Good Bugs, Bad Bugs and Leaky Gut

Intestine Lining in HealthFor thousands of years there have been certain fermented foods eaten by traditional peoples who have regarded them as the secret to their long lives. The Russians and Georgians are renowned for their longevity and many say regular yoghurt eating is the key. The Japanese are also renowned for their long lives and many ascribe them to their fermented plums and radishes, miso and natto. The Koreans eat kimchi. The south-east Asians tempeh, fish sauce and soy sauce. The middle east and India drink variations of kefir fermented from milk. The central Europeans salami, sauerkraut and gherkins. The northern Europeans, surströmming. Poi in Hawaii. Many of these foods are so highly valued, they play a sacred role in the culture used as an offering to the gods or considered essential at the family dinner table.

Intestine Lining in HealthThe Ferment of Probiotics

Is it just coincidence that so many different peoples from around the world value fermented foods so highly? Or was it just because they didn’t have refrigerators and had no choice but to eat food that had gone off? Sure enough, it turns out that fermented foods are very health-promoting. In fact, eating them will make you slimmer, improve your absorption of minerals, make you think more clearly, make you more resistant to getting sick and even protect you from nuclear radiation! It’s such a remarkable list, it almost sounds too good to be true. And yet true it is.

As we all know, bacteria can make food go ‘off’ and eating that food will make you very sick in no time at all. But then food can also ‘ferment’ resulting in all the health benefits above. Your nose will tell you straight away if something is fermented and safe to eat or off. The organisms growing in the fermented foods are called ‘probiotics’ – literally, ‘promoting life’.

My objective in this BluePrint is for you to understand what probiotics are, how they affect your body, and how you ensure you and your family get as much benefit as possible from these amazing foods.

Leaky Gut

Intestine Lining Leaky GutIt turns out that those same organisms that we used to ferment our food actually live inside of us in our digestive tract fermenting away. If these organisms inside us are probiotic, they make us healthy but if they are the kind that make food go off, they make our health go off too. One of the main ways they do this is by affecting the integrity of the lining of the digestive system.

Your digestive system, or gut, forms one continuous tube extending from your mouth to your anus. It does not open to the precious interior of your body at all. In fact, when your body formed inside your mother’s womb, you started as a flat plate that rolled up like a rolled pancake leaving a hollow tube going from top to bottom. That tube is the embryonic precursor to your gut. You can see that the gut is really connected to the outside world and not to the inner organs of your body. This is really important to understand because your gut has to keep a very tricky balance. On the one hand, it needs to maximise absorption of nutrients from your food. On the other hand, it needs to rigorously protect the organs of your body from absorbing toxins that make your body weak and sick.

The part of your body tasked with this monumental feat is a one-cell-thick layer lining your gut made up of cells called enterocytes. These amazing cells are linked together to form a single continuous sheet that covers the entire interior of your intestines. The absorption of nutrients then takes place solely through the bodies of these cells. In this way, the enterocytes can regulate what gets absorbed and what does not. If your body needs more calcium, they absorb more calcium. If not, they don’t. If there is something in your digestive tract that the cells don’t recognise or which they see as harmful, they are left behind to go out with the stool.

For this network to function properly, each enterocyte must be very close to the ones next to it so nothing can slip through the gaps. This is accomplished by ‘Tight Junctions’ made of special proteins produced by the enterocytes for the purpose. These Tight Junctions require a substantial amount of energy to maintain. If the cell gets tired, weak, sick or inflamed for any reason, these tight junctions then start to breakdown and the toxins that should stay inside your intestines can slip right past and into your bloodstream where they can wreak havoc with your health.1

This is called a leaky gut. It’s a very common problem today.

70% of Your Immune System Lines Your Gut

Of course your body is very keenly aware of the fact that your gut is full of potential toxins, bacteria and viruses. To watch out for that, some 70% of your immune system is found clustered along your enterocyte lining constantly on the lookout for nasties that need it to step in. But while this is critical, when you have a leaky gut, this immune response causes even more of the problem. When toxic stuff (food fragments, bacteria, bits of protein, etc.) slips past the weakened enterocyte layer, the immune cells react by making antibodies to fight that stuff which makes more inflammation. This inflammation further weakens the integrity of the barrier making it leak more leading to more immune response and more inflammation.2

Let’s consider an example. Say you eat wheat when your gut lining is in full health. Wheat contains a very large protein called gluten – you’ve probably heard of it. Proteins are made up of series of amino acids (AAs) arranged in a very specific order and in the case of gluten, this chain is over 100,000 AAs long. The gluten gets broken down inside your gut into 1, 2 or 3 AA long segments and then gets absorbed into the enterocyte. Inside, the enterocyte finalises the breakdown of the protein into individual free AAs for the body to use. These are then released into the bloodstream to go to the liver to get packaged up for use by your body. This process is so thorough that in health there is no passage of the whole wheat protein into the bloodstream, only the single amino acids.

Once the Lining is Damaged...

Let’s say now, for the very first time, you have some damage to the lining of your gut. There are many causes, but one is antibiotics which can cause a breach in the barrier so let’s say you have just finished a course of antibiotics. Now when you eat the wheat, it gets partly broken down and fragments of the gluten protein slip right past the enterocytes bypassing the proper processing. You get bits of gluten in your bloodstream. The immune cells are right there and recognize something that doesn’t belong. They make antibodies that track back toward the source of the protein, the gut wall. Attaching there, they release chemicals that cause inflammation because that is what antibodies do. Now the lining of your gut is inflamed not because of the initiating damage, but because of the leaked gluten. If it doesn’t repair in time and you regularly eat bread, this cycle keeps feeding itself and becomes a permanent breach. This is called an allergy and is the cause of food intolerances. You can see how once you get one, it’s easy to get another... and another.3

Remember though, not only gluten and other protein fragments slip past, so do many bacteria. Often when this happens, there are lots of bad bacteria in your digestive tract and these are the ones that come through. The good bacteria are more interested in what’s inside your gut anyway. So your immune system responds to these bacteria too and before long you have your immune system busy trying to mop up the mess of stuff coming from your gut. It’s so busy with this that it doesn’t have time or energy (your immune function uses a lot of energy) to defend against other attacks so now your whole body becomes more susceptible to attack – colds, flu, tummy upsets – catching what’s going around4.

Sound bad? It gets much, much worse. With your immune system working overtime trying to clean up the mess, it generates lots of inflammation – not just at the gut wall but throughout your whole body and chronic inflammation is the root cause of a lot of degenerative disease including arthritis5, psoriasis6, 7, 8, 9 and eczema10. Recent research has now definitely linked leaky gut with heart disease11. This is a very serious problem, and as you know all these are common and serious problems in Australia today.

Leaky Gut Causes Many Modern Diseases

Because your immune system is using up so much of your energy on this impossible task, it leaves you with little energy for other activities. Ever feel low in energy? Left long enough, this low energy state can reach the debilitating level of Chronic Fatigue Syndrome (CFS). Only 20 years ago many doctors doubted the existence of CFS. Now it is recognised by health professionals the world over. Researchers in Belgium have recently (2010) proven that patients with CFS have elevated levels of antibodies against gut bacteria; that healing the leaking gut reduces those antibodies; and that there is a direct correlation between reducing these antibody levels and recovery12, 13. This is very exciting news for the families of anyone with CFS.

These bacterial and protein fragments also affect the function of the brain causing depression, and cloudy thinking14, 15, 16. Depression is one of the single most common reasons that Australian’s make a visit to the doctor. And leaky gut has not been linked only to mild depression but also to major depressive disorder. Leaky gut has even been implicated in the severe brain disorders autism17 and schizophrenia18.

The extra load on the liver causes fatty infiltration. The liver can no longer perform its role of detoxification fully as it is overloaded and clogging up. This in turn leads to skin problems.

Twin studies on obese vs lean twins have shown marked differences in the digestive bacterial population between them. Scientists now believe this is a significant contributor to unwanted weight gain19, 20, 21, 22.

Don’t forget the Children

But new research is suggesting that bad gut flora in infancy and childhood can seriously affect health for a lifetime. Current research is investigating whether inflammatory bowel disease, type 1 diabetes, allergies, asthma, and autism may have their origins in leaky gut23.

Regardless, children with leaky gut are certainly more susceptible to getting sick in general4.

Studies now show that mineral absorption is compromised in an unhealthy gut. Proper probiotic health is important to calcium24, magnesium25 and iron26 absorption. In one study in Houston, Texas, teenagers were followed for one year. Half of them were given supplementation to enhance their gut flora and half were not. After just 8 weeks the teens with the good bugs had measurable increases in mineral absorption. After 12 months, those with the good bugs had fully 35 grams more minerals stored in their bones.

Antioxidants Unavailable Without Healthy Flora

You know how important antioxidants are to your health, well it so happens that without the good bacteria in your gut some of the most powerful antioxidants, the polyphenols, are almost entirely unavailable for your body to absorb.

Having healthy gut flora has been shown to increase antioxidant effectiveness by up to 91%4.

What causes a Leaky Gut?

Anything that damages the healthy bacteria that live inside you will make your enterocytes weak. When too weak to sustain the Tight Junctions, the process of leaking starts. There are three main things that have been shown to damage the healthy bacteria:

  1. Antibiotics (of course – their whole purpose is to kill bacteria and they kill the good as well as the bad.)27
  2. Preservatives in food (again, the whole point of them is to kill or at least block the growth of bacteria.)28
  3. Unhealthy food (Certain foods feed the good bacteria. Not eating them allows the bad to overgrow.)4

Leaky gut can also be caused by certain drugs. Anti-inflammatory drugs (NSAIDs) such as aspirin damage the gut wall so badly they commonly cause bleeding. Many people use aspirin never dreaming it might lead to a cascade of problems29. Alcohol also damages the lining when consumed to excess. Alcohol also damages the liver directly but this is made worse by the damage to the gut wall30.

The Role of your Nervous System

Chiropractors have often seen resolution of many of the symptoms of leaky gut when they correct subluxation in the spine or cranium. They figure this is a result of removal of interference to the nervous system but recent research has uncovered some amazing details of how powerful this effect is.

Remember the enterocyte layer? It is a one-cell-thick sheet of cells bound together with Tight Junctions. Well it turns out that your digestive system has its own nervous system called the enteric nervous system, or ENS, and there is a nerve fibre going from it to every single enterocyte in your gut. Researchers in France discovered that if you damage the ENS, the Tight Junctions break up, the sheet falls apart and the enterocytes start to grow in clumps instead31, 32, 33. Now of course, your ENS is connected to your regular nervous system via nerves from your spine so it stands to reason that subluxation of your spine irritating those nerves could well lead to breakdown of the gut barrier.

But the connection goes even higher up. It has been observed in hospitals that after traumatic brain injury, gut function is impaired. A research team investigating this in Finland discovered that the enterocyte barrier breaks down within 30 minutes of brain injury. 30 minutes!2 Again, we can see that subluxation, perhaps at the base of the brain or even in the cranium itself may well cause leaky gut.

How do you know if you (or your child) have leaky gut?

This is an area of intense current research. Perhaps the best test currently is to measure antibodies to noxious bacteria but so far this has only been used in research settings. Some people have gastrointestinal symptoms but studies show that only 27% of people with actual changes in the gut lining seen in a biopsy report symptoms such as IBS, bloating, diahorrea or constipation. In other words, if you have digestive symptoms, you know there is a problem with your gut, but if you don’t have digestive symptoms, you can’t rule out a problem with your gut.

Classic signs of leaky gut or sick gut flora are:

  • Constant tiredness
  • Skin rashes or itchiness
  • Aches and pains
  • Recurrent sickness
  • Asthma
  • Depression
  • Recurrent thrush / urinary tract infections
  • IBS / IBD (irritable bowel syndrome, inflammatory bowel disease)
  • Allergies – commonly to wheat and/or cows milk
  • Colon cancer
  • Asthma
  • Can’t lose weight
  • Dark circles under the eyes – you commonly see this in kids

How do you fix Leaky Gut and get the good bugs back?

There are four steps:

  1. Kill off the bad bugs
  2. Put in the good bugs
  3. Feed ‘em
  4. Feed your enterocytes

I recommend everyone does the first three. If you avoid the causes of leaky gut I have described above, all you should need as you go into the future is to keep on feeding the good bugs. Regularly eating fermented food is a good idea too. If you are really struggling with CFS, severe allergy, depression or any of the above, step 4 will help speed the process up a lot.

Kill off the bad bugs

Here you mostly need to take care of parasites like pinworms and any other worms you might have. Other than pinworms, parasites are not such a big problem in most parts of Australia but you will get the surest results if you de-worm to be sure. Get worm tablets from the chemist for your whole family or use my recommended non-toxic herbal de-worm capsules. You are best to take them for a month to get the second generation from unhatched eggs.

In Australia, regular de-worming is probably not necessary unless anyone in your family complains of an itchy bottom in which case, do this again.

Put in the good bugs

A healthy lifestyle will maintain the good bugs quite well however it’s best to get on the right foot by putting them in to start with. A healthy gut has a wide spectrum of bugs living in it including bifidobacteria, lactobacillus and even yeasts; and there are many strains of each of these. Many probiotics contain few or even one strain of bugs grown in an artificial medium. They are very concentrated but not so beneficial. I recommend you use a probiotic with a similar wide range of bugs in it to what is found naturally in your healthy gut and one that is based on food.

Take it for about a month. After that, just eat naturally fermented food and feed the bugs. Take the probiotic again if you feel you or your child needs it.

A word of warning, many foods that used to be made by natural fermentation processes are now made by high-speed chemical means which give similar tastes but few of the health benefits. This is common with miso, soy sauce, gherkins, salami, olives and sauerkraut now available in Australia. Shop carefully. You may need to visit a good organic food shop. Even then, shop carefully!

Feed the good bugs

This is the most important step to the long-term healing of your gut. The good bugs love to eat a special kind of carbohydrate called inulin. It’s sometimes called soluble fibre. The best sources you can find easily in Australia are:

  • Leek
  • Onion
  • Garlic
  • Asparagus
  • Jerusalem artichoke

Oat and barley bran contain a different soluble fibre called beta glucan which also feeds the good bugs. As you know, I don’t recommend you eat too much grain. If you do though, oats or barley are probably your best choice.

Be sure to eat something from this group every day.

Feed your enterocytes

The best diet for enterocyte repair is the very diet I recommend for health. Just follow the three food principles. Don’t eat any grains. If you are healing from any of the listed symptoms, cut dairy out too. You can try re-introducing it after a month or so but it may take up to two years before you are fully desensitised. You may also find you are fine with raw dairy even though you react to supermarket dairy.

Nutritional supplements for enterocyte repair are13:

  1. Zinc 40 mg in the morning
  2. L-Glutamine 5 g morning and night
  3. NAC (n-Acetyl Cysteine) 500 mg morning and night

You can get all of these at our online store. Just follow the links above.

Wishing you and your family the best of Health,

Richard Sawyer.

Key Message

  • A healthy digestive system is essential for a healthy body
  • Imbalance causes some or all of the following
    • Constant tiredness
    • Skin rashes or itchiness
    • Aches and pains
    • Recurrent sickness
    • Asthma
    • Depression
    • Recurrent thrush / urinary tract infections
    • IBS / IBD (irritable bowel syndrome, inflammatory bowel disease)
    • Allergies – commonly to wheat and/or cows milk
    • Colon cancer
    • Asthma
    • Can’t lose weight
    • Dark circles under the eyes

Making It Real

  1. Kill off the bad bugs –  Take a natural de-worming product
  2. Put in the good bugs – Take good, real-food probiotics
  3. Feed ‘em – Eat leek, garlic, onion or artichoke (or oat bran) every day

References

1. Ukena SN, Singh A, Dringenberg U, et al. Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity. PloS one. 2007;2(12):e1308. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18074031.

2. Peuhkuri K, Vapaatalo H, Korpela R. Even low-grade inflammation impacts on small intestinal function. World journal of gastroenterology : WJG. 2010;16(9):1057-62. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20205274.

3. Vighi G, Marcucci F, Sensi L, Di Cara G, Frati F. Allergy and the gastrointestinal system. Clinical and experimental immunology. 2008;153 Suppl :3-6. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18721321.

4. Bosscher D, Breynaert A, Pieters L, Hermans N. Food-based strategies to modulate the composition of the intestinal microbiota and their associated health effects. Journal of physiology and pharmacology : an official journal of the Polish Physiological Society. 2009;60 Suppl 6:5-11. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20224145.

5. Orlando A, Renna S, Perricone G, Cottone M. Gastrointestinal lesions associated with spondyloarthropathies. World journal of gastroenterology : WJG. 2009;15(20):2443-8. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19468992.

6. Gyurcsovics K, Bertók L. [Role of bile acids and endotoxins in the pathogenesis and therapy of psoriasis]. Orvosi hetilap. 2000;141(17):915-7. Available at: http://www.ncbi.nlm.nih.gov/pubmed/10827473.

7. Pietrzak A, Jastrzebska I, Chodorowska G, et al. Psoriasis vulgaris and digestive system disorders: is there a linkage? Folia histochemica et cytobiologica / Polish Academy of Sciences, Polish Histochemical and Cytochemical Society. 2009;47(3):517-24. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20164041.

8. Qayoom S, Ahmad QM. Psoriasis and Helicobacter pylori. Indian journal of dermatology, venereology and leprology. 69(2):133-4. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17642857.

9. Ojetti V, De Simone C, Aguilar Sanchez J, et al. Malabsorption in psoriatic patients: cause or consequence? Scandinavian journal of gastroenterology. 2006;41(11):1267-71. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17060119.

10. Singh M, Ranjan Das R. Probiotics for allergic respiratory diseases - Putting it into perspective. Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology. 2009. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19725896.

11. Krack A, Sharma R, Figulla HR, Anker SD. The importance of the gastrointestinal system in the pathogenesis of heart failure. European heart journal. 2005;26(22):2368-74. Available at: http://www.ncbi.nlm.nih.gov/pubmed/15980032.

12. Maes M, Coucke F, Leunis J. Normalization of the increased translocation of endotoxin from gram negative enterobacteria (leaky gut) is accompanied by a remission of chronic fatigue syndrome. Neuro endocrinology letters. 2007;28(6):739-44. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18063928.

13. Maes M, Leunis J. Normalization of leaky gut in chronic fatigue syndrome (CFS) is accompanied by a clinical improvement: effects of age, duration of illness and the translocation of LPS from gram-negative bacteria. Neuro endocrinology letters. 2008;29(6):902-10. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19112401.

14. Maes M, Mihaylova I, Kubera M, Leunis J. An IgM-mediated immune response directed against nitro-bovine serum albumin (nitro-BSA) in chronic fatigue syndrome (CFS) and major depression: evidence that nitrosative stress is another factor underpinning the comorbidity between major depression and CF. Neuro endocrinology letters. 2008;29(3):313-9. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18580855.

15. Maes M, Kubera M, Leunis J. The gut-brain barrier in major depression: intestinal mucosal dysfunction with an increased translocation of LPS from gram negative enterobacteria (leaky gut) plays a role in the inflammatory pathophysiology of depression. Neuro endocrinology letters. 2008;29(1):117-24. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18283240.

16. Maes M. The cytokine hypothesis of depression: inflammation, oxidative & nitrosative stress (IO&NS) and leaky gut as new targets for adjunctive treatments in depression. Neuro endocrinology letters. 2008;29(3):287-91. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18580840.

17. White JF. Intestinal pathophysiology in autism. Experimental biology and medicine (Maywood, N.J.). 2003;228(6):639-49. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12773694.

18. Dohan FC. Genetic hypothesis of idiopathic schizophrenia: its exorphin connection. Schizophrenia bulletin. 1988;14(4):489-94. Available at: http://www.ncbi.nlm.nih.gov/pubmed/2851166.

19. Bäckhed F, Manchester JK, Semenkovich CF, Gordon JI. Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(3):979-84. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17210919.

20. Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature. 2006;444(7122):1022-3. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17183309.

21. Ley RE. Obesity and the human microbiome. Current opinion in gastroenterology. 2010;26(1):5-11. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19901833.

22. Turnbaugh PJ, Hamady M, Yatsunenko T, et al. A core gut microbiome in obese and lean twins. Nature. 2009;457(7228):480-4. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19043404.

23. Liu Z, Li N, Neu J. Tight junctions, leaky intestines, and pediatric diseases. Acta paediatrica (Oslo, Norway : 1992). 2005;94(4):386-93. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16092447.

24. Abrams SA, Griffin IJ, Hawthorne KM, et al. A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young adolescents. The American journal of clinical nutrition. 2005;82(2):471-6. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16087995.

25. Coudray C, Demigné C, Rayssiguier Y. Effects of dietary fibers on magnesium absorption in animals and humans. The Journal of nutrition. 2003;133(1):1-4. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12514257.

26. Tako E, Glahn RP, Welch RM, et al. Dietary inulin affects the expression of intestinal enterocyte iron transporters, receptors and storage protein and alters the microbiota in the pig intestine. The British journal of nutrition. 2008;99(3):472-80. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17868492.

27. Berg RD. Bacterial translocation from the gastrointestinal tract. Journal of medicine. 1992;23(3-4):217-44. Available at: http://www.ncbi.nlm.nih.gov/pubmed/1479301.

28. Kidd PM. Autism, an extreme challenge to integrative medicine. Part 2: medical management. Alternative medicine review : a journal of clinical therapeutic. 2002;7(6):472-99. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12495373.

29. Maino M, Mantovani N, Merli R, et al. Effects of chronic therapy with non-steroidal antiinflammatory drugs on gastric permeability of sucrose: a study on 71 patients with rheumatoid arthritis. World journal of gastroenterology : WJG. 2006;12(31):5017-20. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16937498.

30. Tang Y, Banan A, Forsyth CB, et al. Effect of alcohol on miR-212 expression in intestinal epithelial cells and its potential role in alcoholic liver disease. Alcoholism, clinical and experimental research. 2008;32(2):355-64. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18162065.

31. Toumi F, Neunlist M, Cassagnau E, et al. Human submucosal neurones regulate intestinal epithelial cell proliferation: evidence from a novel co-culture model. Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society. 2003;15(3):239-42. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12787332.

32. Savidge TC, Newman P, Pothoulakis C, et al. Enteric glia regulate intestinal barrier function and inflammation via release of S-nitrosoglutathione. Gastroenterology. 2007;132(4):1344-58. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17408650.

33. Cabarrocas J, Savidge TC, Liblau RS. Role of enteric glial cells in inflammatory bowel disease. Glia. 2003;41(1):81-93. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12465048.

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