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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That 'gut bacteria transplant provokes autistic signs in mice' paper is not perfect but...

The paper by Gil Sharon and colleagues [1] has certainly created headlines and discussion in equal measure (see here and see here and see here and see here). Concluding that: "Mice harboring human ASD [autism spectrum disorder], but not TD [typically developing], microbiomes exhibit ASD-like behaviors", the idea of a gut-brain connection in relation to autism (see here) potentially gains some research traction.

The Sharon study involved transplanting gut bacteria - the gut microbiome - from a small number of participants - "from 5 control volunteers and 11 patients diagnosed with autism spectrum disorder" - into mice lacking a microbiome and breeding said mice. They then analysed the behaviour and other biological parameters of those offspring mice according to whether their mother mice had received a transplant from controls or participants with various 'degrees' of autism. They also looked at 'metabolite profiles' based on "analyses of colon contents from oTD [offspring typically developing] and oASD [offspring autism spectrum disorder] mice."

Results: "colonization with ASD microbiota is sufficient to induce hallmark autistic behaviors." By 'hallmark autistic behaviors' researchers observed that said mice showed "increased repetitive behavior, decreased locomotion, and decreased communication... compared to mice colonized with samples from TD controls (oTD), as tested by marble burying (MB), open-field testing (OFT), and ultrasonic vocalization (USV), respectively." Researchers also observed specific differences across the mouse group gut microbiomes, some of which were consistent with that noted in other independent studies.

Also: "Twenty-seven out of 313 detected metabolites were significantly different in the colon contents of oASD mice, compared to oTD mice." They specifically focused in on two metabolites - taurine and 5-aminovaleric acid (5-AV) - both of which were reported in lower levels in the oASD mice, and how these compounds show a *connection* to GABA, a compound potentially important to autism (see here). Further they showed that supplementation of 5-AV and taurine to another strain of mouse that serves as a 'mouse model of autism' (BTBR T+ tf/J (BTBR) mouse model) resulted in "improved repetitive and social behaviors." I should add the word 'mouse' into the sentence "improved repetitive and social behaviors."

Insofar as the limitations of the Sharon studies and paper, various people have been keen to point out that the results should be viewed cautiously and as preliminary. This on the basis of the number of animals included for study, the reliance on mouse models of autism (and the logical fallacies that can sometimes follow) and some of the generalisations made in the study write-up by the authors. I wouldn't disagree with such cautions, bearing in mind that some mouse models of autism - the valproic acid autism mouse model for example - actually seem to be pretty good at mimicking some facets of (induced) autism. I'd also point out that the metabolomics work undertaken by Sharon and colleagues looks to be pretty wide-ranging (GC-MS and NMR are discussed) and findings related to taurine have also been noted in other independent study (see here). I also observed that there was a research tie-up with Arizona State University in the Sharon study, as the name Dae-Wook Kang is mentioned and 'poo transplants for [some] autism' makes yet another appearance (see here and see here).

"While ours is a limited study, with 16 donor samples from a pediatric cohort, the results support a hypothesis that the human gut microbiota contributes to ASD phenotypes." I'd agree that the Sharon results add a further layer to the idea that the new triad - intestinal permeability, mucosal immunology and intestinal microbiota - could be important to at least some autism. The results offer a road map for further investigation in this area and perhaps eventually, yet another avenue for screening and intervention to complement other recent initiatives (see here); all set with the view of the (plural) 'autisms'.

Finally, I note that another study [2] mentioning the words 'mouse' and 'autism' has been published recently. With some media attention mentioning how: "Exercise reversed autistic behaviors in an animal model of the condition" there didn't seem to be the same 'keenness' to point out the flaws of the Andoh study, despite once again a reliance on 'mouse autism' and all which that entails. It makes me wonder whether the focus on the second brain (gut) and autism detailed in the Sharon study might still have the ability to raise hackles in some quarters?

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[1] Sharon G. et al. Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice. Cell. 2019 May 30;177(6):1600-1618.e17.

[2] Andoh M. et al. Exercise Reverses Behavioral and Synaptic Abnormalities after Maternal Inflammation. Cell Reports. 2019; 27: 10. June 4.

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Probiotics for autism systematically reviewed

"Our review includes two randomized controlled trials, which showed improvement of ASD [autism spectrum disorder] behaviors, and three open trials, all which exhibited a trend of improvement."

So said the findings reported by Jun Liu and colleagues [1] and the results of their "updated systematic review" on the topic of probiotic 'therapy' in the context of behaviour and gastrointestinal (GI) functioning in autism.

The current scientific outlook for probiotic use in the context of autism looked to be pretty good on the basis of the Liu findings. They corroborate quite a few individual study results that have been fodder for this blog (see here and see here) and fit in well with an emerging pattern of research suggesting that the trillions of wee beasties that inhabit the gastrointestinal (GI) tract might be doing a lot more than just helping us digest food (see here and see here).

What else is required? Well Liu et al talk about more "rigorous trials" to answer questions like who on the autism spectrum might be a best responder to this type of intervention and what bacterial species might be most important. I'd also like to see a little more research on the hows-and-whys of such intervention (see here for example) and whether probiotics are as harmless as many have made them out to be.

Still...

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[1] Liu J. et al. Probiotic Therapy for Treating Behavioral and Gastrointestinal Symptoms in Autism Spectrum Disorder: A Systematic Review of Clinical Trials. Curr Med Sci. 2019 Apr;39(2):173-184.

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Lactobacillus plantarum PS128 "ameliorated opposition/defiance behaviors" in boys with autism?

There was something potentially rather special about the results published by Yen-Wenn Liu and colleagues [1] suggesting that use of a probiotic - Lactobacillus plantarum PS128 - might, under "randomized, double-blind, placebo-controlled" conditions, have some important effects with some young people diagnosed with an autism spectrum disorder. Special because, if such results are eventually replicated and borne out, some of the more 'disruptive' behaviours that can sometimes be observed alongside a diagnosis of autism - "opposition/defiance behaviors" - might be amenable to quite a simple intervention. That could be important for many, many different reasons.

The basics: PS128 containing "3 × 1010 CFU/capsule of PS128 with microcrystalline cellulose as the carrier" was the compound under investigation, pitted against a placebo that "only contained microcrystalline cellulose." Eighty participants, all boys diagnosed with an autism spectrum disorder (ASD) were recruited for study; 39 were assigned to receive PS128 and 41 receiving the placebo for a period of 4 weeks. Various different schedules and questionnaires were used to measure behaviour at baseline and week 4 between the groups. With a fairly small attrition rate - data for 36 participants in the PS128 and 35 in the placebo group were analysed - the results were pretty interesting.

Results: first and foremost we are told that no adverse events were reported during the study. That's important. Next, for the vast majority of measures used when straight comparing of PS128 and placebo, no significant difference was noted. The authors even mention how a clinician rated scale, the CGI-I, basically said that "both groups were equivalent to "minimally improved""  between baseline and study end. It was only when results were stratified for age that things started to 'happen' as various behaviours around anxiety, rule-breaking, inattention and opposition/defiance showed something like a 'nominal' reduction in the PS128 group compared with placebo, particularly for those aged between 7-12 years. As per the use of the word 'nominal' to denote a small 'change' the results were not spectacular.

Caveats? Well, this was a 4-week study of boys on the autism spectrum. Not a long time in anyone's book but longer than other studies on other interventions that did show a statistically significant effect (see here for example). The Liu study was also a study that exclusively relied on behavioural observation measures, so we can't say anything about how something like PS128 might have impacted on gut bacteria for example. Other, less methodologically sound studies have been more comprehensive (see here).

But there are strengths to the Liu study; strengths around the design and use of a placebo condition. And if there is a chance that something like Lactobacillus plantarum PS128 or other preparations (see here and see here) or related techniques (see here) *might* help improve quality of life for young and old people on the autism spectrum minus any significant side-effects, they should be explored an awful lot more...

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[1] Liu Y-W. et al. Effects of Lactobacillus plantarum PS128 on Children with Autism Spectrum Disorder in Taiwan: A Randomized, Double-Blind, Placebo-Controlled Trial. Nutrients. 2019; 11: 820.

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"support the hypothesis that early life gut microbiota are associated with neurodevelopmental outcomes in childhood"

Question: "Is the gut microbiome in infancy associated with neurodevelopment in children at preschool age?" Answer: "findings appear to support the hypothesis that early life gut microbiota are associated with neurodevelopmental outcomes in childhood."

That was the about the sum of the findings reported by Joanne Sordillo and colleagues [1] and their analyses of "Ages and Stages Questionnaire, third edition (ASQ-3)" data and "microbiome analysis using 16S rRNA gene sequencing" of stool samples from over 300 infants who were taking part in something called the "Vitamin D Antenatal Asthma Reduction Trial (VDAART)."

The Sordillo paper is open-access so doesn't require any rehashing from me. A few details do however stick out. So: "findings suggest that the infant gut microbiome may be associated with subsequent development of communication, personal and social, and fine motor skills in typical developing 3-year-old children and with odds of possible developmental delays." The authors were specifically drawn to "Clostridiales (Lachnospiraceae genera and other, unclassified Clostridiales taxa)" as being important when it came to their stool analyses. Said bacteria seemed to be *associated* with various ASQ-3 data covering "poorer ASQ-3 communication... and personal and social... scores and with increased odds of potential delay for communication... and personal and social skills."

The word 'autism' is also mentioned in the Sordillo paper, alongside the idea that (1) "poor performance of children on the ASQ-3 (particularly on communication skills) at 16 to 30 months of age has been shown to be sensitive (but not specific) for diagnosis of ASDs [autism spectrum disorders]", and (2) "A number of cross-sectional studies comparing the gut microbiome of neurotypical children with that in children with ASDs have reported increased levels of Clostridiales in the gut microbiome of individuals with ASDs, including higher levels of Clostridium,... Clostridium histolyticum,... and Ruminococcus." Indeed, there's also mention of the Luna study [2] covered on this blog previously (see here).

Obviously there are caveats to the Sordillo findings; not least that this all about looking at two variables (ASQ-3 scores and stool bacterial content) and marrying them together at some quite specific time points. I'd for example, be interested to see whether further follow-up studies saw a continuation of the trends described in this paper perhaps covering examination of multiple stool samples taken over different testing occasions. Also going back to the 'autism' suggestion, the authors note that they "did not have data on clinical diagnoses of ASDs for our analysis" so one has to be a little bit careful with any suggestions there too.

Still, such work is important and further contributes to the idea that the brain probably isn't the only place to look when considering things like cognitive and behavioural development (see here). Indeed, as mentioned previously on this blog (see here), a possible role for inflammation 'impairing' social cognitive processes might not be a million miles away from the Sordillo findings on the basis that the new triad - gut bacteria, intestinal permeability, gut immune function - might be really quite important for lots of processes. And then there is another question to consider: if one is able to 'alter' the gut bacterial make-up at an early age, can one potentially affect behavioural and/or developmental outcomes? I say that in the context that something similar has been talked about before (see here).

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[1] Sordillo JE. et al. Association of the Infant Gut Microbiome With Early Childhood Neurodevelopmental Outcomes. JAMA Netw Open. 2019; 2: e190905.

[2] Luna RA. et al. Distinct Microbiome-Neuroimmune Signatures Correlate With Functional Abdominal Pain in Children With Autism Spectrum Disorder. Cellular and Molecular Gastroenterology and Hepatology. 2017; 3: 218-230.

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A poo(p) transplant for [some] autism? 2 years on with caveats...

The results published by Dae-Wook Kang and colleagues [1] provide some important follow-up work to a study already discussed on this blog (see here) which suggested that: "MTT is safe and well-tolerated in children with ASD ages 7–16 years" and also "led to significant improvements in both GI- and ASD [autism spectrum disorder] -related symptoms" [2]. MTT by the way, refers to Microbiota Transfer Therapy, or in other words a poo(p) transplant. A press release accompanying the recent Kang paper is also available (see here).

The original Kang study included quite a bit more than just a poo(p) transplant as per their use of a 4-stage protocol: "(1) oral vancomycin, (2) MoviPrep, (3) SHGM [Standardized Human Gut Microbiota], and (4) Prilosec" with 18 participants diagnosed with an autism spectrum disorder (ASD). The results on that last occasion were promising insofar as (a) adverse effects being small and fairly limited and (b) some improvements noted in relation to behaviour and gastrointestinal (GI) symptoms. That all being said, one needs to remember that the previous study was an open trial and results were therefore preliminary.

On this latest research occasion, Kang et al followed up their 18 participants "two years after treatment was completed." The follow-up involved "the same GI and behavior tests that we employed previously" which involved the use of various parent- and professional-report questionnaires on behaviour, questionnaire analysis of GI issues and analysis of poo(p) samples: "16 out of 18 original ASD participants provided an additional fecal sample two years after the open-label trial."

Researchers reported that "most improvements in GI symptoms were maintained, and autism-related symptoms improved even more after the end of treatment." They observed something of a possible *relationship* between bowel and behavioural signs and symptoms whereby "GI relief provided by MTT may ameliorate behavioral severity in children with ASD, or vice versa, or that both may be similarly impacted by another factor" which is interesting (see here). They also noted that the bacterial composition of stools analysed at follow-up showed evidence of sustained change "including significant increases in bacterial diversity and relative abundances of Bifidobacteria and Prevotella." In short, things were still looking pretty good after 2 years.

"Despite steady and continuous improvement in behaviors over two years, we must underscore that the original clinical trial and current follow-up study are open-label trials without a control for placebo effect." The authors are frank about the limitations of their studies, and how behavioural and GI symptoms in particular can potentially be influenced by all-manner of different variables. Indeed, they noted that "12 of 18 participants made some changes to their medication, diet, or nutritional supplements" which allied to the waxing and waning of symptoms typically associated with autism (see here), means that one has to be careful about making too many sweeping statements about cause-and-effect.

But in the context that for these 18 participants, a poo(p) transplant was seemingly not associated with too many adverse side-effects and that their behavioural and GI data typically followed a course of improvement, one cannot easily discount the Kang results. The call for further research "with a placebo-control arm" made by the authors should echo throughout the autism research landscape. And with it, further focus on how gut bacterial make-up and the all-important metabolites that specific bacterial species produce seem to be something quite important to at least some autism (see here)...

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[1] Kang D-W. et al. Long-term benefit of Microbiota Transfer Therapy on autism symptoms and gut microbiota. Scientific Reports. 2019; 9: 5821.

[2] Kang D-W. et al. Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study. Microbiome. 2017; 5: 10.

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On gut bacteria and schizophrenia

Hot on the heels of my discussions about the possible *association* between gut bacteria and depression (see here), the results published by Peng Zheng and colleagues [1] (open-access) entered my Twitter feed recently, extending the gut bacteria 'connection' to schizophrenia.

The Zheng study represents yet more good scientific value for money as researchers initially sought to "compare the gut microbial communities of patients with SCZ [schizophrenia] and healthy controls (HCs) to evaluate whether microbiotal dysbiosis was linked with schizophrenic episodes or the severity of schizophrenic symptoms." They also "transferred gut microbiota from patients with SCZ into GF [germ free] mice to test whether SCZ-relevant behavioral phenotypes were transmissible via their gut microbiome". The study findings have been covered quite a few media outlets (see here).

The results: "seminal evidence that SCZ is associated with changes in gut microbiota composition that are both specific to SCZ and correlated with symptom severity." This translated into:

• reduced (alpha) microbial diversity in those with schizophrenia (n=63) compared with "healthy controls" (authors words not mine),
• the identification of certain bacterial differences between the groups: "the most significant deviations between SCZ and HC subjects occurred for the bacterial families Aerococcaceae, Bifidobacteriaceae, Brucellaceae, Pasteurellaceae, and Rikenellaceae",
• a *correlation* between the presence of some bacterial species and the 'severity' of symptoms of schizophrenia,
• behavioural changes in those mice who received a gut bacterial transplant from participants with schizophrenia,
• "Perturbed gut-brain amino acid and lipid metabolism in SCZ microbiota recipient mice." Gut bacteria produce chemicals (for messaging and the like), and those bacteria transplanted into germ-free mice produced a different cocktail of chemicals that showed up "in the SCZ microbiota compared to the HC microbiota recipient mouse samples." In particular: "lower glutamate and higher glutamine and GABA in the hippocampus."

As I said, the Zheng study was pretty good value for money on the basis of the results obtained. The authors note that their findings "provide a novel framework for understanding the mechanisms of SCZ through the MGB [microbiota-gut-brain] axis and may lead to new diagnostic and treatment strategies."

Caveats? Well, fairly small participant numbers is one thing, as was the potential influence of medication being taken by those with schizophrenia. Authors however mention that: "we found that the distributions of global microbial phenotypes did not vary between medicated and unmedicated patients with SCZ." I'll also point out the inherent 'difficulties' when talking about "SCZ-relevant behaviors in GF recipient mice" similar to that noted with regards to other diagnostic labels (see here). People are given labels like schizophrenia not mice.

But it's a good start and complements other work in 'related' areas talking about the gut-brain axis as being potentially pertinent to 'some' schizophrenia (see here and see here). Whether modifying gut bacteria via use of something like diet, probiotics or the horror that is the gut microbial transplant might relieve some of the signs and symptoms of schizophrenia is an area that requires quite a bit more investigation...

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[1] Zheng P. et al. The gut microbiome from patients with schizophrenia modulates the glutamate-glutamine-GABA cycle and schizophrenia-relevant behaviors in mice. Science Advances. 2019; 5: 2.

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On gut bacteria and depression

I'm kinda standing on the shoulders of giants with this post talking about the findings reported by Mireia Valles-Colomer and colleagues [1]. An editorial published in Nature [2] to coincide with the publication of the Valles-Colomer paper says just about everything that needs to be said on this research, which observed that: "Gut–brain module analysis of faecal metagenomes identified the microbial synthesis potential of the dopamine metabolite 3,4-dihydroxyphenylacetic acid as correlating positively with mental quality of life and indicated a potential role of microbial γ-aminobutyric acid production in depression." Yes folks, the idea that "microorganisms in the human gut could influence the brain" is moving from 'wild idea' to "wise pursuit".

The sequencing of DNA from donor stool samples as part of the Flemish Gut Flora Project ('You too are a walking bacteria colony' is the strap line) was the starting point for the Valles-Colomer study; stool samples provided by over 1000 participants. Researchers also accessed data on both self-reported and "physician-diagnosed depression" and set to work looking for any potentially important correlates between bacteria and psychology. The words "with validation in independent data sets (ntotal = 1,070)" are also (importantly) mentioned in the Valles-Colomer paper. Oh, and they also "mined the data to generate a catalogue describing the microbiota’s capacity to produce or degrade molecules that can interact with the human nervous system."

Results: "Butyrate-producing Faecalibacterium and Coprococcus bacteria were consistently associated with higher quality of life indicators." There's that word again: butyrate and yet more positive publicity for this compound (see here) and it's standing reaching almost 'bacterial sainthood'. Researchers also observed that two groups of bacteria were also reduced in those with depression: Coprococcus and Dialister alongside observing that this finding held "even after correcting for the confounding effects of antidepressants [use]." And then there was that 3,4-dihydroxyphenylacetic acid, also called DOPAC, finding, a metabolite of the neurotransmitter dopamine. I have actually mentioned DOPAC before on this blog (see here) in relation to what happens when rats are subjected to 'early immune stimulation' [2]. I don't think there is much overlap between that rat study and the Valles-Colomer paper (that rat paper was looking at DOPAC levels in brain tissue for example) but the suggestion from the authors that DOPAC levels were "correlating positively with mental quality of life" requires quite a bit more investigation.

Caveats? Well, out of their initial 1054 participant cohort, only 121 participants had "GP-reported depression." About half of these participants were taking antidepressants for their depression, the others weren't. The participant numbers aren't exactly tremendous for this portion of the study. Similar to something mentioned in other research (see here), I'm also minded to suggest that future research might perhaps consider looking at multiple stool samples taken over different occasions for the same person. This would perhaps establish whether gut bacterial populations are stable and whether that stability translates into stability of something like depressive symptoms too. I am likewise cautious that we don't jump ahead of ourselves here in terms of important issues like cause-and-effect and for example; whether there may be other important 'influencers' of gut bacteria when it comes to depression (see here and see here).

Lots more study is required on the suggestion of a gut bacterial *link* to depression [3], including that focused on the mechanics of any relationship (e.g. any involvement of the vagus nerve). If the link is further established, there are a number of potentially important implications: the possibility of a 'bacterial transferability hypothesis of [some] depression' (see here), intervention options focused on redressing balance in bacterial colonies (see here) and what such findings might do for the whole 'gut-brain axis' idea.

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[1] Valles-Colomer M. et al. The neuroactive potential of the human gut microbiota in quality of life and depression. Nature Microbiology. 2019. Feb 4.

[2] Editorial. Links between gut microbes and depression strengthened. Nature. 2019. Feb 4.

[3] Cheung SG. et al. Systematic Review of Gut Microbiota and Major Depression. Front Psychiatry. 2019;10:34.

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The gut microbiome and autism... so far (continued)

Building on other reviews of the peer-reviewed science literature looking at the intestinal microbiota in relation to autism (see here), the paper published by Feitong Liu and colleagues [1] provides an updated 'where we're at' position in relation to the "potential evidence for the characteristic dysbiosis of gut microbiota in ASD [autism spectrum disorder] patients compared with healthy controls (HCs)." Just before you say anything, those are the authors words not mine; I'm not a fan of the word 'patients' nor use of the term 'healthy control' to denote not autism - not autistic, but there you go.

Language use aside, the Liu paper covers quite a lot of the peer-reviewed science talking about gut bacteria and autism up to March 2018. Their systematic review took in data from 16 studies - human studies "that compared the composition of gut microbiota in ASD patients and HCs using culture-independent techniques." Researchers had also previously registered their intention to conduct this review as per their PROSPERO entry (see here).

So what did their systematic review reveal? Well, they talked about how most studies looked at the intestinal microbiota via the examination of stool samples, although a couple relied on gut biopsy samples instead. Coincidentally, I spotted a bit of an error in the Liu paper in relation to their study reference numbering in one of the results sections, and how the Luna study (see here) and Williams study (see here) which relied on biopsy samples were replaced by other 'stool as a sample media' studies in the Liu write-up. It's only a small point and doesn't detract from the paper overall. The included studies also covered various different populations in a geographic sense as well as taking into account a mix of participants (with autism) in terms of the presence of gastrointestinal (GI) issues and the use of 'special' diets. Indeed, we are told that: "As restricted diet is very common in ASD patients, we tried to extract the information of eating habit in ASD and control group." Yes it is common, and yes it can have sometimes very negative effects (see here). Finally, all the studies included for review were "identified and assessed as medium (6–7) to high (8) quality" suggesting that the data were pretty reliable in a methodological sense.

Some key points emerged: "Overall, the changed structure of gut bacterial community in terms of β-diversity was observed coherently in ASD patients compared with HCs." Beta-diversity basically translates into 'between samples diversity' and in this case represents autism vs. not-autism controls. Out of the 16 studies included in their systematic review, Liu et al reported that "ten studies analyzed β-diversity (unweighted UniFrac distance, weighted UniFrac distances, and Bray-Curtis)." Six of those 10 studies "consistently reported that the microbiota of ASD patients clustered significantly apart from that of HCs." Ergo, there is evidence - some evidence - that the gut microbiome is significantly 'different' in autism compared with not-autism in a group sense. Some evidence at least.

Also: "Consistently, ASD patients had elevated abundance of Proteobacteria rather than HCs. In addition, Bifidobacterium, Blautia, Dialister, Prevotella, Veillonella, and Turicibacter were consistently decreased, while Lactobacillus, Bacteroides, Desulfovibrio, and Clostridium were increased in ASD patients relative to HCs."  Bear also in mind that Liu et al talked about various other bacterial species that were, in individual studies, elevated or depressed in the groups with autism, and how important such information might be. Such bacterial changes in terms of diversity or individual species predominating might also have knock-on effects as a result of the different chemical messages that different bacteria produce. One example: "Bacteroides is an abundant genus at all ages, from infants to adults. It is the main producer of propionate in the gut, and the abundance of propionate in feces correlates strongly with the abundance of Bacteroides." It probably won't surprise you to hear that propionic acid (propionate) has also got some research history when it comes to autism (see here) and indeed, with caveats, continues to do so [2]. Other examples are included in the Liu paper, including the 'chemical of the moment', butyrate (butyric acid) (see here). This complements other recently published research [3] too. And let's not forget how such bacteria and their chemical messaging also has some important 'effects' on things like gut barrier function and gut immune function, as part of the 'new triad' when it comes to autism and the gut (see here).

"Microbiome reconstitution could be a potential therapy to ASD patients in future." That's another topic raised in the Liu study on how "remodeling gut microbiota with diet, antibiotics, prebiotics, probiotics, and FMT [fecal microbiota transplant]" could be an option. Actually the future is now, as a quick scour of the autism research literature shows that some of these options are already being / have been investigated (see here and see here for examples). We do need a lot more information on the hows-and-whys of such therapeutic options; importantly covering safety, effectiveness and maybe highlighting the possible mechanisms involved, but there is already existing literature in this area.

As well as highlighting some of the shortcomings of the current research literature discussing the intestinal microbiota and autism, the Liu study provides a nice overview of this topic as things currently (up to March 2018) stand. Whether such information can eventually be 'manipulated' to improve things like quality of life in the context of autism remains to be seen...

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[1] Liu F. et al. Altered composition and function of intestinal microbiota in autism spectrum disorders: a systematic review. Translational Psychiatry. 2019; 43.

[2] Shams S. et al. Systemic treatment with the enteric bacterial metabolic product propionic acid results in reduction of social behavior in juvenile rats: Contribution to a rodent model of autism spectrum disorder. Dev Psychobiol. 2019 Jan 28.

[3] Wang M. et al. Alterations in Gut Glutamate Metabolism Associated with Changes in Gut Microbiota Composition in Children with Autism Spectrum Disorder. mSystems. 2019 Jan 29;4(1). pii: e00321-18.

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Lactobacillus (L.) reuteri and mouse-modelled autism: spotlight on the vagus nerve?

I am a little late getting to the findings reported by Martina Sgritta and colleagues [1] but I eventually arrived at them. As per the title of this post - "Lactobacillus (L.) reuteri and mouse model autism: spotlight on the vagus nerve?" - there were a number of important elements to this research covering mouse-modelled autism, the "gut-microbiota-brain axis", and something called the vagus nerve. Pretty good fodder for this blog by all accounts, given some past discussions (see here) including mention of previous research from some of the Sgritta paper co-authors (see here).

So, continuing a theme from this research group suggesting that, in mice at least, some pregnancy 'risk factors' for autism might have a microbial connection [2], authors set out to examine whether the inclusion of a specific bacterial species called Lactobacillus reuteri or L. reuteri for short might have some important effect on the social behaviour of a mouse model of autism. L. reuteri has been the source of quite a bit of study down the years [3] as a function of it's antimicrobial activity and connection to inflammatory conditions. Outside of the Baylor College group research, this bacterial species has also been talked about with autism in mind [4] in other studies, as per conclusions like: "This study identifies bacterial species that are sensitive to an autism-related mutation." I'll say no more on that study for now aside from offering a viewpoint from elsewhere on the web.

Alongside their observation that "treatment with L. reuteri selectively rescues social deficits in genetic, environmental, and idiopathic ASD [autism spectrum disorder] models" Sgritta et al also put a little scientific flesh on what underlying mechanism(s) might be pertinent to such 'rescuing' of social issues. You might think it was something to do with the contribution of L. reuteri to the gut microbiome of the [mouse] host? Well, yes and no, as authors highlighted how "L. reuteri acts in a vagus nerve-dependent manner" so bringing in the notion that bacteria in the gut *talk to* the brain (or the wider central nervous system) via the vagus nerve. The paper by Bruno Bonaz and colleagues [5] provides as good an explanation as any of how bacteria talk to the brain and what this means for the "gut-microbiota-brain axis." Interestingly too, authors mention how when the vagus nerve was 'disrupted' in said mouse-modelled autism, so the the gut-microbiota-brain link was also disrupted, and onward that L. reuteri didn't seemingly work as well at rescuing those social issues. They also talk about a role for oxytocin receptors in their findings similar to their last research occasion, but I'm going to leave that for now. All I will say is that oxytocin has also been mentioned in other autism research circles (see here).

Obviously, there are caveats to the Sgritta findings; not least that mouse-modelled autism is just mouse-modelled autism and probably not totally representative of real-life human autism (see here). Generalisations of the social behaviour of mice raised and living under laboratory conditions to people (children and adults) traversing the big, wide real world always require a degree of caution.

But I am interested in this area and would like to see more study done on the "gut-microbiota-brain axis" with autism in mind. Given that L. reuteri is freely available to anyone and everyone and seems to have quite a good safety record, I'd perhaps suggest that a clinical trial could be something that a young, upcoming researcher or research group might possibly want to consider exploring...

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[1] Sgritta M. et al. Mechanisms Underlying Microbial-Mediated Changes in Social Behavior in Mouse Models of Autism Spectrum Disorder. Neuron. 2018 Dec 3. pii: S0896-6273(18)31009-2.

[2] Buffington SA. et al. Microbial Reconstitution Reverses Maternal Diet-Induced Social and Synaptic Deficits in Offspring. Cell. 2016 Jun 16;165(7):1762-1775.

[3] Mu Q. et al. Role of Lactobacillus reuteri in Human Health and Diseases. Front Microbiol. 2018;9:757. 

[4] Tabouy L. et al. Dysbiosis of microbiome and probiotic treatment in a genetic model of autism spectrum disorders. Brain Behav Immun. 2018 Oct;73:310-319.

[5] Bonaz B. et al. The Vagus Nerve at the Interface of the Microbiota-Gut-Brain Axis. Front Neurosci. 2018;12:49. 

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Middle ear infection and autism (again)

"Autism was more common in children who had had an otitis media infection or who had been treated with antibiotics."

Minus any sweeping generalisations, that was the conclusion reached in the study by Theresa Wimberley and colleagues [1] continuing some important research themes (see here and see here) examining any "interplay among otitis media, antibiotics, and the subsequent risk of developing autism."

Just in case you don't already know, otitis media infection refers to an infection of the middle ear that "causes inflammation (redness and swelling) and a build-up of fluid behind the eardrum." Aside from the use of painkillers, the seriousness of certain otitis media infections can sometimes mean that antibiotics are prescribed to combat any underlying bacterial infection or even in some cases, grommets inserted as treatment.

Wimberley et al report results based on "the entire Danish population", well, over three-quarters of a million children "followed from birth (January 1, 1997 to December 31, 2008) until December 31, 2012." They calculated various 'risk of autism' statistics as a function of a previous medical diagnosis of otitis media and "antibiotic prescriptions redeemed at Danish pharmacies." Yes folks, yet again those big data Scandinavian population registries have been used to good research effect.

Results: "The absolute risk of autism before age 10 was increased among children with otitis media (1.2% for females and 3.3% for males) and in children who had redeemed an antibiotic prescription (0.6% and 2.7% for females and males) compared to children without a history of otitis media and antibiotics usage (0.4% for females and 1.9% for males)." Researchers also reported finding "little evidence of a synergistic effect between otitis media infections and treatment with antibiotics" despite them being over-represented in relation to autism. They also caution that cause-and-effect cannot be inferred from their observational results.

What more can one say about the Wimberley findings? Well, echoing the idea that correlation is not the same thing as causation, I'd say that there is quite a bit more research to do on this topic. Further investigations are required into the possible mechanisms through which autism may manifest at least partially as a result of a history of ear infection (or indeed vice-versa). Mention of antibiotics also brings in areas of additional research interest such as the gut microbiome and what antimicrobials might 'be doing' to the trillions of passengers that are carried in the deepest, darkest recesses of the human body. I'm also minded to suggest that alongside antibiotic use to potentially treat infections like otitis media, researchers might also want to focus in on other medicines that might be accessed in such case such as over-the-counter pain relief in light of other *associations* that have been made (see here).

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[1] Wimberley T. et al. Otitis media, antibiotics, and risk of autism spectrum disorder. Autism Res. 2018 Oct 3.

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"Anti-Candida albicans IgG antibodies in children with autism spectrum disorders"

The quote titling this post - "Anti-Candida albicans IgG antibodies in children with autism spectrum disorders" - reflects the title of the paper by Paul Ashwood & Heather Hughes [1] who set out to "determine if children with ASD [autism spectrum disorder] exhibit elevations in antibodies that target C. albicans, indicating current or previous overgrowth of this fungal species." Such work is based on the still developing idea that "individuals with ASD have significant aberrations in the composition of their gut microbiota, known as dysbiosis" and part of that dysbiosis might also stretch to fungal as well as bacterial species.

Candida albicans also known as C. albicans is described as a 'opportunistic pathogenic yeast' quite readily observed in quite a large proportion of 'healthy adults'. For most people, this yeast does not cause any issues. On occasion however, C. albicans can lead to problems, particularly among those who are described as 'immunocompromised'. This is not the first time that C. albicans has been examined in the context of autism. Granted, the studies so far have been relatively small scale [2] and in requirement of follow-up [3] but this topic is no stranger to the peer-reviewed science literature. The Ashwood & Hughes paper should also be viewed in the context of other science discussions from this authorship group [4]; indeed several [5].

So: "We measured anti-C. albicans immunoglobulin (IgG) in plasma from eighty children enrolled in the UC Davis MIND Institute CHARGE study." IgG antibodies, represent 'immune status' with regards to a history of encountering specific pathogens. So, being positive to "anti-C. albicans immunoglobulin (IgG)" means that someone has been exposed to C. albicans at some point in their lifetime and retained something of an 'immune memory' to it. This subsequently means that your immune system is 'primed' in case that specific pathogen is encountered once again.

Results: "Plasma anti-C. albicans antibody positivity was found in 36.5% (19/52) of children with ASD. Anti-C. albicans antibodies in typically developing controls was (14.3%; 4/28)." I'm sure that you can see the disparity between the groups, bearing in mind that this was not an 'all-or-nothing' finding in relation to the separation of the groups. I should also mention that researchers also reported that gastrointestinal (GI) symptoms, also examined in this cohort, did not seemingly play a role in C. albicans antibody positivity.

Where next for this area of investigation? Well, alongside perhaps taking these results into consideration with other findings from this research group (see here), the authors mention that "exploring fungal composition within the gut as well as metabolic byproducts of yeast species such as d-arabinitol and ethanol, and identifying associations these might have with behaviors in ASD" could be one direction. In light of other independent research (see here), I'd say that was a sensible next step to take.

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[1] Ashwood P. & Hughes HK. Brief Report: Anti-Candida albicans IgG antibodies in children with autism spectrum disorders. Front. Psychiatry. 2018. Nov 26.

[2] Ekiel A. et al. Intestinal microflora of autistic children. Med Dosw Mikrobiol. 2010;62(3):237-43.

[3] Iovene MR. et al. Intestinal Dysbiosis and Yeast Isolation in Stool of Subjects with Autism Spectrum Disorders. Mycopathologia. 2017 Apr;182(3-4):349-363.

[4] Hughes HK. et al. The Gut Microbiota and Dysbiosis in Autism Spectrum Disorders. Curr Neurol Neurosci Rep. 2018 Sep 24;18(11):81.

[5] Hughes HK. et al. Immune Dysfunction and Autoimmunity as Pathological Mechanisms in Autism Spectrum Disorders. Front. Cell. Neurosci. 2018.

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Exclusion diet plus prebiotics for [some] autism?

"To our knowledge, this is the first study where the effect of exclusion diets and prebiotics has been evaluated in autism, showing potential beneficial effects."

So said the results reported by Roberta Grimaldi and colleagues [1] and the findings of their study attempting to "understand the impact of diet on GM [gut microbiota] composition and metabolism in ASD [autism spectrum disorder] children and to investigate the modulating potential of B-GOS® intervention on these parameters."

The study by Grimaldi et al was research registered (see here) and included the 'gold-standard' research design: "A randomised, double-blind, placebo-controlled" study. Researchers divided 30 children diagnosed with an ASD into two groups (A and B), which were subsequently further divided into two groups depending on whether they followed an exclusion diet - "mainly gluten and casein free" - or an unrestricted diet based on the analysis of 4-day food diaries. Within those subdivided A and B groups, half received a placebo supplement of maltodextrin whilst the other half received a "prebiotic B-GOS® mixture" over a 6-week period. As well as including 'run-in', 'beginning of treatment' and 'end of treatment' periods, researchers also included a follow-up period of 2 weeks at the end of the study where various behavioural and physiological measures complemented their use during the experimental period.

Results: there were quite a few results reported as a function of baseline variables such as whether or not participants were following an exclusion diet or not, and as a function of the intervention(s). Behaviourally (probably most importantly), researchers reported that: "Results showed consistent reduction over time in anti-sociability score in children on the combination of the exclusion diet and B-GOS intervention, with the most apparent difference occurring at follow-up." This was measured using the autism 'rising' instrument called the A-TEC (see here) and also complemented other results based on the use of the autism spectrum quotient (AQ). As far as I can make out, all other behavioural and psychometric measures used - the "empathy and systemising quotient (EQ-SQ)... and the Spence’s Children Anxiety Scale-Parent version (SCAS-P)" - did not show any significant changes over the study duration.

Gastrointestinal (GI) symptoms were also examined during the Grimaldi study as per the use of "daily questionnaires for GI function and symptoms" and the utilisation of that fabulous graphical resource, the Bristol stool chart. Authors reported no significant changes/differences as a consequence of intervention, although: "Significantly lower scores of abdominal pain (P < 0.05) and bowel movement (P < 0.001) were reported in children following exclusion diets" at baseline. Interesting, in light of other independent results (see here).

Grimaldi and colleagues also provide quite a lot of data following their examination of fecal and urine samples taken over the course of their investigation. This is perhaps not unexpected given their previous research interests in this area and the proud reputation earned at one of the affiliated institutions. The results? Lots of them. Perhaps the most important intervention-wise was the finding of a "significant increase of Lachnospiraceae family" following B-GOS® intervention. The authors talk about this in terms of the production of butyrate (as per their previous research) and the (positive) reputation this stuff is starting to garner.

Other details? Well, going back to the baseline assessment of samples as a function of the use of an exclusion diet or not, there are some interesting findings. So: "Before prebiotic B-GOS® intervention, we evaluated the nutritional impact of exclusion diets (GFCF) and our results showed deficiency in vitamin D intake, which was significant in children on unrestricted diets." This kinda ties into other research which observed that the 'horror' that is a gluten-free, casein-free (GFCF) diet in relation to autism might actually not be that horrible in nutritional terms (see here). I'm also minded to bring in other recent research which suggested that a GF diet might be a bit of a 'fixer' when it comes to vitamin D deficiency issues under certain clinical circumstances [2]. I say this minus any sweeping generalisations or universal application to the label of autism.

Overall the Grimaldi findings are interesting and suggest further investigations are required on the use of prebiotics and diet in autism; perhaps complementing the still-growing interest in the gut microbiota and autism (see here for one example). Mindful also that diet can itself be seemingly affect gut bacterial composition too [3]. As they stand however, the current results aren't yet a glowing endorsement of B-GOS® intervention 'for' autism, mindful of the small participant group eventually included for study and the relatively short-term nature of the Grimaldi trial. We'll see where this goes... although next time, I'd also like to see a more prominent statement about any harms or side-effects encountered or not during the study period. I assume 'not' in the current study but...

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[1] Grimaldi R. et al. A prebiotic intervention study in children with autism spectrum disorders (ASDs). Microbiome. 2018 Aug 2;6(1):133.

[2] Zingone F. & Ciacci C. The value and significance of 25(OH) and 1,25(OH) vitamin D serum levels in adult coeliac patients: A review of the literature. Dig Liver Dis. 2018 Aug;50(8):757-760.

[3] Berding K. & Donovan SM. Diet Can Impact Microbiota Composition in Children With Autism Spectrum Disorder. Front Neurosci. 2018 Jul 31;12:515.

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A poo(p) transplant for depression and anxiety?

Contrary to the title of this post - "A poo(p) transplant for depression and anxiety?" - I don't think we are yet in a position to say that Fecal Microbiota Transplantation (FMT) is ready to go 'mainstream' as an approved treatment for depression and/or anxiety. I do however, think that the findings reported by Shunya Kurokawa and colleagues [1] provide evidence for a further, more controlled, scheme of research on this topic.

Based on their following a small-ish group of patients diagnosed with "either Irritable Bowel Syndrome (IBS), Functional Diarrhea (FDr) or Functional Constipation (FC) who underwent FMT for the treatment of gastrointestinal symptoms and observation of psychiatric symptoms" authors report results before said poo(p) transplant and after 4 weeks based on ratings on various instruments pertinent to the presentation of depression and anxiety. Alongside "intestinal microbiota were measured" with a particular focus on the level of diversity of species that were present in pre- and post-FMT samples. I might also mention at this point, how something like IBS is not without it's own 'psychological' correlates as per other research (see here and see here).

Following an 'open-trial' methodology and including only a "small sample size with no control group", researchers reported some significant improvements in relation to those depression and anxiety symptom scores for some. Importantly too, they noted that potential FMT effects on mood seemed to be independent of effects on "gastrointestinal symptom change." Similarly: "There was a significant correlation between baseline Shannon index and HAM-D [Hamilton Rating Scale for Depression] score, and a correlation between Shannon index change and HAM-D improvement after FMT." This suggests that bacterial diversity might be something to look at as potentially explaining the psychological effects of FMT.

Reiterating that the Kurokawa findings are preliminary and hence, require quite a lot more further (independent) study, I find this topic to be an interesting one. Although there may be some 'consumer resistance' to the idea of FMT, for some people, this type of intervention is nothing short of life-saving (see here). The idea that a similar type of transplant *might* also hold some benefits for conditions/labels outside of something like Clostridium difficile (C. difficile) infection has already been noted in the peer-reviewed science literature (see here and see here for examples), including conditions characterised by behaviour and psychology. This alongside a growing interest in how mood and temperament might have some important connection to those trillions of wee beasties (the gut microbiome) that call us all home (see here). We'll see where this goes...

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[1] Kurokawa S. et al. The effect of fecal microbiota transplantation on psychiatric symptoms among patients with irritable bowel syndrome, functional diarrhea and functional constipation: An open-label observational study. J Affect Disord. 2018 Apr 12;235:506-512.

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The art of the gut microbiome: 'Excretory Wipings' across 45+ years

Excretory Wipings May 18-October 21, 1970 is a piece of art composed by the artist Billy Apple (more accurately known as Billy Apple®). Due to possible copyright issues et al I don't want to show the actual piece on this blog but will describe to you what it comprises: a conceptual art work including toilet paper with erm, traces of a bowel movement provided during that particular period of 1970.

It's probably not everyone's cup of tea when it comes to art, but believe it or not, it might potentially be a rather important research starting point if say you wanted to characterise the gut microbiome past and present. Indeed, that is exactly what the paper by Thilini Jayasinghe and colleagues [1] describes, as they compared the bacterial colonies present in the 1970 sample(s) with a more up-to-date set of samples provided by the same artist in 2016.

Some media interest in the Jayasinghe results can be seen here. In short, three archived samples sourced from the 1970 art piece were compared with three more recent samples sourced exactly 46 years later (2016). Researchers performed 16SrRNA sequencing to "study bacterial phylogeny and taxonomy" [2] and try and find out whether there were similarities/differences in the types of bacteria present and indeed, whether overall bacterial diversity had reduced/stayed the same/increased between the testing periods.

Following this N=1 study, a few details emerged. So: "We observed that 45% of the microbial species were retained over the 45 year interval." Such a finding has to bear in mind that the artist "was suffering from diverticular disease in 2016" and that such a condition may well impact on the type of gut bacteria that is present and/or predominating. Indeed, the authors talk quite a bit about how "the 2016 microbiomes contained significantly higher level of genus Prevotella... which may be related to the observed diverticular disease by having a negative impact on gut immune system." Interestingly too, the word 'butyrate' appears in the Jayasinghe text, as in lower levels of butyrate-producing bacteria being present in the more recent sample. Butyrate is currently going through a period of bacterial 'sainthood' [3] at the moment...

Next: "The diversity of the microbial species from samples taken when Apple was 80 years of age was lower than that from samples when he was 35." Bacterial diversity is something of real interest these days, as more and more people start talking about gut bacterial diversity (and the loss of it) as potentially being related to all-manner of life-enhancing and life-not-so-enhancing correlations. The recent results are framed by the authors in the context of being "consistent with the idea of a drift towards a core microbiome" where ageing seems to play a role in diversity.

Of course, one shouldn't forget that this was a N=1 study and that gut bacterial populations are subject to all-manner of influences pushing and pulling on what bacteria may or may not be present in a longitudinal sense. These latest results say very little outside of what happened to the bacterial profile of Billy Apple® between the testing occasions.

But I'm still intrigued by such findings and the idea that there may be other resources to 'tap into' when it comes to looking either long-term at gut bacterial profiles or indeed, comparing gut bacterial patterns as a function of time and various ages. Y'know, comparing bacterial profiles of various ages in years gone by with similarly aged participants in more modern times, and trying to determine what various factors linked to modern life might have done (or not) to gut bacterial profiles. I don't doubt that some results might be rather interesting and discussion-provoking...

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[1] Jayasinghe TN. et al. Long-term stability in the gut microbiome over 46 years in the life of Billy Apple®. Human Microbiome Journal. 2017; 5-6: 7-10.

[2] Janda JM. & Abbott SL. 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls . Journal of Clinical Microbiology. 2007;45(9):2761-2764.

[3] Canani RB. et al. Potential beneficial effects of butyrate in intestinal and extraintestinal diseases. World Journal of Gastroenterology : WJG. 2011;17(12):1519-1528.

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Probiotics degrading gluten peptides - part 4

Here we go again. Probiotics degrading gluten peptides part 4 adds to previous posts on this topic (see part 1 here, part 2 here and part 3 here).

The difference this time around? People. People actually eating a a test meal containing gluten - "a porridge containing 0.5 g gluten" - and being given a potential gluten-degrading preparation - "Aspergillus niger-derived prolyl endoprotease (AN-PEP)" - or a placebo whilst their gastric and duodenal content was sampled for gluten concentrations over a 3-hour period after. So described the results published by Julia König and colleagues [1] continuing a research theme from this group [2].

AN-PEP was the product under the research spotlight following some previous scientific findings [3] suggesting that unlike various other digestive enzyme supplements, this stuff showed some pretty good actions/effects on immunogenic gluten peptides. Following a "randomized placebo-controlled crossover study" design, researchers followed 16 participants "with self-reported gluten sensitivity" (but not coeliac disease or wheat allergy) across 3 test days when on each day either a high dose of AN-PEP was given, or a low dose of AN-PEP was given or a placebo was given. Details of the high and low doses included: "The low dose tablets provided 83300 Protease Picomol International (PPI), and the high dose 166700 PPI of AN-PEP enzyme (1 PPI is the amount of enzyme that releases one picomole of p-nitroaniline per second under defined assay conditions)."

This was quite an invasive study as researchers had to gain access to parts of the gastrointestinal (GI) tract of participants and so: "Subjects attended each test day after an overnight fast, and a multi-lumen nasoduodenal catheter was placed with one lumen tip in the gastric antrum and one lumen tip 15 cm lower in the duodenum." It was then a case of drawing off stomach and duodenal samples and analysing for gluten content. Further: "success of AN-PEP in degrading gluten was defined as at least 50% gluten degradation compared to placebo, calculated as area under the curve (AUC) over 180 min."

Results: "It actually works" was a quote from one of the authors of the paper in previous media attention of this study before peer-reviewed publication. "In the stomach, gluten levels were reduced from 176.9 to 22.0 in the high dose and to 25.4 μg × min/ml in the low dose. In the duodenum, gluten levels were reduced from 14.1 in the placebo to 6.3 in the high dose and to 7.4 μg × min/ml in the low dose." AN-PEP appeared to be doing its designated job. Importantly too: "No severe adverse events were reported" over the course of the study period.

Scientific replication is the name of the game following the König results. Replication with larger sample numbers and also potentially looking at whether such a preparation might be useful for those diagnosed with something like coeliac disease or other accepted immune-related pathology linked to gluten consumption. I say that acknowledging that a gluten-free diet is the best that science and medicine currently has for the management of coeliac disease. The authors also note that they "did not perform a double-blinded placebo-controlled gluten challenge as sometimes suggested to confirm the diagnosis of gluten sensitivity" in their participants. This kinda intersects with the continuing discussions about what non-coeliac gluten/wheat sensitivity actually is (see here).

"In conclusion, our study showed that the AN-PEP enzyme is effective in degrading small amounts of gluten as part of a complex meal in the stomach. Even though the use of AN-PEP is not intended to replace a gluten-free diet in gluten-related disorders, it appears to be effective as a digestive aid protecting against the unintentional intake of gluten." I can't argue with that and look forward to seeing more on this topic in future.

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[1] König J. et al. Randomized clinical trial: Effective gluten degradation by Aspergillus niger-derived enzyme in a complex meal setting. Sci Rep. 2017 Oct 12;7(1):13100.

[2] Salden BN. et al. Randomised clinical study: Aspergillus niger-derived enzyme digests gluten in the stomach of healthy volunteers. Aliment Pharmacol Ther. 2015 Aug;42(3):273-85.

[3] Janssen G. et al. Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements. PLoS One. 2015 Jun 1;10(6):e0128065.

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"abnormalities in mitochondrial activity in the lower GI tract of children with ASD"

The findings reported by Shannon Rose and colleagues [1] (open-access) continue a research theme by [some of] this authorship group looking at how mitochondrial dysfunction seems to be part and parcel of at least some autism (see here). Indeed, how when one talks about mitochondrial issues potentially accompanying [some] autism, one really needs to look at it in the context of other issues potentially also 'over-represented' in relation to autism (see here).

This time around, Rose et al set out to "determine whether mitochondrial dysfunction may contribute to GI [gastrointestinal] symptoms in children with ASD [autism spectrum disorder]" on the basis that GI symptoms (whether functional or more pathological) are no stranger to autism (see here). With this in mind, I note the name Tim Buie is included as part of the Rose paper authorship team and so should reference some of the sterling work he and his team have done on the topic of GI issues and autism and its importance down the years.

Researchers analysed mitochondrial function(s) in rectal and cecum mucosal biopsies in a small sample of children diagnosed with ASD (n=10) and compared results with those from "10 children with Crohn’s disease and 10 neurotypical children with nonspecific GI complaints." There are two points for me to make here: first, although it is an invasive procedure to collect them, those biopsies used for study were extracted on a clinical basis as part of "elective diagnostic colonoscopy." This was not a case of 'experimenting' on children for the sake of an experiment; rather that children were already undergoing investigations for their significant bowel issues, save any health inequalities appearing "just 'cos they were autistic" for example. Second, although the authors have chosen to use the term 'neurotypical' to reflect not-autism, I myself still find this terminology to be scientifically problematic (see here) in the context that no brain is seemingly typical or atypical according to current scientific evidence. Not least also on the basis that immune-based conditions such as inflammatory bowel diseases do seem to carry an increased 'risk' of psychiatric issues (see here) and what that might [eventually] mean for those children diagnosed with Crohn's disease (an inflammatory bowel disease) for example. Anyhow, two approaches are described in connection with the study of mitochondria in those biopsy samples looking at both the quantity and activity of various electron transport chain (ETC) complexes. Yet again, I can profess no serious expertise on the various elements of mitochondria but there is some good reading out there in the peer-reviewed science domain on the topic.

Results: "Differences in mitochondrial function were found in children with ASD as compared to the other control groups across several ETC complexes suggesting a difference in overall mitochondrial function rather than a change in one specific mitochondrial enzyme." Accepting the small participant numbers included for study, these are potentially important results. Not least because other work looking at such mitochondrial issues in relation to [some] autism has been predominantly based on activity in muscle; now it appears extending "this observation to altered ETC complex activity in the GI mucosa" too.

Then to some speculation: "The fact that increased ETC complex protein content was primarily seen in the cecum, an area where enteric microbiome fermentation products such as PPA [propionic acid] and BUT [butyrate] are abundant, suggests a role for the enteric microbiome in the evolution of mitochondrial abnormalities in children with ASD." An interesting perspective indeed and in need of some further investigation. Butyrate has, in recent years, been elevated to almost scientific sainthood (see here for example) so one has to perhaps be a little cautious about sweeping statements in the context of autism or any other label. I say this with particular relevance to an 'autism colon' discussed by the authors (see here) which I also think is perhaps a little premature to speculate on.

No mind, the results are what they are and add to the growing literature discussing mitochondria in the context of [some] autism. The implication once again is to screen for such issues within the context that a diagnosis of autism should represent a starting point for further investigations not the finishing line.

And finally, to another author on the Rose paper, I'm still waiting to read about some of your results...

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[1] Rose S. et al. Mitochondrial dysfunction in the gastrointestinal mucosa of children with autism: A blinded case-control study. PLoS One. 2017 Oct 13;12(10):e0186377.

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