S100B protein and autism continued

"Our findings showing an increase in peripheral concentrations of S100B and TNF-α provide limited support to the hypothesis about the roles of altered immune function and S100B in autism spectrum disorder (ASD)."

So said the findings reported by Selin Aktan Guloksuz and colleagues [1] (open-access available here) continuing some discussions a few years back on a possible role for S100B in relation to at least some autism (see here).

S100B - S100 calcium-binding protein B - is a compound involved in quite a few biological reactions not least "as a biomarker of global glial activity." Elevations of the S100B have been reported in relation to several states including that of [traumatic] brain injury. Outside of some research suggesting that elevations of S100B might also be a feature of diagnoses such as schizophrenia (see here), it has also been the topic of investigations with [some] autism in mind [2] too. The name of the game is elevations in S100B in relation to autism and more.

Based on analyses of fasting blood samples from "40 unmedicated children with autism" (where autism diagnoses were confirmed by study researchers) and 35 asymptomatic control children, researchers measured levels of plasma S100B alongside various markers of immune function (cytokines). Among the suite of cytokines examined, levels of "tumor necrosis factor alpha (TNF-α), interferon gamma, interleukin (IL)-1β, IL-4, IL-6, IL-10, and IL-17A" were included. The idea of using unmedicated children with autism stems from the suggestion that at least one medication used for some autism might have the ability to elevate S100B [3].

Results: as per the opening sentence to this post, levels of S100B and TNF-α were 'different' between the groups (both elevated) and this finding remained "after controlling for age, sex, and BMI [body mass index]." Researchers also reported some results looking at whether ASD symptom presentation might show any 'association' with S100B levels. On this topic they reported that: "Plasma S100B concentrations in children with severe ASD symptoms were higher than in children with mild-moderate ASD symptoms" but when again controlling for age, sex and BMI this association did not hold (significantly). As for the other cytokines outside of TNF-α... nothing came up as significant between the groups. This is interesting in light of recent work (see here) and even Guloksuz et al talk about future "prospective longitudinal studies investigating a broad set of immune markers, both in serum and CSF [cerebrospinal fluid], in large samples" and the pros- and cons of looking in CFS.

Where next for this area of investigation? Well, taking into account the link between S100B and brain injury and what that could mean for cognitive processes for example, I'd be minded to suggest that more study is needed looking at the effect of S100B levels in relation to cognition and autism. Take for example the study results from Chen and colleagues [4] who reported that "serum S100B level was an independent contributor to the global cognitive dysfunctions, particularly for the speed of processing, attention/vigilance, visual learning and reasoning/problem solving subscores" in their cohort of participants with schizophrenia. Might similar correlations be present alongside S100B elevations in relation to autism? I'd also be minded to suggest looking at a possible role for comorbidities potentially accompanying a diagnosis of autism as being important for S100B elevations in light of other research on depression for example [5]. Depression (various types) and autism is very much an important area of overlap (see here for example) and might actually offer at least one way to target elevations in S100B.

There is more to do on this topic.

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[1] Guloksuz SA. et al. Elevated plasma concentrations of S100 calcium-binding protein B and tumor necrosis factor alpha in children with autism spectrum disorders. Rev Bras Psiquiatr. 2017 Jan 12:0.

[2] Al-Ayadhi LY. & Mostafa GA. A lack of association between elevated serum levels of S100B protein and autoimmunity in autistic children. J Neuroinflammation. 2012 Mar 16;9:54.

[3] Quincozes-Santos A. et al. Effect of the atypical neuroleptic risperidone on morphology and S100B secretion in C6 astroglial lineage cells. Mol Cell Biochem. 2008 Jul;314(1-2):59-63.

[4] Chen S. et al. Cognitive dysfunction correlates with elevated serum S100B concentration in drug-free acutely relapsed patients with schizophrenia. Psychiatry Res. 2017 Jan;247:6-11.

[5] Rajewska-Rager A. & Pawlaczyk M. The role of S100B protein as a potential marker in affective disorders. Psychiatr Pol. 2016;50(4):849-857.

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Guloksuz SA, Abali O, Aktas Cetin E, Bilgic Gazioglu S, Deniz G, Yildirim A, Kawikova I, Guloksuz S, & Leckman JF (2017). Elevated plasma concentrations of S100 calcium-binding protein B and tumor necrosis factor alpha in children with autism spectrum disorders. Revista brasileira de psiquiatria (Sao Paulo, Brazil : 1999) PMID: 28099628

Autism, luteolin and inflammatory markers

"We further show that the children with ASDs [autism spectrum disorders] in which the elevated serum IL-6 and TNF levels decreased at the end of the treatment period with a luteolin formulation, were the ones whose behavior improved the most."

That was an excerpt from the paper by Tsilioni and colleagues [1] (open-access available here) who looked at some of the potential biological (and behavioural) effects following supplementation with the dietary formulation known as NeuroProtek® [2] containing the flavonoid luteolin. Piggybacking on a previous open trial of luteolin [3] in relation to autism, authors report on levels of various serum cytokines between baseline and post-intervention as well as versus control specimens.

Levels of IL-6 (interleukin 6) and TNF (tumor necrosis factor) were reported to decrease in the autism group (N=38) following supplementation. Further, authors report on "two clusters of ASD children with low and high serum IL-6 and TNF levels indicating two subgroups." Specifically for those children with autism with higher values (n=10), they reported some interesting changes in scores on the Vineland Behavior Scales (VABS) suggesting that: "these children gained 9.73 months in the communication domain, 6.64 months in daily living skills and 8.09 months in the social domain."

These are intriguing results. Accepting the relatively small participant group and the open-trial methodology of the original study from which biological samples were derived, it strikes me that there may be quite a bit more to see from luteolin and related compounds when it comes to at least some autism. I'm not on this occasion going to focus too much on the cytokine results discussed by Tsilioni et al because I think there is more than enough peer-reviewed evidence implicating these various chemical messengers in at least some aspects of some autism (see here). I do think it is interesting however that the authors suggest that analysis of compounds like IL-6 and TNF might be one step towards looking for potential best responders to this type of intervention. Indeed, 'response to intervention' is something I'd like to see discussed a little more when it comes to teasing apart the different types of autism (see here).

I have mentioned luteolin previously on this blog (see here) and the link back to one or two of the authors on the Tsilioni paper including Prof. Theoharis Theoharides (he of mast cells and autism fame). "Luteolin is structurally closely related to 7,8-dihydroflavone, which was shown to have brain-derived neurotrophic factor (BDNF)-like activity" is one of the ways that the authors put forward as potentially being explanatory of their results, bearing in mind how 'mixed up' BDNF is when it comes to autism (see here). That and proposals for delivery via "intranasal administration" tapping into the rise and rise of pharmaceutical technology (see here) and I think we're going to be hearing quite a bit more about luteolin-containing preparations and autism in future research times...

Music: Sons of Pitches and a rather interesting version of MMMBop (in 10 genres).

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[1] Tsilioni I. et al. Children with autism spectrum disorders, who improved with a luteolin-containing dietary formulation, show reduced serum levels of TNF and IL-6. Transl Psychiatry. 2015 Sep 29;5:e647.

[2] Theoharides TC. et al. A case series of a luteolin formulation (NeuroProtek®) in children with autism spectrum disorders. Int J Immunopathol Pharmacol. 2012 Apr-Jun;25(2):317-23.

[3] Taliou A. et al. An open-label pilot study of a formulation containing the anti-inflammatory flavonoid luteolin and its effects on behavior in children with autism spectrum disorders. Clin Ther. 2013 May;35(5):592-602.

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Tsilioni I, Taliou A, Francis K, & Theoharides TC (2015). Children with autism spectrum disorders, who improved with a luteolin-containing dietary formulation, show reduced serum levels of TNF and IL-6. Translational psychiatry, 5 PMID: 26418275

Cortisol and cytokines: a diagnostic tag-team for autism?

A quote from the paper by Chang-Jiang Yang and colleagues [1] begins today's post: "The results of ROC [receiver operating characteristic] analysis indicated the cortisol VAR, IL-6 and TNF-α were potential biomarkers in diagnosis of ASD [autism spectrum disorder]."

With many thanks to Natasa for providing me with a copy of this paper, I'd like to discuss these 'joined up' findings a little further. A few pointers to begin with:

Boot the grime of this world in
the crotch, dear

• Based on quite a bit of previous research looking at the steroid hormone cortisol in relation to autism (see here) alongside an increasingly important body of work talking about cytokines and autism (see here), the authors embarked on assessing the joint role of these compounds as "potential biomarkers in assisting the diagnosis of ASD."
• Based in China, a small-ish group of participants diagnosed with ASD (n=35) aged around 10 years were recruited alongside an asymptomatic group (n=32) "unrelated to the autistic participants".
• Salivary cortisol levels were measured (8 of them) at various points of the day from waking up to just before going to sleep. A fasting blood sample was also provided from each participant for analysis of cytokines. The Childhood Autism Rating Scale (CARS) served as the measure of autism severity.
• Results: the cortisol VAR - "diurnal variation of cortisol" - did show something of a group difference between those with autism and controls exemplified by "reduced diurnal amplitude in... cortisol concentration". Group cortisol concentrations differed particularly at the time of "just before going to sleep", where the ASD group tended to show higher levels than controls.
• Insofar as those cytokines: "There was a significant difference noted in median plasma concentrations of IL-6 and TNF-α between the ASD and control individuals." Both measures were elevated in the autism samples when taking groups as a whole.
• Then came the ROC analysis, and based on those values for cortisol VAR and those two cytokines, various measures of sensitivity and specificity were put forward individually. "The combination of three factors had a sensitivity of 91.43% and a specificity of 96.87% (AUC = 0.97)". Those are pretty good values in anyone's book
• The authors conclude that their study results "may supply a simple clinical approach for aiding the diagnosis of ASD." Importantly however, they make mention of the small participant groups included in their study.

As always, further independent replication of these findings are warranted before anyone goes and gets too excited about the possible implications. That and the fact that 'biomarkers' mentioned in the context of autism perhaps doesn't mean as much as you might think bearing in mind the heterogeneity of the autism spectrum ('the autisms'?) and those all-important comorbidities which I keep going on about. Whether results also translate to other geographic or ethnic populations is another point to be seen.

What I perhaps like best about the Yang paper and results is however the logical simplicity behind their study. As indicated, both areas of cortisol - as part of the HPA axis - and cytokines have something of an important history in autism research [2] which has thus far been seldom looked at together. I acknowledge the paper by Brian Lovell and colleagues [3] (discussed in this post) looking at pro-inflammatory biomarkers and cortisol levels in parents of children with autism or ADHD (attention-deficit hyperactivity disorder) but that was parents, not children with autism.

The obvious next questions after any independent replication are 'why' and what could this mean for potential interventions (if and when required). The 'why' question is probably going to be rather complicated as per the involvement of genetics (see here), epigenetics (see here) and biochemistry (see here) intersecting when it comes to immune function involvement and autism for example. Despite some headlines talking about elements of immune function as being the next frontier for autism research I'd be minded to say that is has been for at least the past 20 years or so, in some circles at least. Oh, and science is also picking up the idea of "an immune-mediated subtype of autism" too [4] perhaps at the centre of any cytokine biomarkers [5].

Insofar as the intervention side of things, well this is where things can get a little more contentious. Accepting that concepts like inflammation are starting to be more readily used in the context of psychiatry (see here), the idea that treating said inflammation might impact on behavioural measures is still something squarely in the research domain at least for now.

But this kind of work does represent an interesting area ripe for further study...

Music to close... and I am the only one transfixed with this 'how to play Heart and Soul' on the piano?

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[1] Yang C-J. et al. The roles of cortisol and pro-inflammatory cytokines in assisting the diagnosis of autism spectrum disorder. Research in Autism Spectrum Disorders. 2015; 9: 174-181.

[2] Ashwood P. et al. Elevated plasma cytokines in autism spectrum disorders provide evidence of immune dysfunction and are associated with impaired behavioral outcome. Brain Behav Immun. 2011 Jan;25(1):40-5.

[3] Lovell B. et al. The psychosocial, endocrine and immune consequences of caring for a child with autism or ADHD. Psychoneuroendocrinology. 2012 Apr;37(4):534-42.

[4] McDougle CJ. et al. Toward an immune-mediated subtype of autism spectrum disorder. Brain Research. 2014. November 13.

[5] Rose D. & Ashwood P. Potential cytokine biomarkers in autism spectrum disorders. Biomark Med. 2014 Oct;8(9):1171-1181.

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Yang, C., Tan, H., Yang, F., Liu, C., Sang, B., Zhu, X., & Du, Y. (2015). The roles of cortisol and pro-inflammatory cytokines in assisting the diagnosis of autism spectrum disorder Research in Autism Spectrum Disorders, 9, 174-181 DOI: 10.1016/j.rasd.2014.10.012

Lenalidomide and autism?

Lenalidomide @ Wikipedia

Of the thousands of medicines which have been or are currently in use, a few really enter the mainstream public consciousness for a variety of different reasons.

That little blue pill which now graces many a man's medicine cabinet worldwide is probably the best example, as any email spam folder can readily confirm.

One drug, suggested to treat morning sickness during pregnancy in the late 1950s, is however synonymous with the darker side of pharmaceutics; illustrative of the failings of appropriate medicines testing: thalidomide.

If you don't know the story of thalidomide this article offers quite a succinct timeline, and how only recently the company responsible for the invention of thalidomide has formally apologised to all those affected by this medicine.

You would perhaps think that after such a torrid history, this drug would and should be consigned to the annals of history, never to be used in medical practice again. You might however want to think again; as thalidomide is re-emerging as a medicine for various conditions including certain cancers, inflammatory diseases and even persistent cough, albeit with a few tweaks and heaps more pharmacovigilance particularly when administered to females of reproductive age.

OK so what's all this got to do with autism? Well, my attention was grabbed by the publication of this proof-of-concept study by Michael Chez and colleagues* (open-access) and the suggestion that lenalidomide, an analogue of thalidomide, might (might) show some promise in cases of autism. I had seen mention of this study, or something like this study, in a previous abstract from IMFAR 2010 (see here). The ClinicalTrials.gov entry for the study can also be seen here.

The keen eyes out there might recognise the name Dr Michael Chez and the Sutter Institute for some other recent publicity regarding the first FDA-approved stem cell trial for autism (see trial details here) which has created some column inches among mainstream media and bloggers alike (including me). It seems that this research group is intent on creating scientific waves.

Back to the paper, which is open-access, for a summary and few comments:

• The basic premise of the trial was the recognition of quite a bit of research which has indicated the immune system and various features of immune function might correlate with the presence of autism in some cases. Mention for example of that dastardly cytokine, interleukin-6 (IL-6) is made, but the main protagonist in this particular study is tumor necrosis factor - alpha (TNF-α), another inflammatory cytokine, and a target for lenalidomide (see here). The authors cite the focus of TNF-α as an outcome measure "because other cytokines were not always available in all patients" referring to tested parameters.
• A daily dose (2.5mgs) of lenalidomide was given for 12 weeks to 7 males (aged 6-12 years) diagnosed with autism and with parental report of regression being associated with symptom onset. Autoimmune "dysfunction" (I assume this means autoimmune disease) was reported in first-degree relatives of all participants, although prospective participants with various diagnosed autoimmune conditions were excluded from the present study.
• Various measures of behaviour and cognitive functioning were used during the study (baseline - 6 weeks - 12 weeks) including ADOS, CARS and the Receptive and Expressive One-Word Picture Vocabulary Test. This accompanied measurement of both serum and CSF levels of TNF-α (CSF levels collected via that most invasive of methods, lumbar puncture - don't click on this link if you are squeamish). 
• To ascertain the pharmacokinetics of the drug (drug metabolism), blood draws at 1, 2, 4 and 8 hours after the first dose of the lenalidomide were administered. 
• Results: well, mixed is probably the best description. Safety-wise, two participants were withdrawn from the study following their developing a rash. Another participant discontinued when their neutrophil count (white blood cells) dipped; albeit transiently. 
• Four participants registered a drop in TNF-α levels in both serum and CSF at study end. Whilst on the surface of things this might be considered an interesting finding, one has to bear in mind that analysis of CSF levels of the cytokine were accepted anywhere up to 8 months before the baseline testing started.
• Behaviourally, there was some suggestion of improvement on the various measures included for study, particularly at 6 weeks, but alas none of these changes were statistically significant. Indeed with the attrition (drop-out) rate (n=3) mentioned at study end, I'm not really surprised that nothing was found to be significantly improved. It would have to be an almost spectacular change in behaviour for 4 participants to register a significant change from baseline to study end.
• Conclusion: some interesting trends from the various data but nothing concrete probably as a function of the small participant group.

The science-y types out there may very well look at this trial and its results and focus on its failings and weaknesses. Whilst not trying to defend the study, I would draw your attention to several keywords in the title like 'pilot study' as a clue to the very preliminary nature of this investigation. Indeed, the authors do mention that this was not a randomised trial (i.e. participants randomly allocated to treatment or not), did not include a placebo group and neither was it double-blind; everyone knew that participants were taking lenalidomide and nothing else. It's more of case series description than a scientific trial.

Before any chatter arises about me somehow endorsing thalidomide derivatives to 'treat' autism by highlighting this study, I just want to say that I'm not. My mind keeps wandering back to the devastating teratogenic effects that thalidomide bestowed, and still I can't help but wonder if this is indeed one of those drugs which given its history perhaps should have been consigned to the great pharmaceutical rubbish tip, never to be used again.

I'll let you form your own opinion on whether this is an area requiring further research with autism in mind or not. Bear in mind however that for some people with certain conditions, lenalidomide has probably had some real benefit for them (see here) and its adoption reflects, quote "the pressing need to develop molecules with enhanced immunomodulatory and antitumor activity". Not that there may not be alternatives as per this study however.

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* Chez M. et al. Safety and observations in a pilot study of lenalidomide for treatment in autism. Autism Research & Treatment. 2012; 291601.
DOI: 10.1155/2012/291601

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Michael Chez, Renee Low, Carol Parise, & Tammy Donnel (2012). Safety and observations in a pilot study of lenalidomide for treatment in autism Autism Research & Treatment DOI: 10.1155/2012/291601