Friday 27 July 2012

Fetal alcohol syndrome

Pregnancy is a special time for both prospective mum and dad. OK, dad's role is a little more 'peripheral' at this stage; more about support and understanding, doing that little more around the house - 'its called a washing machine' - and generally just being there when, for example, the words 'could you nip out and get me some pickled onions darling' are said late at night.

Pregnancy is also an important time for both foetal (English-spelling) and maternal health; as information comes thick and fast about health, diet, nutrition and exercise. So, lots of things to do (exercise, folic acid(!), etc) and lots of things to avoid (smoking, drinking alcohol, eating raw shellfish, old paintworketc). Whilst there is some good evidence behind these dos and don'ts, in recent times some of the suggestions have been the source of some debate. 

Take for example the advice about drinking alcohol during pregnancy. Down the years, there's been an almost operatic battle about what is and isn't a safe level of alcohol consumption during pregnancy. It seems some days expectant mums are advised no alcohol at all during pregnancy, other days reports come out saying a little moderate consumption is probably going to be alright. I can perhaps see how it all could be quite confusing (as per other health advice - see here). 

More confusion has arrived at the time of writing, as several studies have recently emerged looking at alcohol consumption during pregnancy and future offspring development (see here) followed by quite a few headlines such as this one from the BBC "Moderate drinking in early pregnancy branded 'safe'". I might add that I am not endorsing this or any other stance but would also direct readers to this post by the NHS Choices website for some balance regarding the publications in question.

I have to say that I always get a little bit nervous when something is 'branded safe'; thinking back to a certain British Politician and a daughter's hamburger a few years back. Indeed, these latest studies got me thinking about alcohol and pregnancy and how, if it is so safe, do we arrive at something called fetal alcohol syndrome (FAS). I want to talk about FAS with autism in mind but tread very, very carefully here so as not to make connections where none exist.

A short description first. FAS is quite a nebulous term characterised by quite a lot of different symptoms covering everything from cognitive and learning issues to specific facial features to other overlapping features more readily covered under the more general term fetal alcohol spectrum disorder (FASD). Some quite recent research (here) has reiterated the cognitive side of things as being central to the effects of alcohol exposure.

With autism - autistic behaviours - in mind, the research path goes something like this:

  • Various cases of the presence of FAS and autism are dotted around the research landscape. So, this paper by Nanson* seems to be one the earliest, describing six cases of children with FAS who also "fulfill the criteria for diagnosis of autism". Followed in succession by this paper by Harris and colleagues** and a few others.
  • I was drawn to this short reply by Eric Fombonne*** (click 'show summary') on the question of an association between FAS and autism in light of the previous findings. Drawn to it because some good issues are raised here, not least that autism is not universal to all children with FAS (2% of cases is the suggestion) and also that the other features normally accompanying FAS are not normally associated with autism. Indeed, there is a strong argument in this piece that the coincidence of autism in FAS is nothing more than just a chance finding as per the recent autism prevalence figures.
  • Further analysis of the presentation of behavioural features in cases of autism vs. FASD has also revealed some interesting issues. Bishop and colleagues**** described a comparative study based on ADOS and ADI measures in both groups. They suggested that whilst social issues like appropriate behaviour and peer relationships were common between the groups, the core 'social affect' behaviours (as it will become) seem to be firmly rooted in autism not FASD. The lack of other control groups (speech and language delay, learning disability, etc) is a bit of a short-coming to this study but the results still hold up demonstrating differences between autism and FASD.

There are other papers mentioning autism and FAS / FASD but I have to say the suggestion of a link between these conditions is rather unimpressive based on the current data. That's not to say that certain behaviours linked to autism might not also be present in FAS / FASD, but that autism as a diagnostic package is likely not to show strong associations with these conditions.


* Nanson JL. Autism in fetal alcohol syndrome: a report of six cases. Alcoholism, Clinical & Experimental Research. 1992; 16: 558-565.

** Harris SR. et al. Autistic behaviors in offspring of mothers abusing alcohol and other drugs: a series of case reports.. Alcoholism, Clinical & Experimental Research. 1995; 19: 660-665.

*** Fombonne E. Ask the Editor: Is exposure to alcohol during pregnancy a risk factor for autism? JADD. 2002; 32: 243.

**** Bishop S. et al. Re-examining the core features of autism: a comparison of autism spectrum disorder and fetal alcohol spectrum disorder. Journal of Child Psychology & Psychiatry & Allied Disciplines. 2007; 48: 1111-1121.

Sunday 22 July 2012

GABA Dabba Doo

I'm probably setting myself up for a fall with this quite descriptive post on GABA - gamma aminobutyric acid in relation to autism spectrum conditions given my amateur status in the knowledge stakes regarding the various neurotransmitters. I am however going to chance my luck because more and more frequently GABA seems to be cropping up in the peer-reviewed literature on autism.

Indeed I don't know whether it is just me, but other neurotransmitters such as serotonin (5-HT) seem to be experiencing a slight drop in popularity in autism research circles these days, whilst transmitters like GABA and glutamate, are in the ascendancy. As per every post on this blog (a) no medical advice is intended or given, and (b) always treat my observations with a healthy dose of scepticism.

What is GABA?

Well, I've mentioned glutamate already and indeed glutamate is the core material for making GABA alongside the active form of vitamin B6, pyridoxal phosphate (PLP or P5P) used as a cofactor. The enzyme glutamate decarboxylase (GAD) is essential for this transformation from excitatory neurotransmitter (glutamate) to inhibtory neurotransmitter (GABA). Brain synthesis is an important part of GABA given that it finds some difficulty crossing the blood-brain barrier. Keep in mind both PLP and GAD for later on in this post.

What does it do?

Well, it's a neurotransmitter with corresponding GABA receptors dotted around the central nervous system including in the region of the 'second brain' that is the gastrointestinal tract (here) similar to other neurotransmitters (here). GABA is also described as an inhibitory neurotransmitter (here) similar to glycine; a sort of yin and yang to the excitatory neurotransmitters which modulate the firing of neurons; GABA (generally) decreasing their likelihood of firing.

Over-zealous neuronal firing caused by an 'imbalance' between excitatory and inhibitory neurotransmitters is thought to be involved in conditions like epilepsy (here) and the reason why certain compounds that increase GABA levels in the brain also possess anticonvulsant properties (here). There is also some speculation linking the use of a ketogenic diet and increases in brain GABA synthesis to account for the anticonvulsant observations noted (here) for example. Should I mention that GABA might also possess some anti-inflammatory action (here) too?

So a quick recap: glutamate, GAD and PLP in the brain (and other places) make GABA, an inhibitory neurotransmitter which dampens down neuronal firing and potentially the reason why some antiepileptics (and other compounds) which enhance GABA production/action work the way they do.

Let the cherry-picking begin: GABA and autism.

Where to start...mmm. GABA has been looked at from several perspectives in cases of autism falling into some general categories to include: (a) issues with the receptors for GABA in the brain, and (b) issues with GAD involved with the synthesis of GABA from glutamate. There are other areas (such as autoantibodies to GABAergic neurons)  but the lion's share of research seems to fall into one of these two categories.

(a) GABA receptors are basically the bedfellows of GABA. Several types of GABA receptor are currently known about and have been examined with autism in mind.

  • Quite a bit of research has been done in this area so I am indeed cherry-picking. The comorbidity of autism and epilepsy is obviously a point of real interest given the function of GABA and its receptors as described in this paper by Kang & Barnes*.
  • Various authors have reported on issues with GABA receptors in cases of autism; in the majority suggesting reduced receptor numbers in various parts of the brain (see here, here and here).
  • The genetics of GABA receptors have also been the topic of some research although not wholly convincing in providing an effect (see here and here). I don't want to seem unduly biased against any genetic link to GABA receptors and autism so will balance that last sentence by noting the evidence on for example, gene-gene interactions and GABA receptor genes in autism (see this paper by Ma and colleagues** full-text).

(b) GAD has been mentioned in passing previously on this blog in relation to the imbalance seemingly present between glutamate/glutamine in cases of autism. I can see why this is such an attractive area of study with autism in mind, covering not only the possibility of issues with the formation of GABA but also potentially (and partially) accounting for the 'build up' of glutamate thereby unsettling that delicate excitatory/inhibitory balance. That's my take on it anyway. The body of research published so far suggests:

  • GAD production (the isoforms GAD65 and GAD67) might not be optimal in cases of autism. Yip and colleagues*** noted reduced mRNA levels of GAD67 in Purkinje cells of a group with autism (Purkinje cells being a target for previous autism research based on the suggested involvement of cerebellar function). The same group also reported reduced mRNA levels of GAD65**** (here full-text).
  • Overall, genomic studies which have included the examination of genes thought to be involved in the production of GAD have not yielded any wider significant issues in cases of autism as per the studies by Rabionet and colleagues***** and Buttenschøn and colleagues******.
  • Environment has however been proposed as a possible interfering variable affecting GAD production and function as per the suggestion by Nouel and colleagues******* on the prenatal effects of a bacterial endotoxin on GAD67 (in rats). That and the possibility of autoantibodies to GAD65 being found in a small sub-group of children with autism and ADHD as reported by Rout and colleagues******** (similar antibody findings also being reported in the very interestingly named 'Stiff-Person syndrome', a condition characterised by amongst other things heightened sensitivity to stimuli).

I think you can see that for some people on the autism spectrum, GABA, it's receptors and findings around its manufacture - seems to have a few potential issues attached. Stepping back to the PLP / vitamin B6 link with GABA, there is also some interesting evidence emerging there also with autism in mind. Indeed another familiar name, Jim Adams (he of the vitamin RCT among other things) published a paper********* a few years back indicating high levels of plasma vitamin B6 to be present in cases of autism. You're probably thinking to yourself, well high levels of vitamin B6 is good? Er, not exactly, because high levels of vitamin B6 in unsupplemented individuals as this group were, might actually suggest that vitamin B6 is not being optimally metabolised by pyridoxal kinase into PLP for example. I could start to talk about pyridoxal kinase and ATP complexed to zinc (see here) but then we start asking questions about zinc availability and the whole thing gets really, really complicated.

I think I'll stop there for now with the hope that I have done more informing rather than misinforming. To end, a link to something completely different... The Killers and Mr Brightside.

GABA, Dabba, Doo!


* Kang JQ. & Barnes G. A Common Susceptibility Factor of Both Autism and Epilepsy: Functional Deficiency of GABA(A) Receptors. JADD. May 2012.

** Ma DQ. et al. Identification of significant association and gene-gene interaction of GABA receptor subunit genes in autism. American Journal of Human Genetics. 2005; 77: 377-388.

*** Yip J. et al. Decreased GAD67 mRNA levels in cerebellar Purkinje cells in autism: pathophysiological implications. Acta Neuropathologica. 2007; 113: 559-568.

**** Yip J. et al. Decreased GAD65 mRNA levels in select subpopulations of neurons in the cerebellar dentate nuclei in autism: an in situ hybridization study. Autism Research. 2009; 2: 50-59.

***** Rabionet R. et al. Analysis of the autism chromosome 2 linkage region: GAD1 and other candidate genes. Neuroscience Letters. 2004; 372: 209-214.

****** Buttenschøn HN. et al. A population-based association study of glutamate decarboxylase 1 as a candidate gene for autism. The Journal of Neurotransmission. 2009; 116: 381-388.

******* Nouel D. et al. Prenatal exposure to bacterial endotoxin reduces the number of GAD67- and reelin-immunoreactive neurons in the hippocampus of rat offspring. European Neuropsychopharmacology. 2012; 22: 300-307.

******** Rout UK. et al. Presence of GAD65 autoantibodies in the serum of children with autism or ADHD. European Child & Adolescent Psychiatry. 2012; 21: 141-147.

********* Adams JB. et al. Abnormally high plasma levels of vitamin B6 in children with autism not taking supplements compared to controls not taking supplements. Journal of Alternative & Complementary Medicine. 2006; 12: 59-63.

Thursday 19 July 2012

Mouse modeling, immune function and autism

Contrary to the title of this post and any images that it may conjure up of mice parading down a runway in this season's 'hottest looks' whilst pouting to the clicks and flashes of multiple cameras, I'm back to mouse models and autism again(!) and an interesting piece of research by Hsiao and colleagues*.

I am kinda standing on the shoulders of giants with this paper given that it comes from the laboratory of Paul Patterson who has already run with a short description about it on his blog (see here). Whilst not pinning my colours to any mast, Prof. Patterson's blog is one I enjoy reading, not least because of the various links being made between the immune system and the brain (at least in mice). I assume most people would recognise by now that the brain does not run independent of the rest of the body despite our implicit need to compartmentalise anything and everything (see this post on labels).

Drawing heavily on Prof. Patterson's latest blog entry and the paper in question - hopefully without plagiarising - a few factoids:

  • The Patterson team have previously published results based on a mouse model of stimulated immune activation during pregnancy and the resultant behavioural effects on offspring which seemed to overlap with the core symptoms of autism (see this paper by Malkova and colleagues**).
  • In the latest study* the authors report on the profile of immune function in offspring mice of immune-stimulated mothers, suggesting some interesting effects in the area of T regulatory cells and cytokine production. I have recently talked about T-cells in this post on pristine cysteine so will perhaps put that to one side for now. Of just as much interest are elevations in that old favourite IL-6 and similar suggestions for IL-17, again the source of some interest recently on the topic of autoimmunity and autism (here). Roads toward inflammation seemed to be a key part of their findings.
  • Coincidental to these findings is the report of "altered myeloid lineage potential and differentiation" in offspring. I'm not even going to profess to begin to know what this actually means, aside from referring you to quite a nice overview of hematopoietic stem cells (here) showing the distinction between myeloid and lymphoid progenitors. 
  • Then to the big findings and please don't shoot the messenger: irradiating and transplanting "immunologically normal" bone marrow from both affected and non-affected control mice offspring into the offspring of immune stimulated mother mice seemed to correct some of the autism-type behaviours that were exhibited. So repetitive- and anxiety-like behaviours seemed to be reduced bearing in mind that anxiety is not (yet) a core symptom of autism (see here).
  • That and some suggestion that timing might be everything when it comes to programming for immune dysfunction as a result of very few effects being seen when transplanting bone marrow from affected offspring to non-affected offspring over being born into a stimulated maternal immune system environment.

I note that on quite a few sites analysing these latest results, the authors have gone to great lengths to stress that (a) these were mouse findings - I'll say again, these were mouse findings, and (b) at the moment, no-one is suggesting that a bone marrow / stem cell transplant is any kind of 'treatment' for autism given questions for example, about whether the 'irradiation' bit of the procedure might have shown any effect alongside the actual bone marrow transplant. I would most definitely support these statements given both the preliminary nature of this research and also the complications and risks attached to bone marrow transplants (see here).

Having said that this is not the first time that bone marrow transplants and conditions like autism have appeared in the research literature. This paper by Akaho and colleagues*** talks about such transplants in cases of autism (and schizophrenia) occurring alongside leukaemia with a specific focus on maintaining treatment regimes and the anxiety related to the treatment process. Sharma and colleagues**** discussed some rather more direct observations following administration of "autologous bone marrow-derived mononuclear cells" in their quite varied patient group including cases of autism, bearing in mind one tree does not a forest make.

Indeed the concept of stem cell therapy, words which still seem to create quite an emotional response in many people, seems to be occurring more and more often in the research literature on autism as per this review by Siniscalco and colleagues***** (full-text) including a familiar name (Anna Sapone). I know many people might read 'stem cells and autism' and think back to those pop-up ads that seem to appear on various search engines offering some kind of James Bond style 'Die Another Day' rearrangement. Again, no endorsement is intended or given but perhaps what the Patterson lab study is suggesting is that a little more focused research is required in this area just before the door is entirely slammed shut. 

The Hsiao findings do represent another very important preliminary step into the immune-behaviour relationship with conditions like autism in mind. Assuming that the whole is greater than the sum of its parts, these moves towards a more whole body analysis of conditions like autism, where immune function, gut and gut bacterial function and brain function are examined in unison, offer the promise of some truly tantalising insights into autism.

And finally... just in case you are not convinced on the potential for an immune-behaviour link, cast your eye over this recent preliminary report on Alzheimer's disease and the use of IVIg as another area ripe for further inquiry.


* Hsaio EY. et alModeling an autism risk factor in mice leads to permanent immune dysregulation. PNAS. July 2012.
DOI: 10.1073/pnas.1202556109

** Malkova NV. et al. Maternal immune activation yields offspring displaying mouse versions of the three core symptoms of autism. Brain, Behavior & Immunity. 2012; 26: 607-616.

*** Akaho R. et al. Bone marrow transplantation in subjects with mental disorders. Psychiatry & Clinical Neurosciences. 2003; 57: 311-315.

**** Sharma A. et al. Administration of autologous bone marrow-derived mononuclear cells in children with incurable neurological disorders and injury is safe and improves their quality of life. Cell Transplantation. 2012; 21: Suppl 1: S79-S90.

***** Siniscalco D. et al. Autism spectrum disorders: is mesenchymal stem cell personalized therapy the future? Journal of Biomedicine & Biotechnology. 2012; 480289.

--------- Hsiao EY, McBride SW, Chow J, Mazmanian SK, & Patterson PH (2012). Modeling an autism risk factor in mice leads to permanent immune dysregulation. Proceedings of the National Academy of Sciences of the United States of America PMID: 22802640

Wednesday 18 July 2012

Anxious children and autism traits?

I'm always been quite intrigued by studies examining the various behaviours linked to autism presenting in other conditions not necessarily linked to autism as a discrete entity. The various studies done on eating disorders* spring to mind as quite a good example.

This type of research - when it shows some positive correlation - kinda reiterates that when we talk about autism, we aren't so much talking about a single homogeneous condition, but perhaps rather a range of conditions which share some common behavioural characteristics summed up by the diagnostic label autism. I suppose its a bit like calling fizzy drinks (soda) and summer fruits 'sweet' but also realising that soda and fruit are quite different things outside of their sweet taste (if that makes any sense).

With this in mind, the publication of an interesting paper by Francisca van Steensel and colleagues** (full-text) on the presence of autistic traits in children with various anxiety disorders is quite an interesting read. The paper is open-access and suggests that whilst not necessarily fulfilling all the criteria for autism or the autism spectrum, children diagnosed with various anxiety-related conditions do seem to present more frequently with autism traits as described by their previous developmental history and current behaviour. There is another paper from the same authors*** which might also be relevant but I'm going to leave that for this post.

A short summary:

  • Parental interviews made on behalf of 42 children (mean age 12 years) diagnosed with one or more anxiety disorder were compared with interviews for 42 asymptomatic control children (mean age 11 years) based on the Autism Diagnostic Interview-Revised (ADI-R) and Children’s Social Behavioral Questionnaire (CSBQ) probing offspring early and current autism traits respectively.
  • Results: children with anxiety related disorders were reported to show/have shown an increased number of autism related behaviours compared with controls. 
  • Based on the ADI-R algorithm scores for the triad of core autism domains, over a third of children with an anxiety disorder surpassed one or more cutoff points for the presentation of autism-like behaviours during their developmental history. This was compared with none of the control group. Indeed, one child in the anxiety group surpassed thresholds on all 3 domains. Statistically speaking, the anxiety group scored significantly higher across all autism domains than the control group.
  • Based on CSBQ scores, the anxiety group also presented with a greater number of current autism-like behaviours than the control group. Again, nearly a third of children with anxiety related conditions "had scores that fell in the ASD range".
  • Interestingly, when looking at current anxiety symptoms based on the Screen for Child Anxiety Related Emotional Disorders (SCARED-71), several types of anxiety disorder correlated with scores of particularly current autism-like traits including panic disorder, generalised anxiety disorder, social anxiety disorder and separation anxiety disorder (all significant at p equal or less than 0.01).

I don't want to make too much out of this current paper bearing in mind the small participant group included. That being said however, this is not the first time that anxiety disorders have been looked at with autism traits in mind (see here).

Aside from important considerations such as (a) screening for autism in children with anxiety disorder brought about by this kind of research, assuming good validity of the instruments used, and not 'over-screening' and (b) the possibility of shared biochemistry, genes, etc., I started to wonder about what these results might mean for people with an autism spectrum condition. So for example, do the results work in reverse, and those with autism are perhaps more prone to an anxiety disorder? Certainly I don't think I can stress enough the effects that anxiety seems to have on many people with autism. In some cases dare I suggest that it is one of the most debilitating aspects to autism. The question is whether such issues merit a separate diagnosis of anxiety disorder and whether for example, managing the anxiety might have knock-on effects to other more core presentation?

On a final note, I couldn't help but raise a smile at the results presented by Pobbe and colleagues**** (full-text) on anxiety measurement in the Dangermouse that is the BTBR mouse model of autism. Not specifically relevant to the latest work but interesting insofar as the potential usefulness of the BTBR model.

Now, clear some space, break out the air guitar and let Bryan tell you all about his summer of '69.


* Coombs E. et al. An investigation into the relationship between eating disorder psychopathology and autistic symptomatology in a non-clinical sample. The British Journal of Clinical Psychology. 2011; 50: 326-338.

** van Steensel. et alAutism spectrum traits in children with anxiety disorders. JADD. June 2012.
DOI: 10.1007/s10803-012-1575-z

*** van Steensel FJ. et al.  Anxiety and quality of life: clinically anxious children with and without autism spectrum disorders compared. Journal of Clinical Child & Adolescent Psychology. July 2012.

**** Pobbe RL. et al. General and social anxiety in the BTBR T+ tf/J mouse strain. Behavioral Brain Research. 2011; 216: 446-451.

---------- van Steensel FJ, Bögels SM, & Wood JJ (2012). Autism Spectrum Traits in Children with Anxiety Disorders. Journal of autism and developmental disorders PMID: 22733297

Tuesday 17 July 2012

Pristine cysteine-matically done

First of all, sorry for the terrible pun that makes up the post title. What can I say apart from (a) what else rhymes with the amino acid cysteine? and (b) I'd probably make a terrible tabloid newspaper headline maker unlikely to come up with something like this British classic.

In this post I want to focus on a paper by Mostafa Waly and colleagues* (full-text) which includes a couple of notable names on the authorship list including Dick Deth (macroepigenetics and high-fructose corn syrup) and Mady Hornig (carbohydrate digestion and the bacteria which just rolls of the tongue, Sutterella in relation to autism).

The name of the paper's game is cysteine uptake in autism, and how issues with this process may have some interesting connections to "inadequate antioxidant capacity" and onwards affecting prenatal- and postnatal epigenetic programming. I have to admit that this paper does jump around quite a bit in terms of what might impact on what so don't be surprised if I start bringing quite disparate areas into this post. I'll also say now that ultimately this is a paper of mouse models and how an old friend, autoimmuunity, might play some role in cysteine uptake. I'll stress the 'might play some role' before I progress any further.

A few descriptions first:

The amino acid cysteine has cropped up previously on this blog. Not only linked to those very important observations on sulphate (sulfate) levels in various biofluids in cases of autism but also with regards to the growing interest in glutathione (GSH) and autism as a result of cysteine being one of the building blocks of GSH and the various brain revelations published not so long ago.

Epigenetics... well, you could have a look at this post from a few months back introducing epigenetics in relation to autism. The mantra: your genome might not necessarily be your destiny just about covers the science of epigenetics and potentially how epigenetics might resolve some of the issues in the grudge match that is genes vs. environment. I've posted about this elsewhere quite recently (here).

Anyway back to the Waly paper. It is open-access but here are a few of the highlights:

  • Unless I am missing something, it is not immediately clear whether this is a summary paper, an experimental-type paper or some combination of the two. After a few reads, I favour the latter option because aside from introducing the important processes involved in cysteine metabolism and epigenetics, there does appear to be some practical experimentation on various types of cell and tissue derived from animal models; in particular the C57BL/6  and SJL/J mouse models. Unfortunately no room for the BTBR Dangermouse model of autism.
  • Indeed the practical experiment side of things seemed to involve a few things including: (a) extracting things like regulatory (CD4+ CD25+) T-cells from the mouse models to ascertain the expression of EAAT3, a mediator of cysteine uptake in various body sites (see here) (b) analysis of the level of GSH in the frontal cortex of said mouse models treated with or without the mercury-based preservative thiomersal (or thimerosal) which has been the focus of quite a lot of discussion over the years, and (c) analysis of the activity of methionine synthase, the enzyme that converts homocysteine to methionine, again in the cortex of thiomersal treated and untreated mice.
  • A few of the results, but don't quote me on this: GSH levels in the frontal cortex of the SJL/J mice were lower than the C57BL/6 mice. This might make a little more sense if I point you towards some evidence that the SJL/J mouse has been described as quite a good model of autoimmunity, or at least slightly better than the C57BL/6 model.
  • Similarly, levels of methionine synthase activity were described as lower in the SJL/J mice.
  • It appears that thiomersal treatment had very little effect on GSH or methionine synthase activity results.
  • The EAAT3 results, remembering that EAAT3 transports cysteine, cysteine from dietary sources, into cells partly for GSH synthesis. "Expression of EAAT3 was significantly lower in CD4+ T-cells from SJL/J mice versus C57BL6/J mice, suggesting that autoimmunity is associated with impaired capacity for cysteine uptake."

I'll admit that I have scratched my head a few times when reading this paper. The title suggests epigenetic programming to be a core part of the presented evidence but ultimately the data seems to focus more on the speculation around the mouse model differences over any experimental data on a specific epigenetic role tied to autism.

Don't get me wrong, the GSH and methionine synthase expression findings are important and I would love to see how they might compare against the BTBR mouse model of autism bearing in mind its representativeness to autism (see this paper by Pobbe and colleagues** full-text). The additional fact that thiomersal treatment seemed to have very little effect on these parameters in both mouse models is also a potentially important finding.

That being said I almost got the impression that this paper might have been better split into two manuscripts: one on the speculated mechanisms, which provide an excellent overview it has to be said, and another on the fact that C57BL/6 mice don't tend to show as many issues with cysteine, glutathione and methionine pathways as the SJL/J mouse. I caution though that this last finding might not necessarily translate into real life autism.


* Waly M. et alPrenatal and postnatal epigenetic programming: implications for GI, immune, and neuronal function in autism. Autism Research & Treatment. 2012
DOI: 10.1155/2012/190930

** Pobbe RL. et al. Expression of social behaviors of C57BL/6J versus BTBR inbred mouse strains in the visible burrow system. Behavioral Brain Research. 2010; 214: 443-449.

Monday 16 July 2012

Mitochondrial dysfunction and ME/CFS

I continue my interest in research examining chronic fatigue syndrome / myalgic encephalomyelitis (CFS/ME) in this post looking at what might turn out to be quite an important paper by Booth and colleagues* (full-text) on a potential role for mitochondrial dysfunction.

Where to start....

Mitochondria (plural) are not to be confused with the midi-chlorians of a Galaxy far, far away. A few associated words: organelleseukaryotic cell, the power plant of cells, cellular respiration (this link carries a really easy to understand description of this process). In short, mitochondria provide energy to the cell in the form of ATP. Cells like most thing need energy to function properly; where insufficient energy is produced... well, cells don't work as well as they should and the results can be serious and wide-ranging.

I've kinda eluded to mitochondrial function, or rather dysfunction, in posts like this one on lactate levels in autism although I dare say that as some point I will come back to the topic in more detail. Indeed elevations in lactate levels do seem to indicate some potential issue with mitochondrial dysfunction as per studies like this one from Magner and colleagues**.

I digress. Booth et al have previously reported on mitochondrial dysfunction in cases of CFS/ME in this paper*** (full-text); the current study being a sort of extension and elaboration effort. I might add that they are by no means the first to suggest that there may be some mitochondrial 'involvement' in cases of CFS/ME (see here and here). Low carnitine levels? Now where have I heard that before?

Their latest paper is full-text and contains quite a bit of data but a short summary is perhaps in order:

  • The ATP profile features quite strongly in the paper. This is described as containing among other things, ATP concentration in blood neutrophils (in the presence of excess magnesium, deficiencies of which have been linked to CFS) and is a part of a so-called mitochondrial energy score (MES). In their previous paper (***) the authors reported an impressive correlation between MES and CFS Ability as described using the Bell Ability Scale, a rough and ready measure of the level of disability as a consequence of the condition (see here)
  • It looks like there were a few elements to this paper including a reanalysis of the cohort reported in their previous paper - cohort 1 (minus 10 participants who were outside of the age range of controls, again taken from their original publication) and analysis of a new participant group - cohort 2 - made up of 138 participants aged between 18-65, mean age 41 years.
  • Similar to the last paper, the MES showed a pretty good correlation with CFS Ability (correlation coefficient = 0.80). I have to admit that quite a lot of the rest of the results are beyond the limits of my biochemistry as various measures of ATP inhibition, functionality of the translocator proteins (e.g. ANT) and efficiency of oxidative phosphorylation are tested ( I think!). The end result is that the mitochondrial dysfunction noted by the authors in this patient group seems to (a) frequent, very frequent and (b) particularly involves issues with the translocator protein (TL) regulating the passage of ATP and ADP across mitochondrial membranes (see here - no endorsement intended). Other preliminary studies of oxidative phosphorylation capacity in CFS for example, suggested that this is probably not the main reason for mitochondrial dysfunction**** (full-text).
  • The authors conclude: "Taken together, these measurements show that ME/CFS is a serious illness which may affect every cell in the body".

And relax. 

I should point out that my interpretation of the Booth results should not be taken as Gospel or anything like that. I do however believe that these results are important for CFS/ME and potentially represent at least one part of the puzzle that is this debilitating set of conditions. Independent replication of the Booth results is the next step.

I was drawn to the fact that other conditions presenting with a fatigue component such as fibromyalgia have also been suggested to be linked to issues with mitochondria as per this case study by Abdullah and colleagues***** (full-text). The 'solution' to their reported case was supplementation with various compounds including coenzyme Q10, creatine and carnitine among other things. Whilst I am not in a position to endorse anything like such a treatment protocol (please do speak to your healthcare provider first), I note that supplements like coenzyme Q10 have cropped up more than once in relation to CFS/ME as per articles like this one****** from a familiar name to this area of study, Michael Maes and colleagues.

ME/CFS are a heterogeneous set of conditions which, similar to the description of autism, probably include quite a few different paths to the development of symptoms. Whilst the area of mitochondrial dysfunction is an attractive potential marker showing involvement in cases - at least some cases - the question of whether this is a 'core' aspect of the conditions still remains to be seen alongside its connection with other pathways of interest

To finish, Kate Bush sings Wuthering Heights. A lesson in the art of dance and music (and its absolutely fantastic).


* Booth NE. et alMitochondrial dysfunction and the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). International Journal of Clinical & Experimental Medicine. 2012; 5: 208-220.

** Magner M. et al. Elevated CSF-lactate is a reliable marker of mitochondrial disorders in children even after brief seizures. European Journal of Paediatric Neurology. 2011; 15: 101-108.

*** Myhill S. et al. Chronic fatigue syndrome and mitochondrial dysfunction. International Journal of Clinical & Experimental Medicine. 2009; 2: 1-16.

**** Vermeulen RC. et al. Patients with chronic fatigue syndrome performed worse than controls in a controlled repeated exercise study despite a normal oxidative phosphorylation capacity. Journal of Translational Medicine. 2010; 8: 93.

***** Abdullah M. et al. Mitochondrial myopathy presenting as fibromyalgia: a case report. Journal of Medical Case Reports. 2012; 6: 55.

****** Maes M. et al. Coenzyme Q10 deficiency in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is related to fatigue, autonomic and neurocognitive symptoms and is another risk factor explaining the early mortality in ME/CFS due to cardiovascular disorder. Neuro Endocrinology Letters. 2009; 30: 470-476.

Friday 13 July 2012

Brain glutathione redox status, Brussels sprouts and autism?

Sprouts, sprouts, sprouts @ Wikipedia
Glutathione in relation to autism spectrum conditions is a bit of a favourite topic of mine. I've talked about glutathione a few times on this blog (here and here) and how the various reports on a reduced level of functioning of this fantastic part of our antioxidant system seem to show more than a passing connection to cases of autism. That and the fact that consumption of a favourite foodstuff of mine, the Brussels sprout, might have quite a positive influence on some of the inner workings of the glutathione system (see Nijhoff and colleagues*) makes it a compound of some interest.

Up until this point, the collected research looking at glutathione in relation to autism had however tended to be focused on circulating levels of glutathione in its various forms alongside the enzymes supporting its important tasks. Enter then a paper published by Rose and colleagues* (full-text) on glutathione, the brain and autism previously described at IMFAR 2012 (here). You may have spotted a few familiar names on the authorship panel of this paper including Jill James (hypomethylation and autism) and Richard Frye (folate receptor autoantibodies).

Aside from a couple of forays into the world of brain research and the autism spectrum (see here for example), I have tended to keep away from discussing such investigations in too much detail on this blog. My reasoning: adhering to the phrase 'a cobbler should stick to his last', coupled with a view that the various research focused on the brain and autism just seemed so darned complicated. Suffice to say that it all brings back blurred memories of my undergraduate days where I admit to being more than a little confused about what brain region was supposed to do what.

In this post I am going to include some discussion on the findings reported by Rose et al albeit with the caveat that my brain may not be up to exploring all the avenues of potential interest related to these findings and what they mean.

Since I am discussing glutathione, I also want to bring to your attention some slightly more 'preliminary' findings reported in this poster by Cruikshank and Wood** on urinary glutathione in relation to autism. The caveat here being that this is not a peer-reviewed piece of research and hence still requires quite a lot more work before being taken as Gospel (despite the recent media interest).

Back to Dr Rose's paper:

  • This was a study of post-mortem brain specimens. In light of the recent news of a freezer malfunction linked to the destruction of a number of stored brain tissue samples from people with autism, there is a degree of poignancy to this study reiterating how valuable these types of tissue are to furthering autism research.
  • Samples from two areas of the brain - the cerebellum and Brodmann area 22 (BA22) (part of the superior temporal gyrus) - were studied, comparing samples from people with autism (n=15 & n=12 for the two areas) with control specimens. These brain areas have been talked about before with autism in mind (here and here) as the name Eric Courchesne drifts into my consciousness.
  • Levels of various compounds were examined in samples including: reduced glutathione (GSH), oxidised glutathione disulfide (GSSG), 3-nitrotyrosine (3-NT) and 3-chlorotyrosine (3-CT); calculating glutathione redox/antioxidant capacity (GSH/GSSG), oxidative protein damage and oxidative DNA damage (8-oxo-deoxyguanosine; 8-oxo-dG). Aconitase activity was also measured.
  • The results: in both brain regions, all studied compounds were altered at a group level in cases of autism compared to controls. So, levels of glutathione (GSH) were decreased in both brain areas compared to control samples (43% and 32% reductions in cerebellum and BA22 respectively). Levels of oxidised glutathione disulfide (GSSG) were elevated in autism vs. controls in both brain areas (18% vs. 19% elevations respectively) and overall glutathione redox/antioxidant capacity (GSH/GSSG) was significantly different in autism vs. controls. These findings are roughly in line with what has been reported in studies of other tissues in cases of autism.
  • Oxidative stress and oxidative protein damage markers were also significantly elevated in the autism group vs. controls in both brain areas. Aconitase activity was significantly lower in the autism group in the cerebellum but escaped significance in relation to BA22. 
  • The authors note: "decreased glutathione-mediated redox/antioxidant capacity previously observed in plasma and immune cells from children with autism is also significantly decreased in two brain regions previously shown to be affected in autism, the cerebellum and BA22".

There is quite a bit of information to take in from this study and as a result, several important things which will require some external replication with suitable age and sex matched controls. Oxidative stress / damage is something that tends to get banded around quite a lot in these days of the free radical. Rose and colleagues have now provided some very important preliminary flesh on the bones to this story, suggesting "functional consequences" on specific brain areas previously linked to autism following their results. Their findings also pretty much confirm what quite a few others have been saying about glutathione in relation to cases of autism: whether causative or epiphenomenal, there's something amiss with the whole oxidative stress / antioxidant balance in at least a proportion of cases of autism and it may well extend beyond just a casual relationship. 

I note also their findings with regards to glutathione redox/antioxidant capacity (GSH/GSSG) and 8-oxo-dG in the cerebellum. Combining autism cases and control data, the authors report on an important relationship between how well the GSH/GSSH capacity performs and the amount of oxidative DNA damage potentially present. Similar things have been reported in other studies so no real surprises there.

I'm not going to get too far into the findings with regards to decreased aconitase activity in the autism cases and its link to mitochondrial oxidative stress. If you really want some reading on the subject, this paper by Cantu and colleagues**** (full-text) should keep you going for a while. Suffice to say that mitochondrial aconitase inactivation might have some pretty negative implications as per this article by Vasquez-Vivar and colleagues***** (full-text).

Just a couple of more things to add then I'm done. The suggestion of "a chronic neuroinflammatory state" at the brain sites under investigation has to be included in any synopsis. Neuroinflammation in autism is a topic that has cropped up quite a few times in the research literature. To pick out one study that springs to mind, the findings from Vargas and colleagues****** and their introduction of microglia into the mix is a case in point. Microglia is another area not readily touched upon this blog so in this case I will perhaps refer you to a nice blog post by Paul Patterson on the topic of hungry microglia potentially eating synapses in autism. I'm not necessarily saying that this is an essential part of the Rose findings but it could be a potentially important tie-in.

The link between elevated levels of 3-NT and elevated nitric oxide (NO) production described in the Rose paper is a final point. The mechanism for this relationship is explained pretty well here. I've covered some of the work on elevated NO metabolites and autism in a previous post. The net results seeming to indicate that levels of NO metabolites are elevated quite consistently in cases of autism. And then there is the inflammation link which has been covered quite a bit before.

OK that's enough for now. You might have realised that I am fairly interested in this paper by Rose and colleagues despite the preliminary nature of their study and the limitations of my knowledge in the area of the brain in autism. Yet another good reason why glutathione in relation to autism deserves a lot more research interest alongside tentative suggestions on whether we might actually be able to do something about it (see previous post). 

To finish, how very dare they make this song which implies a world without Brussels sprouts. You will eat your phenylthiocarbamide and like it...


* Nijhoff WA. et al. Effects of consumption of Brussels sprouts on intestinal and lymphocytic glutathione S-transferases in humans. Carcinogenesis. 1995; 16: 2125-2128.

** Rose S. et al. Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain. Translational Psychiatry. July 2012.
DOI: 10.1038/tp.2012.61

*** Cruikshank C. & Wood T. Quantitation of glutathione as a urinary autism biomarker (poster).

**** Cantu D. et al. Oxidative inactivation of mitochondrial aconitase results in iron and H2O2-mediated neurotoxicity in rat primary mesencephalic cultures. PLoS ONE. 2009; 4: e7095.

***** Vasquez-Vivar J. et al. Mitochondrial aconitase is a source of hydroxyl radical. The Journal of Biological Chemistry. 2000; 275: 14046-14069.

****** Vargas DL. et al. Neuroglial activation and neuroinflammation in the brain of patients with autism. Annals of Neurology. 2005; 57: 67-81.

---------- Rose S, Melnyk S, Pavliv O, Bai S, Nick TG, Frye RE, & James SJ (2012). Evidence of oxidative damage and inflammation associated with low glutathione redox status in the autism brain. Translational Psychiatry, 2 PMID: 22781167

Thursday 12 July 2012

Autism and the family tree

Of all the great philosophical questions, 'why are we here?', 'what purpose do we serve?', 'how come I get blue fluff in my belly button?', the question of 'where we came from?' is perhaps among the most asked. I'm not so much thinking about the origins of humankind or anything so general, but rather our fascination with digging up the past and finding out about our own distant relatives.

In recent years, I too have started down the genealogy path, discovering my industrial and military roots going back over a hundred years or so. I don't know whether age had anything to do with my desire to undertake this search; that and the realisation that we all end up as old photographs and historical facts and figures in a shoebox eventually, but nonetheless the detective work began.

I wish I was able to tell you that Royal blood courses through my regal veins and I am something like the 1,023 person in line to the British throne. But alas, at least for the moment, all I know is that my relations were apparently all hard-working manual labourers or military types and not in anyway Landed gentry. Some of them actually seem to have met rather unfortunate ends it has to be said.

Why the focus on the family history?

Well, today's post concerns an interesting piece of research by Patrick Sullivan and colleagues* (full-text) looking at familial history of bipolar disorder and/or schizophrenia as a risk factor for autism. The authorship group includes a name, I believe, which has already featured on this blog, Dr Cecilia Magnusson and her very interesting research on migration and autism (see here).

I've talked before about the possibility of various 'intersections' between conditions like autism and schizophrenia. So for example, the appearance of autism or autistic features in cases of schizophrenia (see here) and similarities in some cases with regards to gastrointestinal and immune-related findings (see here). Indeed specifically with the relationship between autism and schizophrenia in mind, the work of the late Curt Dohan seems always to crop up, on a potential role for diet, gluten and casein components of diet, in cases of the two conditions. I will however, for now, just put that to one side, apart from this letter which I will come back to at a later date.

The paper by Sullivan and colleagues is open-access so no real need for me to go through it with a fine-toothed comb. A summary however:

  • This was really three studies all rolled into one based on the examination of various registries held in Sweden (registry #1 and registry #2) and Israel (registry #3). 
  • Cases of autism spectrum disorder (ASD) were initially picked out from the registries based on the ICD criteria. ASD cases were matched with various asymptomatic controls based on several parameters including age and year of birth; from the Swedish registries at a 1:10 ratio in a case-control fashion. The actual total number of people with ASD included for the entire study numbered above 30,000 cases.
  • The rates of schizophrenia and bipolar disorder were inspected among parents of ASD cases (registries #1 & #2) and siblings (registries #1 & #3).
  • Results: based on the Swedish registry data (#1 and #2), the "exposure of schizophrenia in parents" was associated with odds ratios (ORs) of 2.9 for a diagnosis of autism. 
  • Similar 'exposure' of schizophrenia in siblings (#1 and #3) was also linked to an increased risk of autism; the ORs ranging from 2.6 to a staggering 12.1 (#3). Indeed bearing in mind the smaller ASD group included for study based on the Israeli registry (#3) (n=386), the 95% confidence intervals were reported as 4.5 - 32.5.
  • When analysing for any potential effect from comorbid learning difficulties (LD) in ASD cases, the results suggested exposure to schizophrenia in parents and/or siblings seemed to be more powerful in cases where no LD was present.
  • The data on familial bipolar disorder, whether parent or sibling, also suggested some increased risk of autism;  although not to the same extent as with schizophrenia.

The authors seem to be pretty confident in their findings. To quote: "The findings were clear. The presence of schizophrenia or bipolar disorder in first-degree relatives was a consistent and significant risk factor for ASD in all 3 samples". I have to say that allowing for potential issues with the diagnostic criteria used and data collection methods, I'm inclined to agree with their findings.

Bear in mind however some important details reported in the study with for example, regards to the effect of learning disability and dare I say, a degree of protection that it seemed to confer with regards to familial psychiatric history. Does this mean that those cases of autism and comorbid learning disability might have a slightly different pattern of aetiology from the autism cases without LD? I must also link to this press release associated with the study and quote a sentence: "Most people with a family history of one of these disorders actually get nothing - the vast majority in fact".

Then to the question of why, why in some cases there may be a link? As much as I like to tug on the loose threads of the genetics research on autism and schizophrenia as a function of the 'genes are everything' mentality that some people hold, one has to allow for the fact that genes must show some involvement in the current study. I will perhaps temper that last statement by pointing out that the growth in the gene*environment viewpoint and the increasing interest in epigenetics are potentially as important as the 100% heritability argument. I assume for example, that most parents and siblings lived in the same household with the participants identified with autism and so their environmental exposure patterns would be considered similar through things like household environment, drinking water, food and diet, pathogen exposure, etc. Bearing in mind also the possibility of other factors being potentially related to schizophrenia/psychosis such as T.gondii exposure, anti-gliadin antibodies, etc.... too much?

 To end some Top of the Pops 1980s Gold. Level 42 and Running in the Family. No pun intended.

----------- * Sullivan PF. et al. (2012). Family history of schizophrenia and bipolar disorder as risk factors for autism Archives of General Psychiatry DOI: 10.1001/archgenpsychiatry.2012.730

Tuesday 10 July 2012

The trial of Toxoplasma gondii

Although self-directed harm and attempted suicide are probably not most people's favourite topic of conversation, it is a fact of life that they happen, and happen more regularly than many of us might think. The reasons for such behaviours are complex and to a large degree, unique to an individual's circumstances; acknowledging that external forces such as the economic downturn that we are all currently presented with, can play a role (see this post on Greece). It is indeed a sobering thought that even things like the work conditions of a father can conceivably impact on offspring risk of attempted and completed suicide* (full-text); such is the potential effect of the environment around us.

Not being an expert on the psychology of self-harm and suicide, I can't readily provide information on the intricate details of how a person arrives at such a situation. Up until a few years ago, I just assumed that the person themselves represents the starting point, and onwards how they cope (or not) with the situations they are presented with bearing in mind the various issues/conditions which can affect mental health. The suggestion that a person might not have as much control over such behaviours or ideations as we might think, seemed a little bit far-fetched; that is until I read a little more about organisms such as Toxoplasma gondii.

Toxoplasma gondii or T.gondii is one of nature's survivors. I don't want to go through all the details of its survival tricks or what it might conceivably do because they have to some extent already been covered in previous blog posts on rats being attracted to cats and mention of the possible link with cases of schizophrenia. Suffice to say that this is a parasite who knows how to keep its head above the water.

Indeed, having previously discussed some interesting data produced by Pedersen and colleagues** in this post on risk of schizophrenia spectrum disorders and T.gondii infection, I was equally interested in the latest paper from this research group*** (full-text) looking at self-directed violence and T.gondii infection. Sensational headlines like 'Are ‘Cat Ladies’ More Likely to Attempt Suicide?' are to be put to one side for now.

I'm not the first to cover this paper. I don't really want to regurgitate all the ins and outs of the latest study by Marianne Pedersen and colleagues (see Dr Emily Deans' post for a good dissection of the study), which by all accounts seemed to mimic their previous protocol: looking at more than 45,000 women giving birth in Denmark between 1992 and 1995, measuring IgG antibody levels to T.gondii and, on this occasion, seeing how this linked (or not) to the reports of suicide, attempted suicide and self-directed violence via hospital and death registers. Suffice to say that was a mammoth project both in terms of numbers and the work gone into the various studies.

There are several interesting parts to this study and its results which I do however want to mention. From a methodological point of view, the study highlights a potential 'predictive value' from IgG antibody levels to T.gondii and risk of self-directed violence and attempted suicide. Based on their statistics, the link between IgG antibodies and actual suicide showed the strongest relationship with a relative risk of 2.05 (95% CI, 0.78-5.20). Bear in mind however that out of the total 45,000+ women studied, only 18 women committed suicide, and 8 of them were seropositive for antibodies, so less than half.

The authors discuss the possible mechanism to account for the association found. I was interested in a couple of the ideas outlined:

  • Once again those letters and that number, IL-6, crop up as possibly being related to the findings. I wasn't aware that apparently there may be a relationship between IL-6 levels and suicide attempts**** and hence how inflammatory cytokines may show some critical relationship to the presence of T.gondii. I suppose it is what the immune system is designed to do.
  • Keeping with the inflammation theme, some discussion on a role for the enzyme indoleamine 2,3-dioxygenase (IDO) is also provided, taking us back to another previous dinner party guest, tryptophan. It doesn't stop at tryptophan however as kynurenic acid moves into the fray and its link to glutamatergic neurotransmission. I have a post scheduled fairly soon looking at the kynurenic acid hypothesis of schizophrenia***** just to give you a flavour of where this line of thought might lead.

This is an interesting area of work which although requiring a lot more investigation adds another layer to the idea that we might think we are in control of our destiny but perhaps not as much as we would like to be even outside of the various social nudging. As per my previous discussions on T.gondii there is also the issue of what we might be able to do if infection is picked up early enough, and importantly whether such intervention might actually in this case save lives. Again I link to this study by Goodwin and colleagues****** and their suggestion, to quote: "some agents used to treat schizophrenia have the ability to inhibit T. gondii proliferation in cell culture". Makes me wonder how many other medications and other products******* might have similar effects?


* Aleck O. et al. The impact of fathers' physical and psychosocial work conditions on attempted and completed suicide among their children. BMC Public Health. 2006; 6: 77.

** Pedersen MG. et al. Toxoplasma infection and later development of schizophrenia in mothers. The American Journal of Psychiatry. 2011; 168: 814-821.

*** Pedersen MG, Mortensen PB, Norgaard-Pedersen B, & Postolache TT (2012). Toxoplasma gondii Infection and Self-directed Violence in Mothers. Archives of General Psychiatry, 1-8 PMID: 22752117

**** Lindqvist D. et al. Interleukin-6 is elevated in the cerebrospinal fluid of suicide attempters and related to symptom severity. Biological Psychiatry. 2009; 66: 287-292.

***** Erhardt S. et al. The kynurenic acid hypothesis of schizophrenia. Physiology & Behaviour. 2007; 92: 203-209.

****** Goodwin DG. et al. Evaluation of five antischizophrenic agents against Toxoplasma gondii in human cell cultures. The Journal of Parisitology. 2011; 97: 148-151.

******* Kavitha N. et al. In vitro Anti-Toxoplasma gondii activity of root extract/fractions of Eurycoma longifolia Jack. BMC Complementary & Alternative Medicine. 2012; 12: 91.