SHANK3, gut issues and (mouse) autism continued

"We conclude that apart from its well-known role in the CNS [central nervous system], SHANK3 plays a specific role in the GI [gastrointestinal] tract that may contribute to the ASD [autism spectrum disorder] phenotype by extracerebral mechanisms."

So said the findings reported by Ann Katrin Sauer and colleagues [1], and yet more evidence that issues with SHANK3 mentioned in relation to 'some' autism, may well (partly) explain much more than just behaviour (see here and see here).

The Sauer study was yet another mouse study. They specifically focused on the "Shank3αβ KO" mouse, where KO means knock-out, referring to the engineering of this mouse strain to mimic issues with the functioning and availability of SHANK3, "a known scaffolding protein of the postsynaptic density (PSD) of glutamatergic excitatory synapses." Said knock-out mice have been "reported to display ASD-like behavior with abnormal ultrasonic vocalization, repetitive self-grooming, and reduced interest in novel mice." I say this being careful to reiterate that we're talking about a mouse not human beings (see here).

On the basis of the observation that SHANK3 is expressed in the gut as well as brain and that GI issues are no stranger to autism (see here), researchers set about looking at how SHANK3 issues might also manifest as gut issues, and what this *could* mean for some autism. They observed some interesting things:

• "analysis of the GI tract of Shank3αβ KO mice revealed significantly altered gut morphology" which included, among other things, increased levels of ZONULIN1 ("a modulator of tight junctions and alterations"). Zonulin is something that I'm particularly interested in on this blog (see here and see here) on the basis of its *connection* to intestinal barrier function and the misnomer that is 'leaky gut' (see here).
• "The Microbiome of Shank3 KO Mice Is Altered." Bearing in mind the increasing importance of the gut microbiome to autism (see here), researchers reported some interesting difference between "Shank3αβ KO mice" and controls with regards to several different bacterial species. 
• Researchers describe how those gut morphology and gut bacterial differences seemed to be linked to alterations in the "expression of inflammatory markers" too as they talked about "signs of increased immune activation in the periphery and the brain." A familiar cytokine is mentioned - IL-6 - and quite a few avenues for further investigation.

The net result of all this work is to say that, yes, the SHANK3 mouse model *potentially* mimicking some of the behavioural signs and symptoms of autism does also appear to show some significant gut-related issues. No, this does not directly translate into issues for 'all human autism', but it does add further credence to the idea that the gut-brain axis is likely important to at least 'some autism'. Where also SHANK3 issues are identified as coinciding with 'human autism', one might also entertain the idea that gut issues should be screened and treated/managed. And there might be lots of ways to manage them (see here for one example)...

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[1] Sauer AK. et al. Altered Intestinal Morphology and Microbiota Composition in the Autism Spectrum Disorders Associated SHANK3 Mouse Model. Int. J. Mol. Sci. 2019; 20: 2134.

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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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Elevated zonulin levels in ADHD = more hyperactivity and "impairment of social functioning"

"Children with ADHD [attention-deficit hyperactivity disorder] had higher serum zonulin levels and were more impaired in social functioning compared to controls."

So said the findings reported by Gonca Özyurt and colleagues [1] exploring a topic quite close to my research heart, zonulin and the assumption that "the level of zonulin increases when intestinal permeability is impaired."

Before heading further into the Özyurt findings, I'll perhaps refer you to some of my previous musings on the topic of zonulin (see here) and the hows-and-whys of this potentially important compound. It's rooted in the idea that intestinal permeability is perhaps rather more than it should be in some people with some labels (see here) and this *could* have some important implications for biochemistry and beyond; particularly the notion of a 'gut-brain' relationship (see here).

Özyurt et al examined zonulin in the context of attention deficit hyperactivity disorder (ADHD) based on the idea that: "Zonulin has been shown to be associated with social impairment in children with autism spectrum disorder" but such functions (and other attention-related behaviours) have not yet been looked at with ADHD in mind. Based on the examination of serum zonulin levels in some 40 kids diagnosed with ADHD and a similar number of not-ADHD controls, analysed via "enzyme-linked immunosorbent assay", researchers reported that: "Children with ADHD had higher serum zonulin levels and were more impaired in social functioning compared to controls." Also: "The level of zonulin was independently predicted with hyperactivity symptoms and SRS [Social Responsiveness Scale] scores in regression analysis."

Bearing in mind that the Özyurt study was a fairly small scale study that utilised a methodology that has its critics (see here), I'm cautiously interested in the presented findings. I don't want to say anything further about this at the present time; aside that is, from the need for quite a bit more data on this potentially interesting relationship...

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[1] Özyurt G. et al. Increased zonulin is associated with hyperactivity and social dysfunctions in children with attention deficit hyperactivity disorder. Compr Psychiatry. 2018 Oct 29;87:138-142.

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"CFS symptoms resemble a hypothyroid state" but...

I am a little late getting to the paper published by Begoña Ruiz-Núñez and colleagues [1] observing that, at least for some diagnosed with chronic fatigue syndrome (CFS), clinical findings related to thyroid function might "resemble a mild form of “non-thyroidal illness syndrome” and “low T3 syndrome” experienced by a subgroup of hypothyroid patients receiving T4 monotherapy." But I did get here eventually. Before heading into this paper, I'm gonna link to one of the 'already prepared' discussions on the Ruiz-Núñez findings (see here). My analysis is pretty similar but not entirely the same...

So: "We studied 98 CFS patients (21–69 years, 21 males) and 99 age- and sex-matched controls (19–65 years, 23 males)" was the starting point, as participants provided blood samples and 24-hour urine samples onward to various analyses being carried out. This included: "the measurement of routine hematological parameters [Hb, hematocrit, WBC, red blood cells (RBC), and thrombocytes]" and more specifically: "parameters of thyroid function, low-grade inflammation and gut wall integrity..., together with secondary markers of inflammation." Those 'parameters of thyroid function' included various measures of free and total levels of T3 and T4 required to ascertain the presence of "low-T3 syndrome." I was impressed to see that a measure of gut wall integrity was also on the research menu in the form of plasma zonulin levels being included (albeit analysed via ELISA and bearing in mind the issues that have emerged with that particular method).

Results: "Chronic fatigue syndrome patients exhibited lower FT3, TT4, TT3, %TT3, SPINA-GD, and SPINA-GT, lower ratios of TT3/TT4, FT3/FT4, TT3/FT3, and TT4/FT4; and higher %rT3 and rT3/TT3 ratio." These findings were based on 'group' comparisons with those sex-matched not-CFS controls, and point to some 'issues' with thyroid function in general. Coupled to other thyroid related findings, the Ruiz-Núñez suggest that lower levels of thyroid hormones were detected but "distinct from thyroidal disease" typical levels of thyroid-stimulating hormone (TSH) were also reported. TSH is the stuff that tells the thyroid gland to make thyroid hormone (thyroxine (T4)), where T4 is, in effect, the starting material for T3 (triiodothyronine). Where there are suitable levels of TSH but lower levels of T4 and/or T3, one gets the impression that it's more about what's 'happening' to T3 and T4 over and above issues with their production. Indeed, the collected findings led authors to talk about that 'low T3 [triiodothyronine]syndrome' as being potentially pertinent to their findings in relation to CFS. Going back to those plasma zonulin findings, and there is just a sentence from Ruiz-Núñez and colleagues: "Zonulin, a parameter of intestinal permeability... was lower in CFS patients as compared to controls" but not much else.

I'm not particularly au fait with all the details of low T3 syndrome in the context of CFS or anything else so can't really add too much more. From what I gather, this is not a CFS-exclusive condition but does seemingly tap into talk about CFS being reflective of a "hypometabolic state" (see here). Questions about how to 'treat' such thyroid-related issues in the context of CFS remain unanswered, despite authors talking about "trials with, e.g., T3 and iodide supplements" being potentially indicated. I say this bearing in mind that the focus on biochemistry in the Ruiz-Núñez paper could perhaps, have been complemented by a little more on the presentation of clinical symptoms too.

In short, quite a bit more investigation in this area is indicated...

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[1] Ruiz-Núñez B. et al. Higher Prevalence of “Low T3 Syndrome” in Patients With Chronic Fatigue Syndrome: A Case–Control Study. Front. Endocrinol. 2018. Mar 20.

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"children with ASD who experience GI symptoms have an imbalance in their immune response"

The findings reported by Destanie Rose and colleagues [1] piqued my interest recently for a variety of reasons. Not only was there an emphasis on gastrointestinal (GI) issues in relation to autism (see here) but mention of the words 'microbiota composition' and 'impaired gut barrier function' make an important reference to something of a new triad in relation to [some] autism (see here).

First things first, as well as being another welcome research publication from the MIND Institute, the name Destanie Rose has appeared before on this blog with reference to maternal immune activation (MIA) and 'Old World monkeys' (see here). This work illustrated how infection during pregnancy can, under certain circumstances and at critical times, have a bearing on offspring development and behaviour; keeping in mind, that is, the possibility of logical fallacies (see here) when it comes to extrapolating from animal studies.

This latest time around, and including other notable names on the authorship list including Alessio Fasano (zonulin man) and Paul Ashwood (gut and immune system man), the focus shifted to immune function in the context of real life autism with the aim to "determine whether there are biological signatures in terms of immune dysfunction and microbiota composition in children with ASD with GI symptoms."

Four groups of children diagnosed with an autism spectrum disorder (ASD) participated in the study, including those with and without a diagnosis of ASD and with and without "current or previous GI symptoms." Both blood and stool samples were donated by study participants and subject to various analyses pertinent to assessing 'cytokine production' (cytokines are chemical signallers of the immune system) and ahem, the 'microbial composition' of poo(p) samples.

Results: those in the ASD + GI symptoms group showed "increased levels of mucosa-relevant cytokines including IL-5, IL-15 and IL-17" under "Toll-Like receptor (TLR)-4 stimulation" compared with those diagnosed with autism but with no bowel symptoms. TLR-4 is a protein that, as one of its duties, "plays a fundamental role in pathogen recognition and activation of innate immunity." Artificial stimulation of TLR-4 kinda mimics what would happen in real life as and when the body comes across a pathogen such as bacteria and needs to activate those immune defences.

Alongside other findings suggestive of "differences in microbiome composition between ASD and TD [typically developing] children with GI symptoms", authors also observed some interesting findings pertinent to impaired gut barrier function too. So: "The ASDGI also showed an over-representation of the gene encoding zonulin, a molecule regulating gut permeability, compared to the other groups." The gene in question is something called HP or Haptoglobin, and specifically HP2 which refers to a "common polymorphism consisting of two structural alleles: HP1 and HP2" [2]. As per the Vanuytsel paper [2], the HP2 allele is described as a risk allele for things like inflammatory bowel disease (IBD).

Bearing in mind that symptoms such as functional bowel issues (such as constipation and diarrhoea) are not necessarily the same as pathological bowel conditions such as the IBDs, I was interested in one of the figures included in the Vanuytsel paper on how HP2 links into gut permeability issues. In particular how "it is not unlikely that carriers of the zonulin gene (i.e., individuals with genotype HP21 or HP22) could possibly have an increased risk to develop IBD, because of the permeating effect of zonulin on the intestinal barrier." Zonulin has been something else of interest to this blog in the context of autism and so-called 'leaky gut' (see here) hence the interest in "a propensity to impaired gut barrier function which may contribute to their [gastrointestinal] symptoms and clinical outcome."

From what I gather, there was an over-representation of the HP2 allele (HP22 genotype) and under-representation of the HP1 allele in the ASDGI group examined in the Rose study, but things were not [statistically] completely cut-and-dried. This however, has to be set in the context of a seemingly increased risk of IBD as and when autism is diagnosed (see here).

What are the take-away messages from the Rose findings? Well bowel symptoms - functional bowel symptoms - occurring alongside autism probably have quite a complicated series of genetic and biological processes going on behind them. Both the mucosal immune system and the wider immune system are probably going to show 'some kind of relationship' to such chronic symptoms and, unsurprisingly, those trillions of wee beasties known as the gut microbiota are also probably involved/affected. The "propensity to impaired gut barrier function" associated with autism + bowel issues is also mentioned by Rose, and offers further testable hypotheses regarding the possibility of a gut-brain axis in relation to [some] autism (see here) and indeed, what measures might ease the pressures of such bowel issues. Also, whether bowel symptoms *might* show a connection to certain presented behaviour (see here) is another important area of further investigation...

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[1] Rose DR. et al. Differential immune responses and microbiota profiles in children with autism spectrum disorders and co-morbid gastrointestinal symptoms. Brain, Behavior, and Immunity. 2018. March 20.

[2] Vanuytsel T. et al. The role of Haptoglobin and its related protein, Zonulin, in inflammatory bowel disease. Tissue Barriers. 2013;1(5):e27321.

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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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Zonulin (testing): "its clinical utility questionable"

The quote making up part of the the title of today's post is taken from the paper by Aristo Vojdani and colleagues [1] (open-access available here) providing some well needed analysis of a compound of some interest for various clinical areas: zonulin.

Just in case you weren't familiar with all-things zonulin, this is a compound that has found some scientific favour when it comes to the concept of intestinal barrier function being perturbed in several diagnoses [2]. I must admit to being pretty interested in some quite recent research talking about zonulin in the context of 'some' autism (see here) based on the idea that intestinal barrier function might not be 'optimal' for some people diagnosed with an autism spectrum disorder (ASD) and what implications that might have (see here and see here).

Vojdani - who is also no stranger to autism research - cautions that the inevitable testing 'free for all' that has ensued as zonulin has risen up the scientific ranks might not be all good, as they pitted the direct measurement of serum zonulin levels against "antibodies against zonulin" to see which measure might provide the most accurate results. Antibodies against zonulin by the way, meant IgA and IgG antibodies against zonulin and was carried out "using enzyme-linked immunosorbent assay methodology."

Results: based on the analysis of over 70 blood samples from "18 volunteers at intervals of 0, 6, 24, and 30 h[ours]" authors noted that a third of participants (6/18) had low levels of serum zonulin "very close to the detection limit of the assay." We are told over the course of the hours, levels of serum zonulin "did not significantly fluctuate" in their trace amounts in this group. For the other 12 participants, it was a slightly different story as "significant fluctuation in zonulin levels was observed in almost all 12 of these subjects at the 6-, 24- or 30-h blood draws." When it came to those antibodies against zonulin, the clinical picture appeared to be slightly more calm as data showed that "both IgG and IgA antibody levels from blood obtained at 0, 6, 24, and 30 h were highly stable with variations of less than 10%." On that basis, the authors recommend that a single measurement of zonulin itself may not be a suitable indicator "for assessment of intestinal barrier integrity."

There was also another part to the Vojdani study looking at serum zonulin levels in "30 healthy controls along with 30 patients with known celiac disease." Coeliac or celiac disease (CD) is the archetypal gluten-related autoimmune condition and has some connection to zonulin. Results for this part of the study indicated a significant group difference between CD and non-CD groups where serum zonulin levels were higher in those with CD. When comparing serum zonulin levels against those antibodies to zonulin in the CS vs no-CD groups, authors reported "detection of antibodies against zonulin in 67% of patients with CD while zonulin level elevations were detected in only 33%." They suggested that these results could be due to "zonulin fluctuation in the blood and its removal by the immune system."

These types of results are interesting and help to add some 'detail' to big, sometimes sweeping, scientific findings with an emphasis on the technology and techniques used to measure such compounds. In the context of the Esnafoglu paper [3] that was the source material for my blogpost on zonulin and autism, there may be lessons to be learned as per their use of an enzyme-linked immunosorbent assay to analyse for serum zonulin levels in that particular cohort. That being said, a comparison of the range of zonulin levels reported in their autism cohort "(ASD (122.3 ± 98.46 ng/mL) compared with the healthy controls (41.89 ± 45.83 ng/mL)" compared with the Vojdani results (CD mean = 8.5 ng/mL vs. controls mean = 3.7 ng/mL ) shows that there may be quite a bit more to see when it comes to zonulin and [some] autism outside of just testing factors.

Just before I go, I do have one possible suggestion which might help matters in the area of zonulin measurement. Being quite a big fan of techniques such as mass spectrometry over other analytical methods and bringing in other recent data suggesting that *some* immunoassay kits purposed for zonulin analysis might be missing the mark [4], I'm minded to suggest that a more direct analysis of something like serum zonulin in various groups could be warranted based on mass spec and related techniques including those diagnosed with CD and autism (or even both)...

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[1] Vojdani A. et al. Fluctuation of zonulin levels in blood vs stability of antibodies. World J Gastroenterol. 2017 Aug 21;23(31):5669-5679.

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

[4] Scheffler L. et al. Widely used commercial ELISA for human Zonulin reacts with Complement C3 rather than preHaptoglobin2. bioRxiv preprint. 2017. Jun 30.

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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

Coeliac disease risk not affected by early feeding practices

I'd like to bring three papers to your attention, all united by their discussion of coeliac (celiac) disease, that most classic of autoimmune conditions in the most part managed by the use of a lifelong gluten-free diet.

First up are the papers by Elena Lionetti and colleagues [1] and Sabine Vriezinga and colleagues [2] which unfortunately pour cold water on the notion that the risk of developing coeliac disease (CD) can be somehow mitigated via the use of either the early or delayed introduction of gluten nor seemingly affected by breastfeeding habits. Indeed, as science has known about for sometime, genes and specifically the HLA genotype linked to CD, seem to be the important predictors of disease outside of gluten consumption itself.

Both the Lionetti and Vriezinga papers were published in the New England Journal of Medicine and were accompanied by an editorial by Jonas Ludviggson among others (he of the 'not CD but something else potentially linking gluten and some autism'). Both studies were based on randomised controlled trials.

The Lionetti study involved either the introduction of gluten at 6 or 12 months to at-risk infants (those with a first-degree relative with CD) and then examining: "the prevalence of celiac disease autoimmunity and of overt celiac disease among the children at 5 years of age". The numbers of participants involved featured in the hundreds so this was by no means an under-powered trial. The authors concluded that: "Neither the delayed introduction of gluten nor breast-feeding modified the risk of celiac disease among at-risk infants, although the later introduction of gluten was associated with a delayed onset of disease".

The Vriezinga study looked at whether introducing gluten early (between 4-6 months) might also offset the risk of CD. Again based on a pretty impressive participant number (N=944) comprising children with a first-degree relative with CD as well as being "positive for HLA-DQ2 or HLA-DQ8", participants were randomly assigned to either "100 mg of immunologically active gluten daily" or a placebo. Again, biopsy confirmed CD and some of the various serology associated with CD were the outcomes. The authors concluded: "As compared with placebo, the introduction of small quantities of gluten at 16 to 24 weeks of age did not reduce the risk of celiac disease by 3 years of age in this group of high-risk children".

The final paper to discuss today is that published by Elfström and colleagues [3] (open-access here) and the findings of their systematic review and meta-analysis looking at "associations between coeliac disease and type 1 diabetes". Type 1 diabetes, distinct from type 2 diabetes, is an autoimmune condition whereby the insulin producing cells of the body have been destroyed. The Elfström paper coincidentally carrying Dr Ludviggson as a co-author, concluded that: "More than one in twenty patients with type 1 diabetes have biopsy-verified coeliac disease" and onwards that there may be some substance to the idea that birds of an autoimmune feather flock together (see here). Indeed, I've previously covered the possibility of a connection between Type 1 diabetes and CD (see here) and how, for example, some of the research on [General] zonulin might be potentially very informative to this area (see here).

There's little more for me to say in this post aside from reiterating that when it comes to CD, genotype combined with gluten exposure seems to be the most important factors in disease onset and progression. That's not to say that a gluten-free diet might be the only tool in the management arsenal (see here) nor that other variables might not potentially impact on CD risk (see here) but for now, screening and where indicated, gluten avoidance, seem to be the primary measures to be undertaken.

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[1] Lionetti E. et al. Introduction of Gluten, HLA Status, and the Risk of Celiac Disease in Children. N Engl J Med. 2014 Oct 2;371(14):1295-1303.

[2] Vriezinga SL. et al. Randomized Feeding Intervention in Infants at High Risk for Celiac Disease. N Engl J Med. 2014 Oct 2;371(14):1304-1315.

[3] Elfström P. et al. Systematic review with meta-analysis: associations between coeliac disease and type 1 diabetes. Aliment Pharmacol Ther. 2014 Oct 1. doi: 10.1111/apt.12973.

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Lionetti E, Castellaneta S, Francavilla R, Pulvirenti A, Tonutti E, Amarri S, Barbato M, Barbera C, Barera G, Bellantoni A, Castellano E, Guariso G, Limongelli MG, Pellegrino S, Polloni C, Ughi C, Zuin G, Fasano A, Catassi C, & the SIGENP (Italian Society of Pediatric Gastroenterology, Hepatology, and Nutrition) Working Group on Weaning and CD Risk (2014). Introduction of Gluten, HLA Status, and the Risk of Celiac Disease in Children. The New England journal of medicine, 371 (14), 1295-1303 PMID: 25271602



Vriezinga SL, Auricchio R, Bravi E, Castillejo G, Chmielewska A, Crespo Escobar P, Kolaček S, Koletzko S, Korponay-Szabo IR, Mummert E, Polanco I, Putter H, Ribes-Koninckx C, Shamir R, Szajewska H, Werkstetter K, Greco L, Gyimesi J, Hartman C, Hogen Esch C, Hopman E, Ivarsson A, Koltai T, Koning F, Martinez-Ojinaga E, Te Marvelde C, Pavic A, Romanos J, Stoopman E, Villanacci V, Wijmenga C, Troncone R, & Mearin ML (2014). Randomized Feeding Intervention in Infants at High Risk for Celiac Disease. The New England journal of medicine, 371 (14), 1304-1315 PMID: 25271603



Elfström P, Sundström J, & Ludvigsson JF (2014). Systematic review with meta-analysis: associations between coeliac disease and type 1 diabetes. Alimentary pharmacology & therapeutics PMID: 25270960

MAR autism and maternal autoimmune conditions: speculations

The term MAR autism - maternal autoantibody-related autism - whilst still a relatively new addition to the autism research vocabulary, has nevertheless already courted some controversy. This follows a decision to try and commercialise the growing research base in this area (see here) which raised a few eyebrows in various quarters.

Speculating on a dead cat bounce?  @ Wikipedia 

As I indicated on my previous post about said commercialisation, there are a few questions which perhaps need answering before this work starts down the path of becoming any sort of reliable 'autism test'. Some of these questions being particularly important if one is to learn the lessons from another proposed autism test which came across a few problems in replication recently (see here).

Away from such discussions, the notion that a proportion of mums of children with autism spectrum disorder (ASD) "have antibodies in their bloodstream that react with proteins in the brain of their babies" is potentially a very important finding. The evidence for such a process is actually becoming quite consistent (see here) following on from other investigations pointing at some role for the maternal immune system when it comes to at least some cases of autism (see here).

The paper by Lior Brimberg and colleagues* adds to the autism - maternal anti-brain antibodies story with their findings suggesting that "Mothers of an ASD child were four times more likely to harbor anti-brain antibodies than unselected women of child-bearing age (10.5 vs 2.6%)" based on the analysis of collected data from one or two quite large autism study initiatives (Simons Simplex Collection and Autism Genetic Exchange Resource). This in itself would be a worthy confirmatory research finding bearing in mind the number of plasma samples that were analysed as part of the study.

But of perhaps equal importance was the observation that "The analysis of ASD mothers with brain-reactive antibodies also revealed an increased prevalence of autoimmune diseases, especially rheumatoid arthritis and systemic lupus erythematosus". This point was covered by other commentary of this study (see here) including findings related to the detection of anti-nuclear antibodies (ANAs) (53% vs 13.4%) in autism mums with and without the anti-brain antibodies respectively.

Those who regularly visit this blog might know about my interest in all things autoimmunity with autism in mind (see here). The suggestion that the presence of brain reactive antibodies seemed to correlate with an increased frequency of maternal autoimmune conditions or their biological links represents yet another possible connection between autoimmunity and autism, at least some cases of autism.

With my speculating hat firmly in place, I wondered about a couple of things as a result of these findings. I wondered for example, whether the anti-nuclear antibodies were also present in offspring of those mums who tested positive for both brain reactive antibodies and ANAs. I note that ANAs have been previously reported in cases of autism as per the findings of Mostafa and Kitchener** who observed: "Children with autism had a significantly higher percent seropositivity of anti-nuclear antibodies (20%) than healthy children (2.5%; P < 0.01)". That and their suggestion: "Anti-nuclear antibody seropositivity was significantly higher in autistic children with a family history of autoimmunity than those without such history (36.8% and 5%, respectively; P < 0.001)" makes for some interesting connections.

Harking back to the ScienceDaily piece on the Brimberg study (see here) I was also intrigued by the suggestion that a 'leaky' blood-brain barrier may "allow the "anti-brain" antibodies to pass through to the babies' brains, possibly causing autism".

Now just before I get too carried away with this, there has been a bit of debate down the years about just when the blood-brain barrier (BBB) actually becomes effective in the foetus and infant. The more recent discussions suggest that there is a "well developed barrier mechanisms in the developing brain". This contrasts with other reports such as the study by Volodin and colleagues*** suggesting that the final establishment of the foetal BBB, under typical circumstances, is carried out in the latter stages of gestation. I'll leave readers to draw their own conclusions on which is the correct position.

This barrier however, partly physical and partly biochemical, is susceptible to 'damage' under certain circumstances as reported in an older post on this topic (see here). That alongside some of the gatekeeper molecules such as P-glycoprotein which help transport things through the BBB (see here) being potentially susceptible to 'alteration' as a function of various factors, means that BBB permeability is influenced by quite a few fluidic variables.

If one assumes that, as in the example of a potential link between maternal SSRI use during the first trimester of pregnancy and offspring autism risk (see here**** and here for my post), early stage pre-completed formation of the infant BBB is a 'risk' time for the developing foetus and its susceptibility to things like anti-brain antibodies and/or ANAs, one might get a sense of how and when such a process "possibly causing autism" may come about. I hasten to add that I'm still speculating at this point.

With leaky membranes in mind and the still-awaited peer-review publication of some conference proceedings from the lab of Paul Patterson (see here) on the suggestion of leaky gut present in their maternal immune activation offspring mouse model, I'm also wondering whether there may be another connection to be had here too. Various autoimmune conditions have been talked about with gut hyperpermeability in mind; ranging from gastrointestinal conditions such as coeliac disease (see here*****) to type 1 diabetes (see here******). It's not necessarily an all-or-nothing relationship but leaky gut and autoimmunity (with other factors such as gut bacteria also in the mix) is certainly on the scientific map.

In a similar vein to the ANA story, I'm wondering whether there may be merit in looking at whether there is any tie-up between gut permeability issues and (a) those mums where brain reactive antibodies have been reported, and (b) children of those mums with reported brain reactive antibodies and/or ANAs. Indeed, if (and it is still a very speculative 'if') there is some association to be found, whether as per the collected de Magistris work, one might envisage a role for dietary intervention to act on the permeability issue and any knock-on effects that might have with regards to autoimmune processes and presented symptoms? The other potential factor in this relationship being the link between gluten (or rather gliadin) and another barrier gatekeeper molecule called zonulin (see here******* open-access and here for a previous post) which is also deserving of some study with autism in mind.

I know that there's been speculation-a-go-go on this entry and I'm very much exceeding the remit of the paper by Brimberg and colleagues. I apologise. It's an easy thing to do when it comes to a blog, open-access, with no peer-review and full editorial control to the owner (i.e. me). I'd like to think however, that this area of immune activation and autoimmunity potentially opens up a whole range of further research questions ripe for scientific inquiry outside of just the formulation of a test for autism risk or not. In these days of autisms over autism and "autism as fractionable into different, largely independent sets of clinical features" new frontiers for autism research abound, and that's just as true for MAR autism and autoimmunity.

And for those interested in the attached picture included with this post and what a 'dead cat bounce' is, it's not as harrowing as you might think... (see here). Meow.

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* Brimberg L. et al. Brain-reactive IgG correlates with autoimmunity in mothers of a child with an autism spectrum disorder. Molecular Psychiatry. 2013; 18: 1171-1177.

** Mostafa GA. & Kitchener N. Serum anti-nuclear antibodies as a marker of autoimmunity in Egyptian autistic children. Pediatr Neurol. 2009 Feb;40(2):107-12.

*** Volodin NN. et al. Status of the blood-brain barrier in newborn infants of various gestational ages in the normal state and in pathology. Pediatriia. 1989;(3):10-4.

**** Croen LA. et al. Antidepressant use during pregnancy and childhood autism spectrum disorders. Arch Gen Psychiatry. 2011 Nov;68(11):1104-12.

***** Drago S. et al. Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines. Scand J Gastroenterol. 2006 Apr;41(4):408-19.

****** Bosi E. et al. Increased intestinal permeability precedes clinical onset of type 1 diabetes. Diabetologia. 2006 Dec;49(12):2824-7.

******* Fasano A. Zonulin and its regulation of intestinal barrier function: the biological door to inflammation, autoimmunity, and cancer. Physiol Rev. 2011 Jan;91(1):151-75.

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Brimberg L, Sadiq A, Gregersen PK, & Diamond B (2013). Brain-reactive IgG correlates with autoimmunity in mothers of a child with an autism spectrum disorder. Molecular psychiatry PMID: 23958959

Coeliac Disease - a training post

You're probably wondering why, with all the reams of autism research being produced every day, that I'm dedicating a post to describing coeliac (celiac) disease on this blog? Well, the answer is simple; I've talked about coeliac disease (CD) quite a bit in relation to autism (here) and schizophrenia (here) and other conditions (here) but I'm mindful that not everyone actually knows what it is or what we think we currently know about it.

Gluten @ Wikipedia  

So in future occasions when I talk about CD in relation to some wonderful new study, I've got this training post as mine and yours go-to reference for the condition.

If you want the long and complicated story of CD, there are plenty of peer-reviewed papers which I could suggest you read such as this one from Kagnoff* (open-access) or this one from Meresse and colleagues** (open-access). There are lots of other papers on the topic too but if you want the Mr Men version, read on.

Gluten protein and peptides
CD is a condition governed by genes and environment in pretty equal measure. I suppose it all starts with foods containing the protein gluten. Actually gluten is a bit of a catch-all word because it combines two types of protein: gliadin and glutenin. Gluten is a protein which consists of long chains of amino acids. When ingested, various enzymes go to work on chopping up the protein into those nutritious rich amino acids that our bodies so rely on. But digestion does not just see the gluten protein immediately exploded into its constituent amino acids but rather breaking the protein down chunk by chuck to form small chains of amino acids called peptides along with way.

If you're a follower of autism research and in particular the whole gluten- and casein-free dietary intervention thing, you'll probably have heard about peptides as per the opioid-excess theory*** (open-access) put forward as one explanation for why diet might 'work' for some on the autism spectrum. It's still a little bit contentious but that's perhaps a topic for another day.

Anyhow, gluten is not an easy protein to digest as per the presence of certain amino acids such as glutamine and proline in that protein chain, the chemistry of which don't like being degraded easily. So what you potentially get are quite a few peptides swimming around our gastrointestinal (GI) tract which are not completely degraded into their simplest building block form, the amino acids.

Gut access
These gluten, sorry gliadin, peptides however don't just stay in the gut; some of them are also able to gain access to a part of the gut barrier called the lamina propria. Once there, something rather interesting seems to happen in cases of CD. The peptides come across something called tissue transglutaminase (tTG) something else which has cropped up on this blog with autism in mind (see here). The clue is in the name about tTG (also called TG2) and what it can do: -ase means it's an enzyme and the glutaminase bit means that it does things to the amino acid glutamine. The specific duty it does is a process called deamidation which basically involves the conversion of glutamine to glutamic acid (otherwise known as glutamate). Without getting too much into the chemistry of this process, the newly deamidated gliadin peptide is now 'super-charged' (neutral into negatively charged amino acids) in terms of its attraction (binding affinity) to molecules of the almighty MHC - major histocompatability complex or HLA in humans (see here).

DQ2 and DQ8
OK, so a quick recap. Gluten protein digested into gluten peptides. Said peptides meet and greet tTG and funny things start to happen to them.

Next in the process chain of CD is how these newly enhanced peptides from an immunogenicity point of view are met by the cells of the MHC and the sparks that fly as a result. Just in case you didn't click on my link talking about the MHC, it's all about how things are presented to the immune system and in particular, the tricky task of making sure that 'self' is not confused with 'other' by the immune system.

The genetics of CD represent the important part of this next stage of proceedings as per the HLA-DQ2 and DQ8 heterodimers; in effect the genes of CD. It's all about inheritance patterns as to whether or not a person will have two or one or no copies of these genes as a consequence of genetic zygosity.

HLA DQ2 or DQ8 molecules are part of the antigen presenting cells (APCs). Those newly enhanced gluten peptides fit nicely into the 'pocket' of the DQ2 and/or DQ8 molecules and once there activate T cells or more specifically a Th1 CD4+ response**** (open-access) focused on gliadin. This eventually leads to the release of cytokines such as IFN-γ (see here also) and TNF which then go on to damage the gut mucosa as a function of their important role in the process of inflammation.

This is quite a simplistic overview of the main processes involved in CD. As per the discussions on the Kagnoff and Meresse papers, there are still quite a few unknowns about the whole process of CD. There's also the relatively newer work coming into the science of CD such as a role for zonulin (see this post) and its 'gatekeeper' role in relation to the gut barrier and things like the wheat amylase trypsin inhibitors (thanks Jad).

The gluten-free diet
As you'll probably already know, management of CD is primarily via the use of a gluten-free diet. The theory being that if there is still no starting material (gluten) to form those peptides, even though the genetics may be there, there is nothing or only little material for tTG or the DQ2/DQ8 molecules to go to work on.

That being said, you'll probably also see a few other potential areas where other interventions might also be developed***** (open-access). So how about helping to degrade those gluten peptides? What about stopping those peptides from meeting tTG? Blocking DQ8 and DQ2 molecules? Or even reducing the release or blocking the effects of those cytokines? And the good things is that research is underway in some of these areas.

Testing for coeliac disease
Just before you go it might also be worthwhile mentioning about how one goes about testing for CD in light of some confusion in this area over the years. It's worth pointing out that an accurate diagnosis of CD relies on more than one test (see here) covering serology, gut biopsy and on occasion, genetic testing. One of the more recent professional consensus statements on testing can be seen here****** (open-access).

And finally.... please don't take my word for it, do some research yourself.

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* Kagnoff MF. Celiac disease: pathogenesis of a model immunogenetic disease. J Clin Invest. 2007 Jan;117(1):41-9.

** Meresse B. et al. Celiac disease: from oral tolerance to intestinal inflammation, autoimmunity and lymphomagenesis. Mucosal Immunol. 2009 Jan;2(1):8-23. doi: 10.1038/mi.2008.75.

*** Whiteley P. et al. How Could a Gluten- and Casein-Free Diet Ameliorate Symptoms Associated with Autism Spectrum Conditions? Autism Insights 2010:2 39-53.

**** Nilsen EM. et al. Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology 1999; 115: 551-563.

***** Bakshi A. et al. Emerging Therapeutic Options for Celiac Disease: Potential Alternatives to a Gluten-Free Diet. Gastroenterol Hepatol (N Y). 2012 Sep;8(9):582-588.

****** Husby S. et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition Guidelines for the Diagnosis of Coeliac Disease. JPGN. 2012; 54: 136-160.

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Meresse B, Ripoche J, Heyman M, & Cerf-Bensussan N (2009). Celiac disease: from oral tolerance to intestinal inflammation, autoimmunity and lymphomagenesis. Mucosal immunology, 2 (1), 8-23 PMID: 19079330