General Zod? No, General Zonulin

I hold my hands up and admit that gut hyperpermeability - leaky gut - is a bit of an obsession of mine. I know to some the mere mention of leaky gut conjures up images of 'alternative medicine' and all things tree-hugging. Just for the record I've never knowingly hugged any tree and am a meat eating, petrol car driving, house dwelling regular guy who has yet to dabble in anything 'alternative' assuming that the odd vitamin D tablet and probiotic counts as regular. Still I remain very interested in how abnormal gastrointestinal (GI) permeability might be tied into quite a few conditions.

One aspect of GI permeability in particular has surfaced quite recently on my research radar, an interesting compound called zonulin.

A description first. Zonulin enjoys quite a special place in the science of the tight junctions. Tight junctions (TJs) among other roles, serve quite an important barrier function in lots of parts of the body; so making sure that things stay in and other things stay out. Zonulin seems to be part and parcel of the chemistry of tight junctions and in particular sharing quite an important relationship with the enhanced permeability of TJs.

With the gut in mind, zonulin has found quite a bit of interest. A familiar name to this blog, Dr Alessio Fasano, seems to have been present right at the beginning of interest in zonulin, with a particular focus on gut permeability tied into the presence of coeliac (celiac) disease as per this article* and write-up.

Ever since then, zonulin has just been making wave** (full-text) after wave*** (full-text) after wave**** as per its 'disassembly' activity when it comes to TJs. The initial link with coeliac disease is an interesting one given that later work suggested that gluten, or rather the gliadin fraction of gluten, has the propensity to induce zonulin release***** (at least under certain laboratory conditions).

But coeliac disease was just the starting point for zonulin, as more recent research has suggested a potential role for this protein in relation to gut permeability in type-1 diabetes (here and here), obesity (here) and potentially quite a few other conditions (here) with a specific focus on autoimmune conditions. General Zod? No, General Zonulin.

Accepting that there still remains some work to do on zonulin with regards to the methods and mode of action of zonulin on gut permeability******* this is a very interesting protein.

With my 'wondering' hat on, I have a few questions:

• Assuming the link between type-1 diabetes, gut permeability and zonulin holds up, does this mean that a gluten-free diet might be 'advantageous' for at least some people with type-1 diabetes? I'm thinking about this recent case study******* as a template. I would also add that no medical advice is given or intended by  this question.
• Gut hyperpermeability, leaky gut, has been documented in other conditions including one close to my research heart, autism spectrum conditions (see here). Again noting the suggestions by de Magistris and colleagues (here) on how a gluten- & casein-free diet seemed to affect measures of gut permeability in their cohort, is it perhaps time to look at zonulin with regards to conditions like autism? How about schizophrenia also? 
• Finally(!), the amino acid glutamine and its proposed tie up with gut permeability. Might glutamine affect zonulin production or even the other way around? Or am I just confusing things and heading out a step too far? 

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* Fasano A. et al. Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease. Lancet. 2000; 355: 1518-1519.

** Wang W. et al. Human zonulin, a potential modulator of intestinal tight junctions. Journal of Cell Science. 2000; 113: 4435-4440.


*** Fasano A. Intestinal zonulin: open sesame! Gut. 2001; 49: 159-162.


**** El Asmar R. et al. Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure. Gastroenterology. 2002; 123: 1607-1615.


***** Clemente MG. et al. Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function. Gut. 2003; 52: 218-223.


****** Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Annals of the New York Academy of Sciences. 2012; 1258: 25-33.


******* Sildorf SM. et al. Remission without insulin therapy on gluten-free diet in a 6-year old boy with type 1 diabetes mellitus. BMJ Case Reports. June 2012

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Does diabetes start in the intestines?

The findings of an interesting paper by Wei and colleagues* (full-text) pose a question: are the origins of diabetes in the intestines?

For those that don't know too much about diabetes, here is a link that should help. The concise version (if there is such a thing) is that diabetes normally manifests as either type-1 diabetes or type-2 diabetes with insulin being the key compound in controlling blood sugar, and corresponding issues either with its production or when resistance is built up to it.

The crux of the paper by Wei et al is that an insulin-responsive super enzyme called fatty acid synthase (FAS) involved in lipogenesis is also involved in gut barrier regulation through its action on Mucin 2 (Muc2), a gel-forming component of mucus. The authors' suggestion is that becoming resistant to insulin is associated with issues with FAS and correspondingly problems with mucus in the gut, inflammation and diabetes. No pressure then.

The paper summarised (deep breath):

• Several groups of mice were included for study: (a) mice with chemically-induced (tamoxifen induction of Cre recombinase) decreases of FAS protein and mRNA, (b) mice bred with inactivated FAS in the intestine and (c) control germ-free mice. For group (b) mice, diabetes was induced by administration of streptozotocin, a toxin to the beta cells which produce insulin in the pancreas.
• Assays looking at gut bacteria, intestinal permeability, cytokine release and protein S-palmitoylation were used to investigate various parameters.
• The findings: a chemically-induced deficiency of FAS in mice started a cascade of events linked to inflammation. One of the primary cytokine markers of this inflammation was elevated levels of TNF-α although animals were also noted to show weight loss and other gastrointestinal symptoms. A quarter of these mice actually died within 14 days.
• The authors deduced that although some changes were noted to the intestinal bacterial makeup of FAS reduced mice, these changes were not enough to cause the inflammation observed but rather were as a result of the inflammation. They demonstrated this via a previously discussed method on this blog, bacterial transplantation; in this case to the germ-free mice (group c) who did not show the accompanying inflammation as a result of their donor bacteria. That is not however to say that gut microbiota did not have some effect, as per the reduction in inflammation noted in the FAS deficient mice following administration of the antibiotics ciprofloxacin and metronidazole.
• The link between FAS deficiency and Muc2 was evidenced by the lower levels of Muc2 shown in FAS deficient mice and reduced inner mucus layer thickness in the colon of affected mice. 
• Looking at the inactivated FAS (group b) diabetic mice, a similar pattern of issues with Muc2 and reductions in the mucus layer was seen alongside penetration of bacteria indicating intestinal hyperpermeability (leaky gut). Interestingly, insulin supplementation seemed to positively affect some of the permeability issues.

This is quite a complicated paper and so please do not take my summary as gospel. It is intriguing that inflammation is at the heart of their theory and in particular, inflammation as a result of not having enough FAS present in the gut with the knock-on effects on gut permeability. Indeed not for the first time has it been suggested that diabetes and leaky gut are connected as per articles like this one. Makes you wonder also about any other possible dietary inter-related connections?

* Wei X. et al. Fatty acid synthase modulates intestinal barrier function through palmitoylation of mucin. Cell Host & Microbe. February 2012.
DOI:  10.1016/j.chom.2011.12.00

Probiotics, probiotics, probiotics

Do you ever have one of those days where a certain word seems to keep cropping up again and again no matter where you turn? Well, today is one of those days for me, and today's word is probiotics. Like a good dose of influenza (if there is such a thing as a good dose), I just can't seem to shake that word today.

So here are the papers with that word:

Delzenne and colleagues* (open-access) report on an interesting relationship between gut bacteria and obesity with a specific focus on studies looking to 'alter' the gut microbiota via pre- and probiotics and the various reported outcomes based on anthropometric and biochemical parameters linked to obesity. I talked about something similar a few months back on a sister blog.

Min Tan and colleagues** (open-access) report on the quite positive effects to patients recovering from traumatic brain injury in intensive care following administration of probiotics. Based on a relatively small patient group, patients (n=52) were randomised into either a nutrition + probiotics group or a nutrition alone group. Feeds were administered via a nasogastric tube initially which then progressed to the 'by mouth' route when patients were well enough. Various serum cytokines and related immune markers were analysed over the course of the study which lasted for 21 days.

Unfortunately some of the patients did not make it following their group allocation as sadly might be expected following a serious brain trauma. Other patients developed complications following their accident which did not seem to differ in quantity statistically between the groups aside from the number of pathogens potentially related to infections: fewer pathogens in the probiotic group. The probiotic group (the probiotic including Bifidobacterium longum, Lactobacillus bulgaricus, and Streptococcus thermophilus) did however show an altered immune profile to the control group in terms of indications of the Th1-Th2 slant to the immune system. Although perhaps a little simplistic, Th1 represents the fighting infection side of the immune system and Th2 is the production of antibodies side of things. The probiotic group showed an immune profile more representative of the Th1 response, speculated to be important for their speedier recovery and their less frequent use of antibiotics.

Although the results were not totally astounding in this study, there is an important clinical lesson from this trial in that patients spent statistically less time in intensive care and relied on fewer antibiotics during their recovery as a result of probiotic administration. For patients and physicians alike, this has got to be a good thing.

Finally, ScienceDaily carries an interesting statement from the Annual Scientific Meeting of the American College of Gastroenterology (ACG) regarding probiotics and the potential anti-inflammatory properties of certain types of probiotic. I like to sound of the collected press conference for the studies listed in this release: "Good, bad and ugly bugs: Mother Nature as a treatment for better health in the GI tract". As per the release there are a few headlines including:

• A meta-analysis of studies looking at the use of probiotics used to reduce cases of antibiotic-associated diarrhoea (diarrhea) concluded that yep, probiotics, and particularly that most lovable to yeasts Saccharomyces boulardii, do a pretty good job at curbing your risk of this quite unpleasant condition.
• The use of Bifidobacterium infantis 35624 for those suffering from recurrent abdominal bloating and discomfort did not fare too well in a new randomised-controlled trial. B.infantis 35624 had previously shown some interesting positive results for those with irritable bowel syndrome (IBS).
• Having said that, it was not all bad news for B.infantis 35624 as per another trial which suggested that the probiotic might have some pretty good anti-inflammatory properties when it comes to those dastardly pro-inflammatory cytokines. Indeed that most common of inflammatory markers, C-reactive protein seemed also to be reduced in cases of psoriasis, ulcerative colitis and even in chronic fatigue patients following probiotic administration.

What then can we assume from these collected studies. Well, at least in the short-term under various controlled conditions, probiotics, various probiotics, might just be able to influence our health and ill-health particularly in certain conditions/states. As per my previous post, our collected gut bacteria is a complicated organism which talks to our immune system in ways we are only starting to understand. Like every married couple, sometime the talk is positive and healthy; other times the talk is slightly less healthy... (not in front of the kids!).

Whether in the longer-term, supplementing with probiotics offers any added benefit, I don't know. Speculation (and it is only that) would perhaps suggest that the way probiotics are delivered (those all important enteric coatings), how our immune system recognise bacteria as 'self' or 'not-self' and good old homeostasis (the body's drive to keep harmony) are all factors potentially affecting the ability to make a more permanent shift in our not-so-good bacterial species to those preferred choices. Indeed this last point on what is good and bad gut bacteria perhaps needs a little more research in terms of the effects of individual species, etc on health and wellbeing.

* Delzeene NM. et al. Targeting gut microbiota in obesity: effects of prebiotics and probiotics. Nature Revs. November 2011.

** Min Tan. et al. Effects of probiotics on serum levels of Th1/Th2-cytokine and clinical outcomes in severe traumatic brain-injured patients: a prospective randomized pilot study. Critical Care. December 2011.

Crohn's disease and the LOFFLEX diet

A short post this one following a very interesting series airing here in the UK called the Food Hospital. 'Let food be your medicine' was the mantra of a certain Greek gentleman called Hippocrates (or perhaps according to Bill and Ted, 'Hippo-crates') and that is exactly what this series is suggesting.

In the programmes so far we have seen conditions ranging from migraine to obesity discussed. Tonight's programme featured a particularly brave young woman called Laura who presented with quite a serious case of Crohn's disease. For those who want more information about Crohn's see here. The long and short of it is inflammation, discomfort and quite a few 'peripheral' symptoms; no recognised cure and a life of medication and possibly surgery. Certainly one of the more serious gastrointestinal (GI) conditions.

Anyhow true to the name, the Food Hospital investigated whether it was possible the manage some of the symptoms of Crohn's disease through dietary changes. The plan in question is highlighted here and included a 3-step dietary intervention beginning with a cessation of food intake and reliance instead on a liquid feed containing all the necessary nutrients, followed a few weeks later by the implementation of the LOFFLEX (LOw Fibre, Fat Limited EXclusion) diet finally followed by dietary challenge/rechallenge in the hope of identifying problem foods related to symptoms.

It was interesting to see that some success was reported whilst on the regime in terms of symptom reduction following a flare-up. This is not proof of anything but certainly an interesting case study (n=1). Looking at the scientific literature for the LOFFLEX diet, things seem to be a little bit sparse at the moment. I found this trial from Addenbrookes Hospital which was the first place to look at the diet (I think). I also noted an interesting name on the authorship list, Prof. John Hunter, who is quite an expert on all things gastroenterology and particularly a role for gut bacteria in various bowel conditions.

I await more research on this very interesting regime and its potential applications to other conditions.

Cytokines and gut motility

A relatively short post this one.

I think most people would understand why gastrointestinal motility is important to our health and wellbeing. Too fast or too slow a transit time is likely to lead to some pretty uncomfortable symptoms and indeed could signal the presence of one of a number of complications.

A recently published review paper on motility in gastrointestinal disorders by Akiho and colleagues* (full-text here) caught my eye. I was interested in this paper because it reviews the association between gastrointestinal motility and the expression of cytokines as a function of which way the immune system might be skewed.

I should perhaps back-up slightly here and provide some commentary on the way the immune system can be poised (according to our current knowledge) and in particular the concepts of Th1 and Th2. A good overview of the T helper cells is here. In brief, it's all to do with different kinds of immunity and how our immune system attempts to strike a balance between cell-mediated immunity (Th1) and humoral immunity (Th2) depending on what particular pathogen the immune system is up against.

The Akiho review paper lists a number of the most common GI disorders currently in the medical dictionary and details what particular types of response and cytokines are tied into the disease state. So for coeliac (celiac) disease and Crohn's disease there is a predominantly Th1 skewed cytokine profile either associated with disease onset or perpetuation. In ulcerative colitis, it is more of a Th2-like response in terms of cytokines associated with the condition. The authors do make mention also about Th17-mediated inflammation (possibly linked to autoimmunity) but this is still very much an emerging area of investigation.

The authors then proceed to review the evidence that Th1-related cytokines seem to show more of an affinity with hypocontractality of inflamed intestinal smooth muscle (slowing down) and Th-2 show a more hypercontractility (speeding up).  This is perhaps too simplistic a view to take, one linked to one but not the other, given the number of cytokines tied into various GI states and the complexity of the whole thing. But their analysis of the current evidence base is an interesting one.

What work like this serves to show is that the our immune system is a fantastic piece of engineering constantly trying to strike a balance between fighting off pathogens and infections and invaders, whilst at the same time keeping the host (us) in working order, trying not to destroy us also. The presentation of GI conditions, many GI conditions, seem to reflect the inner workings (and malfunctions) of the immune system and when establishing how the immune system manifests itself in individual conditions, offers some tantilising insights into potential therapies.

* Akiho H. et al. Cytokine-induced alterations of gastrointestinal motility in gastrointestinal disorders. World J Gastrointest Pathophysiol. October 2011

I, claudin

There is no way that I can blog about the gastrointestinal (GI) tract without making mention of that wonderful barrier separating gut contents from the other recesses of our body. Without being too dramatic, I could probably say that I owe my livelihood to the gut barrier; as a consequence of much of my research career heading towards some involvement for the gut barrier at least in some cases of autism spectrum conditions.

A recent paper brought all the years of reading on this subject back to me with the suggestion of a link between intestinal inflammation and claudin-1. The paper by Poritz and colleagues* describes how key tight junction proteins like occludin and zonula occludens-1 (ZO-1) are decreased in inflammatory bowel diseases and how treatment of intestinal epithelial cells with the inflammatory cytokine TNF-alpha (sorry about the lack of Greek characters) seems to increase permeability (leaky gut). With all due respect to the authors, these findings are nothing new.

What was slightly more interesting from Poritz however were the findings in relation to the ratio of claudin-1 and occludin (C:O) as a consequence of cell treatment with TNF-a and what this ratio looked like in real life when comparing different intestinal diseases like Crohn's disease (CD) and ulcerative colitis (UC). Answer: decreased occludin and increased claudin-1 in diseased UC vs. non-diseased UC and normal colons and nothing spectacularly different in different disease-states of CD or controls.

Even to an interested amateur like me, this data indicate some things potentially very important: (a) ratios between multiples rather than independent statistics might yield more diagnostic information, and (b) there may be some underlying differences in the way that tight junctions might be affected in UC vs. CD.

I will come back to the leaky gut and various other things related to it at some point in the future no doubt. But for now if you really, really want some extra credits bedtime reading, here is an article that I always found invaluable on the subject (long yawn, adjust reading glasses and reach for that glass of scotch on the bedside table).

* Poritz LS. et al. Increase in the tight junction protein claudin-1 in intestinal inflammation. Dig Dis Sci. October 2011.

Parasitic infection, coeliac disease and inflammation

My day-to-day job does not normally include giving lectures and presentations but today was an exception. I was asked to give a short talk on autism, and specifically some of the more 'somatic' research aspects to the condition. One of the issues that cropped up during that talk was some discussion on how we might think that we are in charge of our behaviour, but more often than not external environmental forces might also influence us. By environment, I mean the physical environment we can see and interact with but also the more hidden aspects. Words like Toxoplasma gondii and schizophrenia were used as examples, but also some speculative suggestions about how other 'parasitic' infections might affect our health and wellbeing.

Such discussion brings me neatly to a recent paper by Henry McSorley and colleagues from Australia published in PLoS ONE. The crux of the paper (which is open-access) is that purposeful infection with hookworm might have some interesting positive effects on various aspects of inflammation in relation to coeliac (celiac) disease (CD).

I must admit that I have covered parasitic infection in a previous post on autism (here); the conclusion being that our modern-day obsession with hygiene leads to the eradication of certain parasitic species which have evolved alongside us, and with it, a suggested increase in things like inflammation and conditions manifesting inflammation.

It does appear that similar findings were reported by McSorley and colleagues, where:

• A 2-stage clinical trial was undertaken. Trial 1: 20 participants in total with CD and on a gluten-free diet; 10 were randomly infected with the larvae of the hookworm (Necator americanus) administered via the skin, the controls were given topically administered chilli pepper. At 20 weeks post-infection (with a top-up at 5 weeks), a gluten-challenge was given. Trial 2: a follow-up control participants, infecting 7 of them with hookworm, top-up infection and gluten challenge.
• Levels of duodenal interferon-gamma (IF-y) (sorry about the lack of Greek gamma symbols) and the cytokine IL-17A were significantly decreased in the hookworm infected group from Trial 1 post gluten challenge. This and other results led the authors to assume that helminth therapy might skew the immune response in CD towards a Th2 phenotype; in effect, a more anti-inflammatory response (see this article for a better description of Th1 and Th2).

I must point out that I am in no way advocating such parasite therapy for anyone. I am also under no illusion that these results suggest a simple relationship between parasite and inflammation because they do not. I am however drawn (again!) to the notion that such parasites might carry some important evolutionary function for human health and as such, our recent leanings towards cleanliness (next to Godliness apparently) and sterility might just be to the detriment of our health.

* McSorley HJ. et al. Suppression of inflammatory immune responses in celiac disease by experimental hookworm infection. PLoS ONE. September 2011.