to bringing relief to those who suffer from cancer or chronic medical conditions by raising awareness of:

• Commercially unsponsored medications.
• Off-label or secondary uses for approved medications.
• Specialized tests that enable treatments to be tailored to the individual.

MedInsight's co-founder, Moshe Rogosnitzky, is an expert in the clinical application of Low Dose Naltrexone (LDN), Opioid Growth Factor (OGF) and related therapies, which have made incredible inroads with Crohn’s disease, cancer and other serious diseases. He collaborates in research with the discoverers of LDN and OGF, Dr. Ian S. Zagon and Dr. Patricia J. McLaughlin of Hershey Medical Center, Penn State University, as well as with Dr. Jill P. Smith of Hershey Medical Center, who conducts clinical research into LDN and OGF.

This site is intended to provide you with general information on the background, use of and evidence forLDN, OGF and related therapies, and to answer any questions on these therapies and our work. If you cannot find the answer to your particular question, please submit your question and we will respond to you as soon as possible.

To become a much-valued supporter of this site or of our life-changing research into LDN, OGF and related therapies, please visit: Join Our Supporters.

Thank you,

The LDNscience™ Team

Tuesday, January 8, 2013

Research Article-IL-13 Promotes Collagen Accumulation in Crohn’s Disease Fibrosis by Down-Regulation of Fibroblast MMP Synthesis

Plos One Research Article. Fibrosis is a serious consequence of Crohn’s disease (CD), often necessitating surgical resection. Research study examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment. (Not written in layman terms. Written using medical terminology which may be confusing for some to understand)

PLOS ONE: IL-13 Promotes Collagen Accumulation in Crohn’s Disease Fibrosis by Down-Regulation of Fibroblast MMP Synthesis: A Role for Innate Lymphoid Cells?:

Abstract

Background

Fibrosis is a serious consequence of Crohn’s disease (CD), often necessitating surgical resection. We examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment.

Methods

Factors potentially modulating collagen deposition were examined in intestinal tissue samples from fibrotic (f) CD and compared with cancer control (C), ulcerative colitis (UC) and uninvolved (u) CD. Mechanisms attributable to IL-13 were analysed using cell lines derived from uninvolved muscle tissue and tissue explants.

Results

In fCD muscle extracts, collagen synthesis was significantly increased compared to other groups, but MMP-2 was not co-ordinately increased. IL-13 transcripts were highest in fCD muscle compared to muscle from other groups. IL-13 receptor (R) α1 was expressed by intestinal muscle smooth muscle, nerve and KIR⁺ cells. Fibroblasts from intestinal muscle expressed Rα1, phosphorylated STAT6 in response to IL-13, and subsequently down-regulated MMP-2 and TNF-α-induced MMP-1 and MMP-9 synthesis. Cells with the phenotype KIR⁺CD45⁺CD56^+/−CD3⁻ were significantly increased in fCD muscle compared to all other groups, expressed Rα1 and membrane IL-13, and transcribed high levels of IL-13. In explanted CD muscle, these cells did not phosphorylate STAT6 in response to exogenous IL-13.

Conclusions

The data indicate that in fibrotic intestinal muscle of Crohn’s patients, the IL-13 pathway is stimulated, involving a novel population of infiltrating IL-13Rα1⁺, KIR⁺ innate lymphoid cells, producing IL-13 which inhibits fibroblast MMP synthesis. Consequently, matrix degradation is down-regulated and this leads to excessive collagen deposition.

Citation: Bailey JR, Bland PW, Tarlton JF, Peters I, Moorghen M, et al. (2012) IL-13 Promotes Collagen Accumulation in Crohn’s Disease Fibrosis by Down-Regulation of Fibroblast MMP Synthesis: A Role for Innate Lymphoid Cells? PLoS ONE 7(12): e52332. doi:10.1371/journal.pone.0052332

Editor: Niels Olsen Saraiva Câmara, Universidade de Sao Paulo, Brazil

Received: August 8, 2012; Accepted: November 12, 2012; Published: December 31, 2012

Copyright: © 2012 Bailey 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: This work was supported by Action Medical Research, SP4260 (http://www.action.org.uk); National Association for Colitis and Crohn’s disease, M/07/5 (http://www.nacc.org.uk); Swedish Fund for Strategic Research (http://www.stratresearch.se/en/), and Edith Lidiard Private Bequest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: John.Tarlton@bris.ac.uk

# These authors contributed equally to this work.

Introduction

Inflammation-induced fibrosis – pathologic accumulation of extracellular matrix (ECM) – in the intestine represents a serious complication of inflammatory bowel diseases (IBD). In ulcerative colitis (UC), ECM may accumulate in the mucosa and submucosa (SM) contributing to stiffening of the colon, whereas in Crohn’s disease (CD) the excess ECM, particularly fibrous collagen, deposited transmurally, leads to stricture and loss of normal function[1]. Up to one third of patients with CD develop debilitating intestinal fibrosis.

Inflammation in CD may lead to fibrosis by up-regulation of pro-fibrogenic factors, such as TGF-β [2]. Fibroses in lung, liver and kidney have been linked to T cell synthesis of IL-13 [3]. This can promote collagen synthesis independently of TGF-β [4]; and cause smooth muscle cell proliferation [5], [6] and contraction [7], which may contribute to fibrotic stricture formation. IL-13 activates many other cells, including macrophages, mast cells, B cells and nerve cells (reviewed [8]), potentially contributing to pathology.

IL-13 signals predominantly via the low affinity IL-13Rα1 which forms dimers with IL-4Rα and subsequently activates the JAK1/STAT6 pathway. IL-13Rα2, originally considered to act as a decoy receptor, has recently been shown capable of signalling [9] and can occur in cell surface and soluble forms. TNF-α, in conjunction with IL-13, was shown to increase IL-13Rα2 synthesis in macrophages leading to TGF-β synthesis [9]. In a mouse model of intestinal fibrosis, blockade of IL-13Rα2 and TGF-β signalling reduced levels of colonic IGF-I and collagen deposition [10]. IL-13 promotes transcription of matrix metalloproteinase (MMP)-2, 9, 12 and 14 [11]; decreases MMP-1 synthesis [12]; and synergises with TGF-β to increase fibroblast tissue inhibitor of metalloproteinase (TIMP)-1 [13] – a pro-fibrotic mechanism. IL-13 has recently been linked to fistula formation in CD [14]. Consequently, both IL-13 and IL-13Rα2 are considered potential therapeutic targets in fibrotic diseases and in other CD pathologies [9], [14].

Fibrosis occurs when extracellular matrix (ECM) synthesis exceeds degradation. Breakdown of collagen, the principle ECM molecule deposited in fibrosis, is mediated by proteolytic enzymes. Of these, MMPs are principally involved: collagenase (MMP-1), cleaves mature collagen fibres [15]: and MMP-2 is co-ordinately regulated with collagen synthesis via TGF-β but also through common promoter elements such as AP-2 and SP1, and probably serves to remodel nascent collagen molecules to allow correct fibril formation [16], [17], [18]. Other MMPs may play a role in inflammation-induced fibrosis, for example MMP-9, which mediates leukocyte migration, and MMP-14, which may promote fibrosis via up-regulated TGF-β signalling [19]. MMP activity is controlled by specific inhibitors, the TIMPs, levels of which are modulated in disease processes. Therefore it is important to attempt to understand the complex interplay between these mediators which determine the level of collagen deposition.

Much of the work identifying IL-13 as a fibrotic mediator has been carried out in mouse models and in tissues other than the intestine. In order to identify the IL-13 pathway as a relevant therapeutic target in CD, it is important to understand the processes occurring in human intestine. We have therefore investigated the hypothesis that CD fibrosis results from an IL-13-mediated imbalance in collagen synthesis and degradation. The results indicate that IL-13 is elevated in fibrosis, resulting in decreased MMP-2, MMP-9 and MMP-1 activity which would promote collagen deposition.

Methods

Ethics Statement

Samples were collected from Bristol Royal Infirmary and written informed consent was obtained by all patients. The study was carried out under Ethical Approvals E4896 (Central and South Bristol Ethics Committee) and 07/H0205/44 (Somerset Research Ethics Committee).

Reagents

All chemicals and reagents were obtained from Sigma (Poole, UK) unless stated otherwise.

Patients and Samples

Intestinal tissue was collected from CD patients undergoing surgery for bowel obstruction (n = 19, 12 men and 7 women, age range 18–73 y, average age 35 y), including uninvolved areas; ulcerative colitis (n = 8, 6 men and 2 women, age range 18–62 y, average age 32 y), including uninvolved areas; or colorectal cancer (n = 13, 7 men and 8 women, age range 56–88 y, average age 72 y), described in Table S1. Tissues were examined macroscopically and palpated to determine areas of fibrosis, and subsequently examined by microscopy and allocated to “uninvolved” and “fibrotic” groups. Tissue evaluations were carried out independently by two “blinded” observers (MM and PB). From each sample, full-thickness tissue was frozen for immunohistology. As the excessive collagen deposition occurs in the submucosa and outer muscle layers, tissue was separated into muscle-enriched (referred to as “muscle”) and mucosa-enriched (“mucosa”) fractions. The muscle was pinned and the mucosa dissected out below the muscularis mucosa. In some fibrotic samples, the separation was carried out within the submucosa approximately 1–1.5 mm from the luminal surface, carefully excluding contamination of muscle by mucosa. Fractions were snap frozen in liquid nitrogen and stored at −80°C; or in RNA later, pending biochemical analyses. A sample of each fraction was orientated in OCT before freezing in liquid nitrogen. Image and qPCR analysis were carried out on a subset of patients (n = 8 for each group for cancer, inflamed UC, uninvolved CD and fibrotic CD; n = 3 for uninvolved UC).

Sample Preparation

Frozen tissue fractions were pulverised in a liquid nitrogen-cooled freezer mill and homogenised in immuno-precipitation (IP) lysis buffer (300 mM NaCl, 0.05 M Tris pH 7.4, 1% Triton X-100) or in RIPA buffer (1% NP40, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 2 mM EDTA, 0.01 M phosphate buffer pH 7.2) for 1 h on ice, with 10 strokes of the homogeniser. Extraction buffer contained protease inhibitor cocktail (P8340) and phosphatase inhibitors cocktails 1 (P2850) and 2 (P5726).

Cell Culture

Primary intestinal fibroblasts, isolated from uninvolved intestinal muscle tissue taken from CD and UC patients, were cultured as previously described [20], with some modifications. Briefly, 2×2 mm separated muscle were incubated in 100 u/ml type IV collagenase for 2–3 h and fibroblasts allowed to adhere overnight in DMEM containing 10% FCS, non-essential amino acids, vitamins, L-glutamine, nystatin and double strength antibiotics (penicillin 200 u/ml, streptomycin 200 µg/ml, amphotericin 0.25 µg/ml, gentamicin 100 µg/ml). Fibroblasts were transferred to 1× antibiotics after 7 days. This medium was then used for all experiments and cell culture, except when cells were starved and cytokine-treated, when the medium contained 0.1% FCS and 250 µg/ml ascorbate-2-phosphate, essential for collagen synthesis. Cells (2500/well of a 96-well plate) were cultured for 48 h in 10% FCS, before starvation for 48 h, and then incubated in TGF-β (2 ng/ml, AbDSerotec, Kidlington, UK), TNF-α (10 ng/ml, AbDSerotec), or IL-13 (20 ng/ml, AbDSerotec) for 72 h. Collagen, TGF-β and MMPs were assayed from frozen supernatants. For each line and each experiment, a titration of cell number was performed and standard curve prepared on cells grown for 16 h and assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Millipore, Watford, UK). For signalling analyses, cells were added to 12-well plates, cultured until 80% confluent, starved for 48 h and then cytokines added in starvation media for 0.5 to 72 h.

Explant Culture

Muscle fragments (2×2 mm), prepared as above, were washed in serum-free medium and 3–5 fragments per well were placed in 12-wellplates and covered with 1 ml of starvation medium, with or without 20 ng/ml IL-13. Explants were cultured for up to 24 h and then snap frozen in liquid nitrogen with or without OCT.

ELISA

Pro-MMP-1 was measured in culture supernatants by ELISA (R&D Systems, Abingdon, UK); TGF-β1 was measured in culture supernatants by ELISA (in-house) [21]; and collagen synthesis was measured in culture supernatants by ELISA for type I collagen C-terminal pro-peptide (CICP) (Technoclone, Dorking, UK). Assays were run as 5 well replicates. Tissue extracts were analysed in duplicate for CICP and MMP-1 using the same assays as culture supernatants, and IL-1β and TIMP-1 were analysed using R&D Systems ELISA kits. Data were normalized to soluble protein for tissue extracts or to cell number for culture supernatants. Levels were determined using standards and data expressed as the ratio to unstimulated cultures.

Western Blotting and Zymography

Adherent cells were washed in cold TBS and lysed in RIPA buffer (40 µl/well). Explants were weighed and homogenised in RIPA buffer (500 µl:100 mg wet weight). Lysates or tissue extracts were centrifuged and supernatants containing equal amounts of soluble protein (cells 0.01 mg/lane; explants 0.05 mg/lane; original extract for IP samples 3 mg/lane) were subjected to SDS-PAGE (8%) and proteins transferred to Immobilon P. Blots were blocked and proteins detected using rabbit antibodies to STAT6 (1:1000), or PSTAT6 (1:1000) (Table S2) and donkey anti-rabbit peroxidase (Jackson, 1:20000) by chemiluminescence (ECL plus, GE Healthcare, Little Chalfont, Bucks, UK) and Biomax light film. MMP-2 and MMP-9 were assayed by gelatin-zymography as described previously [22]. Bands were quantified using NIH image.

RNA Isolation

Muscle fragments were disrupted using a TissueLyser (Qiagen Ltd, Crawley, UK) followed by treatment with proteinase K (6 mAU/ml, 55°C/15 mins) and RNA extracted using Macherey-Nagel NucleoSpin RNA II Isolation Kits (ABgene, Epsom, UK). The resulting RNA was assessed for quantity and quality by automated gel electrophoresis (Experion, Bio-Rad Laboratories, Hemel Hempstead, UK) and stored at −80°C before use.

Primer and Hydrolysis Probe Design

Primers and probes were designed using Primer 3 [23] (http://frodo.wi.mit.edu/primer3) and M-Fold using the human specific GenBank sequences for IL-13, IL-13Rα2, type I collagen and TGF-β1 primer sequences, GenBank accession numbers and housekeeper genes (primers as [24]) are given in Table S3a and Table S3b. Primers and probes were synthesised by Metabion International AG (Munich, Germany).

Quantitative RT-PCR (qPCR)

Duplicate RNA samples, confirmed as free from genomic contamination, were subject to reverse transcription using random hexamers and ImProm-II Reverse Transcription System (Promega Corporation, Southampton, UK). Quantitative PCR was performed using HotStarTaq Master Mix (Qiagen). Gene specific amplification was performed using 4.5 mM MgCl₂, 0.2 µM primer, 0.1 µM probe or SYBR Green 1 (1/100,000) and cDNA equivalent to 35 ng RNA. Sample incubations were performed in an MxPro3005P (Stratagene, California, USA) at 95°C for 15 minutes and then 45 cycles of 95°C for 15 seconds, and 60°C for 30 seconds. When SYBR Green I was used, samples were heated from 75°C to 95°C in 0.5°C increments with a dwell time at each temperature of 10 seconds. Threshold cycle (Ct) values were calculated when the sample exceeded baseline fluorescence mean ±10 SD. A negative control of nuclease-free water and a positive control sample with a known Ct value were included with all sample runs.

In order to determine the most appropriate housekeeper genes for the study, all 8 potential genes were quantified in all samples. Relative copy number was calculated using the E^ΔCt method (E: reaction efficiency) with ΔCt values calculated from the sample with the largest Ct (fewest gene copies). The geNorm VBA applet for Microsoft Excel was used to determine the most stable genes. The four most stable genes for muscle fractions were (in order of stability) RPL13A, YWHAZ, SDHA and GAPDH. These genes were then used for the analyses.

Relative expression data for each of the gene targets was calculated using the qBase applet for Microsoft Excel (http://medgen.ugent.be/qbase/). This applet calculates a relative copy number for each sample, normalised against the four stably expressed housekeeper genes, using the methods described by Vandesompele et al. [24]. All samples were normalised to the sample with the latest Ct value (assigned a relative copy number = 1).

Histology and Immunofluorescence

Formalin fixed tissues were stained with Haematoxylin van Giesons (HVG) stain for collagen and examined for the presence of mucosal inflammation and the presence of fibrosis.

Acetone-fixed frozen sections (5 µm) of full thickness tissue or cytokine-treated explants were blocked in 10% normal goat and/or donkey serum followed by avidin/biotin blocking (Vector Laboratories, Peterborough, UK). Sections were incubated overnight at 4°C with primary antibodies (Table S2). The isotype-specific secondary antibodies to mouse IgG (all used at 1:100 from Southern Biotechnology) were all raised in goat and were conjugated to biotin, Texas red, FITC, AF633, or TRITC, and combined according to primary antibodies applied. Donkey anti-rabbit IgG (1:400), streptavidin-Texas red (1:100) and streptavidin-Dylight488 (1:200) were obtained from Stratech (Newmarket, Suffolk, UK). Species and isotype-specific negative controls were used for each experiment. Finally, sections were mounted using Vectashield (Vector Laboratories), viewed using a Leica fluorescence microscope and digital images captured using Image Pro Plus software (Media Cybernetics, Silver Spring, MD).

Laser Capture Microscopy

Eight-micron frozen sections were collected onto polyethylene naphthalate (PEN)-coated slides (Leica Microsystems). A modified staining procedure was used to maximize RNA recovery from captured cells: sections were fixed in ice-cold ethanol; primary mouse anti-KIR and secondary goat anti-mouse FITC were combined in a buffer containing RNALater and applied to sections for 2 h on ice; after a brief wash in PBS containing RNALater, sections were dehydrated in ethanol, viewed without a coverslip and held on dry ice before LCM. KIR⁺ cells were identified by fluorescence imaging in the Zeiss PALM Microbeam, cut using minimal laser beam diameter and minimal profile diameter and catapulted into adhesive caps of microtubes. 500 KIR⁺ cells and 500 KIR⁻ adjacent tissue fragments were captured in each experiment and immediately processed for RNA extraction using a Qiagen RNeasy Micro RNA extraction kit. cDNA was generated and amplified by Whole Genome Amplification (Qiagen), followed by RT-qPCR using the Roche Light Cycler 480 with primers as detailed above. Fold differences in IL-13 transcription were assessed against the GAPDH housekeeper. Due to the low levels of RNA extracted using this technique it was only possible to normalize against one housekeeper gene, GAPDH, one of the most stably expressed genes.

Image Analysis

Images, 5–10 per section, were captured at ×20 and analysed using ImageJ software as previously described[25]. Amount of contaminating submucosa attached to tissue fractions was assessed by analysing the area of submucosa as a percentage of the whole fragment area in the subset of tissues described in “patients and samples”.

Statistics

The data were tested for normality using P-P plots and then analysed by bivariate analysis and Pearson correlation on samples paired by patient, or ANOVA on untransformed or log transformed data for all five tissue groups, followed by post-hoc analysis. Cell culture data were analysed by ANOVA and post-hoc analysis. Data were considered significant below p = 0.05.

Results

Strictures in Crohn’s Disease Involve Increased Collagen Deposition in Gut Layers External to Mucosa

In histologically normal cancer controls, uninvolved CD or UC tissues, collagen was distributed in thin dense bands between muscle bundles, throughout the loose connective tissue of the submucosa, and in the serosa (Figure 1). A similar pattern was observed in inflamed UC. Fibrotic CD intestine, from both small and large bowel, was defined as a sample from a CD patient receiving surgery for a diagnosed stricture which, on histopathological assessment, had increased and/or disrupted collagen deposition, with or without obvious muscle hyperplasia. Most (13/19) fibrotic CD samples had evidence of inflammation in the mucosa and/or muscle. In fibrotic CD, collagen deposition was variable; the submucosa and/or serosa were often expanded, with dense collagen between the muscle bundles. In some strictures, the intestinal wall was considerably thickened. Dense collagen deposition was not observed within the mucosa.

Download:

Figure 1. Increased collagen synthesis in muscle and submucosa in fibrotic CD tissue.

Full thickness sections of formalin fixed tissue stained with Haematoxylin van Geisens. Collagen is stained bright pink and nuclei are grey. (A) Cancer control (colon), (B) Inflamed UC (C) Uninvolved Crohn’s disease (terminal ileum) and (D) Crohn’s disease with stricture (terminal ileum) (stitched image). Arrows show dense collagen deposits in outer muscle layers. Dotted line defines the outer muscle/sub-mucosa (sm) border. Images captured with ×2.5 objective. Bars represent 1 mm.

doi:10.1371/journal.pone.0052332.g001

To examine changes in factors within the intestine where excessive collagen is deposited, namely the gut layers external to the mucosa, without confounding results with elements derived from the leukocyte and epithelial-rich mucosa, we separated mucosa away from muscle layers. After separation, there was no contamination of muscle samples by mucosa, as judged by microscopy (Figure S1). The boundary between the submucosa and muscle layer was less distinct in fCD than in other groups, so this was defined as the line where discernable muscle bundles ended. The submucosa remained attached in variable amounts to muscle and mucosa fractions and, by image analysis, there was a relatively larger amount of contamination of the mucosa fraction (Figure S1A,B). There was no significant difference between the percentage of submucosa attached to the muscle when fCD was compared to cancer (p = 0.06), uUC (p = 0.08), uCD (p = 0.05) or iUC (p = 0.05). When muscle data from all groups were combined, the amount of attached submucosa significantly correlated with collagen synthesis, pro-MMP-1 and TIMP-1 (Data in this section does not show. See original content to view)

In Fibrotic Muscle, Collagen Synthesis is Increased without a Corresponding Increase in the Remodelling Enzyme, MMP-2

Type I collagen synthesis was significantly greater in fibrotic CD muscle compared to all other samples (Data in this section does not show. See original content to view)

, Tables S4 and S5a). ProMMP-2 was not increased in fibrotic CD muscle compared to other groups (p>0.33 all comparisons) and the levels of proMMP-2 did not correlate with collagen synthesis in the muscle (Data in this section does not show. See original content to view)

Download:

Figure 2. Fibrogenesis is increased in CD muscle and mucosa tissues, without concomitant MMP-2 synthesis in muscle.

Tissue fragments were extracted and analysed by ELISA (collagen CICP synthesis, IL-1β, MMP-1, TIMP-1, TIMP-2) or zymography (MMP-2, MMP-9). Data are expressed as the ratio of the group mean for uUC, iUC, uCD or fCD to the mean for cancer control tissue for each parameter. Data are derived from 14–15 cancer controls, 3 uUC, 8 iUC, 8–12 uCD and 14–21 fCD patients. See Table S4 for actual means and SEM for each parameter. (Data in this section does not show. See original content to view) doi:10.1371/journal.pone.0052332.g002

By comparison, in mucosa from fibrotic CD (Figure 2 C,D), there was a significant increase in collagen synthesis and MMP-2 compared to cancer controls (Data in this section does not show. See original content to view)Table S5b

). There was also a significant increase in TIMP-1 and pro-MMP-1, compared to uCD. In mucosal tissue from inflamed UC (Figure 2C,D), collagen synthesis, pro-MMP-1, TIMP-1, pro-MMP-9 and IL-1β were significantly increased (Data in this section does not show. See original content to view)

Figure 2 shows the ratio of the various parameters to cancer control tissue, Table S4 shows the means and SEM of the data, Table S5a shows the trends and levels of significance and Table S5b shows the correlations between collagen synthesis and various parameters.

Raised IL-13 and IL-13Rα2 Transcription in fCD Muscle

In tissue extracts by qPCR, IL-13 mRNA (Figure 3A) was increased in fibrotic CD muscle, and this was significant compared to cancer (Data in this section does not show. See original content to view)Figure 3B

). It was not possible to quantify IL-13 or receptors in any tissue extracts because of endogenous inhibitory factors, and IL-13 Rα1 transcription was not determined.

Download:

Figure 3. IL-13 transcription, but not IL-13Rα2 transcription, is increased fibrotic muscle.

(A) IL-13 mRNA and (B) IL-13 Rα2 mRNA in muscle tissue lysates. RNA was extracted from tissue fragments and processed for qPCR. Results were normalized to four stably expressed housekeeper genes. Data are derived from 14–15 cancer controls, 2 uUC, 8 iUC, 8–12 uCD and 14–21 fCD patients. Significant differences from fibrotic CD, (Data in this section does not show. See original content to view) doi:10.1371/journal.pone.0052332.g003

Phenotype of IL-13Rα1 and IL-13Rα2⁻ Expressing Cells

In all groups, IL-13Rα1 and Rα2 were co-expressed (Figure 4A) by neurofilament⁺ (Figure 4B) spindle-shaped cells in the muscle layers and IL-13Rα1 was expressed at low levels on smooth muscle cells (Figure 4A). IL-13Rα1 was highly expressed on a population of mononuclear cells in the muscle in CD (Figure 4A). These Rα1⁺cells expressed variable levels of CD45 (Figure 5A) but did not express mast cell tryptase (Figure 5B), CD3 (Figure 5C), mast cell chymase (data not shown) or NKG2D (data not shown). Anti-NKp46 antibody stained smooth muscle cells in all groups even at high dilution, such that no evidence of co-localisation with Rα1 was obtained. Most of these Rα1⁺ cells co-expressed KIR (Figure 5 panel D and E), and a proportion expressed CD56/NCAM (nerve cell adhesion molecule) (Figure 5 panel E) to a variable extent. The majority of cells expressing KIR also expressed IL-13Rα1 (KIR⁺ Rα1⁺ cells, mean 77% ±23%SD; KIR only, mean 10% ±10%SD; Rα1 only mean 13% ±9%SD, of cells/x20 field (data from 4 patients and 3 fields per tissue)). These IL-13Rα1⁺cells were highly significantly increased (Data in this section does not show. See original content to view)Figure 6A

) and were rarely observed in muscle tissue from cancer control or inflamed UC intestine (Figure 4 panel A, Figure 6A) and there was an uneven distribution of KIR⁺ cells in fibrotic muscle tissue, with the highest numbers in the inner muscle, and significantly fewer in the mucosa compared to all other regions (Data in this section does not show. See original content to view)Figure 6B). Variable levels of IL-13 co-localised with the IL-13Rα1⁺⁺ cells (Figure 7) and there was no evidence IL-13 expression by other cells in the muscle or submucosa. It was not possible to analyse whether IL-13 co-localised with KIR as the primary antibodies were both IgG1. In all experiments, isotype matched control antibodies were used, and no staining was observed in muscle tissue.

Download:

Figure 4. IL-13 receptors are expressed on smooth muscle cells and neurofilaments in CD strictures.

Frozen sections were processed for double immunoflorescence. (Panel A) double labeling for IL-13 Rα1 and Rα2 in intestinal muscle tissue, arrows show double-stained cells, arrowheads show cells expressing Rα1 only at a very high level. Boxed area is shown as the colour image and its monochrome split. (B) Co-staining of IL-13Rα1 and neurofilament (NF) (green) and the monochrome split images. A,B, ×20 images.

doi:10.1371/journal.pone.0052332.g004

Download:

Figure 5. Mononuclear cells expressing very high levels of IL-13Rα1 co-express CD45, KIR and CD56.

Images of frozen, immunostained tissue were captured using a 12 bit monochrome camera and images from the different colour channels combined to form two or three colour images. Multicoloured composites D and E, have been split into their original monochrome images to aid interpretation. Arrows show Rα1 co-localising with other markers. 13Rα1 co-localises with CD45 (A) but not mast cell tryptase (B) or CD3 (C). Panel D, 13Rα1 (green), 13Rα2 (red) and KIR (blue), plus split images for 13Rα1, 13Rα2 and KIR, note that 13Rα2 does not colocalise with 13Rα1⁺KIR⁺ cells (arrows). Panel E, 13Rα1 (green), KIR (red) and CD56 (blue). Split image shows a cell expressing all three markers (arrow), note there is also a strongly KIR-expressing cell (bottom left of image) which does not express 13Rα1 or CD56 A–C, ×40 images, D and E, ×64 images. Representative images from 8 patient samples.

doi:10.1371/journal.pone.0052332.g005

Download:

(See Original Article to view/read this figure)Figure 6. IL-13Rα1 and KIR expressing-cells are increased in fibrotic CD, particularly in the muscle, by image analysis of immunostained frozen tissue sections.

(A) Total number of mononuclear cells/field expressing very high levels of Rα1 and no co-expression of Rα2 in muscle tissue "very">doi:10.1371/journal.pone.0052332.g006

Download:

Figure 7. IL-13 is co-localises with IL-13Rα1 in fCD muscle.

Double labeling, in frozen fibrotic CD tissue sections, for IL-13 (green) and IL-13Rα1 (red) and split monochrome images (×20).

doi:10.1371/journal.pone.0052332.g007

By image analysis, the highest levels of IL-13Rα1 were observed in fibrotic CD muscle (Figure S2A) and total IL-13Rα2 was significantly increased (Data in this section does not show. See original content to view)Figure S2B

). The highest double positive (IL-13Rα1⁺Rα2⁺) area was observed in the muscle layers of fibrotic CD (Figure S2C).

KIR⁺ Cells Synthesis IL-13

While the majority of KIR⁺ cells expressed high levels of IL-13Rα1, and IL-13Rα1⁺ cells expressed cell surface IL-13, it was not known whether KIR⁺ cells also produced IL-13. Therefore, a protocol was developed for the isolation, by laser capture microscopy (LCM), of KIR⁺ and adjacent KIR⁻ cells from fibrotic muscle tissue. Preliminary to LCM analysis, comparison was made between transcription of IL-13 in fibrotic CD tissue in two patients, and non-fibrotic CD tissue or uninvolved UC tissue, using whole tissue section extracts, as used in the LCM protocol (Table 1). IL-13 transcripts were readily detected in tissue from CD fibrotic gut, but were at a significantly lower level in tissue from non-fibrotic CD or UC gut (Figure S3). Levels of IL-13 transcript were consistently greater in KIR⁺ cells compared to adjacent KIR⁻ cells from each of these fCD muscle samples but both samples had similar GAPDH transcript levels (Table 1, Figure S3). Overall, IL-13 transcript levels were 114.8+/−3.4 times greater in KIR⁺ cells than in KIR⁻ cells. Interferon-γ from NK cells has been shown to be a regulator of liver fibrosis [26]. However, analysis of frozen tissue sections of fibrotic intestine from these two patients, both of which had high level muscle infiltration by KIR⁺ cells, showed that IL-13 transcription was readily detectable (Table 1, Figure S3), whereas IFN-γ was undetectable in both samples. In summary, the KIR⁺ cells we have described within CD fibrotic muscle are a major source of IL-13, but do not transcribe IFN-γ.

Download:

Table 1. KIR⁺ cells in fibrotic muscle tissue synthesise IL-13.

doi:10.1371/journal.pone.0052332.t001

Explanted Muscle Tissue Responds to IL-13

Treatment of explanted muscle tissue with IL-13 induced phosphorylation of STAT6, with maximum activation after 2–4 h (Figure 8A). PSTAT6 was not detected at time 0 (Figure 8B), but was observed in the nucleus of many cells after 2 h (Figure 8C). When fibrotic CD muscle was treated with IL-13, STAT6 was activated in smooth muscle cells, but not in KIR⁺ cells (Figure 8 panel D). Indeed, KIR⁺ cells did not appear to express STAT6 (Figure 8 panel E), despite co-localisation with IL-13Rα1 in explanted muscle (Figure panel F).

Download:

Figure 8. IL-13 activates smooth muscle cells, but not KIR⁺ cells, in tissue explants.

Tissue explants were cultured with or without IL-13 for 1–24 h. Explants were then snap frozen and processed for Western blotting or immunoflorescence. (A–C) IL-13 signaling in explanted intestinal muscle tissue. (A) Time course for activation of STAT6 by Western blotting or (BC) by immunofluorescence in cancer control tissue. Arrowheads indicate positive nuclei, arrow indicates autoflorescence. (D–F) Double labeling for KIR and PSTAT6 (Panel D), STAT6 (Panel E), or Rα1 (Panel F) in fibrotic CD explanted tissue after 2 h incubation with IL-13. Arrows show KIR⁺ cells which colocalise with IL-13Rα1 but not with PSTAT6 or STAT6. A and C, ×64, B, ×20.

doi:10.1371/journal.pone.0052332.g008

In vitro Fibroblast Response to IL-13

Primary muscle-derived mesenchymal cell lines derived from uninvolved regions from both UC and CD, expressed vimentin, type I collagen and prolyl-4-hydroxylase, but less than 1 in 100 cells expressed α-smooth muscle actin (SMA) at passage 3 (Figure S4A–D), and this did not increase with passage number. These cells were, therefore, predominantly fibroblasts and not smooth muscle cells. Cells cultured on microscope slides expressed IL-13Rα1 and IL-13Rα2 (Figure S4E–F). When cultured with IL-13, all primary cell lines phosphorylated STAT6, with maximum activation at 1 h (Figure 9A).

Download:

Figure 9. TGF-β drives co-ordinate collagen synthesis and pro-MMP-2 synthesis in intestinal muscle fibroblasts, whereas IL-13 has no effect on collagen synthesis and suppresses proMMP-2, in lines derived from uninvolved CD or UC tissue.

(A) Western blots showing phosphorylation of STAT6 by primary fibroblasts in response to exogenous IL-13. Representative blot from 6 experiments. (B) Effect of IL-13, TNF-α and/or TGF-β on collagen, MMP-2 or TGF-β synthesis in primary fibroblasts, Data represent the mean+SEM of the ratio of treated to untreated cultures from a minimum of 8 separate experiments. (Data in this section does not show. See original content to view)doi:10.1371/journal.pone.0052332.g009

As shown in Figure 9B, there was no significant difference between CD and UC lines in their response to cytokine additions. TGF-β significantly stimulated collagen and proMMP-2 (Figure 9B) synthesis by fibroblasts compared to untreated cultures (Data in this section does not show. See original content to view)Figure 9C

(Data in this section does not show. See original content to view)Figure 9D).

Discussion

Intestinal strictures form as a result of chronic inflammation in CD and diverticulitis, but are rarely a clinical problem in UC. Although increased extracellular matrix deposition, including collagen, is driven by inflammation in the mucosa as part of the wound healing response, it is thought that fibrotic strictures in CD result from dysregulated inflammation within the submucosal and muscularis microenvironment, leading to excessive collagen deposition in these tissues [1].

IL-13 has been implicated in both lung and liver fibrosis, though studies examining levels of IL-13 in UC and CD intestine have had contradictory results [27], [28], [29]. No studies have been performed in fibrotic human intestine or in intestinal muscle in IBD. We show here that IL-13 and its receptors are present in the gut, and that they are increased in the outer layers (predominantly muscle) in fibrotic CD intestine compared to other groups, suggesting a role for IL-13 in CD fibrosis. As IL-13 has been proposed as a gut fibrogenic factor [9]–promoting fibrosis by increasing collagen synthesis [4] or TIMP-1 [30], or by decreasing MMP-1 [12]–this is a novel and important finding.

A striking influx or accumulation of IL-13Rα1⁺⁺ KIR⁺ cells, into fibrotic CD muscle tissue was observed. This was a heterogeneous population of cells expressing variable levels of KIR and IL-13Rα1, but the majority of cells expressing these receptors were double positive. The absence of CD3 indicates that these cells are potentially an NK cell subset and not NKT cells. These cells potentially lie on the phenotypic and functional spectrum of type 2 innate lymphoid cell (ILC), a group of lineage-negative lymphoid cells with effector and/or regulatory function, NK characteristics, with plasticity of function, and with cytokine potential biased towards IL-13 and IL-5 [31]. Further studies are underway to fully define their relationship to this lineage and to confirm whether they are contributing to, or responding to, fibrosis. In liver fibrosis, there is evidence for NK cell regulation of fibrosis in early disease[26] through mediation of cytotoxicity against activated collagen-producing stellate cells [32] via IFN-γ, but also enhancement of late-stage disease through secretion of pro-fibrotic factors, including IL-13 [33]. We have consistently observed high level transcription of IL-13 with almost undetectable IFN-γ transcription co-existing in whole fibrotic muscle tissue sections and this coincides with the IL-13⁺, IFN-γ⁻ transcriptional profile of fCD muscle KIR⁺ ILC. Despite cell surface IL-13Rα1 expression, we did not detect IL-13 signalling in stricture KIR⁺cells, but did find evidence of cell-surface IL-13. Transcription of IL-13 in fibrotic muscle KIR⁺ cells was consistently greater than in adjacent KIR⁻ cells. At present our data point to a role for KIR⁺ cells as a major source of IL-13 and probable net contributors to advanced gut fibrosis in CD.

IL-13 transcription was increased in fibrotic muscle fractions compared to controls. In control tissues, highest levels of collagen was deposited, and therefore synthesised, in submucosa (HVG histology data). In fCD, the submucosa was expanded with a concomitant increase in collagen deposition but there was also a noticeable increase in collagen deposition in the muscle layers. As the amount of muscle-associated submucosa correlated with collagen synthesis but not IL-13 synthesis, and extract IL-13 did not correlate with collagen synthesis, this indicates that muscle is the major source of IL-13 and not the submucosa. As KIR⁺ cells were significantly increased in the muscle layers compared to the submucosa, and IL-13 was expressed by IL-13Rα⁺ KIR⁺ cells, we propose that the increased collagen deposition in the muscle layers is part mediated by IL-13 derived from KIR⁺ cells, acting by downregulation of fibroblast MMP synthesis. The observation that MMP-2 and collagen synthesis correlate in the mucosal fractions, suggest that newly synthesised collagen is remodelled in the mucosa, so preventing a damaging accumulation. A contributory factor to the above scenario may be an increased TIMP-1/MMP-1 ratio in fCD muscle compared to fCD mucosa.

Cultured gut fibroblasts from uninvolved tissue expressed IL-13Rα1 and responded to IL-13 by activation of STAT6. IL-13 did not directly stimulate collagen synthesis, as has been shown for dermal fibroblasts [4]. However, IL-13 may interact differently with fibroblasts derived from fibrotic or inflamed tissue, which were not examined in this study. IL-13 has pro-fibrotic effects via its actions on MMPs, for example inhibiting IL-1β-induced MMP-1 synthesis [12], a key enzyme in initiating degradation of fibrillar collagen. IL-13 significantly reduced both TNF-α-induced MMP-1 and MMP-9, and reduced MMP-2 synthesis by cultured fibroblasts in our study. This is a novel finding, and may indicate the biological consequence of elevated IL-13 in muscle from fibrotic gut, as we observed that MMP-2 synthesis was not co-ordinately up-regulated with collagen synthesis in fibrotic CD muscle. Lack of remodelling does not account for this result, as the correlation was between active collagen synthesis (CICP levels) and MMP-2 levels. Newly synthesised collagen is remodelled by MMP-2, hence the tight coordination of their gene activation, and reduced MMP-2 has been linked to collagen accumulation in liver [34].

The consequence of reduced MMP-1 would be that mature collagen, such as that found in fibrotic lesions, could not be degraded. In extracts, MMP-1 was increased in fibrotic CD muscle but not to the expected level, as the ratio of MMP-1 to collagen synthesis was much higher in iUC and fCD mucosa than in fCD muscle. There was also a significant increase in TIMP-1 in fibrotic muscle tissue, which would have inhibited MMP activity. MMP-9 was decreased in fibroblast cultures exposed to IL-13, but in tissue extracts there was a general increase in all groups compared to cancer controls. MMP-9 is associated with inflammation and leukocyte infiltration, therefore increased leukocyte MMP-9 in diseased tissue extracts may account for this discrepancy.

IL-13Rα1 has been shown to be on epithelial, smooth muscle and nerve cells [35] and to be important in muscle hypercontractility via STAT6 signalling in neurons of the myenteric plexus [7] in mice. In our study, nerve cells and smooth muscle cells (SMC) expressed IL-13Rα1, and SMC signalled in response to exogenous IL-13. IL-13, derived from KIR⁺ cells, may drive muscle hyperplasia in CD by stimulating proliferation of SMC [6], so contributing to stricture formation [5]. We have also shown that nerve cells express IL-13 receptors and are increased in active UC and CD. It is possible that this represents increased innervation of the bowel musculature in IBD, as has been demonstrated for Substance P⁺ nerves [36]. As IL-13 has been shown to act on enteric nerves to stimulate smooth muscle contraction [37], it is likely that increased IL-13 in fibrotic CD muscle promotes both smooth muscle proliferation and contraction, increasing stress within the stricture, further driving fibrogenesis [38] and potentially increasing pain and distress to patients.

In mouse gut, IL-13Rα2 is expressed by mucosal fibroblasts [39] and by cells in the muscle layers [35]. We have shown that Rα2 is expressed at a high level by nerve cells in the muscle, with no obvious expression by other cells in the muscle layers. In fCD muscle, both Rα2 mRNA and protein were significantly increased and may be acting as a either a soluble decoy or stimulatory receptor. We did not find convincing evidence for IL-13Rα1 or Rα2 expression on macrophages in CD muscle, a prerequisite for the IL-13Rα2- profibrotic mechanism proposed by Fichtner-Feigl [9]. However, it is possible that analysis of macrophages by flow cytometry and qPCR, may yield supporting data, as shown in mice [40].

In the muscle layers, our data demonstrate that IL-13 is a potential pro-fibrotic factor in CD, acting by suppressing collagen degradation. We provide evidence of a positive feedback loop, whereby KIR⁺ cells are recruited, by an unknown mechanism, into the muscle layers in CD and then synthesise IL-13, further promoting collagen accumulation and stricture formation. These suggest that drugs targeting the IL-13 pathway should be explored in patients with Crohn’s disease to try to reduce the potential for fibrotic strictures.

Supporting Information
Figure S1.

Muscle and mucosa fractions after separation. Representative images of mucosa and muscle fractions from cancer (AB), inflamed UC (CD) and fibrotic CD (EF, E, stitched images). All images captured with x5 objective. Submucosa indicated (sm), dotted line (E) shows muscle/submucosa border in fCD.

(TIF)

Figure S2.

IL-13 receptors are quantitatively increased in strictured muscle. Image analysis for positive pixel area of (A) total single Rα1⁺, (B) total single Rα2⁺, (C) total Rα1⁺ Rα2⁺ double stained cells. C, Cancer control, uUC, uninvolved UC, iUC, inflamed UC, uCD, uninvolved CD, fCD, fibrotic CD. Data are derived from 14–15 cancer controls, 2 uUC, 8 iUC, 8–12 uCD and 14–21 fCD patients (A, C) or from mucosa (B), * (p<0 .05=".05" and="and" p="p">

(TIF)

Figure S3.

Transcriptional analysis of fibrotic and non-fibrotic tissues by laser capture microscopy. A, IL-13 transcripts were assayed in whole frozen tissue sections from fibrotic CD intestine from two patients; B, transcription of IL-13 was compared in KIR⁺ and KIR⁻ cells (500 of each per assay) retrieved by laser capture microscopy from fibrotic CD muscle; C, transcription of IL-13 was compared to transcription of IFN-γ in whole frozen tissue sections from fibrotic CD intestine Representative data from at least three identical assays.

(TIF)

Figure S4.

Primary cell lines are IL-13Rα1⁺ IL-13Rα2⁽⁺⁾ fibroblasts. All cell lines expressed vimentin (AB), prolyl-4-hydroxylase (P4H) (C) and type I collagen (D), with little or no detectable smooth muscle actin (SMA) (A–C). Tissue sections were used as positive controls for SMA stain. Cultured cells also expressed IL-13Rα1 (EG), but IL-13Rα2 was generally expressed at a lower level.

(TIF)

Table S1.

Patients and samples.

(DOCX)

Table S2.

Primary Antibodies used in immunohistology and Western blotting.

(DOCX)

Table S3.

a) Gene accession numbers and primer sequences. b) Housekeeping genes.

(DOCX)

Table S4.

Mean values for parameters measured in tissue extract data presented in Figure 2 .

(DOCX)

Table S5.

a) Summary of data for proinflammatory and profibrotic parameters in muscle and mucosa from fibrotic Crohn’s disease intestine compared to expression of these parameters in muscle and mucosa in controls. b) Correlation between collagen synthesis and other parameters.

(DOCX)

Acknowledgments
We would like to thank Gabriella Koning and Amanda M. Williams for technical assistance

Author Contributions

Conceived and designed the experiments: CVW PWB CSJP JFT JRB. Performed the experiments: CVW JRB. Analyzed the data: CVW JRB PWB MM. Contributed reagents/materials/analysis tools: JFT MM PAS IP. Wrote the paper: CVW JRB PWB JFT CSJP.

References

Burke JP, Mulsow JJ, O’Keane C, Docherty NG, Watson RW, et al. (2007) Fibrogenesis in Crohn’s disease. Am J Gastroenterol 102: 439–448. doi: 10.1111/j.1572-0241.2006.01010.x. Find this article online
Mourelle M, Salas A, Guarner F, Crespo E, Garcia-Lafuente A, et al. (1998) Stimulation of transforming growth factor beta1 by enteric bacteria in the pathogenesis of rat intestinal fibrosis. Gastroenterology 114: 519–526. Find this article online
Wynn TA (2003) IL-13 effector functions. Annu Rev Immunol 21: 425–456. Find this article online
Oriente A, Fedarko NS, Pacocha SE, Huang SK, Lichtenstein LM, et al. (2000) Interleukin-13 modulates collagen homeostasis in human skin and keloid fibroblasts. J Pharmacol Exp Ther 292: 988–994. Find this article online
Koukoulis G, Ke Y, Henley JD, Cummings OW (2001) Obliterative muscularization of the small bowel submucosa in Crohn disease: a possible mechanism of small bowel obstruction. Arch Pathol Lab Med 125: 1331–1334. Find this article online
Bosse Y, Thompson C, Audette K, Stankova J, Rola-Pleszczynski M (2008) Interleukin-4 and interleukin-13 enhance human bronchial smooth muscle cell proliferation. Int Arch Allergy Immunol 146: 138–148. doi: 10.1159/000113517. Find this article online
Akiho H, Blennerhassett P, Deng Y, Collins SM (2002) Role of IL-4, IL-13, and STAT6 in inflammation-induced hypercontractility of murine smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 282: G226–232. Find this article online
Hershey GK (2003) IL-13 receptors and signaling pathways: an evolving web. J Allergy Clin Immunol 111: 677–690; quiz 691.
Fichtner-Feigl S, Strober W, Kawakami K, Puri RK, Kitani A (2006) IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis. Nat Med 12: 99–106.Find this article online
Fichtner-Feigl S, Young CA, Kitani A, Geissler EK, Schlitt HJ, et al.. (2008) IL-13 signaling via IL-13R alpha2 induces major downstream fibrogenic factors mediating fibrosis in chronic TNBS colitis. Gastroenterology 135: 2003–2013, 2013 e2001–2007.
Lee PJ, Zhang X, Shan P, Ma B, Lee CG, et al. (2006) ERK1/2 mitogen-activated protein kinase selectively mediates IL-13-induced lung inflammation and remodeling in vivo. J Clin Invest 116: 163–173. doi: 10.1172/JCI25711. Find this article online
Leonardi A, Cortivo R, Fregona I, Plebani M, Secchi AG, et al. (2003) Effects of Th2 cytokines on expression of collagen, MMP-1, and TIMP-1 in conjunctival fibroblasts. Invest Ophthalmol Vis Sci 44: 183–189. doi: 10.1167/iovs.02-0420. Find this article online
Zhou X, Trudeau JB, Schoonover KJ, Lundin JI, Barnes SM, et al. (2005) Interleukin-13 augments transforming growth factor-beta1-induced tissue inhibitor of metalloproteinase-1 expression in primary human airway fibroblasts. Am J Physiol Cell Physiol 288: C435–442. Find this article online
Scharl M, Frei S, Pesch T, Kellermeier S, Arikkat J, et al.. (2012) Interleukin-13 and transforming growth factor beta synergise in the pathogenesis of human intestinal fistulae. Gut.
Visse R, Nagase H (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 92: 827–839. doi:10.1161/01.RES.0000070112.80711.3D. Find this article online
Qin HW, Sun Y, Benveniste EN (1999) The transcription factors Sp1, Sp3, and AP-2 are required for constitutive matrix metalloproteinase-2 gene expression in astroglioma cells. Journal of Biological Chemistry 274: 29130–29137. doi: 10.1074/jbc.274.41.29130. Find this article online
Goffin L, Seguin-Estevez Q, Alvarez M, Reith W, Chizzolini C (2010) Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts. Arthritis Research & Therapy 12.
Kim ES, Sohn YW, Moon A (2007) TGF-beta-induced transcriptional activation of MMP-2 is mediated by activating transcription factor (ATF)2 in human breast epithelial cells. Cancer Letters 252: 147–156. Find this article online
Krantz SB, Shields MA, Dangi-Garimella S, Cheon EC, Barron MR, et al. (2011) MT1-MMP cooperates with Kras(G12D) to promote pancreatic fibrosis through increased TGF-beta signaling. Mol Cancer Res 9: 1294–1304. Find this article online
Rogler G, Gelbmann CM, Vogl D, Brunner M, Scholmerich J, et al. (2001) Differential activation of cytokine secretion in primary human colonic fibroblast/myofibroblast cultures. Scand J Gastroenterol 36: 389–398. doi: 10.1080/003655201300051216. Find this article online
Whiting CV, Williams AM, Claesson MH, Bregenholt S, Reimann J, et al. (2001) Transforming growth factor-beta messenger RNA and protein in murine colitis. J Histochem Cytochem 49: 727–738. Find this article online
Tarlton JF, Whiting CV, Tunmore D, Bregenholt S, Reimann J, et al. (2000) The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis. Am J Pathol 157: 1927–1935. doi: 10.1016/S0002-9440(10)64831-6. Find this article online
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386. Find this article online
Vandesompele J, De Paepe A, Speleman F (2002) Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR. Anal Biochem 303: 95–98. doi:10.1006/abio.2001.5564. Find this article online
Inman CF, Rees LE, Barker E, Haverson K, Stokes CR, et al. (2005) Validation of computer-assisted, pixel-based analysis of multiple-colour immunofluorescence histology. J Immunol Methods 302: 156–167. doi: 10.1016/j.jim.2005.05.005. Find this article online
Notas G, Kisseleva T, Brenner D (2009) NK and NKT cells in liver injury and fibrosis. Clin Immunol 130: 16–26. doi: 10.1016/j.clim.2008.08.008. Find this article online
Kadivar K, Ruchelli ED, Markowitz JE, Defelice ML, Strogatz ML, et al. (2004) Intestinal interleukin-13 in pediatric inflammatory bowel disease patients. Inflamm Bowel Dis 10: 593–598. doi: 10.1097/00054725-200409000-00014. Find this article online
Vainer B, Nielsen OH, Hendel J, Horn T, Kirman I (2000) Colonic expression and synthesis of interleukin 13 and interleukin 15 in inflammatory bowel disease. Cytokine 12: 1531–1536. doi:10.1006/cyto.2000.0744. Find this article online
Inoue S, Matsumoto T, Iida M, Mizuno M, Kuroki F, et al. (1999) Characterization of cytokine expression in the rectal mucosa of ulcerative colitis: correlation with disease activity. Am J Gastroenterol 94: 2441–2446. Find this article online
Zhou X, Hu H, Huynh ML, Kotaru C, Balzar S, et al. (2007) Mechanisms of tissue inhibitor of metalloproteinase 1 augmentation by IL-13 on TGF-beta 1-stimulated primary human fibroblasts. J Allergy Clin Immunol 119: 1388–1397. Find this article online
Spits H, Di Santo JP (2011) The expanding family of innate lymphoid cells: regulators and effectors of immunity and tissue remodeling. Nat Immunol 12: 21–27. doi: 10.1038/ni.1962. Find this article online
Radaeva S, Sun R, Jaruga B, Nguyen VT, Tian Z, et al. (2006) Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners. Gastroenterology 130: 435–452. doi: 10.1053/j.gastro.2005.10.055.Find this article online
Jeong WI, Park O, Suh YG, Byun JS, Park SY, et al. (2011) Suppression of innate immunity (natural killer cell/interferon-gamma) in the advanced stages of liver fibrosis in mice. Hepatology 53: 1342–1351.Find this article online
Radbill BD, Gupta R, Ramirez MC, DiFeo A, Martignetti JA, et al. (2011) Loss of matrix metalloproteinase-2 amplifies murine toxin-induced liver fibrosis by upregulating collagen I expression. Dig Dis Sci 56: 406–416. doi: 10.1007/s10620-010-1296-0. Find this article online
Morimoto M, Morimoto M, Zhao A, Madden KB, Dawson H, et al. (2006) Functional importance of regional differences in localized gene expression of receptors for IL-13 in murine gut. J Immunol 176: 491–495. Find this article online
Straub RH, Grum F, Strauch U, Capellino S, Bataille F, et al. (2008) Anti-inflammatory role of sympathetic nerves in chronic intestinal inflammation. Gut 57: 911–921. doi: 10.1136/gut.2007.125401.Find this article online
Zhao A, McDermott J, Urban JF, Jr., Gause W, Madden KB, et al.. (2003) Dependence of IL-4, IL-13, and Nematode-Induced Alterations in Murine Small Intestinal Smooth Muscle Contractility on Stat6 and Enteric Nerves. 948–954.
Bishop JE (1998) Regulation of cardiovascular collagen deposition by mechanical forces. Mol Med Today 4: 69–75. doi: 10.1016/S1357-4310(97)01193-3. Find this article online
Kawashima R, Kawamura YI, Kato R, Mizutani N, Toyama-Sorimachi N, et al. (2006) IL-13 receptor alpha2 promotes epithelial cell regeneration from radiation-induced small intestinal injury in mice. Gastroenterology 131: 130–141. Find this article online
Fichtner-Feigl S, Fuss IJ, Young CA, Watanabe T, Geissler EK, et al. (2007) Induction of IL-13 triggers TGF-beta1-dependent tissue fibrosis in chronic 2,4,6-trinitrobenzene sulfonic acid colitis. J Immunol 178: 5859–5870. Find this article online

PLOS ONE: IL-13 Promotes Collagen Accumulation in Crohn’s Disease Fibrosis by Down-Regulation of Fibroblast MMP Synthesis: A Role for Innate Lymphoid Cells?:

'via Blog this'

Pages

Tuesday, January 29, 2013

Vitamins for Crohn's Disease

Crohn's Disease Nutrition

Crohn's Disease Nutrition: What's Missing?

Vitamins for Crohn's Disease: Diet vs. Supplements

Crohn's Disease Nutrition: Working With Your Doctor

Friday, January 25, 2013

Restoring Bacteria

Wednesday, January 23, 2013

You are being lied to about probiotics ingredients

Check your probiotics label right now

How to really avoid GMOs in probiotics

Sunbiotics certified USDA organic chewable probiotics

No soy, no dairy, no GMO, no wheat, no refined sugars, no corn, no nonsense!

Friday, January 18, 2013

Fecal transplants cure diarrhea, modulate testosterone levels

Nutella was not used as a placebo control.

Fighting bacteria with bacteria

Bacteria, sex, and immunity

Uceris Approved for Active Ulcerative Colitis

FDA Approves Uceris

Important Safety Information About Uceris

Wednesday, January 9, 2013

Clinical Trials

Tuesday, January 8, 2013

Abstract

Background

Methods

Results

Conclusions

Introduction

Methods

Ethics Statement

Reagents

Patients and Samples

Sample Preparation

Cell Culture

Explant Culture

ELISA

Western Blotting and Zymography

RNA Isolation

Primer and Hydrolysis Probe Design

Quantitative RT-PCR (qPCR)

Histology and Immunofluorescence

Laser Capture Microscopy

Image Analysis

Statistics

Results

Strictures in Crohn’s Disease Involve Increased Collagen Deposition in Gut Layers External to Mucosa

In Fibrotic Muscle, Collagen Synthesis is Increased without a Corresponding Increase in the Remodelling Enzyme, MMP-2

Raised IL-13 and IL-13Rα2 Transcription in fCD Muscle

Phenotype of IL-13Rα1 and IL-13Rα2− Expressing Cells

KIR+ Cells Synthesis IL-13

Explanted Muscle Tissue Responds to IL-13

In vitro Fibroblast Response to IL-13

Discussion

Author Contributions

Phenotype of IL-13Rα1 and IL-13Rα2⁻ Expressing Cells

KIR⁺ Cells Synthesis IL-13