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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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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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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A poo transplant for [some] autism?

I've talked about 'fecal microbial transplants' a.k.a the poo(p) transplant before on this blog (see here). That previous entry was about the more typical (and potentially life-saving) use of a poo transplant - where stool from one person is extracted, 'repackaged' and transferred to another person - albeit with caveats in terms of possible long-term side-effects. Now it appears that poo transplants are being investigated with something rather more central to the typical contents of the blog...

The paper by Dae-Wook Kang and colleagues [1] (open-access) has already been picked up by some media (see here) and it seems, also has a following from likely proponents and detractors particularly on social media. Including one James Adams on the authorship list, someone who is quite well-known in autism research circles (see here and see here for examples) alongside some other notable inclusions (Alessio Fasano, Thomas Borody, etc), the authors describe the results of small open-label study - I repeat, a small open-label trial - of 18 participants diagnosed with an autism spectrum disorder (ASD) who underwent a 10-week program characterised by the use of antibiotics, a bowel cleanse and then regular poo transplants for approximately 8 weeks. Additional information about the study and its results can be found here or if you wish, you can see the ClinicalTrials.gov entry here.

Tapping into a growing interest in how the gut (and its contents) might be important for at least some autism (see here for example) the aims of the trial were to "follow gut microbiota in healthy and treated children with ASD longitudinally as well as to evaluate an investigational new treatment, MTT [Microbiota Transfer Therapy], for its effectiveness in children with ASD in treating both GI [gastrointestinal] symptoms (primary outcome) and ASD-related symptoms (secondary outcomes), and to determine the effect of MTT on the gut microbiome."

The study included children diagnosed on the autism spectrum - ADI-R diagnosed - aged between 7-16 years old. All presented with moderate to severe functional bowel issues alongside their autism (something not unusual it seems). The authors also report using a control group of "20 age- and gender-matched neurotypical children without GI disorders" who were monitored but not treated as part of their study design.

The study first involved the administration of the antibiotic vancomycin for 2 weeks (something that has, on its own, some peer-reviewed research history with autism in mind [2]) used to 'profoundly suppress' pathogenic bacteria. Prilosec, the brand name for omeprazole was also administered towards the end of the bacterial washout phase initiated by the use of vancomycin. Prilosec is a medicine traditionally used to suppress stomach acid secretions and was used to "remove most remaining gut bacteria and vancomycin" and aid the passage and survival of the donor stool to the wider gastrointestinal (GI) tract. I say all that knowing that such medicines can affect the composition of the gut microbiota. Then came the bowel cleanse (Moviprep) complete with a fasting from food day, followed by the main [research] event: oral or rectal administration of donor stool and an initial high dose followed by maintenance doses. I know some people might be slightly uncomfortable with the idea of the rectal administration of medicines but there are some common-sense reasons behind this form of medicines delivery particularly where oral dosage forms (tablets, capsules) might not be well tolerated. As for the initial oral dosage form: "the participants began either oral administration of SHGM [Standardized Human Gut Microbiota] (2.5 × 1012 cells/day) mixed in a chocolate milk, milk substitute, or juice for 2 days (divided into three daily doses)." I have to say that whilst I initially envisaged Austin Powers and his 'tastes a bit nutty' scene, this was very much NOT how things actually were.

Alongside the donor stool formulation being trialled predominantly with regards to safety and initial efficacy, researchers also surveyed participants in relation to (i) effects on their gut microbiota (diversity and species present), (ii) the presentation of bowel symptoms/habits and (iii) behavioural outcomes covering autism-specific issues (via the CARS) and more general adaptive behaviours (via the Vineland scales for example). I was also happy to see a section included in their paper labelled 'virome bioinformatics' hat-tipping the idea that gut bacteria are not the only passengers we carry in our deepest, darkest recesses.

Results: well something certainly seemed to happen when looking at before, during and after results of this case series trial. First and foremost adverse effects were small and limited (hyperactivity, irritability) meaning that in the short term at least, the poo transplants and pre-poo transplant protocols were tolerated quite well. This is also evident in the 0% study attrition rate (i.e. everyone who started the study stayed in the study).

So: "Substantial changes in GI and ASD symptoms were observed. GI symptoms, as assessed by the GSRS [Gastrointestinal Symptom Rating Scale], significantly improved for abdominal pain, indigestion, diarrhea, and constipation." The authors report some significant differences in scores over the course of the intervention period such that: "The average GSRS score dropped 82% from the beginning to end of the treatment and remained improved (77% decrease from baseline) even 8 weeks after treatment stopped." That is a helluva placebo effect! Indeed, only 2 participants from the cohort were classified as 'non-responders' on the basis of their GSRS scores over the course of the study.

Also: "Beyond these GI improvements, ASD-related behavior also improved following MTT." The sorts of changes to CARS scores being reported were in the region of a 20% reduction in 'core ASD' symptoms at 8 weeks compared to baseline reports. Further, 8 weeks after the intervention had been completed the behavioural gains ("relative to baseline") were still evident based on CARS scoring. These ratings also did not depend on whether the poo transplant was administered orally or rectally.

The authors also discuss some not unexpected changes to gut bacterial profiles in their cohort over the intervention period. At baseline: "gut bacteria were significantly less diverse in children with ASD than neurotypical controls." This finding is in line with other study results from the authors (see here). Bacterial diversity did (slowly) change over the intervention period to a point where at 18 weeks after baseline median richness "was statistically indistinguishable between the ASD and control groups." This was noted in 16 of the 18 participants with ASD.

Finally: "Specific genera that significantly changed in their relative abundances with treatment included Bifidobacterium, Prevotella, and Desulfovibrio." The authors have chosen to zoom in on Prevotella as a consequence of their results - "the increase in the relative abundance of Prevotella after MTT is consistent with their potentially beneficial role in the gut of children with ASD" - but I'd like to see a little more work done on this before any grand claims are made. As to the virome/phage side of things: "phage richness and evenness did not significantly change following MTT given the timeframe of this study" so again, there is more science needed in this area too as to hows and why (nots).

OK, I think it is worth stressing again that this was an open-label study not a gold-standard, double-blind, randomised-controlled trial or anything like that. What this means is that these results are preliminary - very preliminary - and although quite promising, need scientific follow-up for a longer period. Whilst such results are seemingly pretty good evidence for the whole 'gut-brain axis' thing with regards to autism (see here) there are still questions that need answering, not least with regards to possible mechanisms linking gut bacteria changes to behaviour changes. Do these results, for example, point to some role for intestinal permeability changes given what is already being said about 'leaky gut' in the context of [some] autism (see here) and the growing interest in a relationship between gut bacteria and gut barrier integrity? How do gut bacteria 'communicate' with the grey-pink matter floating in the skull? Questions aplenty there be. There is also a certain degree of 'yuck factor; to contend with as a result of the use of a poo transplant in this context and what this might mean for future trials and possible 'rolling out' of such an intervention assuming effectiveness is confirmed. I can just see the headlines now...

But I don't want to take anything away from these novel findings and [with caveats] the significance of the results. When one considers how many people on the autism spectrum are suffering (yes, suffering) with bowel complaints, whether functional or something rather more pathological (see here), efforts to reduce pain and any additional burden they bring should be welcomed from wherever they emerge...

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[1] 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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Kang, D., Adams, J., Gregory, A., Borody, T., Chittick, L., Fasano, A., Khoruts, A., Geis, E., Maldonado, J., McDonough-Means, S., Pollard, E., Roux, S., Sadowsky, M., Lipson, K., Sullivan, M., Caporaso, J., & Krajmalnik-Brown, R. (2017). Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study Microbiome, 5 (1) DOI: 10.1186/s40168-016-0225-7