Friday 30 November 2012

T.gondii and dietary gluten joining forces?

Looking back at the way this blog has taken shape over the nearly two years that its been running, certain topics have tended to predominate which I freely admit that I knew very little about before getting into this blogging malarky.
Cold cat @ Wikipedia  

Had anyone suggested that one day I would be reading and writing about critters like Toxoplasma gondii (T.gondii to its friends), I previously would have replied 'yer what?' with puzzling facial expression to match.

Nevertheless I have found myself drawn into discussing research on this survivor and in particular how it might do so much more than just infest quite a few people around the world with particular focus on its potential role in conditions like schizophrenia, or at least some cases.

Enter then another study by a familiar name to this blog, Emily Severance and colleagues* (open-access) discussing T.gondii and some interesting possibilities on how it might be able to alter the immune response to dietary gluten, in mice, with potential implications for schizophrenia and even autism.

The research of Dr Severance and colleagues, as mentioned, has appeared twice on this blog so far as per posts on gastrointestinal (GI) inflammation in cases of schizophrenia (see here) and C1q activation (see here). I was particularly impressed with the GI inflammation paper it has to be said, not least because it introduced a really interesting investigative tool (anti-Saccharomyces cerevisiae IgG antibodies) which I thought could easily be looked at with autism in mind.

To the more recent paper:

  • Aside from one of our papers getting a mention (thank you!), the study aimed to look at how infection with T.gondii might impact upon the way the immune system recognises and deals with the dietary protein gluten in a mouse model, in a sort of infection-changing-immune-system type way.
  • Balb/C mice were infected with T.gondii via one of three routes: intraperitoneal (IP), peroral (PO) (via oral-diet), or prenatally.
  • Antibodies (IgG) to T.gondii, gluten and C1q were measured.
  • Results: I seem to say this everytime, but lots of results were generated. Perhaps the most important was the finding that infection with T.gondii was 'convincingly' (author word not mine) related to the production of gluten antibodies. 
  • Second in importance was the finding that prenatal exposure to T.gondii as in the offspring of female mice who were infected, showed an elevation of antibodies to T.gondii when mummy mouse was seropostive for T.gondii which also coincided with very significantly elevated antibodies to gluten and C1q. Also, the "female sex is more severely affected following T. gondii infection".
  • Finally(!) there is a suggestion of some involvement for gut hyperpermeability (the so-called leaky gut) in this story, as per the question of "how T. gondii strains gain access to systemic circulation, but a para-cellular route affecting epithelial tight junction proteins is suspected". And even those words "bacterial translocation" and "zonulin" are mentioned as part and parcel of how T.gondii might be able to open up the gut membrane and potentially allow gluten peptides to come into contact with the immune system. Not a million miles away from what's been talked about with autism in mind previously.

And rest. So, we are presented with lots of potentially important statements here concerning how infection might combine to promote an immune response to an important dietary component like gluten. We are also given a hint that maternal infection might also lead/cause/be associated with an immune response to dietary gluten in offspring. Sounds to me like some quite important leads there and more than a passing relationship with other work looking at immune activation models of conditions like autism (hint: Paul Patterson and colleagues). I also wonder whether there may be other, wider considerations of maternal immune activation as per those findings on maternal antibodies to foetal brain and the transglutaminase research in autism.

With the old science hat on, I have to reiterate that this was a study of mice not humans, and as far as I can remember these results are slightly at odds with the other C1q activation and T.gondii data previously presented**. Independent replication is also required, but I'm not going to take anything away from these very, very interesting findings which perhaps mirror other findings of infection triggering antibody production*** (many thanks to Natasa for this last link).

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* Severance EG. et al. Anti-gluten immune response following Toxoplasma gondii infection in mice. PLoS ONE. 2012; 7(11): e50991.

** Severance EG. et al. Complement C1q formation of immune complexes with milk caseins and wheat glutens in schizophrenia. Neurobiol Dis. 2012; 48: 447-453.

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ResearchBlogging.org Emily G. Severance, Geetha Kannan, Kristin L. Gressitt, Jianchun Xiao, Armin Alaedini, Mikhail V. Pletnikov, & Robert H. Yolken (2012). Anti-Gluten Immune Response following Toxoplasma gondii Infection in Mice PLoS ONE : doi:10.1371/journal.pone.0050991

Thursday 29 November 2012

Metal measuring and autism

Now and again, two papers of a similar ilk are published temporally close together in the great autism research melting pot so as to cumulatively make for interesting reading.
Congratulations, it's a baby T-1000 @ Wikipedia  

In the case of this post, I am referring to the paper by Jim Adams and colleagues* and the paper by Yahya Al-Farsi and colleagues** which both examined the burden of metals present in cases of autism from the perspective of two very different geographical areas of the world. Both papers are published in the same journal too.

I should say that I've covered metals, heavy and toxic, before on this blog with reference to the background to this whole area (see here) and some research on that most controversial of areas, mercury and autism (see here). As with just about every other instance of autism research, the message is a messy one, with no 'one-size-fits-all' finding, which often wrongly gets translated as either 'it must be a universal effect' or 'there must be no effect' with seemingly no middle ground where the autisms are concerned.

Anyhow, a few details about the papers:

The Adams paper:

  • The hypotheses were: (a) "children with autism would have higher levels of some toxic metals in their blood and urine" (n=55) compared with asymptomatic controls (n=44), and (b) the severity of symptoms would be associated with the toxic metal burden.
  • This current study formed part of a wider body of work undertaken and published by the authors*** (open-access) looking at children aged 5-16 years. This follows other work in a similar vein**** (open-access).
  • Morning blood and urine samples were eventually analysed by an old favourite method (ICP mass spectrometry) and the severity of autistic symptoms assessed by various means including the ATEC.
  • Results: there were quite a few of them but most notably levels of lead in red blood cells (RBC) and urine were significantly elevated in the autism group compared with the control group, alongside urinary thallium, urinary tin and urinary tungsten.
  • In terms of the measured severity of autism correlating with the metals findings, a slightly complicated picture emerges which suggested that whole blood and RBC mercury concentrations showed "possibly" significant correlations with the three assessment instruments used.
  • The authors conclude that their results suggest that either there is increased exposure to these metals, increased absorption of these metals or decreased (fecal) excretion of these metals or some combination of these explanations. 

The Al-Farsi paper:

  • A very similar starting point by all means, testing the hypothesis that "children with ASD will show variations and deregulated levels of heavy metals and essential minerals when compared to non-ASD controls".
  • Based in the Sultanate of Oman, hair samples from children with DSM-IV autism (n=27) were analysed again by ICP-MS for the presence of various heavy metals and compared with results from age- and sex-matched asymptomatic controls (n=27).
  • Results: again, quite a few but generally speaking levels of heavy metals were elevated in the autism group compared to controls, and included cobalt, cadmium, chromium, aluminium and also our old friend lead.
  • When also looking at levels of essential minerals, a slightly more mixed picture emerges with lower levels of calcium and copper to be present but higher zinc, iron and sulphur (to name but a few).

OK, there are a few differences to point out between these studies outside of just the ethnicity of their autism cohorts and the possible differences in geographical exposure patterns. The Adams paper looked at blood and urine, the Al-Farsi paper looked at hair. The mean ages of the groups examined were also slightly different (Adams: autism = 5.3 years; Al-Farsi: autism = 10 years). So we are not able to directly transpose results one on top of another.

That being said, there were some similarities to these results not least about those findings related to lead. Indeed, I have quite an interest in all things lead for quite a few reasons, not least that even Superman had problems with lead so why wouldn't children exposed to the stuff, and those figures from last year (2011) which listed lead reduction as being one potential candidate among many as to why the US crime figures were falling. Exposure to lead is generally speaking not a great thing for developing or developed brains/bodies.

I'll admit to not being a great expert on how hair, blood and urine samples compare when looking at metals or any other kind of chemical/compound so I can't offer too much in the way of information about the functionality of these measures and the extent to which they reflect levels of these metals or indeed evidence of storage and any on-going physiological activity / effect. I am for some reason drawn back to the very recent CHARGE findings on air pollution and autism (bearing in mind that the reduction of lead in petrol we've seen over the past few decades) as an interesting variable potentially relevant to these results but will say no more than that at this time.

Accepting the issue of hair analysis, the Al-Farsi findings revealed some interesting trends with reference to the trace minerals examined. Higher zinc, higher iron and higher sulphur raise questions about what this means in light of other research indicating lower hair zinc (see this post) for example, and indeed that body of research on iron levels in autism (see this post and this post). The curse of messy heterogeneous autism research?

Interestingly both papers seem to wade back to similar biochemical pathways in terms of the potential significance of their findings. So glutathione pops up (see this post on where glutathione sits in the current autism research world) and how glutathione is a key component in both the processes of antioxidation-oxidative stress as well as being tied into the removal of things like metals. The feeling is that the consistently lower levels of glutathione (GSH) noted in cases of autism is probably not going to be a great thing for the biological processing of such toxic metals.

Both papers end with calls for further research into this complicated (and often controversial) area. I'd like to second that call given that this is a topic which seems to be cropping up more regularly in recent years. I know that when people start talking about toxic metals and autism, specific toxic metals, some conversations instantly move into areas which make some people a little uncomfortable. Irrespective of what causes such issues to become apparent, the questions must surely be (i) what effect, if any such burdens have contributing to the presentation of autism or indeed any of its potential comorbidities and (ii) whether there is something that can be done to ease any burden and what, if any, effects this might have on symptoms.

To close, how can I not make reference to some great music following a post about metal. Take it away AC/DC.

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* Adams JB. et al. Toxicological status of children with autism vs. neurotypical children and the association with autism severity. Biol Trace Elem Res. November 2012.

** Al-Farsi YM. et al. Levels of heavy metals and essential minerals in hair samples of children with autism in Oman: a case-control study. Biol Trace Elem Res. November 2012.

*** Adams JB. et al. Nutritional and metabolic status of children with autism vs. neurotypical children, and the association with autism severity. Nutr Metab (Lond). 2011; 8: 34.

**** Adams JB. et al. The severity of autism is associated with toxic metal body burden and red blood cell glutathione levels. J Toxicology. 2009; Article ID 532640. doi:10.1155/2009/532640

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ResearchBlogging.org Adams, J., Audhya, T., McDonough-Means, S., Rubin, R., Quig, D., Geis, E., Gehn, E., Loresto, M., Mitchell, J., Atwood, S., Barnhouse, S., & Lee, W. (2012). Toxicological Status of Children with Autism vs. Neurotypical Children and the Association with Autism Severity Biological Trace Element Research DOI: 10.1007/s12011-012-9551-1

ResearchBlogging.org Al-Farsi, Y., Waly, M., Al-Sharbati, M., Al-Shafaee, M., Al-Farsi, O., Al-Khaduri, M., Gupta, I., Ouhtit, A., Al-Adawi, S., Al-Said, M., & Deth, R. (2012). Levels of Heavy Metals and Essential Minerals in Hair Samples of Children with Autism in Oman: a Case–Control Study Biological Trace Element Research DOI: 10.1007/s12011-012-9553-z

Wednesday 28 November 2012

Targeting mitochondrial dysfunction in ME/CFS?

Consider this post a bit of a follow-up to a previous one based on some interesting observations by Booth and colleagues looking at mitochondrial dysfunction in cases of myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS).
Join the Stone Age dots? @ Wikipedia  

Same authorship group but this time around presenting the results of an audit of patients who underwent examination based on the ATP profile (no endorsement given) and what they did about it published by Sarah Myhill and colleagues* (open-access).

I will at this point just reiterate my caveat about not giving advice, medical or otherwise, on this blog and hence not advocating anything based on discussions about the Myhill paper. I'm just talking, nothing else.

So here goes:

  • The paper is based on the same 138 participants diagnosed with ME/CFS as described on their previous paper and how, when a multi-faceted intervention regime was put in place, some of the participants fared in terms of their mitochondrial profiles and presented symptoms.
  • I say that this paper describes some of the original cohort; in essence this boils down 34 of them who had more than one ATP profile "separated by some months" of which 30 followed the intervention regime with vigour and 4 participants who were a little more lax. Bear in mind this was reported as an audit not a clinical trial.
  • What was the treatment regime? Well, (a) a stone age diet which I assume is similar to a Paleolithic diet(?), (b) a good sleeping routine, (c) a supplemental vitamin/mineral/other regime and (d) assuming an appropriate work-rest balance; pacing but not PACE.
  • Elements of these interventions were specifically tailored for participants based on their mitochondrial and other tests of nutritional status. Interestingly the authors report on some familiar issues as per often finding "deficiencies in glutathione (GSH) and glutathione peroxidase (GSH-PX) which are needed  to protect cells from oxidative damage and to detoxify xenobiotics". Other elements that turned up on the various testing panels were slightly more contentious as per the suggestion that "High levels of heavy metals can be tackled using selective chelation therapy".
  • Results: there are some case histories reported and quite a bit of data per participant per testing occasion. I'll let you draw your own conclusions from what is described but in essence, the authors observed that there was some degree of responsiveness to the interventions shown on the repeat testing profiles for some of the participants. The caveat being that there was as expected, some degree of variability in response potentially affected by lots of variables, not least because individuals are individuals, and also probably due to the intervention regime itself.
  • The authors report that "All 30 of the multiple test patients who followed the treatment protocol have increases in the major parameters of mitochondrial function and in the Mitochondrial Energy Score".
  • The co-factors involved in ATP production - niacinamde, L-carnitine, coenzyme Q10 - also saw some interesting changes between baseline and post-intervention testing as a function of "% within normal range" though not presented in absolute mean values (pre- and post).
  • All that being said, there is little in the way of formalised description as to the presentation of symptoms, and whether and how these overlapped with such changes (perhaps reiterating the audit nature of this paper).

For those who might be rather sceptical of this area of functional medicine, mitochondria being involved in ME/CFS or even members of the authorship group themselves, it would be easy to dismiss this paper as being nothing more than an advert for the clinical services of the authors. It is not a formal study of mitochondrial function being assessed or 'modified' in cases of ME/CFS, it focuses on individuals not groups, and there are plenty of scientific holes in how and what results have been presented (or not).

But step back a little and consider a few things. ME/CFS is probably in an even worse clinical position that autism for example, is at the moment. Whereas autism (mostly) enjoys the recognition of being a biologically-based neurodevelopmental condition, ME/CFS is still, in some quarters, thought to be a purely psychiatric condition, where sufferers - yes, people do suffer from ME/CFS - are thought to be either mentally ill or perhaps just as bad, to be hypochondriacs. Labels such as yuppie flu didn't really help matters either.

The next hurdle to be faced is the fact that the presentation of ME/CFS is heterogeneous. Even the diagnostic criteria for the condition has not been fully agreed upon, or at least as a condition with universal diagnostic consensus. That even before we start talking about comorbidity and the like.

When taking these factors into account, it does start to make a little more sense as to why initial, preliminary investigations should reflect these factors. So that mitochondrial issues might be part and parcel of ME/CFS or at least some cases, that reporting and correcting mitochondrial dysfunction might involve different strategies for different people, and that sticking to an intervention regime is not always possible in an intention-to-treat analysis manner, particularly when you are dealing with a condition which impacts so centrally on a person. Don't get me wrong, I'm not standing up for the use of audits over clinical trials - randomised, controlled clinical trials - but do consider this paper, and the previous publications from the authors, to be a good first step of where further investigation should be carried out.

Of course I would very much like to see more formal study not just on what happened to biochemistry as a result of the interventions discussed by Myhill and colleagues but also on overt signs and symptoms and importantly overall quality of life (QoL) outside of just clinical measures. Dietary changes such as those discussed are no laughing matter and can, on occasion, negatively impact on QoL as a consequence of their restrictiveness. Indeed as per other mentions of diet and ME/CFS on this blog, one wonders whether there may be either certain elements of the Stone Age diet which might be more usefully connected to cases, or indeed whether underlying clinical comorbidity such as coeliac disease or non-coeliac gluten sensitivity might be important to symptoms and outcomes.

Whatever your views on this area of research or more generally the definition of what ME/CFS is or isn't, this is peer-reviewed research and therefore represents an addition to the scientific literature and a challenge to the scientific community to undertake further independent replication save any repeat of XMRV...

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* Myhill S. et al. Targeting mitochondrial dysfunction in the treatment of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) – a clinical audit. Int J Clin Exp Med 2013;6 : 1-15.

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ResearchBlogging.org Myhill S,, Booth NE,, & McLaren-Howard J (2013). Targeting mitochondrial dysfunction in the treatment of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) – a clinical audit International Journal of Clinical & Experimental Medicine, 6 (1), 1-15

Monday 26 November 2012

CHARGE, traffic pollution and autism

The headline reads "Traffic fumes linked to autism". The sub-heading describes how babies who were exposed to traffic air pollution during their first year of life "are more likely to become autistic". Welcome to the study by Heather Volk and colleagues* on traffic pollution, particulate matter and risk of autism.
Motorbiking... @ Wikipedia  

Before heading into the paper, the discerning autism research reader will no doubt remember that this is a topic which has already cropped up in autism research circles, and indeed by the same authorship group** (open-access).

The difference being that on the previous occasion, the focus was on in-utero proximity to traffic-related air pollution "as a surrogate for air pollution exposure", whereas the current study looked at both gestation and first-year estimates of exposure.

A few details of the most recent study:

  • Another CHARGE-related investigation, which estimated air pollution and air quality based on mum's address for the various stages of pregnancy and first year of life for 279 children diagnosed with an autism spectrum disorder (ASD) compared with 245 typically-developing controls.
  • The US Environmental Protection Agency (EPA) datasets on regional air pollution were cross-indexed with addresses, and some statistical wizardry applied.
  • Results: compared with control participants, exposure to traffic-related air pollution by residence was highest for the autism group particularly during the first year of life (adjusted odds ratio = 3.1). Exposure to particulates (sub 10 and 2.5 micrometres) based on regional exposure measures were also associated with autism during the first year of life.
  • The authors conclude: "Exposure to traffic-related air pollution, nitrogen dioxide, PM2.5, and PM10 during pregnancy and during the first year of life was associated with autism". The possible effect being one of inflammation (quite a regular visitor to autism shores it has to be said).

One obviously has to be quite careful with such studies of association and all that 'correlation does not equal causation' jazz. On that basis I'm not going to start any great fanfare about this research despite the intricate datasets which have been compared, and the previous body of research that it follows.

That being said, neither am I going to discard these results as being just coincidence. Air pollution is a serious environmental issue related to health; potentially acting in a multitude of ways depending on what type of pollution is present, the level of exposure*** and the age of the exposee. We've seen hints that air pollutants might play some role in cases of autism as per another study of association on meteorological factors and autism recently discussed on this blog. Definitive data is however somewhat lacking including more direct measurement of airborne pollutants and any residual traces that they may leave.

Autism is also not alone in its purported link to air pollution as exemplified by this paper by Siddique and colleagues looking at air pollution and ADHD****. Other research has attempted to link air pollution to issues with cognitive abilities*****. The primary question arising from such research is to ask whether the recent Volk data may reflect any of these comorbidities/issues above and beyond an exclusive link to autism?

Outside of just traffic-related air pollution, the air that we breathe is home to lots of other things too; some things which just don't even bear thinking about (dog poo bacteria, yep you heard me right). Questions are being asked about air quality in our cities on quite a grand scale nowadays and in conditions like autism, fast becoming a 'health priority' (and not before time), the research net should be cast as wide as possible to determine the contribution of environment (if any) to the astounding number of cases of autism estimated and being diagnosed.

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* Volk HE. et al. Traffic-related air pollution, particulate matter, and autism. Arch Gen Psychiatry. November 2012.

** Volk HE. et al. Residential proximity to freeways and autism in the CHARGE study. Environ Health Perspect. 2011; 119: 873–877.

*** Valavanidis A. et al. Airborne particulate matter and human health: toxicological assessment and importance of size and composition of particles for oxidative damage and carcinogenic mechanisms. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2008; 26: 339-362.

**** Siddique S. et al. Attention-deficit hyperactivity disorder in children chronically exposed to high level of vehicular pollution. Eur J Pediatr. 2011; 170: 923-929.

***** Suglia SF. et al. Association of black carbon with cognition among children in a prospective birth cohort study. Am J Epidemiol. 2008; 167: 280-286.

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ResearchBlogging.org Volk, H. (2012). Traffic-Related Air Pollution, Particulate Matter, and AutismAir Pollution, Particulate Matter, and Autism Archives of General Psychiatry DOI: 10.1001/jamapsychiatry.2013.266

Sunday 25 November 2012

Matrix metalloproteinases and autism

Peptidases. The enzymes that split apart peptide bonds, have always been of some interest to me. Not only resulting from my continued interest in peptides as being one potential suggestion why a gluten- and/or casein-free diet might affect symptom presentation in some cases of autism but also because of their relationship to the building blocks of functional life, the amino acids, which are really starting to make some waves in autism research.
Just split @ Wikipedia  

With all this in mind, it was perhaps inevitable that I would arrive at a post on a specific class of peptidases, the matrix metalloproteinases (MMPs) and some very preliminary suggestions of potential involvement in cases of autism and beyond. Please note that not all peptidases work on the same types of peptides, just in case you wondered.

It was the paper by Abdallah and colleagues* which spurred me into writing this post, and their suggestion that analysis of amniotic fluid samples indicated that levels of MMP-9 were elevated in cases later diagnosed with an autism spectrum disorder (ASD). The fact that they also mentioned BDNF was a bonus interest.

Perhaps I should back up a little and provide a quick overview of the MMPs. So with my Twitter limit: zinc-dependent, embryonic development, removal of extracellular matrix, immune function and inflammation, synaptic plasticity**, etc. There are a number of very good review articles on the MMPs including this one by Birkedal-Hansen and colleagues*** (open-access).

The specific MMP discussed by Abdallah et al, MMP-9, has been the topic of quite a lot of research interest over the years. The degradation of collagen is an obvious starting point for all the MMPs, hence a link with connective tissue disorders such as rheumatoid arthritis**** (open-access). It is however with the brain and various neuropsychiatric conditions in mind, that I find myself drawn to the potential effects of MMP-9.

Domenici and colleagues***** (open-access) for example, reported on the possibility that elevated plasma MMP-9 levels may (alongside other compounds) be a biomarker for depression in their cohort. Indeed the genetics of MMP-9 have been similarly linked to bipolar and related disorders. There remains however the chicken-and-egg situation of which came first, MMP-9 or symptoms, not really answered by studies such as this one by Rybakowski and colleagues****** relying on MMP-9 levels to predict staging of bipolar disorder.

Outside of the Abdallah study, there is not presently a great wealth of research done on the MMPs with autism in mind. Accepting the fact that Abdallah was looking in amniotic fluids samples and therefore not functional levels per se, I did turn up this paper by Siller and Broadie******* (open-access) looking at MMPs in Fragile X syndrome (FXS). Regular readers might remember a few papers discussed on this blog in recent times with FXS as the target condition (see here and here) presenting with autistic features. Siller and Broadie asked whether administration of the antibiotic minocycline might actually serve to inhibit MMP (activity or formation?). The answer: it might as per other work in this area but please note that this is not to be construed as medical advice.

I was likewise interested to read the paper by Jang and colleagues******** on how everyone's favourite sleeping aid, melatonin, might also have some important effects on MMP-9 levels, at least in a rat model of stroke. That and the possibility(!) that this relationship might also involve blood-brain barrier (BBB) disruption really got the grey matter working. Once again a pharmacological effect that you won't see on the drug packaging insert.

We have yet to see all there potentially is about MMPs with autism in mind. With all the current interest in excitotoxicity in cases of autism linked to things like glutamate and compounded by the whole microglia story, I'm sure it is a topic that will gain in research popularity and perhaps even open up new avenues for intervention********* for specific people and/or groups should perturbed levels be found and replicated. Given also the possibility of a viral link to elevations in MMP9********** one has to assume that environment may also play a hand in some presentations and potentially tie into those models of maternal infection as being a risk factor for offspring autism?

To finish, the King at his best...

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* Abdallah MW. et al. Amniotic fluid MMP-9 and neurotrophins in autism spectrum disorders: an exploratory study. Autism Res. September 2012.

** Huntley GW. Synaptic circuit remodelling by matrix metalloproteinases in health and disease. Nature Reviews Neuroscience. 2012; 13: 743-757.

*** Birkedal-Hansen H. et al. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993 ;4: 197-250.

**** Distler JHW. et al. The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell microparticles. PNAS. 2005; 102: 2892-2897.

***** Domenici E. et al. Plasma protein biomarkers for depression and schizophrenia by multi analyte profiling of case-control collections. PLoS One. 2010; 5: e9166.

****** Rybakowski JK. et al. Increased serum matrix metalloproteinase-9 (MMP-9) levels in young patients during bipolar depression. J Affect Disord. August 2012.

******* Siller SS. & Broadie K. Matrix metalloproteinases and minocycline: therapeutic avenues for fragile X syndrome. Neural Plast. 2012: 124548

******** Jang JW. et al. Melatonin reduced the elevated matrix metalloproteinase-9 level in a rat photothrombotic stroke model. J Neurol Sci. 2012. pii: S0022-510X(12)00524-2

********* Leonardo CC. & Pennypacker KR. Neuroinflammation and MMPs: potential therapeutic targets in neonatal hypoxic-ischemic injury. J Neuroinflammation. 2009; 6: 13.

********** Kolb SA. et al. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in viral meningitis: upregulation of MMP-9 and TIMP-1 in cerebrospinal fluid. J Neuroimmunol. 1998; 84: 143-150.

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ResearchBlogging.org Abdallah MW, Pearce BD, Larsen N, Greaves-Lord K, Nørgaard-Pedersen B, Hougaard DM, Mortensen EL, & Grove J (2012). Amniotic Fluid MMP-9 and Neurotrophins in Autism Spectrum Disorders: An Exploratory Study. Autism research : official journal of the International Society for Autism Research PMID: 23008271

Friday 23 November 2012

Stop that phenylalanine now!

I'm going slightly off-piste with this post not strictly related to autism; however remaining true to my interest in all things amino acids, and in particular one of those most interesting aromatic amino acids, phenylalanine.

Phenylalanine @ Wikipedia
A few months back Chemistry World carried a very interesting article by Jon Evans* on how the amino acid phenylalanine might very well have the capacity to form amyloid-like fibrils classically related to conditions like Alzheimers disease. The article was based on this study by Adler-Abramovich and colleagues** which reported on a few pretty important observations.

I've done amyloid or rather amyloid precursor protein (in relation to autism) on this blog before (see here). Fibrils as their name suggests, are fibre-like structures. Without trying to plagiarise the article or study, a few points are worth noting:

  • The starting point was the metabolic condition phenylketonuria (PKU), probably the most famous of the inborn errors of metabolism.
  • PKU... the problems with metabolising phenylalanine as a result of issues with phenylalanine hydroxylase leads to a build up of the amino acid which has some particularly nasty effects on the developing body and brain. Said phenylalanine levels normally controlled by lifetime dietary restriction of phenylalanine but also potentially another intervention (BH4).
  • Adler-Abramovich et al observed that phenylalanine in solution tended to 'clump' together forming something that looked like amyloid-like fibrils. Further when such phenylalanine fibrils were added to cell lines, the fibrils showed some degree of toxicity. Even further, rabbits injected with said fibrils started to generate antibodies against them. Finally, after imaging the phenylalanine fibrils, there was a match between what they saw in the laboratory and what they saw in an engineered mouse model of PKU and indeed in real people diagnosed with PKU.
  • Ipso facto: phenylalanine can form amyloid-like fibrils and such fibrils might account for the tissue damage observed in PKU.

I admit to being really quite excited about this work. Modern science knows quite a bit about PKU following the very astute observations made by Dr. Asbjørn Følling. One area however that has always required a little more study was exactly how the build-up of phenylalanine caused the problems that it does; maybe part of that question has been answered with this work.

My mind also goes back to those other aromatic amino acids like tryptophan and tyrosine and whether similar fibrils could be formed from these compounds. The answer, according to the published scientific literature so far, is a very possible yes; as per studies like this one from Cecchini and colleagues*** although quite a lot of the investigations in this area goes well above my state of knowledge.

So, once again amino acids do their stuff. It makes you wonder whether Alzheimer's disease, at least some cases, might also reflect issues with phenylalanine. Logically also whether interventions like a low phenylalanine diet or even BH4 supplementation (see here) might reflect possible areas of intervention requiring much further study?

To finish, I'm sure many of you had the same 'could try harder' reports from school as I did when I was a kid. But to get something like this.... from your Dad?

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* Evans J. Anti-social amino acids gang up. Chemistry World. August 2012.

** Adler-Abramovich L. et al. Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria. Nat Chem Biol. 8: 701-706.

*** Cecchini P. et al. The role of tryptophan in protein fibrillogenesis: relevance of Trp7 and Trp14 to the amyloidogenic properties of myoglobin. PEDS. 2012; 25: 199-203.

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ResearchBlogging.org Adler-Abramovich L, Vaks L, Carny O, Trudler D, Magno A, Caflisch A, Frenkel D, & Gazit E (2012). Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria. Nature chemical biology PMID: 22706200

Wednesday 21 November 2012

More mice, more autism-like behaviours corrected

I'm sure that by now you're all used to hearing about mouse models and autism and the relative ease it seems, that researchers are able to reverse autistic behaviours in certain strains of mice. I've posted entries about such marvels of modern-day science a few times including reference to the work of Paul Patterson and bone marrow transplants and the reports on the use of arbaclofen in the Fragile X syndrome (FXS) / autism behaviours mouse model. All this bearing in mind that mice are mice and not people, and as yet the mouse ADOS has not, to my knowledge, been standardised.
Gimme five... @ Wikipedia  

Enter then yet another 'we can reverse them' mouse study by Christos Gkogkas and colleagues* (a splendid Greek surname reflective of the heavy pronunciation of 'G', as in Gogas) published in the premier journal Nature.

An accompanying news piece also published in Nature by Dan Jones** delivers the headline: "Autism symptoms reversed in mice" and quite a nice summary of the research results. In light of this I'm not going to do a clinical dissection of the Gkogkas study, but rather summarise hopefully without plagiarising. Note also to Dan Jones, the current CDC estimates for autism is 1 in 88 not 1 in 110. Sorry to be pedantic.

So assuming I understand correctly, take a mouse - a specially bred mouse - which lacks a certain gene, Eif4ebp2, which codes for a certain protein (4E-BP2), which moderates the translation of certain mRNA, which potentially impacts on the production of neuroligins (NLGNs) as in increases production of them, which in turn effects synapses. The end result according to the Gkogkas study is synaptic hyperconnectivity and autistic-like symptoms. Don't quote me on this by the way.

More than that however was the suggestion that such symptoms might be reversed by the the use of 'an experimental cancer drug' which works by reducing protein synthesis. Said drug which I can't actually name yet, seemed to do the trick and positively affected certain social-based symptoms. "The team extended these findings by directly suppressing the translation of NLGN proteins using short interfering RNAs [via a non-replicating virus vehicle]" according to Jones, and hey presto, similar effects.

Neuroligins are synaptic cell-adhesion molecules which together with neurexins form a complex which are involved in synapse development and maturity. The term 'shake hands' tends to be used quite a bit when talking about these molecules; quite a good overview being this one from Thomas Südhof*** (open-access). Gkogkas reports that one specific neuroligin seems to be a primary target of their research: neuroligin 1. Other research on neuroligins and autism tends not to be so specific, bearing in mind that this is still a growth area of investigation.

As with the other autism-mouse research these are interesting results. The synaptic focus of this work will probably find quite a lot of support from quite a large contingent of the autism research community given the focus on (a) the brain and (b) the excitatory-inhibitory balance in brain cells; said balance already having surfaced in other autism research (see here).

Translating such research into benefiting real people with autism is the next step but likely however not to be a quick next step given that the experimental drug used to reduce protein synthesis was toxic (thanks Jon Brock!) and the requirement for looking at real people with autism (sorry, autisms) and all the other comorbidities which are more than likely to be present. As far as I am aware issues with 4E-BP2 in autism for example is still more of an idea than an actuality aside from papers like this one from De Rubeis & Bagni**** (open-access) looking again at autism-like behaviours in relation to FXS. The Gkogkas study at least offers a few more directions for autism research to take and demonstrating once again the invaluable contribution of animal models to autism research.

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* Gkogkas CG. et al. Autism-related deficits via dysregulated eIF4E-dependent translational control. Nature. November 2012.

** Jones D. Autism symptoms reversed in mice. Nature. November 2012.

*** Südhof TC. Neuroligins and neurexins link synaptic function to cognitive disease. Nature. 2008; 455: 903-911.

**** De Rubeis S. & Bagni C. Regulation of molecular pathways in the Fragile X Syndrome: insights into Autism Spectrum Disorders. J Neurodev Disord. 2011; 3: 257-269.

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ResearchBlogging.org Gkogkas, C., Khoutorsky, A., Ran, I., Rampakakis, E., Nevarko, T., Weatherill, D., Vasuta, C., Yee, S., Truitt, M., Dallaire, P., Major, F., Lasko, P., Ruggero, D., Nader, K., Lacaille, J., & Sonenberg, N. (2012). Autism-related deficits via dysregulated eIF4E-dependent translational control Nature DOI: 10.1038/nature11628

Tuesday 20 November 2012

Regression and autism continued

Regression.

Definition: "relapse to a less perfect or developed state".

Apologies for the very dramatic and very scientifically 'cold' description of regression. But as has been mentioned in the scientific literature, defining regression where it does occur in cases of autism has been a bit of a sticking point in some quarters.

Indeed, the topic of regression in relation to autism onset has graced this blog before. In that post, the main message was that after a bit of a lack of initial appreciation, regression is now understood to be part and parcel of at least some cases of autism, albeit with some way to go in understanding the precise hows and whys.
"Can you play any other tunes?" @ Wikipedia 

In this post I'm continuing on with regression in light of a few papers which I've recently been able to get hold of full-text.

So the papers by Barger and colleagues* meta-analysing some of the collected works on the prevalence and onset of regression in autism and Ekinci and colleagues** on the phenomenology of regression in autism and finally the paper by Westphal and colleagues*** on Theo Heller's Über Dementia Infantilis.

I've picked these papers to discuss because between them there is about as good and contemporary opinion about regression and autism as one could ask for. Indeed also some interesting ideas which also require a little more follow-up and perhaps a little bit of an autism history re-write too. Without further ado, some points I've picked out:

Barger study:

  • Based on data for a combined nearly 30,000 people with an autism spectrum disorder (ASD), the prevalence of regression - a loss of previously established skills - is approximately 32%. The data used to arrive at this figure was to some degree cobbled together given the different ways of defining regression and sampling data. 
  • They also reported no significant relationship between the child's age and the prevalence of regression, although there was a relationship between sample age and timing of regression similar to that work discussed on a telescoping effect (see here). 
  • Autism as a diagnosis over ASD was more predictive of regression being reported. 
  • Finally, the average age at which regression occurred was reported to be 1.78 years chronological age (21.3 months by my calculation). This average age, with confidence intervals between 20-22 months, was seemingly not dependent on the type of regression.

Ekinic study:
  • A slightly(!) smaller sample of children (N=57, aged 2-17 years) diagnosed with a DSM-IV TR ASD were included for study. 
  • Various measures were used during the study including some very detailed probing of the presence of regression via interviews, hospital files and videos to classify any history of autistic regression (AR). 
  • Based on the quite strict criteria for regression, over 50% of their sample was reported to have a history of AR (56.1%). 
  • Regression was defined one of two ways: Type 1: regression following 'normal' development, or Type 2: regression as a worsening of previous 'autistic features'.
  • Parent-reported gastrointestinal issues/diseases seemed to be more commonly noted in the Type 2 regression over Type 1, bearing in mind the absolute participant numbers were low.

Westphal study:
  • For anyone really interested in Heller's syndrome (Childhood Disintegrative Disorder, CDD), a little bit of important history has been translated. In many respects, it's kinda like reading Kanner's original case studies describing autism in that real lives are charted which share common characteristics. The translation from German is also reminiscent of Uta Frith translating Hans Asperger.
  • The authors suggest that Heller in this 1908 work actually seemed to be describing the core 'triad' of impairments that make up the diagnostic criteria for autism without using the word autism. It makes you think 'what if...'.
  • The prodromal state - early signs and symptoms similar to what prediabetes is to diabetes - of Heller's syndrome is very interesting. Characterised by various 'affective dysregulation', Heller described things like motor restlessness, night terrors and states of extreme agitation as being apparent. Combined with reports of things like tic-like movements, stereotypic behaviours and loss of speech, I'm sure one can see overlap with quite a few behaviourally defined conditions and perhaps even some clues as to the potential connection with things like a pathogenic insult (PANDAS?).  

Cumulatively there is quite a bit of information to take from these papers. First and foremost is the answer to the question, can regression occur in autism... yes. Indeed one wonders whether the recent interest in diagnostic stability and models predicting longer-term outcome might also be affected by instances of regression. 

Next question and a little more of a fuzzy answer: might regression also 'correlate' with other signs and symptoms 'around' the diagnosis of autism? Possibly is the answer based on the Ekinci data bearing in mind we don't actually know what comes first: regression or something like gastrointestinal issues and whether one causes the other or not. That being said, regression might not be totally predictive of other comorbidities like epilepsy as per the paper by Eriksson and colleagues****.

Final question, and an even more fuzzy answer: based on what is known about Heller's syndrome and his original description apparently quite close to what we know as autism, is it possible that somewhere not quite Heller's syndrome but more readily seen in autism, there might be shared clues to the regression that is noted in terms of onset and variables potentially involved? Mmm, I'll leave you to ponder that final question.

To close, a classic Breakfast Club tune about um, forgetting or not...

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* Barger BD. et al. Prevalence and onset of regression within autism spectrum disorders: a meta-analytic review. JADD. August 2012.

** Ekinci O. et al. The phenomenology of autistic regression: subtypes and associated factors. Eur Child Adolesc Psychiatry. 2012; 21: 23-29.

*** Westphal A. et al. Revisiting regression in autism: Heller's Dementia Infantilis. JADD. June 2012.

**** Eriksson MA. et al. Medical conditions affect the outcome of early intervention in preschool children with autism spectrum disorders. Eur Child Adolesc Psychiatry. July 2012.

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ResearchBlogging.org Barger BD, Campbell JM, & McDonough JD (2012). Prevalence and Onset of Regression within Autism Spectrum Disorders: A Meta-analytic Review. Journal of autism and developmental disorders PMID: 22855372

Sunday 18 November 2012

Human Endogenous Retroviruses (HERVs) and autism

Please don't be put off by the title of this post or the jargon it contains. I merely offer some discussion on a paper recently published at Emanuela Balestrieri and colleagues* (open-access) on the expression of Human Endogenous Retroviruses (HERVs) in cases of autism.

The Martians are a comin' @ Wikipedia  
HERVs are probably best described as the genetic remnants of viruses stored in the human genome. Consider this: over the course of the evolution of humankind, we have been in contact with quite a few pathogens. This includes bacteria, parasites and viruses. Over the generations, our exposure to these pathogens leaves a mark, little genetic pieces of things like viruses are left in our genome and are passed on down the generations (germline), seemingly with no useful function (seemingly!). In effect we are all part virus.

I think about the words of Morgan (More Than) Freeman at the end of the film War of the Worlds - the Tom Cruise version - and his description of humans earning their right to exist as a consequence of our evolutionary battle with such pathogens. We adapt, we earn protection (immunity) from such pathogens as the generations pass by, and along the way we also carry and transmit a little piece of them with us. And all that recent chatter about junk DNA probably not just being junk? Well, in among that junk are likely to be HERVs (about 8% of the genome apparently).

So then a few choice details from the Balestrieri paper despite it being open-access:

  • Blood samples drawn from 28 children (aged 3-9 years) were analysed and compared against 28 gender- and age-matched asymptomatic controls.
  • Examining peripheral blood mononuclear cells (PBMCs) immediately and after 72 hours of stimulation in culture, the appearance of retroviral mRNAs derived from 4 HERV families (E, H, K, W) were analysed by qualitative RT-PCR. Real time quantitative PCR was also used to look at the expression of the env sequence for HERV-H and HERV-W (there's a clue there as to what they actually found).
  • Results: samples from their cohort diagnosed with autism - ASD - showed an increased frequency of HERV-H and HERV-W positive samples under both fresh and stimulated conditions. That being said, the significance of the results were not entirely impressive.
  • Expression levels of the env sequence of the 2 HERVs (H & W) suggested some interesting results. HERV-H was more prominently over-expressed in ASD samples for both time periods but significantly so for the fresh samples. HERV-W expression by contrast was significantly lower in the ASD groups compared with controls across both time periods.

The meaning? Well aside from suggesting that HERV-H might, just might, serve as a candidate biomarker for autism (accepting small details like the small current sample size, comorbidity influence and the autisms not necessarily lending themselves to generic biomarkers), I have to say that I'm equally interested in what HERV over- and under-expression might mean to genetics and biology.

I note for example that the authors suggest that "HERVs might actually be considered as emerging pathogens and can be seen as spanning the bridge between genetic predisposition and environmental factors" on the basis that HERVs are part and parcel of human mobile retrotransposon families, the so-called jumping genes. The story goes that HERVs have the capability to alter the structure and functioning of other genes depending on where they land in the genome. Most of the time however HERVs are kept in check by epigenetic means, as per the relationship between methylation and genomic stability. In cases where hypomethylation might be present the mantra: hypomethylation = more genomic instability or less is more (see the paper from Li and colleagues** open-access) seems to apply and HERVs might get more of chance to start expressing. This blog post discussing ERVs in relation to some other research on alcoholism neatly sums up the story bearing in mind its all been boiled down to its very basic elements.

I can't profess to know all the details about HERVs and their potential effects but have easily found related research on, for example, a possible relationship between HERV-W and schizophrenia as per this paper by LeBoyer and colleagues*** bearing in mind the possible implications of certain medication**** (open-access). Indeed the Diem study**** linked to above suggesting that certain medication/s might impact on HERV expression builds upon (a) the growing interest in how certain pharmacotherapies might possess epigenetic-modifying characteristics (see the press release), and (b) again, the regulation of HERV expression by epigenetic means. I'm also minded to included some information about the relationship between HERVs and autoimmune conditions***** if one takes on board some connection between autoimmunity and cases of autism (see this previous post).

It all makes for a really quite interesting hypothesis: that we should ideally have a balanced epigenetic homoeostatic mechanism in place which suppresses those ancient remnants of viruses present in our genome, but when things go awry with epigenetics, we might start to see those viral genes expressing mRNA with potential onward effects. Another far cry from the genes are set model of not so long ago and indeed illustrative of how autism is a very, very, very complicated condition, variably influenced by both genes and environment.

Watch this space on this one.

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* Balestrieri E. et al. HERVs expression in autism spectrum disorders. PLoS ONE. 2012; 7: e48831.

** Li J. et al. Genomic hypomethylation in the human germline associates with selective structural mutability in the human genome. PLoS Genetics. 2012: 8: e1002692.

*** LeBoyer M. et al. Human endogenous retrovirus type W (HERV-W) in schizophrenia: A new avenue of research at the gene-environment interface. World J Biol Psychiatry. September 2011.

**** Diem O. et al. Influence of antipsychotic drugs on human endogenous retrovirus (HERV) transcription in brain cells. PLoS One. 2012; 7: e30054.

***** Brodziak A. et al. The role of human endogenous retroviruses in the pathogenesis of autoimmune diseases. Med Sci Monit. 2012; 18: RA80-R88.

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ResearchBlogging.org Balestrieri E, Arpino C, Matteucci C, Sorrentino R, Pica F, Alessandrelli R, Coniglio A, Curatolo P, Rezza G, Macciardi F, Garaci E, Gaudi S, & Sinibaldi-Vallebona P (2012). HERVs Expression in Autism Spectrum Disorders. PloS one, 7 (11) PMID: 23155411

Friday 16 November 2012

Putting Money on autoimmunity and autism

Go to Jail, go directly to jail... @ Wikipedia  
I owe a debt of gratitude to Natasa who brought me to the very short and very pay-walled communication by McDougle & Carlezon* discussing neuroinflammation and autism. Whilst the open-accessing of research is seemingly discussed in bulk these days, we are not quite there yet in making science free and accessible to all hence I can't refer you to the full-text paper at this time unfortunately.

No mind, the McDougle communication is brief but discusses some interesting themes, some of which have appeared before on this blog including:

I'm not 100% sure if it is the same person or not but the psychologists out there might recognise the name John Money in relation to the very sad case of the late David Reimer. A quick scan through the scientific literature based on the name Money and other authors listed on the the 1971 paper leads me to believe it is the same person who undertook work on gender identity and who championed the chemical castration of certain types of offenders. I offer no opinion on these ideas by the way.

I've previously done a review of some older research before on this blog and in particular the wonderfully descriptive article by Mary Goodwin and colleagues (see this post) on the very earliest days of the gut-brain axis in cases of autism. Readers should consider this post of a similar ilk and indeed published in the same year and by the same journal.

Money and colleagues present a family study with a few interesting points noted:

  • The particular family was one seen at The Johns Hopkins Hospital in Baltimore, USA.
  • It presents on a case of autism presenting in a boy called Tommy also diagnosed with Addison's disease and moniliasis - fungal infection related to Candida to you and me.
  • Tommy had several brothers (x3), 2 of whom also showed various signs and symptoms of autoimmune diseases including those found with Tommy. 
  • The early presentation of Tommy has some interesting features. He was overdue, presented with jaundice, had poor weight gain, issues with bone age and quite a few issues related to his diagnosis of Addison's disease. Tonsilitis with convulsions and coma with seizures "attributed to hypogylcaemia" were all in his clinical notes. 
  • His psychological development is equally interesting, going from a child who reportedly smiled at 3 weeks (remember he was overdue) and cooed at 5 weeks to a child with "an air of preoccupation and a secret smile".
  • Tommy's siblings were not diagnosed with autism but similarly presented with some notable traits. One of the siblings, Peter, was mis-diagnosed with coeliac (celiac) disease though later diagnosed with malabsoprtion in addition to hypomagnesia. He also presented with rapid hair loss aged 16 years. His psychological report mentions quite a lot of attention-seeking behaviour as well as him being prone to depressive episodes and ideations about suicide. 
  • Another sibling, Theodore, was described as presenting with hypocalcemia and eventually diagnosed with diabetes. Behaviour was described as 'autistic' at some points in his development, as well as being reported as "hyperactive, disruptive, short in attention span and imitative". 
  • All this was set against a family history of ulcerative colitis (UC) (maternal).

I know some people will probably read through all this and perhaps think its all just coincidence. One family history which, in the grand scheme of things, means very little to the millions of cases of autism past, present and future. Indeed also with only one of the brothers formally diagnosed with autism. I have to say however that accepting the heterogeneity present in autism(s), I'm not one of those people. Simply because quite a few elements of the cases described by Money et al have been mentioned in subsequent research literature with autism in mind pointing to some possible connections.

Don't believe me? Well have a look at papers like this one from Mouridsen and colleagues on maternal UC linked to autism*** and this one from Paul Ashwood and Judy Van de Water**** on autoimmunity appearing alongside cases of autism. Indeed Money and colleagues were also seemingly not convinced that these patterns of presentation in the family was merely a random act either, accepting that science is all about probability rather than absolutes. 

I was also drawn to some discussion made in the paper which referenced the work of the late Curt Dohan, he of the gluten-schizophrenia hypothesis. It appears that Money and colleagues might have had some personal communication with Dohan about their results and indeed how Dohan had "reported finding autoimmune antibody formation to brain and gut tissues in patients with schizophrenic-like symptoms and celiac disease". I don't want to make mountains out of molehills but one does wonder whether there might be a link with cases of autism as per the more recent tissue transglutaminase antibodies findings and perhaps even the folate receptor autoantibodies research. Although by no means am I suggesting autism is schizophrenia or vice-versa, we do know that the two conditions have been known to overlap and there's no reason to suspect that biology, genetics and even epigenetics might not also.

I'm going to finish with another quote from the Money paper: "it is quite possible that the patient's progressive withdrawal into autism, after a perhaps auspicious beginning, represents a developmental deterioration dependent on internal autoimmunizing factors rather than psychosocial factors". Sentiments that at least McDougle & Carlezon seem to be taking quite seriously.

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* McDougle CJ. & Carlezon WA. Neuroinflammation and autism: toward mechanisms and treatments. Neuropsychopharmacology. 2013; 38: 241-242.

** Money J. Autism and autoimmune disease: a family study. J Autism Child Schizophr. 1971; 1: 146-160.

*** Mouridesen SE. et al. Autoimmune diseases in parents of children with infantile autism: a case-control study. Dev Med Child Neurol. 2007; 49: 429-432.

**** Ashood P. & Van de Water J. Is autism an autoimmune disease? Autoimmun Rev. 2004; 3: 557-562.

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ResearchBlogging.org Money J, Bobrow NA, & Clarke FC (1971). Autism and autoimmune disease: a family study. Journal of autism and childhood schizophrenia, 1 (2), 146-60 PMID: 5172389