"the first nationwide population-based study to investigate the risk of CFS in patients with IBD"

The quote titling this post - "the first nationwide population-based study to investigate the risk of CFS [chronic fatigue syndrome] in patients with IBD [inflammatory bowel disease]" - comes from the research published by Shin-Yi Tsai and colleagues [1].

Inflammatory bowel disease (IBD) covers quite a bit of diagnostic ground, but typically refers to ulcerative colitis (UC) or Crohn's disease (CD). There are many similarities between the conditions, but also some important differences too (see here). Both conditions manifest in the bowel (inflammation) and have pathological effects; both also typically show functional bowel symptoms too.

Utilising that fabulous (but sadly now defunct) research resource that was the National Health Insurance Research Database (NHIRD) in Taiwan, researchers set out to "evaluate the subsequent risk of CFS in patients with IBD" on the basis of "possible common pathophysiology between IBD and CFS" among other things. One of those 'pathophysiological' mechanisms quite prominently featuring in the Tsai article is "a similarity to the impaired intestinal mucosa of IBD." Interesting (see here).

So, from a starting population of a million people (or medical insurance records of a million people), authors whittled the figures down to the thousands in two groups: an IBD group (n=2163) and a non-IBD group (n=8652). All were "newly diagnosed" with IBD apparently; and none had a previous diagnosis of CFS "before the index date." Then: "Both groups were followed from the index date until the diagnosis of CFS, withdrawal from the NHI program, or December 31, 2011" with said CFS diagnosis following the Fukuda/CDC criteria.

Results: before heading into the CFS frequency figures according to group, there was another important observation made, potentially pertinent to a gut-brain connection: "The prevalence of depression, anxiety, [and] sleep disorder... was higher in the IBD group than in the non-IBD group." Indeed, the increased frequency of sleep disorder in the IBD group *might* have some important 'connection' with another diagnosis where sleep and 'gut issues' has been mentioned (see here and see here). And I might as well also mention depression and anxiety in that context too (see here).

Continuing: "After adjustment for age, and comorbidities, the risk of CFS was higher in the IBD group than in the non-IBD group (adjusted HR, 2.25; confidence interval [CI], 1.70–2.99)." There was also a possible sex-linked relationship too: "we identified male sex, advanced age, absence of comorbidities, and CD as the predictors of increased CFS risk." There's a pretty little diagram to accompany the Tsai findings (see here) outlining what *could* be going on with regards to IBD and CFS. Terms like 'bacterial translocation' and 'immunoinflammatory pathways' are used, in line with some other research in this area [2]. There's no mention of any psychobabble 'biopsychosocial' or the like in the Tsai paper which is always a good thing.

What else is there to say? Well authors go on to mention about the possibility of "intrinsic defects in IBD patients that precipitates CFS" which could have some quite profound implications for at least some cases of CFS. They added that future work might want to have a look at what certain immunotherapies indicated for some IBDs * might* mean for CFS and it's potential *treatment* too. We'll have to wait and see.

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[1] Tsai S-Y. et al. Increased risk of chronic fatigue syndrome in patients with inflammatory bowel disease: a population-based retrospective cohort study. Journal of Translational Medicine. 2019; 17:55.

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A role for viral infection in the aetiology of coeliac disease?

"In this longitudinal study, we found that a higher frequency of enterovirus infections was associated with increased risk of coeliac disease."

So said the findings reported by Christian Kahrs and colleagues [1] (open-access available here) based on the results of their: "Case-control study nested within Norwegian birth cohort recruited between 2001 and 2007 and followed to September 2016."

Following the screening of some 47,000 infants for the 'genetics of coeliac disease' - HLA genotype DR4-DQ8/DR3-DQ2 - over 900 children were entered on to the study. This group were followed "with repeated blood and faecal samples from the age of 3 months" for quite a few years. Just over 500 participants who quite regularly donated blood samples, were asked if they wanted a screen for coeliac disease. Two hundred and twenty of them (or their parents/guardians) said 'yes please' and the study results were drawn from this group. Twenty seven of the 220 participants with those all-important coeliac risk genes were diagnosed with the condition. Twenty five of those 27 diagnosed with coeliac disease were matched against 50 of the no coeliac disease participants; matching was done "for duration of follow-up, date of birth, and county of residence." It was then just a case of looking at all the biological data that had been accrued from those blood and fecal samples to ascertain things like (a) "the time interval when cases seroconverted for coeliac disease markers" (i.e. when the antibodies diagnostic of coeliac disease began to be present) and (b) if and when enterovirus was detected in some of the samples (stool samples) via PCR (polymerase chain reaction).

Results: as shown in the supplementary material, the mean age at first presence of coeliac disease (CD) antibodies in the CD diagnosed sample (n=25) was around 42 months (when the first positive sample was recorded). Symptoms 'debut' was around an average age of 73 months and CD diagnosis was received on average at 87 months. Enterovirus was reported in both CD (n=25) and non-CD groups (n=49). Some 20% of the stool samples from the CD group were positive for some kind of enterovirus exposure compared with 16% of controls. Slightly more enterovirus positive stool samples were observed in the CD group (median 4 positive samples per child) than the control group (median 3 positive samples per child).

As per the opening quote to this post: "Enterovirus was found in 370 (17%) of 2135 samples and was significantly more frequent in samples collected before development of coeliac disease antibodies in cases than in controls." Further: "The association was restricted to infections after introduction of gluten." In other words, there seemed to be some evidence of a possible temporal connection between enterovirus and the development of CD, suggesting that enterovirus exposure and not gluten was the more important trigger for CD.

Mechanisms? Well, there's some speculation about that in the Kahrs paper and a: "plausible explanation is that enterovirus causes impaired barrier function, which in turn increases the risk of coeliac disease." Enterovirus causing impaired intestinal (gut) barrier function eh? Interesting, does that mean 'leaky gut' might have a viral origin in some cases? Mmm, that could have lots of implications...

Obviously more investigation is required in this area. The Kahrs study has some strengths in terms of the sample collection protocols and frequency and potentially establishing a temporal *link* between CD and enterovirus exposure. But there's still more to do: "unmeasured confounding factors or residual confounding can never be entirely ruled out in non-randomised studies." But don't let that take anything away from the potential importance of these findings.

And on the topic of prospectively following children who might be prone to develop coeliac disease, the study findings published by Lionetti and colleagues [2] are equally interesting. In particular, based on 23 of their 26 children who received a "potential diagnosis of CD" but nonetheless "continued a gluten-containing diet... 19 (83%) became antibodies negative at 1 year from the first biopsy and remained negative up to 10 years of follow-up." Could there be a tie-up with the Kahrs findings perhaps?

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[1] Kahrs CR. et al. Enterovirus as trigger of coeliac disease: nested case-control study within prospective birth cohort. BMJ. 2019 Feb 13;364:l231.

[2] Lionetti E. et al. Long-Term Outcome of Potential Celiac Disease in Genetically at-Risk Children: The Prospective CELIPREV Cohort Study. J Clin Med. 2019 Feb 5;8(2). pii: E186.

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Gluten, mimicry and schizophrenia

The findings reported by Daniela Čiháková and colleagues [1] provide the rather long blogging fodder today, and some interesting observations on how the immune system 'reacting' to gluten in some cases of schizophrenia might have some pretty far-reaching consequences when it comes to other proteins with a similar chemical structure.

OK, first things first, gluten is a protein. It's made up of long chains of amino acids; the building blocks of proteins. As gluten is digested in the gastrointestinal (GI) tract, various enzymes get to work on the protein to break it down into it's constituent amino acids, forming peptides (short chains of amino acids) along the way. The protein gluten and its components like gliadin has a characteristic shape like every protein has. For reasons that aren't yet completely understood, the immune system of some people can sometimes 'mark' normally fairly harmless proteins like gliadin as something that needs dealing with. It does this via the production of antibodies. Antibodies usually mark pathogens like bacteria or viruses, and by doing so, set off a cascade of biological processes to [try and] ensure that such invaders don't take hold and also to remind the immune system of what to look out for. Marking something like gliadin out (which is neither bacteria nor virus!) probably means that the body is detecting gliadin in places it shouldn't be; something that I'll come back to in a minute.

It's not beyond the realms of possibility that when the immune system marks a specific protein as something to keep an eye on, it can make mistakes. If for example, two proteins 'look' similar to each other in a chemical arrangement sense, despite being different proteins with different functions, the immune system can sometimes become a little confused and start to mark both as being an invader on the basis of one already having 'special interest' status. Several descriptions describe such a process: "cross reactivity or mimicry." This accidental marking can sometimes have important repercussions, where such a process is thought to be a basis for autoimmunity.

Čiháková et al detail findings suggesting that just such a process - mimicry - could well be pertinent to some cases of schizophrenia. As already mentioned, they started with the observation that some people diagnosed with schizophrenia have high levels of specific antibodies to gliadin (see here and see here). This follows quite a lot of history linking gluten and schizophrenia together (see here). They wanted to see if as well as presenting with antibodies to gliadin, a cohort of people diagnosed with schizophrenia might also present with elevated antibodies to something called GRINA - Glutamate Ionotropic Receptor NMDA type Subunit Associated with protein 1. They focused in on GRINA because it has a "similar protein structure to gliadin representing a potential target for cross reactivity or mimicry." GRINA also links into glutamate system functioning, something which has also already been mentioned with schizophrenia in mind (see here).

There's another detail about the Čiháková study which relates to a point I touched upon earlier, on the possible hows-and-whys of gliadin antibodies being found in cases of schizophrenia: enhanced gut permeability a.k.a leaky gut. Researchers also analysed serum samples for the presence of something called Anti-Saccharomyces Cerevisiae antibodies (ASCA) which they say are "related to gut permeability." This follows other research in a similar vein (see here) and the suggestion that something like abnormal gut permeability *could* be implicated in some cases of schizophrenia.

Results: looking at serum samples of 160 people diagnosed with schizophrenia and 80 not-schizophrenia controls, researchers observed "a higher prevalence of positivity to ASCA IgA... and IgG" in those with schizophrenia. This tallies with the leaky gut hypothesis. They also reported that "GRINA IgG was higher in schizophrenia patients than in healthy controls." Putting these results together, they concluded that the mimicry hypothesis might well be pertinent to some schizophrenia.

There is a lot more work required in this area for sure. This will need to involve further investigation of the hows-and-whys of any such mimicry, and whether such a process could be a potential target for intervention. Indeed, in that intervention vein, I'm wondering whether use of a gluten-free diet for some with schizophrenia who have such antibodies (to gluten and other things like GRINA) might be an option. There's also merit in looking further at the issue of gut permeability and schizophrenia; whether again adoption of a gluten-free diet (which can positively affect gut permeability measurements) might be indicated, perhaps alongside other therapeutic targets.

But this area of research is interesting, and adds to the quite long research history linking food components and some behavioural / psychiatric labels...

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[1] Čiháková D. et al. Gut permeability and mimicry of the Glutamate Ionotropic Receptor NMDA type Subunit Associated with protein 1 (GRINA) as potential mechanisms related to a subgroup of people with schizophrenia with elevated antigliadin antibodies (AGA IgG). Schizophr Res. 2019 Jan 23. pii: S0920-9964(19)30007-6.

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Serum zonulin testing via ELISA: be very careful

I appreciate that the findings reported by Mary Ajamian and colleagues [1] probably aren't going to set many research hearts racing. Their observations that "current commercial zonulin assays are not detecting the actual protein as prehaptoglobin-2" is not exactly 'change the world' science, but that doesn't mean that they aren't important findings.

So, zonulin is the name of the research game. A protein described with properties "capable of reversible tight junction disassembly and, therefore, is implicated in the regulation of mucosal permeability" means that zonulin and dysregulation of the zonulin pathway has found a home in the science of 'gut permeability' a.k.a leaky gut. And it is with mention of the misnomer called 'leaky gut' and it's *association* with some autism (see here) that I gravitated towards the Ajamian findings. Indeed, zonulin has already made a mark in autism research too (see here). We'll come back to this shortly.

Researchers zoomed in on some of the commercially available methods currently available to 'test for zonulin' - "commercially-available ELISA assays" - and whether they are cutting the scientific mustard. And before I go on I should mention that Ajamian et al aren't the only ones who have looked at this issue [2]. Two ELISA assays were examined: "from CUSABIO (Wuhan, China) and Immundiagnostik AG (Bensheim, Germany)" and pitted against each other and various other analytical techniques to assess "whether the assays are reliably detecting zonulin as prehaptoglobin-2 and if not, what they may be detecting instead." I note the words 'mass spectrometric analysis' are also used in the Ajamian paper, which is music to my analytical ears.

Results: "Serum samples were collected from well-characterised patients and healthy individuals between the ages of 16 and 70 years living in Melbourne, Australia." Those 'well-characterised' participants included those diagnosed with non-coeliac wheat sensitivity (NCWS), coeliac disease, and ulcerative colitis (N=93) and their results were compared with nearly 50 asymptomatic controls. "The majority of study participants were zonulin-producers" as haptoglobin phenotype (see here) was also described in the Ajamian study.

Then to the serum [purported] zonulin levels as measured by those commercial assays: "Compared with the cohort of healthy individuals with a median (IQR) of 0.00 (0.00) ng/mL, patient median (IQR) values for purported zonulin were elevated (all p<0.0001) at levels of 0.032 (0.90) ng/mL in NCWS, 0.07 (1.27) ng/mL in coeliac disease, and 1.73 (2.17) ng/mL in ulcerative colitis" using the CUSABIO assay. Unfortunately, when compared with the other commercial assay (the Immundiagnostik assay), there was apparently little relationship observed between the two when it came to [purported] zonulin levels. And things didn't get any better when for example we are told that "2 of 19 participants who were zonulin non-producers had levels detected by CUSABIO assay."

Various other experiments were carried out and reported on in the Ajamian paper. These included attempts to find out what else might be being picked up by those ELISA assays. Unfortunately, even with the notable analytical prowess of something like mass spectrometry, no definitive compound(s) emerged. Something called complement C3 is discussed, as are other potential matches: "haptoglobin, and albumin." But again unfortunately: "neither complement C3 nor haptoglobin, despite both being candidate target proteins as determined by mass spectrometry, was detected by the CUSABIO assay." So we're not really any further forward when it comes to what might be being detected by such assays.

"In conclusion, the current commercial zonulin ELISA assays investigated in this study detect different proteins, neither of which was zonulin. Therefore, there can be no value of circulating concentrations in assessing intestinal mucosal barrier dysfunction and permeability until the target proteins are indeed identified." A harsh conclusion but faithful to the results observed. What this means is that the literature already published talking about zonulin levels in this, that and t'other label/diagnosis/condition (see here) need to be treated with some caution. And yes, that includes studies that have looked at zonulin levels in autism (see here) and related labels like attention-deficit hyperactivity disorder (ADHD) (see here).

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[1] Ajamian M. et al. Serum zonulin as a marker of intestinal mucosal barrier function: May not be what it seems. PLoS One. 2019;14(1):e0210728. Published 2019 Jan 14.

[2] Scheffler L. et al. Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family. Front Endocrinol (Lausanne). 2018;9:22.

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'Mucosal healing' in treated coeliac disease "may come at a cost of an increased risk of mood disorders"

I was really quite intrigued by the findings reported by Jonas Ludvigsson and colleagues [1] attempting to "examine the relationship between anxiety, depression and mucosal healing in coeliac disease." Intrigued because such findings tap into a couple of potentially important areas such as the whole gut-brain 'being linked' bit (see here and see here) and how gut permeability (a.k.a leaky gut) might be something quite important to quite a few different labels/diagnoses/states (see here).

I should perhaps briefly go through a few choice terms before starting. Coeliac disease (sometimes written as celiac disease) is the archetypal 'food affects physiology' condition as per it's description encompassing the words 'gluten', 'autoimmune disease' and 'gastrointestinal'. Part of the biological profile of coeliac disease (CD) is involvement of the gut mucosal membrane and the characteristic 'flattening' of the mucosal surface reducing the capability to properly absorb various nutrients. This is known as villous atrophy and is measured using the Marsh criteria. Luckily, for most people with CD, when a gluten-free diet is installed to treat/manage CD, the gastrointestinal (GI) pathology linked to villous atrophy abate and mucosal healing can take place as long as gluten remains excluded from the diet.

Ludvigsson - a name not unfamiliar to all-research-things coeliac disease - and colleagues sought to look at the presentation of certain extra-gastrointestinal symptoms as a function of mucosal healing (through use of a gluten-free diet) or persistent villous atrophy. They analysed data collected over nearly 40 years "on all small intestinal biopsies with villous atrophy from Sweden's 28 pathology departments." They also garnered data on the presence of a diagnosis of depression and/or anxiety, and married the two data sets together...

Results: "During follow-up, 123 (2.8/1000 person-years) individuals with mucosal healing had developed anxiety, compared to 94 (2.1/1000 person-years) with persistent villous atrophy." What this means is that whilst the presentation of anxiety was spread both across those who did and did not seemingly biologically benefit from the use of a gluten-free diet, those who showed gut healing were seemingly at "a higher risk of future anxiety." A similar trend was also noted when it came to a diagnosis of depression. This led researchers to highlight how "the goal of mucosal healing may come at a cost of an increased risk of mood disorders."

I was truly in head-scratching mode when it came to these findings. I don't know if it's just me, but I would have thought that persistent villous atrophy akin to not-quite-complete mucosal healing would have been more pertinent to the expression of anxiety and depression. I say that from the point of view that micro nutrients (and specifically, the lack of them) seem to have some important relationships with mood states and related conditions (see here and see here) under other circumstances. I'd also mention that a patient with CD knowing that the gluten-free diet wasn't 'doing what it's supposed to' in relation to managing CD at a biological level might also be a cause for concern for them and possibly lead to more complicated 'issues'?

So what could be the reason for the Ludvigsson findings? Well, subject to independent replication of the findings and perhaps also looking at other variables potentially pertinent to the presentation of anxiety and depression in CD (see here for example based on the 'birds of an autoimmune feather' hypothesis), one might surmise a few things. First, that the biological expression of CD 'hides' underlying issues such as anxiety and depression, and only through (successful) treatment do such mood disorders become 'unmasked'. Anecdotally and without any supporting evidence, I remember back to some of my research days looking at gluten- and casein-free diets being used in the context of [some] autism, and how some parents / autistic people reported that the diet seemed to 'reveal' anxiety issues for example, at the costs of helping to resolve certain behavioural and somatic (bowel) issues they faced. Could similar mechanisms apply to CD bearing in mind the effect that dietary gluten removal has on gut barrier function?

Next possibility: gluten peptides. It's pretty well known that the gluten peptides - the intermediate compounds that form during the digestion of the gluten - have some important biological action insofar as them possessing potential opioid activity. The gluteomorphins as they're known, share similar structural data to other well known opioid compounds such as morphine. It's probably one of the reasons that we've had such an enduring relationship with such foodstuffs. Could the rise of mood disorders be related to such biochemistry? Y'know, removal of gluten peptides and the mucosal healing affecting transport to the wider central nervous system meaning that patients aren't getting 'their fix'?

One other possibility is also worth mentioning and investigating: Eyes should also perhaps turn to what other effects a dietary change such as gluten removal might have on physiology; specifically on the trillions of wee beasties that call our GI tract home: the gut microbiota. Does a healed mucosa have implications for the gut microbiota?

Questions, questions, questions...

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[1] Ludvigsson JF. et al. Anxiety after coeliac disease diagnosis predicts mucosal healing: a population-based study. Aliment Pharmacol Ther. 2018 Oct 4.

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Bone health and autism continued

It's been a while since I last wrote about the topic of bone health and autism (see here). On that particular occasion, it was the work by Ann Neumeyer and colleagues [1] that provided the blogging fodder and the observation that: "BMD [bone mineral density] is lower in peripubertal boys with ASD [autism spectrum disorder]." BMD is important because of a possible association between lower BMD and risk of fracture or indeed, something more pathological.

Today I continue with this topic as per further work from Neumeyer and colleagues [2] looking to "examine macro- and micronutrient intakes and self-reported physical activity in boys with ASD compared to TDC [typically developing controls] and the relationship of these variables with BMD."

Based on data from nearly 50 boys aged 8-17 years of age (25 diagnosed with ASD and 24 not-autism controls), researchers once again relied on the technique known as dual-energy x-ray absorptiometry (DXA) for the measurement of bone mineral density. Various measures were taken from various parts of the body -"whole body less head, hip, and spine." Alongside, food diaries provided a rough-and-ready measure of food intake, self-reported physical activity (that's self-reported) did what it said on the tin, and fasting levels of 25(OH) vitamin D and calcium were garnered. I'll come back to some of the pros-and-cons of some of these measures shortly.

Results: consistent with the peer-reviewed data that has come before, BMD z scores at the lumbar spine, femoral neck, total hip, and whole body less head were lower in those with autism compared with control participants. A BMD z score by the way, is basically a comparison of BMD with that of standardised data (i.e. an average person of the same sex and age). Added to such results, authors also observed that less calorie intake was present in the ASD group (again compared with controls) and a "lower proportion of ASD participants were categorized as "very physically active" (27% vs 79%; P<0.001)." Interestingly however: "Body mass index and serum vitamin D and calcium levels were similar."

I was rather intrigued by the Neumeyer results. Not least that vitamin D and calcium levels were 'similar' in the autism and control groups. As I've discussed before on this blog, there have been calls for preferential screening for these biological parameters as and when an autism diagnosis is received (see here) in light of other findings (see here). The Neumeyer observations perhaps reflect a wider need for such screening.

The use of self-report as a measure for physical activity, whilst useful, is slightly outdated in these times of actigraphy. Wearable technology to measure activity and rest cycles is cheap and abundant these days and, as I've mentioned on other occasions, really should be the research industry standard. I'm not saying people might not be accurate in reporting their short-term physical activity but...

I do think there are some additional 'where next?' things to consider when it comes to future work looking at BMD and autism. Noting for example, that both dietary and malabsorptive issues seem to be able to influence BMD [3] there are additional parameters to be looked at. Given previous peer-reviewed reports on lactose issues being present in relation to autism (see here), this could feature in future work. Although still possessing the ability to furrow brows in certain quarters, the observation of issues with intestinal permeability ('leaky gut') in relation to some autism (see here) also could be an additional parameter to examine. I daresay also that some initial chatter about a compound called zonulin potentially serving as a 'biomarker of impaired gut barrier function' in relation to some autism (see here) might also be revealing. And then there is the important issue of epilepsy / seizure disorder often being comorbid with autism (see here), and how certain [important] intervention measures for said issues might affect parameters such as vitamin D (see here) and what that might mean for long-term bone health...

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[1] Neumeyer AM. et al. Bone density in peripubertal boys with autism spectrum disorders. J Autism Dev Disord. 2013 Jul;43(7):1623-9.

[2] Neumeyer AM. et al. Nutrition and Bone Density in Boys with Autism Spectrum Disorder. J Acad Nutr Diet. 2018 Feb 3. pii: S2212-2672(17)31749-5.

[3] Di Stefano M. et al. Lactose malabsorption and intolerance and peak bone mass. Gastroenterology. 2002 Jun;122(7):1793-9.

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Antibodies against gluten in autism don't correlate with leaky gut markers

The paper by Jan Józefczuk and colleagues [1] provided some intriguing observations pertinent to quite a few topics previously discussed on this blog. Not only was there mention of anti-gliadin antibodies (AGA) and more specifically the finding that increased IgG-AGA was found in a quarter of their 77 participants with autism included for study, but also the important point: "An increased production of antibodies related to gliadin and neural TG6 [neural transglutaminase 6] in ASD [autism spectrum disorder] children is not related to serological markers of an impaired intestinal barrier."

It's worthwhile breaking down some of the details of the Józefczuk findings and what they might mean. I'll warn you that this is likely to be a bit of a long-read blog post so please, get comfy and read on...

So, AGA and more specifically, IgG-AGA represent the immune system 'recognising' gliadin, an important part of the protein gluten. IgG-AGA are typically found in many people diagnosed with the archetypal gluten-related autoimmune condition called coeliac (celiac) disease (although this measure is not considered diagnostic) but also alongside other more 'non-coeliac' gluten sensitivity conditions too (see here). The finding of elevated IgG AGA in cases of autism is by no means a new one (see here) (yes, this data came from the AGRE program [2] so no quibbling about the diagnosis of autism or anything like that). Other data has indicated that the presence of such antibodies seems to be a good reason to attempt a gluten-free diet (see here) which is music to my autism research ears (see here) and a good evidence-based reason to quiet down those who might 'challenge' such dietary intervention in the context of [some] autism. Then also is the idea that the presence of IgG AGA in conditions not totally unrelated to autism (see here) *might* play an important role in something like 'peripheral' inflammation [3] and whether the same could be true for [some] autism...

Next: "antibodies against neural transglutaminase 6 (TG6)" described by the authors as present in about 5% of their cohort is also an important finding. Turning up in a variety of different conditions [4] (although not yet considered 'mainstream' in certain quarters), some of the most interesting, and potentially relevant, labels where TG6 might be present include something called gluten ataxia [5], a neurological 'sign' characterised by a 'lack of voluntary coordination of muscle movements that includes gait abnormality.' Again, the data is compelling insofar as the use of a gluten-free diet as an intervention option [6] where gluten ataxia is diagnosed. With specific regards to autism and TG6 antibodies, I think the Józefczuk paper provides the first research outing for the two together (at least in a PubMed search). Other transglutaminase antibodies however, have been reported in the context of autism (see here for example) with the need for lots more investigations including with reference to the overlap between autism and coeliac disease (see here). At this point, I'll also note that gluten ataxia has not yet been linked to autism despite ataxia potentially showing some connection to some cases [7].

Moving on and we have the finding that: "Mean levels of zonulin and I-FABP [intestinal fatty acid binding proteins] in ASD [autism spectrum disorder] patients were similar to those found in healthy controls." Further: "Serum concentrations of zonulin and I-FABP showed no statistically significant association with antibody positivity." OK, zonulin is another topic of interest to this blog particularly in light of other recent findings with autism in mind (see here). Still the topic of considerable debate, zonulin has been described as "a biomarker of impaired gut barrier function for several autoimmune, neurodegenerative, and tumoral diseases" [8]. The data so far seems to indicate that gliadin - that component of gluten - 'induces' zonulin release [9] hinting that diet may be an important variable when it comes to 'impaired gut barrier function' otherwise known as intestinal hyperpermeability (or more imprecisely, leaky gut). I-FABP is something I'm a little less sure about in any context. It has been mentioned in the peer-reviewed research arena with autism in mind [10] but I can claim not expertise on this specific marker.

The Józefczuk findings report that zonulin levels in their cohort with autism were similar to "to those found in healthy controls" which is contrary to those previous findings in autism published by Erman Esnafoglu and colleagues [11]. They (Esnafoglu et al) concluded that: "Serum zonulin levels were significantly higher in the patients with ASD (122.3 ± 98.46 ng/mL) compared with the healthy controls (41.89 ± 45.83 ng/mL). " Forgetting (but not excusing) the incorrect use of 'healthy controls' in that paper, there is something of a difference between the Esnafoglu and Józefczuk results. One could argue that this is simply reflective of 'conflicting' autism research more generally (see here) but one might also question things like the analytical ways-and-means of assaying for something like zonulin too (see here).

The observation that zonulin (and I-FABP) levels showed 'no statistically significant association with antibody positivity' whilst informative is something I've been thinking about quite a bit. I don't have any easy answers as to why they found what they found aside from assuming that such data is evidence for how "increased immune reactivity against gluten" might not be specifically related to "the effect of intestinal barrier abnormalities" in relation to autism. This relationship assumes that abnormal gut permeability is the route through which gluten fragments (peptides) gain access to the wider central nervous system (CNS) which then elicits that immune response. It is a little surprising that no relationship was found given that one of the ways that zonulin is released is ingestion of gliadin and the assumption that gluten needs to be present in the diet for antibodies to be formed against it. But there you go. I suppose one might entertain the possibility that zonulin as a biomarker of impaired gut barrier function might not be the optimal way of measuring gut barrier function and including other more direct measures [12] could be the way forward to resolving this issue further.

Either way, research on immunological responses to gluten and notions of atypical gut permeability in the context of 'some autism' are seemingly not going away any time soon. Indeed, even medical professionals are seemingly not adverse to prescribing a gluten-free diet in the context of [some] autism [13]...

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[1] Józefczuk J. et al. The Occurrence of Antibodies Against Gluten in Children with Autism Spectrum Disorders Does Not Correlate with Serological Markers of Impaired Intestinal Permeability. J Med Food. 2017 Oct 26.

[2] Lau NM. et al. Markers of Celiac Disease and Gluten Sensitivity in Children with Autism. PLoS One. 2013 Jun 18;8(6):e66155.

[3] Kelly DL. et al. Anti Gliadin Antibodies (AGA IgG) Related to Peripheral Inflammation in Schizophrenia. Brain Behav Immun. 2017 Oct 23. pii: S0889-1591(17)30476-2.

[4] Gadoth A. et al. Transglutaminase 6 Antibodies in the Serum of Patients With Amyotrophic Lateral Sclerosis. JAMA Neurol. 2015 Jun;72(6):676-81.

[5] Hadjivassiliou M. et al. Autoantibodies in gluten ataxia recognize a novel neuronal transglutaminase. Ann Neurol. 2008 Sep;64(3):332-43.

[6] Hadjivassiliou M. et al. Dietary treatment of gluten ataxia. Journal of Neurology, Neurosurgery, and Psychiatry. 2003;74(9):1221-1224.

[7] Ahsgren I. et al. Ataxia, autism, and the cerebellum: a clinical study of 32 individuals with congenital ataxia. Dev Med Child Neurol. 2005 Mar;47(3):193-8.

[8] Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Annals of the New York Academy of Sciences. 2012;1258(1):25-33.

[9] Clemente MG. et al. Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function. Gut. 2003 Feb;52(2):218-23.

[10] Pusponegoro HD. et al. Maladaptive Behavior and Gastrointestinal Disorders in Children with Autism Spectrum Disorder. Pediatr Gastroenterol Hepatol Nutr. 2015 Dec;18(4):230-7.

[11] Esnafoglu E. et al. Increased Serum Zonulin Levels as an Intestinal Permeability Marker in Autistic Subjects. Journal of Pediatrics. 2017; 188: 240-244.

[12] Bischoff SC. et al. Intestinal permeability – a new target for disease prevention and therapy. BMC Gastroenterology. 2014;14:189.

[13] Rubenstein E. et al. The prevalence of gluten free diet use among preschool children with autism spectrum disorder. Autism Res. 2017 Nov 20.

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Extended release melatonin for sleep issues in autism

"PedPRM was efficacious and safe for treatment of insomnia in children with ASD [autism spectrum disorder] with/without ADHD [attention-deficit hyperactivity disorder] and NGD [neurogenetic disorders]."

So said the results published by Paul Gringras and colleagues [1] (open-access) covering a topic that is bound to create some excitement/interest given that the diagnosis of autism is no stranger to the issue of sleep problems (see here). Indeed, some have talked about sleep issues/problems in the context of autism as being an important influencing variable on parental well-being too [2].

From a starting population of over 250 children diagnosed with an ASD who were reported to have sleeping issues "(minimum 3 months of impaired sleep defined as ≤6 hours of continuous sleep and/or ≥0.5-hour SL from lights-off on 3 of 5 nights based on parent reports and patient medical history)" but not necessarily a sleep disorder, some 95 children / young adults completed the randomized, double-blind, placebo-controlled element of the trial. PedPRM - Paediatric Prolonged-Release Melatonin - was the active treatment option, compared against a placebo given over 13 weeks. "The a priori primary endpoint was SND [Sleep and Nap Diary]-reported total sleep time (TST) after 13 weeks of treatment." Melatonin, by the way, is one of the treatment options of choice when it comes to sleeping issues across various different clinical scenarios.

"The study met the primary endpoint demonstrating statistically significant effects of PedPRM vs. placebo on change from baseline in mean SND-assessed TST after 13 weeks of double-blind treatment." In other words, it worked. Indeed, when researchers set a standard of 45 minutes or more extra total sleep time as an indication of clinical response to PedPRM, almost 40% of their cohort taking the drug fitted that category compared with only 16% of those taking the placebo. "The 21.5% difference in percentage between groups equals NNT [number needed to treat]=4.7 for any additional responder above placebo."

When first looking at the study design presented by Gringras et al I was about to comment on a potential weakness based on the [subjective] use of caregiver report when it came to total sleep time. I'm not saying that this may not have provided a good rough-and-ready measure of TST but rather that it cannot provide actual physiological data on something like sleep duration and 'intensity' for example. As it happens however: "Actigraphy monitors (Actiwatch) were included in the study as a secondary objective measurement tool for the primary (TST) and first secondary (SL) sleep parameters." They weren't used throughout the whole study duration and were subject to some consumer resistance - "Despite major efforts to ensure adherence, actigraphy monitoring was challenging in this population, and a majority of participants (75% in the PedPRM and 77% in the placebo group) refused to wear the device during one or both periods and/or took it off some time during the night" - but did appear to show some effect in favour of PedPRM on TST and sleep latency (the time taken to fall asleep) for those who wore them during the study period. I guess the challenge for future studies is to make research actigraphy more 'autism-friendly'...

On the important issue of side-effects, the authors also report some observations. Treatment-emergent adverse events (TEAEs) were reported in not-insignificant numbers in both active (PedPRM) and placebo groups: "TEAEs judged by the clinician as treatment-related occurred in 12 (20.0%) participants in the PedPRM and 11 (16.9%) in the placebo group (28 and 20 events respectively)." Somnolence defined as sleepiness and drowsiness was one of them(?) but on the whole, the cost-benefit balance of the intervention seemed to favour use over non-use of PedPRM. There are a few other details included in the Gringras findings but I'm satisfied that the trial showed quite a good profile for the preparation in terms of efficacy and safety. Given also that melatonin is one of the more widely used medicines when it comes to autism, such results are perhaps not unexpected.

I do think more needs to be done in this area, not least on the hows-and-whys of melatonin working on sleep and other physiological functions. Yes, I know the whole biological pathway of melatonin (I've blogged about it before) but like many other medicines, melatonin has other many effects too, not least potentially affecting something else potentially important to some autism - intestinal barrier integrity a.k.a the leaky gut (see here). I wonder therefore if, when it comes to looking at potential responders and non-responders to such melatonin use, researchers might consider measuring gut permeability and perhaps other related compounds (see here) as part of their clinical research scheduling?

And whilst talking about sleep issues in the context of autism, I'll be coming to the findings reported by Len McCue and colleagues [3] on gut and sleep issues potentially intersecting in autism quite soon...

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[1] Gringras P. et al. Efficacy and Safety of Pediatric Prolonged-Release Melatonin for Insomnia in Children With Autism Spectrum Disorder. Journal of the American Academy of Child & Adolescent Psychiatry. 2017. Sept 19.

[2] Bourke-Taylor H. et al. Relationships between sleep disruptions, health and care responsibilities among mothers of school-aged children with disabilities. J Paediatr Child Health. 2013 Sep;49(9):775-82.

[3] McCue LM. et al. Gastrointestinal dysfunctions as a risk factor for sleep disorders in children with idiopathic autism spectrum disorder: A retrospective cohort study. Autism. 2017 Nov;21(8):1010-1020.

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SHANK3 and intestinal barrier function might have implications for some autism...

According to the Spectrum Wiki, SHANK3 - SH3 and multiple ankyrin repeat domains 3 - is described as "a leading autism candidate gene, with mutations occurring in between 1 and 2 percent of individuals with autism spectrum disorder."

Providing instructions for making the SHANK3 protein, alterations to the structure or function of the SHANK3 gene can have some quite far-reaching consequences, notably including disrupting communication between neurons in the brain.

A recent paper published by Shu-Chen Wei and colleagues [1] provides some further discussion on how issues with SHANK3 may very well extend beyond just 'brain function' and indeed, may overlap with reports of an over-representation of bowel disease in the context of autism (see here). In effect, a *possible* genetic association between autism and inflammatory bowel disease. Possibly...

Wei et al initially relied on a mouse model of SHANK3 disruption, something that has also reached autism research [2]. Said mouse model was artificially exposed to dextran sulfate sodium, a compound that creates experimental colitis mimicking human inflammatory bowel conditions such as ulcerative colitis. Various measures were employed to explore the interaction between colitis in the SHANK3 knockout mice pertinent to the expression of intestinal permeability, hyperpermeability of which is also known as 'leaky gut' in some quarters. Gut permeability issues are 'on the radar' when it comes to at least some autism (see here). Researchers also looked at SHANK3 expression in a cohort of human participants diagnosed with an inflammatory bowel disease called Crohn's disease.

Results: "SHANK3 knockout resulted in a leaky epithelial barrier phenotype, as demonstrated by decreased transepithelial electrical resistance, increased paracellular permeability, and increased Salmonella invasion." Going back to the idea that genetic issues identified as being potentially pertinent to some autism might extend beyond just 'effects on the brain' this is an important finding. Much like in other identified genetic conditions manifesting autism or autistic traits, the indications are that intestinal issues might be part and parcel of some autism where SHANK3 issues have been identified (see here and see here for other examples under other genetic conditions).

Further: "Overexpression of SHANK3 enhanced ZO-1 expression, and knockdown of SHANK3 resulted in decreased expression of ZO-1." ZO-1 refers to zonula occludens-1, something called a tight junction protein which serves an important function in intestinal barrier biochemistry. In effect, ZO-1 and other tight junction proteins seal the space - paracellular space - that is part of the intestinal barrier. The implication being that under expression of SHANK3 seems to have a detrimental effect on metabolites involved in intestinal barrier integrity; something also noted when it came to Wei and colleagues looking at "colonic tissue of patients with Crohn's disease" with ZO-1 in mind.

I used the words 'might have implications for some autism' in the title of this post but hasten to add that much more investigation is still required. Yes, SHANK3 seems to have a place in the aetiology and pathology of 'some autism' but further confirmatory research is required. Not least that, as far as I am aware, no-one has actually looked at intestinal barrier function directly in cases of Phelan-McDermid syndrome a primary outcome of SHANK3 genetic issues, despite some chatter about gastrointestinal issues potentially being no stranger to such a diagnosis [3]. I would also like to see a little more done on the measurement of something like zonulin where SHANK3 is mentioned in the context that zonulin seems to have a connection to intestinal barrier integrity. Whether, similar to other preliminary work looking at autism and zonulin (see here), there may be merit in testing when SHANK3 issues are likewise identified.

And while we're on the topic of mouse models and possible connections to autism, it's worth noting the findings reported by Groves and colleagues [4] talking about what vitamin D deficiency might do to certain mouse behaviours in the context that vitamin D has some autism research history too...

Music to close, and not to make light of SHANK3 but it does conjure up the sound of quite a famous song...

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[1] Wei SC. et al. SHANK3 Regulates Intestinal Barrier Function Through Modulating ZO-1 Expression Through the PKCε-dependent Pathway. Inflamm Bowel Dis. 2017 Oct;23(10):1730-1740.

[2] Yoo J. et al. Shank mutant mice as an animal model of autism. Philosophical Transactions of the Royal Society B: Biological Sciences. 2014;369(1633):20130143.

[3] Kolevzon A. et al. Phelan-McDermid syndrome: a review of the literature and practice parameters for medical assessment and monitoring. Journal of Neurodevelopmental Disorders. 2014;6(1):39.

[4] Groves NJ. et al. Adult vitamin D deficiency exacerbates impairments caused by social stress in BALB/c and C57BL/6 mice. Psychoneuroendocrinology. 2017 Sep 6;86:53-63.

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"a leaky gut may play a critical role in the development of age-related inflammation and frailty"

The quote heading this post is taken from the findings reported by Yanfei Qi and colleagues [1] who set out to investigate whether "an aging-associated leaky gut is linked to the age-related inflammation and frailty."

'Leaky gut' is still something of a contentious claim in medical and scientific circles despite some increasing evidence to say that it is a real phenomenon and potentially relevant to several conditions (see here) including some parts of the autism spectrum (see here). Although leaky gut is perhaps a slight misnomer - we all have leaky guts to some degree - the more accurate term 'intestinal hyperpermeability' suggests that for several different reasons, parts of the gastrointestinal (GI) tract may, at times or more chronically, be slightly more permeable that they typically should be. This in turn means that the contents of the GI tract - food derivatives, gut bacteria, etc - might be more readily exposed to parts of the body that they really shouldn't be and onward, potentially pathogenic.

Qi et al carried out some important analysis on two cohorts of differing ages (18-30 years old vs. 70 years and over) with regards to serum samples provided by participants. Markers of immune function, specifically inflammatory related compounds (e.g. tumour necrosis factor (TNF)-α and interleukin (IL)-6)) were assayed for, alongside levels of zonulin "a marker for leaky gut". Zonulin is something that I've covered quite recently on this blog in relation to autism research (see here). This biological data gathered by Qi and colleagues was also complemented by physiological measures such as "strength of plantar flexor muscles and number of steps taken per day."

Results: serum concentrations of zonulin were quite a bit - 22% - higher in the older participants compared to younger ones. Researchers also reported that levels of "high-mobility group box protein (HMGB1, a nuclear protein triggering inflammation)" were elevated in the older participants group too. They observed that zonulin levels also seemed to tie into concentrations of TNF-α and IL-6 (albeit not necessarily impressively). Zonulin levels also showed a potentially important *relationship* with "habitual physical activity" (those 'steps per day' data that was collected). The conclusion: "Serum zonulin was associated with both systemic inflammation and 2 key indices of physical frailty. These data suggest that a leaky gut may play a critical role in the development of age-related inflammation and frailty."

This is interesting stuff. It kinda accords with other independent data suggesting that generally speaking, gut barrier function does not necessarily have to deteriorate with age, but under certain circumstances such as age + inflammation, there might be effects to had [2]. Certain classes of medication might also show some involvement in this process too [3]. Although by no means a universal connection, I was interested in these results in the context of autism. Specifically how those Esnafoglu et al findings [4] reporting on 'significantly higher' serum zonulin levels in their cohort with autism, might also tie into reports of inflammatory markers being elevated in at least a subgroup of those on the autism spectrum (see here for example). I'd like to see the Qi study replicated with quite a few different labels and subgroups within labels...

In relation to the suggestion that serum zonulin levels negatively correlated with habitual physical activity (steps per day), again, there is a whole other research agenda to be [cautiously] followed. It's already known that certain patterns of exercise can affect (increase) intestinal permeability [5] although the specifics still require quite a bit more investigation (hint: exercising for 2 hours plus at a time is probably not great for gut function). As with everything in life, there is a balance to be struck between too little and too much of a good thing...

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[1] Qi Y. et al. Intestinal Permeability Biomarker Zonulin is Elevated in Healthy Aging. J Am Med Dir Assoc. 2017 Jul 1. pii: S1525-8610(17)30297-9.

[2] Valentini L. et al. Small intestinal permeability in older adults. Physiol Rep. 2014 Apr 22;2(4):e00281.

[3] Meier J. & Sturm A. The intestinal epithelial barrier: does it become impaired with age? Dig Dis. 2009;27(3):240-5.

[4] Esnafoglu E. et al. Increased Serum Zonulin Levels as an Intestinal Permeability Marker in Autistic Subjects. J Pediatr. 2017 May 11. pii: S0022-3476(17)30487-0.

[5] Costa RJS. et al. Systematic review: exercise-induced gastrointestinal syndrome-implications for health and intestinal disease. Aliment Pharmacol Ther. 2017 Aug;46(3):246-265.

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Welcoming zonulin into autism research

I was VERY happy to read the paper published by Erman Esnafoglu and colleagues [1] suggesting that: "zonulin, which regulates intestinal permeability, plays a role in the development of symptoms of ASD [autism spectrum disorder]."

Zonulin - something that "can be used as a biomarker of impaired gut barrier function for several autoimmune, neurodegenerative, and tumoral diseases" [2] - is a compound that I've been interested in for a while on this and other blogs (see here). The primary reason for the interest is that connection to intestinal permeability and how 'leaky gut' may well show some relevance to some autism (see here and see here). The thing that was up-to-now missing from the research chatter about intestinal hyperpermeability and autism was the measurement of zonulin on the basis that elevated levels of zonulin show a connection to dietary elements such as gliadin (a facet of gluten) [3]. This is particularly relevant because previous data has observed a possible link between use of a gluten-free diet and a reduction in intestinal permeability in relation to autism [4]. All this is [peer-reviewed] research music to my ears (see here)...

Esnafoglu et al set about measuring serum levels of zonulin in 32 participants diagnosed with an autism spectrum disorder (ASD) compared with 33 not-autism controls. Yet again, the words 'healthy controls' are used by the authors to define the control group and yet again, the assumption is that those participants with autism are somehow 'unhealthy'. Researchers, please just call it what it is: not-autism controls (the term 'neurotypical' also tells us nothing about control groups either). Measurement of zonulin was via ELISA (enzyme-linked immunosorbent assay) and researchers also threw in a measure of autism severity based on use of the Childhood Autism Rating Scale (CARS).

Results: well, the results seemed to be in the expected direction: "Serum zonulin levels were significantly higher in the patients with ASD (122.3 ± 98.46 ng/mL) compared with the healthy controls (41.89 ± 45.83 ng/mL)." Authors also identified a fairly healthy correlation between the CARS score and zonulin levels. These results imply that issues with intestinal permeability - leaky gut - seem to be present in relation to at least some autism. A shocker, I know.

Obviously there is more research to do in this area; not least to increase the sample size, look at dietary intake/status as a function of zonulin measurement and explore the possibility that the genetics of zonulin production might also be *involved* in some autism [5]. I might add that other research on zonulin in relation to diagnoses not necessarily uncommon to autism might also be revealing (see here).

Insofar as what to do about elevations in zonulin as and when detected in cases of autism, well the dietary link to zonulin production implies that the horror that is a gluten-free (GF) diet might be something to consider. The suggestion of a 'bacterial link' to zonulin production also suggests another possible intervention target in these days of gut microbiomes and autism (see here) although I think we have to be slightly careful about the use of some preparations. There is also another avenue for research speculation based on the development of zonulin (receptor) inhibitors such as Larazotide acetate [6] (otherwise known as AT-1001). With no medical or clinical advice given or intended, the evidence base for this zonulin-affecting compound is looking promising [7] with much more to come...

In conclusion, zonulin has arrived on the autism research scene, and I'm expecting to see more peer-reviewed science on this topic in future times. Intestinal hyperpermeability, diet and [some] autism looks to be squarely back on the research agenda.

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[1] Esnafoglu E. et al. Increased Serum Zonulin Levels as an Intestinal Permeability Marker in Autistic Subjects. J Pediatrics. 2017. May 11.

[2] Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Annals of the New York Academy of Sciences. 2012; 1258(1) :25-33.

[3] Lammers KM. et al. Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3. Gastroenterology. 2008 Jul;135(1):194-204.e3.

[4] de Magistris L. et al. Alterations of the intestinal barrier in patients with autism spectrum disorders and in their first-degree relatives. J Pediatr Gastroenterol Nutr. 2010 Oct;51(4):418-24.

[5] Tripathi A. et al. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proc Natl Acad Sci U S A. 2009 Sep 29;106(39):16799-804.

[6] Fasano A. Intestinal Permeability and its Regulation by Zonulin: Diagnostic and Therapeutic Implications. Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association. 2012;10(10):1096-1100.

[7] Leffler DA. et al. Larazotide Acetate for Persistent Symptoms of Celiac Disease Despite a Gluten-Free Diet: A Randomized Controlled Trial. Gastroenterology. 2015; 148: 1311-1319.

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Esnafoglu, E., Cırrık, S., Ayyıldız, S., Erdil, A., Ertürk, E., Daglı, A., & Noyan, T. (2017). Increased Serum Zonulin Levels as an Intestinal Permeability Marker in Autistic Subjects The Journal of Pediatrics DOI: 10.1016/j.jpeds.2017.04.004

Gut barrier integrity meets blood-brain barrier integrity with autism in mind

"In the ASD [autism spectrum disorder] brain, there is an altered expression of genes associated with BBB [blood-brain barrier] integrity coupled with increased neuroinflammation and possibly impaired gut barrier integrity."

Although pretty enthused to see research linking names like Anna Sapone, Tim Buie and Alessio Fasano in the recent paper published by Maria Fiorentino and colleagues [1] (open-access), I was slightly less impressed with the use of the term 'the ASD brain' in their paper potentially joining two concepts that I've been quite interested in down my research years: gut barrier and blood-brain barrier function in the context of autism. Yes, I accept that those most precious of resources, donated brains from the deceased, represented some of the 'material' under scientific scrutiny, but if science has learned anything about autism down the years, it is that sweeping generalisations such as terms like 'the autism brain' don't reflect what the existing research tells us about the heterogeneity under the label. I might just as well use the term 'blogger brain' to denote some of my activities, but such a label tells you nothing about me aside from my pastime.

After that little rant, the paper from Fiorentino is an interesting one in that the goal was to "investigate whether an altered BBB and gut permeability is part of the pathophysiology of ASD." To do this, tissue from both brain and gastrointestinal (GI) tract donated by a small number of deceased and non-deceased participants who were diagnosed with autism, schizophrenia or nothing related (not-autism controls) were analysed "for gene and protein expression profiles." This work was undertaken on the basis of "the interconnectivity of the gut–brain axis, [that] suggests that inappropriate antigen trafficking through an impaired intestinal barrier, followed by passage of antigens or activated immune complexes through a permissive blood–brain barrier (BBB), can be part of the chain of events leading to neuroinflammation and thereby subsequent disease." I might add that the use of the word 'disease' in that sentence is, I think, aiming to describe the physiological effects of 'leaky barriers' not the diagnosis of autism. It is unfortunate however that 'disease' still continues to be banded around in the context of autism [2].

I think it's important to stress that the Fiorentino study was in effect two studies: one that looked at brain samples from one participant group who had died, and one that looked at GI samples from those who were still living (at the time of sample collection) and who presented with "GI symptoms undergoing esophagogastroduodenoscopy (EGD) for clinically indicated reasons." This was not a study where biological samples - brain and gut - came from the same person but rather a mash-up. Keep that in mind for now. The sorts of genes that were focused in on were those "associated with the formation, integrity, and function of the BBB and neuroinflammation" and included the claudins and something called MMP-9 and MMP-2 that have been discussed previously on this blog (see here) with leaky barriers in mind. The key words are 'barrier integrity' when it comes to the list of compounds that were under inspection.

Results: well it was good to see the authors list details of each of the participants from which tissue were used in their study. Brain tissue from the deceased with autism for example, is subject to quite a few factors that can influence the outcome of any results obtained; not least whether specific comorbidity accompanied their autism diagnosis and the nature of their death. Indeed, looking through the various case report numbers, I'm struck by how young many participants, particularly those diagnosed with autism, were at the time of their death. This ties into other discussions and debates (see here).

"Our molecular analysis of the BBB integrity and function shows an altered BBB in the ASD subjects evaluated." This was evidenced by elevations in the gene expression of MMP-9 and its proposed connection to disturbances of BBB integrity. Further: "Of the four claudins (i.e., CLDN-1, -3, -5 and -12) that to date are thought to be incorporated in the BBB... we found that two were significantly more expressed in the ASD brain as compared in HC [healthy controls]." Once again I might suggest the term 'healthy controls' is not an inappropriate one when it comes to determining not-autism or not-schizophrenia.

Then to analysis of those [independent] gut biopsy samples: "results, showing increased expression levels of pore-forming (66% of the ASD samples) and decreased levels of barrier-forming (75% of the ASD samples) TJ [tight junction] components in the duodenal samples, suggest an impaired gut barrier and serve as a proof of concept to support the hypothesis of a gut–brain axis dysfunction in a subgroup of ASD patients." So, those compounds linked to making the gut barrier more 'leaky' were seemingly increased in expression, and those linked to making the gut barrier less 'leaky' were reduced in quite a few of the samples from those diagnosed with autism. Mmm...

There is quite a bit more science included in the Fiorentino study but I think I've gone on long enough in this post. Suffice to say that the whole gut-brain axis thing with autism in mind gets a boost but more work is indicated, not least with larger sample groups and perhaps combining tissues from gut and brain from the same person. I would also like to see a little more done on this topic with some 'interventions' in mind, based on the other autism research that potentially links the authors (see here). Drawing for example, on a paper written by Prof Fasano titled: 'Zonulin, regulation of tight junctions, and autoimmune diseases' [3] suggesting that "gliadin, a storage protein present in wheat and that triggers celiac disease in genetically susceptible individuals, also affect the intestinal barrier function by releasing zonulin" one might see how far from being a set-in-stone state of affairs, dietary changes for some on the autism spectrum, might actually set in motion a host of biological changes pertinent to this area of work. And such changes might not be just confined to accepted gluten-related conditions either...

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[1] Fiorentino M. et al. Blood–brain barrier and intestinal epithelial barrier alterations in autism spectrum disorders. Molecular Autism. 2016; 7:49.

[2] Simms MD. When Autistic Behavior Suggests a Disease Other than Classic Autism. Pediatr Clin North Am. 2017 Feb;64(1):127-138.

[3] Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Annals of the New York Academy of Sciences. 2012;1258(1):25-33.

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Fiorentino, M., Sapone, A., Senger, S., Camhi, S., Kadzielski, S., Buie, T., Kelly, D., Cascella, N., & Fasano, A. (2016). Blood–brain barrier and intestinal epithelial barrier alterations in autism spectrum disorders Molecular Autism, 7 (1) DOI: 10.1186/s13229-016-0110-z

Dietary fibre deficiency and gut barrier integrity

"Dietary fiber deprivation, together with a fiber-deprived, mucus-eroding microbiota, promotes greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium."

So said the findings reported by Mahesh Desai and colleagues [1] meriting an editorial in the publishing journal [2] as the sentiments of 'eating your greens' applies to some rather interesting [mouse] findings.

Fibre (UK spelling) comes in various different forms typically categorised as soluble and insoluble depending on their relationship with water. Using a "gnotobiotic mouse model" - where mice were "colonized with a synthetic human gut microbiota composed of fully sequenced commensal bacteria" - Desai et al reported on the effects of different diets with different fibre content. Their results make for important reading as a fibre-deprived gut was associated with the rise of some rather potent bacteria that seemed to enjoy dining out on the "colonic mucus barrier, which serves as a primary defense against enteric pathogens." Yes, mice gut barriers - or some of their components at least - were being eaten by the very bacteria they contain. Enjoy your lunch.

I'm not dwelling too much on the Desai findings, bearing in mind their focus on mice not humans, but I do want to raise a couple of potentially relevant points. First is the focus on the intestinal barrier and how that so-called 'leaky gut' seems to show a connection to dietary fibre intake. Yet more research bringing this woo-like term in from the scientific cold (see here). Next is the idea that if the Desai results are transferable from mouse to humans, they could be relevant to quite a lot of people who perhaps don't enjoy as much dietary fibre as they should. Further, there may be particular groups of people who might be particularly prone to a poor diet [3] (see here too) where the already discussed term 'leaky gut' is also relevant (see here); also bringing in the idea of a role for those trillions of wee beasties (the gut microbiota) that call us home.

I'll be watching for how this research area pans out...

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[1] Desai MS. et al. A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility. Cell. 2016 Nov 17;167(5):1339-1353.e21.

[2] Gazzaniga FS. & Kasper DL. Veggies and Intact Grains a Day Keep the Pathogens Away. Cell. 2016 Nov 17;167(5):1161-1162.

[3] Bandini LG. et al. Changes in Food Selectivity in Children with Autism Spectrum Disorder. J Autism Dev Disord. 2016 Nov 19.

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Desai MS, Seekatz AM, Koropatkin NM, Kamada N, Hickey CA, Wolter M, Pudlo NA, Kitamoto S, Terrapon N, Muller A, Young VB, Henrissat B, Wilmes P, Stappenbeck TS, Núñez G, & Martens EC (2016). A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility. Cell, 167 (5), 1339-2147483647 PMID: 27863247