No significant difference in circulating cytokines in autism vs controls?

"As compared with 54 typically developing controls, we found no evidence of differences in the blood profile of immune mediators supportive of active systemic inflammation mechanisms in participants with autism."

That was the unexpected research bottom-line published by Carlos Pardo and colleagues [1] (open-access) examining whether various immune-related chemicals - "cytokines, chemokines, or growth factors in serum and cerebrospinal fluid" - might be linked to autism following longitudinal assessment. By longitudinal I mean that: "Up to four serum samples and up to two CSF samples were obtained from participants, at intervals ranging from 9–24 months, and stored until simultaneous laboratory analysis."

"Participants were drawn from a longitudinal study of autism" we are told, the aim of which was 'to learn more about autism and its subtypes'. Indeed, some of the research attached to this cohort has been previously discussed on this blog (see here) and for example, the suggestion that the horror that is a gluten- and/or casein-free diet used in the context of autism might not be as horrible as many people might think [2]. This time around serum samples were available for over 100 children diagnosed with autism and some 54 not-autism controls. Sixty-seven of the children with autism also provided a cerebrospinal fluid (CSF) sample taken via a lumbar puncture. The authors note: "Ethical constraints prevented lumbar punctures in the TYP [control] group" so make of that what you will.

Bearing in mind that no participants had a history of immunodeficiency or autoimmune disorder (important concepts to some autism) but that "Food, environmental, and seasonal allergies were present in a minority of participants, but were more common in AUT [participants with autism]" the results are interesting. First, when comparing results based on the analysis of CSF samples and serum samples researchers noted that there were "striking differences in the expression of selected cytokines, immune-related growth factors, and chemokines in the CSF compartment compared to the circulating bloodstream compartment." So basically what goes on in serum might not necessarily be the same as that going on in CSF in a biochemical sense.

Next and as per the title and headline of this post: "we found no evidence for major differences in the expression of circulating cytokines and chemokines between children with autism and typically developing controls." This contrasts with quite a bit of other research in the area of immune-related compounds and autism (see here for example) but one has to be a little careful with the wording here, specifically the term 'major differences'. I say that because the authors do report that EGF - epidermal growth factor - did come out as 'different' between the groups (greater in the autism group) for example. EGF has been mentioned before in the context of autism but levels of the stuff have tended to be lower in autism not higher (see here). Puzzling.

This is important work not least because of the cautions highlighted by the authors: "about the lack of relationship between central and peripheral immune markers, signaling that caution should be taken when interpreting the available studies implicating current immune dysfunction in the phenomenology of ASD [autism spectrum disorder], as few have included direct measures of CNS [central nervous system] status." Bearing in mind that there were no CFS comparison samples from controls included in this study (quite a big research flaw by all accounts) it is something else to suggest that if one really wants to see what is going on with immune function and autism, one needs to be looking to a far more invasive sample media. That some of this research group have some 'form' when it comes to the immune system potentially being linked to autism [3] and even more invasive tissue types is also worth noting as further investigations are very carefully merited...

The immune system and autism continues to intrigue.

----------

[1] Pardo CA. et al. Serum and cerebrospinal fluid immune mediators in children with autistic disorder: a longitudinal study. Molecular Autism. 2017. 8: 1.

[2] Graf-Myles J. et al. Dietary adequacy of children with autism compared with controls and the impact of restricted diet. J Dev Behav Pediatr. 2013 Sep;34(7):449-59.

[3] Vargas DL. et al. Neuroglial activation and neuroinflammation in the brain of patients with autism. Ann Neurol. 2005 Jan;57(1):67-81.

----------

Pardo, C., Farmer, C., Thurm, A., Shebl, F., Ilieva, J., Kalra, S., & Swedo, S. (2017). Serum and cerebrospinal fluid immune mediators in children with autistic disorder: a longitudinal study Molecular Autism, 8 (1) DOI: 10.1186/s13229-016-0115-7

Systemic low grade inflammation and bowel issues in autism?

The paper from Katarina Babinská and colleagues [1] (open-access here) presents an interesting, if preliminary take on two potentially important issues linked to at least some cases of autism: gastrointestinal (GI) issues and inflammation (see here and see here respectively).

Detailing the examination of plasma levels of a compound called high mobility group box 1 protein (HMGB1), a protein which has the apparent ability to 'bend DNA' and has some pretty potent immune effects [2] (one paper talked about HMGB1 as being a 'nuclear weapon in the immune arsenal'), authors reported results based on the examination of 31 people on the autism spectrum compared with 16 asymptomatic controls. As well as finding as a group, that those with autism presented with significantly higher levels of plasma HMGB1, they also reported that those with some of the highest levels of HMGB1 were more likely to present with GI issues. Ergo: "Results of the study support the involvement of the systemic low-grade inflammation in the pathomechanisms of autism and its possible association with GI symptoms."

Reiterating that this was a small study in terms of participant groups and that among control group participants were "10 siblings of the individuals with autism", these are interesting results. Whilst I might disagree with some of the terminology used by the authors in their paper such as the concept of 'low-functioning autism' and the term 'mental retardation' as a descriptor of the cognitive status of their participants with autism, I believe that there may be quite a bit more to do in this research area.

A quick trawl through some of the other literature where autism and HMGB1 are mentioned reveals that this is not the first time that elevations in HMGB1 have been reported. The paper from Emanuele and colleagues [3] for example, looking at a similarly small number of participants reported that: "HMGB1 levels may be affected in autistic disorder". Further: "Increased HMGB1 may be a biological correlate of the impaired reciprocal social interactions in this neurodevelopmental disorder." I'm not overly sure that based on data from 22 adults with autism one can make such statements about a single biological parameter being linked to one of the core traits that makes up a diagnosis of autism, but certainly this paper adds to the Babinská data. The paper from Russo [4] on epidermal growth factor (EGF) and HMGB1 with autism in mind has been previously discussed on this blog (see here). In that entry, I also linked to a piece of research correlating HMGB1 to up-regulation of something like MMP-9 (which again has been discussed here before). Follow-up work from this author [5] has also been published.

What's more to say about HMBG1 and autism? Well, one might entertain the idea of adding HMBG1 to further research on markers of immune function in autism (see here) not forgetting the adhesion molecules too (see here). In light of the idea that there may be a link between the genetics of immune function and [some] autism (see here) one might also look at the gene producing HMBG1 and perhaps some of the other genetic/biological drivers controlling or moderating HMBG1 production.

Assuming that some people on the autism spectrum are in a state of "systemic low-grade inflammation" (accepting that this description probably covers most people with and without a diagnosis at some point in their lives) and that initial correlation noted with GI issues, one might also entertain the idea of looking at what happens to HMGB1 levels as and when bowel issues are 'treated'. I say this acknowledging that bowel issues and autism can mean quite a few things (see here and see here) and that science is not quite there yet in understanding how such bowel issues come about and what one might be able to do about them. Aside that is, from the Buie papers back in 2010 (see here and see here)...

Music: Dreaming of You by The Coral. You may not know their name, but you'll probably have heard the song before...

----------

[1] Babinská K. et al. Increased plasma levels of the high mobility group box 1 protein (HMGB1) are associated with a higher score of gastrointestinal dysfunction in individuals with autism. Physiol Res. 2015 Feb 10;63 Suppl 4:S613-8.

[2] Bianchi ME. & Manfredi AA. High-mobility group box 1 (HMGB1) protein at the crossroads between innate and adaptive immunity. Immunol Rev. 2007 Dec;220:35-46.

[3] Emanuele E. et al. Increased serum levels of high mobility group box 1 protein in patients with autistic disorder. Prog Neuropsychopharmacol Biol Psychiatry. 2010 May 30;34(4):681-3.

[4] Russo AJ. Decreased Epidermal Growth Factor (EGF) Associated with HMGB1 and Increased Hyperactivity in Children with Autism. Biomark Insights. 2013 Apr 4;8:35-41.

[5] Russo AJ. Increased Epidermal Growth Factor Receptor (EGFR) Associated with Hepatocyte Growth Factor (HGF) and Symptom Severity in Children with Autism Spectrum Disorders (ASDs). J Cent Nerv Syst Dis. 2014 Sep 9;6:79-83.

----------

Babinská K, Bucová M, Ďurmanová V, Lakatošová S, Jánošíková D, Bakoš J, Hlavatá A, & Ostatníková D (2015). Increased plasma levels of the high mobility group box 1 protein (HMGB1) are associated with a higher score of gastrointestinal dysfunction in individuals with autism. Physiological research / Academia Scientiarum Bohemoslovaca, 63 Suppl 4 PMID: 25669692

Epidermal growth factor and autism continued

I've talked before about epidermal growth factor (EGF) in relation to autism spectrum disorders (ASDs) on this blog. In that entry (see here) the suggestion was that plasma levels of EGF were 'generally' (and I use the word very loosely) reported to be lower in grouped cases of ASD.

Backing up a little, EGF is as its name suggests, a fairly abundant growth factor involved in various cell growth. Obviously that description of its function covers quite a lot of ground, so let me then focus your attention on areas like its role in relation to the gastrointestinal (GI) tract for example, and its connections (or not) to things like intestinal barrier function in certain circumstances. You can perhaps already see where I might be going with all this GI chatter in light of some corresponding interest in the GI tract with cases of autism in mind.

Enter then even more study on EGF levels in autism by way of the paper by Anthony Russo* (open-access). Dr Russo had already received a mention on my previous post on EGF with regards to some work done on another growth factor, HGF - hepatocyte growth factor - being decreased in cases of autism and comorbid GI issues. In the latest paper, the suggestion was that group plasma EGF levels were once again lower in cases of autism compared to asymptomatic controls. That and one or two correlations reported with other biological values and presented symptoms.

As per my usual cop-out, I'm not going to heavily into the paper because it is open-access and you can check out the details yourself. I would direct you to Figure 1 of the paper showing the comparisons between the groups (which included both an autism group and a wider autism spectrum - pervasive developmental disorder - group) illustrating just how different control values were from both the autism and PDD (that's PDD, not PPD!) values accepting the relatively small participant numbers included for study.

Russo also points to a positive correlation between EGF levels in the autism group with another compound included for investigation, HMGB1 - high-mobility group protein B1 - and a negative correlation with GABA (see here). Accepting that correlation doesn't necessarily mean anything more than correlation, I was quite interested in HMGB1 and the inflammation link; even more so the suggestion of a toll-like receptor 4 (TLR-4) and matrix metalloproteinase-9 (MMP-9) link** (at least in a mouse model of ischemic stroke).

I'll admit to being a little confused about the paper's link between EGF and HMGB1 though.

There was a suggestion that despite HMGB1 levels not being reported in the paper: "We have also found significantly increased plasma HMGB1 in our autistic group (P = 0.02; unpublished data)." So unpublished but higher levels. But just before the paper reported: "EGF levels correlated with HMGB1 levels (r = 0.5; P = 0.009)". The direction of the correlation is positive; albeit with an r-value not exactly as much as one would like, so one would assume that lower EGF would correlate with lower HMGB1, and higher EGF would correlate with higher HMGB1. Or am I not seeing something here?

Indeed I was slightly annoyed to see quite a few other references to 'unpublished' data in this paper specifically with regards to the statement of no difference in EGF values between GI associated and non-GI associated autism groups. I'd prefer to see the data included in the paper please (even as supplementary material), particularly when followed by a statement like "decreased EGF may be associated with increased inflammation but not necessarily increased inflammation often associated with GI disease in children with autism".

I have a few other issues with this paper too: the use of 'we' where there is only one author listed who seems to have done all the work which means sample blinding was not possible, and a summary sentence: "We suggest that decreased EGF is associated with increased plasma GABA levels".. Er, yes and no, given that the p-value for the correlation escaped significance coming in at p=0.06. Trend, perhaps. Association, hmmm.... assuming, that is, you adhere to the magical p=0.05.

Sorry if I've come across as being a bit pedantic about all this. Take no notice of me, it's been a long week: school half-term, catching up with work in between and oh, did I mention that I'm also involved in writing a book (TBC)? The important point to take from the Russo paper is that of the lower plasma EGF values and how they add to the existing literature on EGF in cases of autism.

Now we just have to work out what it all means.... answers on a postcard please.

To finish, Bastille and their very catchy tune 'Pompeii'.  And please, no questions about what the Romans ever did for us?

----------

* Russo A. Decreased Epidermal Growth Factor (EGF) Associated with HMGB1 and Increased Hyperactivity in Children with Autism. Biomark Insights. 2013; 8: 35-41.

** Qui J. et al. High-Mobility Group Box 1 Promotes Metalloproteinase-9 Upregulation Through Toll-Like Receptor 4 After Cerebral Ischemia. Stroke. 2010; 41: 2077-2082.

---------

Russo AJ (2013). Decreased Epidermal Growth Factor (EGF) Associated with HMGB1 and Increased Hyperactivity in Children with Autism. Biomarker insights, 8, 35-41 PMID: 23645980

Epidermal Growth Factor and autism

Stanley Cohen @ NIH

I'm sure that regular readers of this blog are getting pretty used to (replace with 'sick and tired of') me talking about cytokines in relation to autism and other research areas.

Cytokines are those little chemical messengers which are able to stimulate both arms of the immune system (innate and adaptive) having various effects for example, in mediating inflammation. Yes sirree, these little critters perform some pretty critical tasks related to our health and wellbeing.

Although there is still some debate with regards to terminology, cytokines - some cytokines - represent a very particular type of growth factor in that they are potentially "capable of stimulating cellular growth, proliferation and cellular differentiation".

It is with this 'growth factor' description in mind that I turn my attention to an interesting paper by Charity Onore and colleagues* (open-access) and some observations related to plasma levels of epidermal growth factor (EGF) in cases of autism. The picture accompanying this post by the way is of Stanley Cohen who received the Nobel Prize in Physiology and Medicine in 1986 for the discovery of EGF.

You might recognise the authorship group for this latest  paper as being linked to the MIND Institute and their seemingly endless supply of pretty interesting findings related to autism including a recent entry I made on their work on immune cell adhesion. I know some people (rightly) question some of the work coming out of the MIND Institute in terms of generalisability to all autism and the old correlation-causation conundrum. I myself, believe that there is a plethora of extremely valuable information being published in their repertoire of papers ripe for further investigation and replication including important work on endophenotypes. Just my opinion.

Anyhow, back to the Onore paper and a few factoids about EGF and the paper for your attention:

• Epidermal growth factor, or EGF to its friends, is one of a number of important growth factors. As far as I can ascertain, it is involved in quite a few important processes which include: (a) the growth and proliferation of neurons and glia (stimulating DNA synthesis), (b) aspects of wound healing, and (c) moderating gastric acid secretions. I would also draw your attention to an interesting paper on how EGF might also abate some of the more detrimental effects of testosterone on gastric ulceration just in case you were interested in that area of research.
• As part of the Autism Phenome Project, plasma levels of EGF and another growth factor, hepatocyte growth factor (HGF) were investigated in 49 children with autism (ages: 2-4 years old) and 31 asymptomatic control children. ELISA was the name of the testing game, and all samples were analysed in duplicate.
• Results: plasma levels of EGF were significantly lower in the autism group compared to controls (p=0.003). Plasma levels of HGF were marginally lower from a group point of view in the autism cohort but not significantly so.

As with many aspects of contemporary autism research, this is not the first time that reduced plasma EGF levels have been reported in cases of autism. Suzuki and colleagues** reported lower levels of EGF in their small participant group comprised of adults with 'high-functioning' autism. 

But before you get too carried with 'this technological terror you've created' the literature on EGF and autism is not all one way. Işeri and colleagues*** reported on increased serum levels of EGF in their cohort of children with autism. Indeed, this is not the only occasion that elevations in EGF been noted as per this study by Tobiasova and colleagues**** who further noted that the effects of the antipsychotic drug risperidone seemingly did little to affect EGF levels.

So what does it all mean? A good question and at the moment, I don't have a good answer. The various differences in levels of EGF noted among the studies indicate that EGF is probably not universally 'abberrant' in cases of autism, allowing for differences in age, ethnicity, comorbidity, etc. among the various cohorts studied. I'm not immediately surprised by that fact given that there is a growing appreciation that autism is more likely to be autisms (plural) and at the moment universal constants to describe the condition (outside of symptom classification) are few and far between.

The authors do talk about the possibility of a gastrointestinal element to their findings based on preliminary investigations which showed that "EGF levels are associated with increased bloating in children with ASD". When one considers the volume of work suggestive of both functional and more pathological gastrointestinal (GI)-related conditions being correlated with cases of autism (see here and here respectively), this suggestion takes on an interesting meaning. As far as I can see however, none of the other studies on EGF levels in autism reported on any potential somatic correlates like GI factors, so we can't readily verify this suggestion at the moment.

Drawing however on other research 'around' growth factors, I did stumble on this paper by Russo and colleagues***** (open-access) which seemed to indicate that decreased HGF in cases of autism might correlate with the presence of GI dysfunction in the form of an inflammatory bowel-like disease. Interestingly Russo et al also tied this back to another old friend, MET gene function - discussed here - and its important role in cortical development and gastrointestinal repair. Whether or not this might also tie into EGF is not immediately clear.

EGF and the other growth factors represent an interesting area for further study for autism research. Keeping in mind the other states and conditions also associated with issues with EGF levels like foetal growth (see here), aplastic anaemia (see here) and even cognitive decline in Parkinson's disease (see here), one can perhaps envisage some potential role for EGF in specific endophenotypes of autism. The trick is to find out what that endophenotype might be composed of...

---------

* Onore C. et al. Decreased levels of EGF in plasma of children with autism spectrum disorder. Autism Research & Treatment. 2012; 205362.

** Suzuki K. et al. Decreased serum levels of epidermal growth factor in adult subjects with high-functioning autism. Biological Psychiatry. 2007; 62: 267-269.

*** Işeri E. et al. Increased serum levels of epidermal growth factor in children with autism. JADD. 2011; 41: 237-241.

**** Tobiasova Z. et al. Risperidone-related improvement of irritability in children with autism is not associated with changes in serum of epidermal growth factor and interleukin-13. Journal of Child & Adolescent Psychopharmacology. 2011; 21: 555-564.

***** Russo AJ. et al. Decreased serum hepatocyte growth factor (HGF) in autistic children with severe gastrointestinal disease. Biomarker Insights. 2009; 4: 181-190.

----------

Onore C, Van de Water J, & Ashwood P (2012). Decreased levels of EGF in plasma of children with autism spectrum disorder. Autism research and treatment, 2012 PMID: 22937258