Don't panic! Folate receptor autoantibodies and autism

Don't panic @ Paul Whiteley

Those of you who, like me, enjoyed the adventures of Arthur Dent in the Hitchhiker's Guide to the Galaxy will know of the classic phrase associated with the book/series/film - 'Don't panic'. In the case of this post, I don't want readers to get too stressed or put off by the title and perceived content, as I attempt not to blind everyone with 'the appliance of science' while discussing a rather interesting piece of research. Let's see how I do..

The paper in question is by Richard Frye and colleagues* (open-access) part of the research tag-team that is Rossignol and Frye discussed in other posts. As a point of interest, the word on the cyberstreet is that Dr Rossignol has started a new programme with autism in mind called MAPS focused on the biomedical aspects of autism (no promotion intended).

I digress.

Alongside some other quite distinguished names in autism research including Jill James, Dr Frye reports on (a) the prevalence of folate receptor autoantibodies in a group of children diagnosed with an autism spectrum condition, (b) various measures compared with autoantibody negative children with autism and (c) what happens when folinic acid (leucovorin) is given. Folinic acid should not be confused with folic acid which whilst being an essential supplement during pregnancy has had a slightly bumpy ride these past few days following the publication of this meta-analysis.

OK softly, softly catchee monkey.

A good description of cerebral folate deficiency (CFD) syndrome is here. Basically diagnosis is based on low levels of 5-methyltetrahydrofolate (5MTHF) in cerebrospinal fluid (as Wikipedia when not in blackout describes it, the stuff the brain floats in). There are lots of behavioural manifestations of CFD syndrome which aid its description as a neurodevelopmental condition; autism has also been mentioned in some cases of CFD syndrome. Management of CFD syndrome is via the use of folinic acid (leucovorin) to normalise levels of 5MTHF by-passing folate receptors.

Based on the above paragraph coupled with the already suggested link between autism and possible issues with MTHFR (methylentetrahydrofolate reductase) (the enzyme which converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, 5MTHF) you can perhaps see why this study was done.

So:

• Ninety-three children (~ 7 years old) diagnosed with autism were screened for folate receptor autoantibodies (FRAs). FRAs are a slightly less invasive way of identifying people who might be at risk of CFD outside of doing a lumbar puncture. The folate receptor is a form of folate binding protein used to transport folate across the blood brain barrier. If you imagine that folate is essential for DNA synthesis, methylation and influences levels of homocysteine, you start to appreciate how important this chap is. FRAs impair the function of the folate receptor which might have knock-on effects similar to more genetically-linked effects on receptors. 
• Lots of other information was collected including whether or not participants were following a casein-free (milk-free) diet, given that such diets have been known to reduce FRAs (very, very interesting).
• Looking at both blocking (blocking folate transport) and binding FRAs, 56/93 (60%) of children were positive for blocking FRAs and 41/93 (44%) positive for binding FRAs. Twenty-nine percent of children were positive for both FRAs and 70/93 (75%) positive for at least one FRA. Interestingly, when it came to looking at rates of autoimmune conditions in the family history, more instances were found in those who did not have FRAs than those who did (60% vs. 24%). No other measures significantly differed between positive and negative autoantibody groups (aside from age at testing).
• A lumbar puncture was performed on 16 children included for study and levels of 5-methyltetrahydrofolate were found to be below the normative mean in every case (although not falling below the lower limit of normal though).
• A subgroup of 44 children from the 70 positive for at least one FRA were treated with leucovorin calcium tablets for at least 1 month (mean treatment time was 4 months). Based on parental responses to the Clinical Global Impression Scale - Improvement subscale on various items of cognition and behaviour, approximately two-thirds of children were deemed to have obtained some benefit.

There is a lot of detail to this paper which unfortunately I have neither the time nor space to go through in this post. Allowing for the fact that quite a few inferences were made during the study i.e. the 5MTHF levels found for those who underwent lumbar puncture would also be noted in those who were not tested, combined with the less than impressive way that the clinical effects of folinic acid were reported, there are some valuable details to be had from this report.

The rate of FRAs (blocking and/or binding) noted in the autism group (75%) is high and suggests that quite a bit more study is due in this area not least in comparison to other developmental conditions and control populations. I note for example that studies of blocking FRAs in Rett syndrome suggest that a smaller proportion of this group might be affected (24%). I do also wonder about whether there is any link with the timing of symptom onset, and in particular whether reported regression in behaviour has any effect on the likelihood of FRAs being present or not as per what happens in CFD. Speaking of regression and autism, how about this recent article?

Much like the recent findings from Jyonouchi and colleagues on SPAD and autism, the message yet again is don't stop looking for other things just because a diagnosis of autism is received (bearing in mind that I am not advocating invasive procedures like lumbar puncture unless clinically directed). The potential usefulness of a milk-free diet in cases where FRAs are present also opens up a whole new world of possibilities.

Hopefully you have not panicked and I have not left you dazed and confused with this post. If I have, I apologise; and paying homage to the classic Richard Linklater film Dazed and Confused I offer some light metal relief. Take it away Mr Alice Cooper... (he's such a nice guy).

* Frye RE. et al. Cerebral folate receptor autoantibodies in autism spectrum disorder. Molecular Psychiatry. January 2012.

Autism and SPAD

The NIH very recently issued a press release about some exciting advances being made in the field of cold urticaria, an allergic disease brought about in response to cold temperatures. The release got my attention because it highlighted the role of the immune system and in particular immune deficiency, with findings which might have implications far beyond the original condition under investigation.

Connected, the latest paper from Harumi Jyonouchi and colleagues* alluded to in my recent Sutterella post has, at the same time, been popping in and out of my mind over the past few days. Dr Jyonouchi's research has been discussed before on this blog and this latest offering is along similar lines with a couple of important additions, most notably on a condition called specific polysaccharide antibody deficiency (SPAD). For those like me who are perhaps not acquainted with SPAD, a good overview is included as part of this paper. In short it is immunodeficiency (hence the tentative connection with the NIH research); the inability to make antibody to polysaccharide encapsulated organisms and is normally treated by IVIg infusion. I might add that Jyonouchi seemingly has an interest in SPAD and has discussed SPAD and autism previously.

The paper in question is open-access but a few interesting points are summarised:

• Eight children diagnosed with autism and comorbid SPAD were compared against children with ASD but not SPAD (n=39), asymptomatic controls (no ASD and no SPAD, n=37) and children with SPAD but not ASD (n=12).
• A range of measures were analysed linked to innate and adaptive immune response and cytokine production by PBMCs in response to various stimuli.
• The results: some interesting data on something called food protein induced enterocolitis syndrome (FPIES), a non-IgE-mediated food hypersensitivity primarily linked to milk or soy. FPIES was present in 5/8 (62%) of the ASD/SPAD group and by coincidence, 25/40 (62%) of the ASD/no-SPAD group. The ASD/SPAD group were screened for coeliac (celiac) disease and other GI conditions and everything came out negative. 
• Participants in the ASD/SPAD group seemingly produced different patterns of cytokines compared with the other groups. So lower in response to agonists of Toll-like receptors, were IL-6, IL-23 and IL-1b (beta) (pro-inflammatory cytokines) compared to all other groups. IL-10 levels in the absence of a stimuli were lower in both ASD groups compared to controls. 
• Transcriptional profiling of PBMCs showed that over 300 genes were either up- or down-regulated in the ASD/SPAD group compared to controls. Gene expression of CCL2 (noted in their previous study) may point to a constant activation signal potentially linked to things like inflammation.
• The conclusions drawn from the data are (a) there may be a subset of children with autism whose behavioural presentation may be linked to underlying issues with the immune system and how it reacts to challenges and (b) SPAD can coexist alongside autism.

There are a few comments to make on the collected data presented outside of just the relatively small participant group. Whilst not being an expert on FPIES, I do wonder about the overlap between this condition and the previous findings from Tim Buie for example on lactose intolerance in some cases of autism. I'm not saying that they are one and the same because they are not; just that more generically issues with milk and its components seem to follow autism as per the studies on use of a milk-free diet and predictors of functional bowel problems like constipation.

I touched upon IL-10 in relation to autism in the Sutterella post and that as an anti-inflammatory cytokine, IL-10 is already in the frame in autism research including its potential link to MET gene variants. IL-6 showed up recently in a post looking at the immune profile during pregnancy and risk of autism among other things; IL-23 takes us back to some recent MIND Institute findings linked to social behaviours. I am slightly cherry-picking here with these associative studies but there is some interest there.

The use of IVIg in cases of autism has some history and on the back of studies like this, I suspect it will gain even more clinical favour as time goes by (although note that I am not giving medical or any other advice). Looking around at what else is included in the medicine cabinet in relation to autism, I am struck by how an old favourite like melatonin might also have some interesting immune effects in relation to the current and other studies. So those upregulated CCL2 chemokines.. melatonin might just do something here, increasing levels of IL-10... maybe something here too. Like all medication, the clinical use discussed on the PIL does not mean that is all it might do.

Immune function in relation to autism is a very complicated thing. What studies like this show is that autism is not protective against developing other conditions and that just because someone is diagnosed with autism does not mean that clinical investigations should cease and desist.

To finish a spot of Pavement bringing back some Gold Soundz. Cue the Santas, bows and arrows.

* Jyonouchi H. et al. Immunological characterization and transcription profiling of peripheral blood (PB) monocytes in children with autism spectrum disorders (ASD) and specific polysaccharide antibody deficiency (SPAD): Case study. Journal of Neuroinflammation. January 2012.

Autism and Sutterella

Barely a few days into 2012 and like the tulips in my garden sprouting very early this year because of our exceptionally mild winter, the first research papers of the new year are popping up through the scientific soil. Two studies have so far caught my eye: the first by Harumi Jyonouchi and colleagues* (full-text) looked at some potential overlap between cases of autism spectrum condition and specific polysaccharide antibody deficiency (SPAD); the second by Brent Williams and colleagues** (full-text) reported on some of the differences in gastrointestinal (GI) bacteria present in children with autism compared with controls.

I am going to leave the Jyonouchi paper for now, except for pointing out quite an interesting detail from the paper suggesting that levels of the anti-inflammatory cytokine IL-10 seemed to be an issue (lower) for many participants with autism compared with asymptomatic controls. IL-10 has cropped up before in autism research circles and might yet hold some interesting clues about things like inflammation.

The last paper that I discussed on this blog from Brent Williams holds a special place for me in terms of it uniting gut bacteria, carbohydrate metabolism and GI issues in some cases of autism. His latest offering was therefore always going to be one to watch for me. Before progressing with the summary, it is interesting to note the involvement of Prof. Ian Lipkin in this research. I have quite a bit of time for Prof. Lipkin and his pretty extensive repertoire of research that he has published on ranging from maternal infection inducing subtle neuropathology in offspring to the continuing saga of viral infections 'associated' (?) with conditions like CFS/ME. This is a chap who is not afraid of a little controversy.

The paper is open-access but just in case:

• Biposy samples from 23 participants diagnosed with autism and gastrointestinal (GI) symptoms were compared with 9 samples from control participants with GI symptoms. Ages ranged from 3-10 years old.
• Same as last time, pinch biopsies were taken from the terminal ileum and cecum. Various methods were used to analyse these samples including a novel PCR method used to detect and quantify bacteria from the Sutterella genus.
• Following on from the previous findings of a high abundance of sequences from the Alcaligenaceae family in quite a few of the samples from the autistic group, further more detailed analysis showed that "all sequences of Alcaligenaceae found in AUT-GI patients’ biopsy samples were classified as members of the genus Sutterella". Sutterella presence was confirmed by their super-duper PCR method and the presence of various strains of Sutterella are reported for individual participants. Wikipedia has a more in-depth discussion about categorising bacteria on the basis of their taxonomic rank.
• Plasma antibodies against a specific strain of Sutterella (Sutterella wadsworthensis) were also detected in the autistic group (IgG) (IgM antibodies in one case) confirming its presence. 
• In total 12/23 (52%) of the autism group were PCR positive for Sutterella compared with none of the control group. This is the point that most of the media so far have focused on.

Sutterella, apparently named in memory of one Vera Sutter, who as the linked article suggests worked with Sydney Finegold (remember this paper) is a bacteria that I've not come across before. This paper describing the discovery of S.wadsworthensis explains it as a gram-negative bacteria thriving in low oxygen or anaerobic conditions. Sutterella has been previously linked to inflammatory bowel disease such as Crohn's disease although the more recent data has cast some doubt on the exclusivity of this link.

Whilst the methods and findings used in the current study are interesting, I wait to see where this work will go. It is interesting that in the discussion, the authors talk about abnormal intestinal permeability (the so-called leaky gut) and the potential resulting consequences of the the immune system meeting GI bacteria in places where they shouldn't really be meeting. In this study they specifically looked at plasma antibodies against S.wadsworthensis. The question is: what other antibodies might be present in cases of autism against other GI bacterial companions and what the net effect of this antibody load might be on a person. Interesting that increased epithelial IgG density has been noted before in cases of autism and bowel enteropathy.

I remain very attentive to this whole area of gut bacteria and autism (and other conditions) and hope to see a few more papers like this one in 2012 further probing the various goings-on of our bacterial masters. To finish, a song about a different type of bloom.

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* Jyonouchi H. et al. Immunological characterization and transcription profiling of peripheral blood (PB) monocytes in children with autism spectrum disorders (ASD) and specific polysaccharide antibody deficiency (SPAD): Case study. Journal of Neuroinflammation. January 2012.

** Williams B. et al. Application of novel PCR-based methods for detection, quantitation, and phylogenetic characterization of Sutterella species in intestinal biopsy samples from children with autism and gastrointestinal disturbances. mBio. January 2012

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Williams BL, Hornig M, Parekh T, & Lipkin WI (2012). Application of novel PCR-based methods for detection, quantitation, and phylogenetic characterization of Sutterella species in intestinal biopsy samples from children with autism and gastrointestinal disturbances. mBio, 3 (1) PMID: 22233678

1% of UK school children with chronic fatigue?

I am ashamed to admit that 'wagging school' is something I did on more than one occasion as a teenager. It wasn't that I didn't like school or anything like that but some days I just couldn't face double maths or that sprightly cross country run over fields and mountains in the pouring rain that was PE. Don't get me wrong, I was no Ferris Bueller when it came to excuses and there was most certainly no 'Save Paul' campaign when I was absent from school. I might add that as a [supposedly] responsible adult, I don't condone such behaviour - "kids, stay in school" (he says in his best Mr T voice).

Nowadays I wonder if it would be so easy for me to miss school if I was still a child. With the advent of mobile phones and even mobile emailing, parents of school-absent children can be contacted much more easily and informed about any unscheduled absences. Indeed here in the UK, and no doubt other parts of the world, schools are getting tough on student absenteeism.

With this in mind, a paper published late last year by Crawley and colleagues* has been on mind for a few weeks now. The paper suggested that previously unidentified Chronic Fatigue Syndrome/myalgic encephalomyelitis (CFS/ME) might be an important reason why some children are absent from school. CFS/ME in adults has appeared before on this blog with regards to issues with identification and other more contentious areas which came to a head recently. The T-Rex identification post(?) on CFS reported a prevalence of 0.2% for all areas of the UK. The Crawley study suggested that in children, the prevalence of CFS/ME was 1%.

The paper is open-access as per the journal title, but in case you'd like a summary:

• A new service set up in Bath, UK designed to reduce the numbers of student absences formed the basis of the study. Three schools in the locality with a student body totalling 2855 children aged 11-16 years old were screened for non-attenders who had missed 20% or more of a 6-week term.
• When known medical reasons, a single stint of absence due to illness and truanting were excluded, 112 identified children attended a school clinic to discuss why school had been missed.
• Forty-eight of these 112 children (42.9%) described significant fatigue as a reason for their absence, which when assessed, turned out to fulfil criteria for CFS/ME in 23 cases. Combined with the known cases of CFS/ME in the participant group, 28 cases of CFS/ME were recorded, which in the total study population (2855) equates to a prevalence just shy of 1%. The authors suggest that this is likely to be an underestimate given the children who did not attend the school absenteeism clinic.

As with every study finding, there are caveats to this study which have been covered very succinctly by the NHS Choices website (here).

I don't know what it is about the 1 in 100 figure when applied to heterogeneous conditions with very little universal knowledge on why they occur and how to diagnose them outside of just looking at behaviour. I am sure that someone somewhere probably has a good explanation of this (outside of just being a chance finding) and possible clues on where we should be looking in terms of genetics and environment.

To end, a song which came back into my music collection very recently after a few years absence, being one of the all-time best karaoke classics... livin' on a prayer.

* Crawley EM. et al. Unidentified Chronic Fatigue Syndrome/myalgic encephalomyelitis (CFS/ME) is a major cause of school absence: surveillance outcomes from school-based clinics. BMJ Open. December 2011.

Where is autism research heading in 2012?

To quote everyone's favourite breakdown truck Mater, 'Ain't no need to watch where I'm goin'; just need to know where I've been'. In answer to the title of this post, I don't exactly know where autism research is heading in 2012 but if the previous trends are anything to go by, I might be able to provide you with a few possible clues.

Vasilopita (New Years Day bread) @ Paul Whiteley

The biggest clue comes from this review paper* published in December 2011 by Dan Rossignol and Richard Frye (open-access). Viewers of my recent post on 'I am a HBOT' will have seen the name Rossignol there. The more science-y types out there who enjoy reading the odd edition of Molecular Psychiatry will also remember Dr Rossignol for his quite comprehensive review of possible mitochondrial involvement in some cases of autism (open-access here).

The author's review of autism research is again open-access so no need for me to go into too much detail (what a cop-out!). Comprehensive... well, yes; as with many reviews and meta-analyses, which can often involve more work than writing 'go out there and collect data' experimental papers up. A few points worth mentioning:

• The rise of research into immune issues and autism spectrum conditions, inflammation, mitochondrial dysfunction and environmental exposures was a key point noted in this search of the PubMed literature on autism between 1971 and 2010.
• Moving from a period before 1986 where such factors were only discussed in a handful of papers, the 1990s were a growth time for such biological research and the past 5 years have truly witnessed an explosion in these research areas. Importantly the strength of the evidence, based on how studies were done, is encouragingly quite resilient.
• Genetic research still holds the Lion's share in terms of numbers of papers produced, probably as a result of the pretty large sums of money being pumped into things like genome-wide analysis studies and also accounting for the very complicated genetics paper and database discussed in a recent post.
• Pity the poor Theory of Mind (ToM). From autism research superstar in the 1980s to that unloved embarrassing 1985 Christmas jumper kept at the back of the wardrobe. Even one of ToM's main proponents has decided to head the 'metabolomics' way**. I don't want to be mean to ToM but lets face it, the writing has been on the wall for some time for ToM being the be-all-and-end-all of autism research. I'm thinking last years Nature series on autism...

I have to say that with my research hat on, I am pretty buoyed by the results of this current research review paper. By saying this I am not trying to belittle the reality of what autism means to many, many people on a day-to-day basis or anything like that. Nor do I want to be seen as just another researcher sat in their ivory tower shouting about things which ultimately still do very little to help improve quality of life for many people on the spectrum. It is heartening however to see that research does seem to be going in one of the directions which quite a few parents of, and people with autism have thought and been saying for a long time. Importantly also realising that the behavioural presentation of autism may be but one facet of a complex and heterogeneous condition.

I did say that I would give you clues (plural) as to where I thought autism research was heading in 2012 and beyond. A final clue harks back to another recent post on the study of biological phenotypes in autism; that is, looking at slightly more homogeneous sub-groups outside of just a group of children/adults 'with autism'. If it is somehow possible that autism research can look at the strong evidence findings isolated in the Rossignol and other reviews, source a decent enough sized participant group and apply some of the phenotypic overlay, thinking of the Nordahl study....

I end with an ode to ToM by a person whose life has probably changed beyond all recognition after singing a song... I dreamed a dream in time gone by...

* Rossignol DA. & Frye RE. A review of research trends in physiological abnormalities in autism spectrum disorders: immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures. Molecular Psychiatry. December 2011.

** Schwarz E. et al. Sex-specific serum biomarker patterns in adults with Asperger's syndrome. Molecular Psychiatry. December 2011.

I am a HBOT

Happy New Year. A slightly belated welcome to Questioning Answers in 2012. My second calendar year of blogging (good grief, when is he going to stop..!).

Demetrious Haracopos

I start this new year post with a short note to remember the recent passing away of Dr Demetrious Haracopos previously of the Center for Autisme in Denmark. To those that knew him Demetrious was one of the warmest, friendliest and inquisitive men you could know; a perfect combination for his work on autism, ranging from sexuality and autism to dietary intervention for autism. Rest in peace Demetri from all those whose lives you touched.

To task. I must confess that I was slightly reluctant to write this first post of the new year. Reluctant because I am not an expert on hyperbaric oxygen therapy (HBOT) as a proposed intervention for autism and related conditions. Nevertheless there is a research base to HBOT and autism (albeit currently quite limited), and given that this is a blog supposedly about autism research, the time has come to put pen to paper so to speak.

I got to this post via an interesting report on whether or not a US hospital should be offering HBOT as an intervention option for children with autism. Opinions abound on this topic it seems.

The first thing that comes to my mind (emphasis on the 'my') when talking about anything related to hyperbaric is a scene in the James Bond film, License to Kill. Mr Bond, played by Timothy Dalton, convinces the main villain to pop another villain into a hyperbaric chamber leading to quite a nasty depressurised end. I appreciate that this may not be the best advert for the hyperbaric chamber, but unfortunately that is my recollection, no doubt influenced by my 1980s movie mindset.

The more realistic view of HBOT for treating things like decompression sickness is thankfully a little safer and controlled where the proper expertise is on hand. The technique involves administering oxygen at pressures greater than one atmosphere (atm). Outside of the more traditional uses for HBOT, the theory is that whole body saturation with oxygen administered at pressure might have some onward positive effects for things like brain function particularly where blood flow is impaired and oxygenated levels might be poor. Looking at the evidence base with regards to things like traumatic brain injury and acute migraine, one can perhaps see some promise of effect from HBOT.

Given all the interest in the brain in relation to autism spectrum conditions, it is little wonder that HBOT has been touted as a possible intervention option. More than that however is the potential link between autism and inflammation in all its forms, which might provide another therapeutic target for HBOT.

The research base is, as I said, currently quite small for HBOT use in autism. It started with a few speculative papers published in the journal Medical Hypotheses (here and here). These have been subsequently followed by a few open and more controlled trials on HBOT with some encouraging but mixed results. This paper for example by Dan Rossignol, who seems very much to have lead the advancement of HBOT for autism (more on in him in future posts), suggested some effect on behavioural measures and inflammatory markers (C-reactive protein). The study was an open one and had no control group (either asymptomatic or receiving a sham intervention). Same with this study and same results (behavioural) and this more recent trial which found behavioural improvements but no significant changes to cytokine levels. Other studies have not found any significant effect.

Where a randomised-controlled design has been used, the results did indicate some potential for HBOT for at least some cases of autism. This study came to the conclusion that hyperbaric treatment at 1.3 atmospheres (24% oxygen) over a number of sessions produced a significant effect compared with normal atmosphere, normal oxygen levels. To reiterate my non-professional status with HBOT, I don't know enough about it to say what an increase of 3% over and above normal air oxygen levels administered at 1.3 atmospheres might do. It is difficult to match hyperbaric results with studies for other conditions because parameters can be different (how much oxygen is used and at what pressure). Studies of HBOT for cerebral palsy in children for example, have noted no overall difference between groups when greater oxygen levels were delivered at 1.75 atmospheres compared with only slightly pressurised air being delivered (results were beneficial in both cases). Other studies looking at using HBOT to tackle radiation-induced necrosis in children for example, reported on conditions of 100% oxygen delivered at 2-2.4 atm pressure and noted some potential effect.

On balance, the results obtained for HBOT for autism so far (stress: so far), whilst limited, do tend to suggest that it might be a beneficial intervention option for some. I hasten to add that I am in no way endorsing such an intervention given factors like the accepted uses for HBOT, the price of HBOT and the requirement for multiple sessions. The potential longer-term safety aspects also require a little more study.

To end, a song to reinvigorate you after the post-party/hangover season (although not for any tongue phobics)... "we'll drive you wild.."