SHANK3, gut issues and (mouse) autism continued

"We conclude that apart from its well-known role in the CNS [central nervous system], SHANK3 plays a specific role in the GI [gastrointestinal] tract that may contribute to the ASD [autism spectrum disorder] phenotype by extracerebral mechanisms."

So said the findings reported by Ann Katrin Sauer and colleagues [1], and yet more evidence that issues with SHANK3 mentioned in relation to 'some' autism, may well (partly) explain much more than just behaviour (see here and see here).

The Sauer study was yet another mouse study. They specifically focused on the "Shank3αβ KO" mouse, where KO means knock-out, referring to the engineering of this mouse strain to mimic issues with the functioning and availability of SHANK3, "a known scaffolding protein of the postsynaptic density (PSD) of glutamatergic excitatory synapses." Said knock-out mice have been "reported to display ASD-like behavior with abnormal ultrasonic vocalization, repetitive self-grooming, and reduced interest in novel mice." I say this being careful to reiterate that we're talking about a mouse not human beings (see here).

On the basis of the observation that SHANK3 is expressed in the gut as well as brain and that GI issues are no stranger to autism (see here), researchers set about looking at how SHANK3 issues might also manifest as gut issues, and what this *could* mean for some autism. They observed some interesting things:

• "analysis of the GI tract of Shank3αβ KO mice revealed significantly altered gut morphology" which included, among other things, increased levels of ZONULIN1 ("a modulator of tight junctions and alterations"). Zonulin is something that I'm particularly interested in on this blog (see here and see here) on the basis of its *connection* to intestinal barrier function and the misnomer that is 'leaky gut' (see here).
• "The Microbiome of Shank3 KO Mice Is Altered." Bearing in mind the increasing importance of the gut microbiome to autism (see here), researchers reported some interesting difference between "Shank3αβ KO mice" and controls with regards to several different bacterial species. 
• Researchers describe how those gut morphology and gut bacterial differences seemed to be linked to alterations in the "expression of inflammatory markers" too as they talked about "signs of increased immune activation in the periphery and the brain." A familiar cytokine is mentioned - IL-6 - and quite a few avenues for further investigation.

The net result of all this work is to say that, yes, the SHANK3 mouse model *potentially* mimicking some of the behavioural signs and symptoms of autism does also appear to show some significant gut-related issues. No, this does not directly translate into issues for 'all human autism', but it does add further credence to the idea that the gut-brain axis is likely important to at least 'some autism'. Where also SHANK3 issues are identified as coinciding with 'human autism', one might also entertain the idea that gut issues should be screened and treated/managed. And there might be lots of ways to manage them (see here for one example)...

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[1] Sauer AK. et al. Altered Intestinal Morphology and Microbiota Composition in the Autism Spectrum Disorders Associated SHANK3 Mouse Model. Int. J. Mol. Sci. 2019; 20: 2134.

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Pioglitazone for autism?

Enjoyed Christmas? Welcome back. Onward...

"Pioglitazone is well-tolerated and shows a potential signal in measures of social withdrawal, repetitive, and externalizing behaviors."

So said the findings reported by Lucia Capano and colleagues [1] (open-access) describing preliminary efforts to "elucidate the maximum tolerated dose, safety, preliminary evidence of efficacy, and appropriate outcome measures in autistic children ages 5–12 years old" taking the hypoglycemic medicine called pioglitazone. The results obtained from this phase II pilot study - "a 16-week prospective cohort, single blind, single arm, 2-week placebo run-in, dose-finding study of pioglitazone" - suggest that further research on this medicine in the context of autism is warranted.

Capano et al provide quite a lot of information about the whys-and-wherefores of pioglitazone use in the context of autism. They talk about immune system 'issues' and inflammation being no strangers to autism research. They talk about various findings in relation to immune signalling and autism, drawing on data from several investigations that have looked at compounds like the cytokines and chemokines. Pioglitazone fits into this story by way of it being "an agonist of peroxisome proliferator activated receptor (PPAR)-ϒ." Activation of PPAR-ϒ leads to "insulin sensitization and enhances glucose metabolism." It also seemingly has an anti-inflammatory role to play too. Through the use of  pioglitazone ramping up the action of PPAR-ϒ, so researchers opined that this could be useful for some people diagnosed with autism where immune system and behaviour might meet...

Unlike other trials of pioglitazone in the context of autism [2], the Capano study was more exploratory than 'gold-standard' in it's design. It did however include both behavioural and biological components, where various behavioural outcome measures were included alongside the describing of various "research bloodwork" that included various cytokines ("IL1-β, IL-10, and TNF-α in plasma; IL-6 in serum") that have been discussed in other studies with autism in mind (see here for example).

Alongside those initial results mentioned in the opening sentence of this post, there are a few other important points to make. So: "Overall, pioglitazone was well tolerated." Welcome news indeed. Researchers also noted that: "There were no serious adverse events (SAEs) in any of the doses within the range tested (0.25 mg/kg, 0.5 mg/kg, and 0.75 mg/kg)." This is important in the context that all medicines have the propensity for 'adverse effects' for some people, and pioglitazone is no different. Given also the focus on medication and weight gain in the context of autism (see here for example), it's a bit of relief to see that, for the study period at least, authors reported that: "BMI [body mass index] did not change significantly during the study."

I'm not going to go to heavily into the behavioural changes noted over the study period on this occasion, because these are preliminary and one has to be careful with any interpretation. I do however want to mention some of the biological results; namely: "Significant changes with treatment occurred with both IL-6 and IL-10" and "IL-1β and TNF-α did not change significantly with treatment." The authors note that the IL-6 and IL-10 findings - "decreasing IL-6 and increasing IL-10" - were "consistent with the known effect of PPAR-gamma agonists like pioglitazone." This is an interesting finding.

Cumulatively, such results suggest that quite a bit more research focus is needed on pioglitazone in the context of [some] autism. But for now, it looks quite promising (again [3])...

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[1] Capano L. et al. A pilot dose finding study of pioglitazone in autistic children. Molecular Autism. 2018; 9: 59.

[2] Ghaleiha A. et al. A pilot double-blind placebo-controlled trial of pioglitazone as adjunctive treatment to risperidone: Effects on aberrant behavior in children with autism. Psychiatry Res. 2015 Sep 30;229(1-2):181-7.

[3] Boris M. et al. Effect of pioglitazone treatment on behavioral symptoms in autistic children. J Neuroinflammation. 2007;4:3.

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"findings suggest a protective effect of CRP" on schizophrenia risk?

Science is often a puzzling endeavour. Sometimes, just when you think that you've got something nailed down, scientific results appear that 'trash' long held, cherished beliefs. So it was with the publication of the results by Fernando Pires Hartwig and colleagues [1] who presented findings looking at "the effect of inflammatory markers on schizophrenia risk" based on the use of "a mendelian randomization (MR) design."

MR, by the way, is a interesting technique based on the principle that "genetic variants that either alter the level of, or mirror the biological effects of, a modifiable environmental exposure that itself alters disease risk should be related to disease risk to the extent predicted by their influence on exposure to the environmental risk factor." It's a technique that has already been applied to inflammatory markers in the context of schizophrenia on more than one occasion (see here and see here). Those inflammatory markers studied have included ones which were covered by the Hartwig paper. Specifically: "Genetically elevated circulating levels of C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1Ra), and soluble interleukin-6 receptor (sIL-6R)." I should also point out that Hartwig and colleagues have some research form in the area of applying MR to various aspects of medical science (see here).

As per an accompanying editorial on the Hartwig paper [2], the long-and-short of it was that researchers "used 2-sample MR to test for a potentially causal relationship between inflammation and schizophrenia and to improve inference for the association between genes, inflammatory biomarkers, and risk of developing schizophrenia." I can't claim any specific expertise in the use of MR (see here for a good overview [3]) but it appears that data on single-nucleotide polymorphisms (SNPs) in relation to those inflammatory markers was used to test whether said markers might be linked 'causally' to risk of schizophrenia. Their results were interesting: "we did not find strong evidence that lifelong exposure to increased action of these proinflammatory cytokines increases schizophrenia risk, as previously hypothesized" and indeed that: "blockade of IL-6 effects and low CRP levels might instead increase schizophrenia risk." This is contrary to quite a lot of other research in this area (see here for example).

There are a few words of caution to attach to the Hartwig results that need mentioning not least the primary tenet on which analyses are based: genetic variants (SNPs) affecting something like CRP are of primary importance to schizophrenia. I don't for example, see anything in the data looking at gene function/expression being affected as a result of non-structural changes to the genome via something like epigenetic 'alterations' for example (and there is such a thing as epigenetic Mendelian randomization y'know). I say this on the basis that other genes involved potentially involved in processes linked to DNA methylation have also been *associated* with cases of schizophrenia (see here). The authors also caution that their analyses are based on "lifelong exposure to elevated cytokine and CRP levels" and that exposure during 'critical windows' might be the important issue when it comes to any change in schizophrenia risk. Similarly they note that "it is possible that IL-6 and CRP effects on schizophrenia risk are related to a maternal effect (eg, maternal susceptibility to infections during pregnancy), so that our findings are explained by the correlation between maternal and offspring genotypes." This final point is based on the idea that maternal infection during pregnancy (or the biological consequences of) seems to be quite a big risk factor for at least some presentations of schizophrenia (see here) as well as [cautiously] other labels (see here). Here, the importance of a reprogrammed immune system during pregnancy might also come into play alongside any maternal 'susceptibility'.

Personally I'm not yet ready to totally trash the idea that the immune system, and specifically elevations in inflammatory markers such as CRP and other pentraxins, might not be important to some schizophrenia risk in a more detrimental way. I appreciate that one has to be careful when talking about immune system markers and their inflammatory direction (see here for some chatter on IL-6 and its pro- and anti-inflammatory natures) but the existing data is too evident to just discard on the basis of one new study, despite it's scientific prowess. I don't however doubt that there may be several confounding variables linked to increases in CRP in schizophrenia; not least the impact of something like increased body mass index (BMI) that seems to follow some cases of schizophrenia [4]. These variables need to be further explored, particularly in the context of what side-effects pharmacological management of schizophrenia might have (see here). And I also hat-tip the paper by Manu and colleagues [5] applying the Bradford Hill's guidelines on 'causation' to this area and concluding that (upto 2014) "there is insufficient evidence that the replicated, strong association between schizophrenia and elevated inflammatory markers has etiopathological relevance"...

For now however, the Hartwig findings reiterate that science is an ever-changing, ever-evolving process...

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[1] Hartwig FP. et al. Inflammatory Biomarkers and Risk of Schizophrenia: A 2-Sample Mendelian Randomization Study. JAMA Psychiatry. 2017. Nov 1.

[2] Byrne E. et al. Inference in Psychiatry via 2-Sample Mendelian Randomization—From Association to Causal Pathway? JAMA Psychiatry. 2017. Nov 1.

[3] Sheehan N. et al. Mendelian Randomisation and Causal Inference in Observational Epidemiology. PLoS Med. 2008; 5(8): e177.

[4] Fernandes BS. et al. C-reactive protein is increased in schizophrenia but is not altered by antipsychotics: meta-analysis and implications. Mol Psychiatry. 2016 Apr;21(4):554-64.

[5] Manu P. et al. Markers of inflammation in schizophrenia: association vs. causation. World Psychiatry. 2014 Jun; 13(2): 189–192.

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Childhood inflammation and hypomanic symptoms in young adulthood?

"Higher levels of systemic inflammatory marker IL-6 in childhood were associated with hypomanic symptoms in young adulthood, suggesting that inflammation may play a role in the pathophysiology of mania."

That was the conclusion reached by Joseph Hayes and colleagues [1] (open-access available here) who drew on data derived from the excellent resource that is ALSPAC ("Charting the health of 14,500 families in the Bristol area to improve the health of future generations"). I'll be talking about other ALSPAC-derived data in upcoming posts too.

Based on a cohort of some 4600 children who provided blood samples (analysable blood samples no less) at aged 9 years old and "who completed the Hypomania Checklist (HCL-32)" at aged 22 years, researchers looked at how blood levels of C-reactive protein (CRP) and IL-6 might link in with later self-reported hypomanic symptoms.

Bearing in mind the focus on "immune activity in healthy individuals" (quite a few participants were excluded from the study as a result of reporting an infection in the week before samples were taken), researchers reported some interesting associations. As per the opening sentence: "Higher IL-6 levels in childhood were associated with adult hypomania features in a dose-response fashion." That being said: "Higher serum IL-6 levels at age 9 years were associated with female sex, non-white British ethnicity, lower SES, higher past psychological and behavioural problems and higher BMI" too. When it comes to BMI (body mass index), the observation that various immune system markers might be elevated is not necessarily a new idea.

Although childhood IL-6 levels were associated with later hypomanic symptoms (even when adjusted for the various potentially confounding variables), blood levels of CRP did not seem to show the same kind of relationship: "There was no evidence of an association between CRP levels and hypomanic symptoms." Similarly, when authors looked at the possibility of an association between the presence of atopic disease and hypomanic symptoms based on the inclusion of a parent-response question when their child participant was 10 years old - "Has a doctor ever actually said that your study child has asthma or eczema?" - the authors reported little indication of any connection.

At least one of the co-authors on the Hayes paper has some prior interest in the possible psychiatric manifestations of childhood inflammation as per other entries on this blog (see here). As much as I am intrigued by this area of research, you don't have to be a rocket scientist to understand the potential flaws in such a study where one or two biological variables are mapped on to a self-report questionnaire quite a few years later potentially excluding a myriad of factors not readily taken into account in a study like this. It would for example, be preferable to have seen multiple measures of CRP and/or IL-6 during childhood (say, every year) to see if any potential association between childhood inflammation and hypomanic symptoms holds. That also only IL-6 seemed to show some sort of connection maybe also implies that the generalised idea that 'inflammation' might tie into later psychiatric symptoms is perhaps a little bit too generalised. I could go on i.e. CRP is not the only pentraxin, IL-6 is not necessarily just a pro-inflammatory cytokine, etc.

In short, some interesting observations and yes "immunological understanding of major mental illness could potentially lead to novel approaches to diagnosis, prevention and treatment" but I think we need a lot more quality data on this topic.

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[1] Hayes JF. et al. Childhood interleukin-6, C-reactive protein and atopic disorders as risk factors for hypomanic symptoms in young adulthood: a longitudinal birth cohort study. Psychol Med. 2016 Aug 1:1-11.

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Hayes JF, Khandaker GM, Anderson J, Mackay D, Zammit S, Lewis G, Smith DJ, & Osborn DP (2016). Childhood interleukin-6, C-reactive protein and atopic disorders as risk factors for hypomanic symptoms in young adulthood: a longitudinal birth cohort study. Psychological medicine, 1-11 PMID: 27476619

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

Cytokines activating the kynurenine pathway in schizophrenia?

I'm a bit of a fan of tryptophan biochemistry on this blog. This quite remarkable aromatic amino acid and it's off-shoot metabolites, which appear to have no end of biological uses, have taken quite a bit of my blogging time down the years. Most recently was the suggestion that a metabolite slotting in between serotonin (5-HT) and melatonin might require quite a bit more investigation when it comes to at least some cases of autism (see here).

See ya later, President Fartfeathers.

The findings reported by Lilly Schwieler and colleagues [1] (open-access here) add to the scientific interest and their assertion that: "IL-6 [interleukin-6] induces the KYN [kynurenine] pathway, leading to increased production of the N-methyl-D-aspartate receptor antagonist KYNA [kynurenic acid] in patients with schizophrenia." IL-6 by the way, is a cytokine (chemical messenger of the immune system) which is normally taken to be a pro-inflammatory cytokine (see here). Kynurenine and it's metabolic relations, are yet another set of compounds derived from tryptophan. The kynurenic hypothesis of schizophrenia (see here) hints at some of the research history this compound (and metabolites) has with the condition.

The Schwieler paper is open-access but a few pointers might be in order:

• Looking at a "well-characterized cohort of olanzapine-treated patients with chronic schizophrenia" researchers set about looking at cerebrospinal fluid (CSF) levels of various cytokines compared to a small participant group of asymptomatic controls "free from current signs of psychiatric morbidity or difficulties in social adjustment at the time of sampling".
• Previously measured levels of "tryptophan metabolites of the KYN pathway" were also included in the study bundle. Researchers also looked at a possible 'interplay' between IL-6 and kynurenic acid in human astrocyte cultures. This involved stimulation of said cultures with IL-6 and measuring KYNA using triple quadrupole mass spectrometry.
• Results: "The CSF IL-6 concentration was elevated in patients with chronic schizophrenia compared with controls." No real surprises there considering what has been reported previously in this area of schizophrenia research [2] and the growing idea of inflammation and psychiatry being linked. 
• CSF levels of kynurenine and kynurenic acid were also elevated in the schizophrenia group compared to controls, but no significant differences were noted in the starting material (tryptophan) between the groups. Authors also confirmed that IL-6 did indeed significantly raise levels of kynurenic acid (KYNA) in astrocyte cultures.
• They conclude that "The increased production of KYNA in fetal human astrocytes following exposure of IL-6 shows that this cytokine is able to induce the activity of the KYN pathway." This process may also pertain to schizophrenia.

Aside from the limitations already pointed out by the authors in terms of some analytical issues and the spot sampling methodology employed, I might also point out that whilst participants with schizophrenia were all taking olanzapine (and other meds in some cases), the asymptomatic controls were "free from medication for at least 1 month". Granted olanzapine is not generally thought to directly impact on levels of IL-6 for example [3] but one can't discount that other, more indirect effects might come into play. Indeed, I'm going to be talking about olanzapine, gut bacteria and weight gain (see here) early in the New Year.

I'd like to introduce the paper by Johansson and colleagues [4] at this point, and their observations related to kynurenic acid and related metabolites in "cultured skin fibroblasts obtained from patients with bipolar disorder, schizophrenia or from healthy control individuals." Looking at cells specifically from participants (with all their biological heterogeneity), they similarly concluded that there was an "increase in ratio between neurotoxic 3-HK [3-hydroxykynurenine] and neuroinhibitory/neuroprotective KYNA following exposure to cytokines" in the bipolar and schizophrenia groups compared to controls. The 3-HK finding might be of even greater interest to schizophrenia given the suggestion of a link with redox modulation [5] and the idea that oxidative stress might be a factor to the condition [6].

What's more to say on this topic? Well, not much more aside from the fact that there may be a complicated relationship between immune function - immune signalling - and amino acid biochemistry which may very well impinge on presented behaviour. Such links also offer some interesting prospects for potential intervention too...

And to some music: Lower Than Atlantis - Here We Go.

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[1] Schwieler L. et al. Increased levels of IL-6 in the cerebrospinal fluid of patients with chronic schizophrenia - significance for activation of the kynurenine pathway. J Psychiatry Neurosci. 2014 Dec 2;39(6):140126.

[2] Kunz M. et al. Serum levels of IL-6, IL-10 and TNF-α in patients with bipolar disorder and schizophrenia: differences in pro- and anti-inflammatory balance. Rev Bras Psiquiatr. 2011 Sep;33(3):268-74.

[3] Hori H. et al. Effects of olanzapine on plasma levels of catecholamine metabolites, cytokines, and brain-derived neurotrophic factor in schizophrenic patients. Int Clin Psychopharmacol. 2007 Jan;22(1):21-7.

[4] Johansson AS. et al. Activation of kynurenine pathway in ex vivo fibroblasts from patients with bipolar disorder or schizophrenia: cytokine challenge increases production of 3-hydroxykynurenine. J Psychiatr Res. 2013 Nov;47(11):1815-23.

[5] Colín-González AL. et al. The Janus faces of 3-hydroxykynurenine: Dual redox modulatory activity and lack of neurotoxicity in the rat striatum. Brain Res. 2014 Nov 17;1589:1-14.

[6] Flatow J. et al. Meta-analysis of oxidative stress in schizophrenia. Biol Psychiatry. 2013 Sep 15;74(6):400-9.

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Schwieler L, Larsson MK, Skogh E, Kegel ME, Orhan F, Abdelmoaty S, Finn A, Bhat M, Samuelsson M, Lundberg K, Dahl ML, Sellgren C, Schuppe-Koistinen I, Svensson C, Erhardt S, & Engberg G (2014). Increased levels of IL-6 in the cerebrospinal fluid of patients with chronic schizophrenia - significance for activation of the kynurenine pathway. Journal of psychiatry & neuroscience : JPN, 39 (6) PMID: 25455350

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

An inflammatory autism subtype?

The paper from Harumi Jyonouchi and colleagues [1] (open-access) continues a theme from this author with their suggestion of "an imbalance in the production of inflammatory (IL-1ß and IL-6) and counterregulatory (IL-10) cytokines by ‘flare’ ASD-IS [autism spectrum disorder - inflammatory subtype] monocytes".

Panic on the streets of Birmingham...

'Flare' ASD-IS in this case refers to a coding given to a small participant group (n=24) who were: "defined as those with a history of fluctuating behavioral symptoms following immune insults (mainly microbial infection)" and who experienced: "worsening behavioral symptoms following immune insults, despite the resolution of acute conditions such as viral syndrome (that is, the resolution of other infectious symptoms if associated with a microbial infection, lack of fever, and no other acute physical symptoms associated with immune insults)".

Innate immune system functions (yes, cytokines again) were measured in flare ASD-IS and compared with results from other groups: (i) controls with autism with a history of "non-IgE mediated food allergy (NFA)" (n=20), (ii) ASD/non-NFA controls (n=20), and (iii) "three groups of non-ASD controls (non-ASD/NFA subjects (N =16), those diagnosed with pediatric acute onset-neuropsychiatric syndrome (PANS, N =18), and normal controls without NFA or PANS (N =16))". For those unfamiliar with the term PANS - pediatric acute onset-neuropsychiatric syndrome - this is a term originating from PANDAS (see here) and denotes an important condition bridging the link between infection and psychiatric symptomatology [2]. As if you needed telling...

Authors concluded that: "‘Flare’ ASD-IS PB Mo [peripheral blood monocytes] produced higher amounts of inflammatory cytokines (IL-1β and IL-6) without stimuli than ‘non-flare’ ASD-IS cells". They concluded that their findings: "support parental impression of worsening behavioral symptoms in the ‘flare’ state following immune insults" on the basis of the immune findings also linking in with behavioural descriptions at the time of sample collections.

Whilst including relatively small participant groups, it is the spread of presentations (not just based on diagnosis) which makes this paper stand out. I note that the authors also report that their results may: "indicate a possibility that monocytes from ASD-IS children also have intrinsic defects in regulatory mechanisms of IL-10 production". This is again, potentially important. Although there is still some misconception that cytokines are binary in function (either pro-inflammatory or anti-inflammatory) when the emerging data are suggesting it is very much more complicated than that (see here), the authors are hinting that inflammation with regards to autism may actually be down to issues with the opposing anti-inflammatory response over and above an upregulated pro-inflammatory response. Sort of like a fire tender not carrying enough water to put out a blaze. Indeed, work from this research team had previously hinted as much [3] (see also my previous post on this paper) as have other studies [4].

I am looking forward to seeing this research independently followed-up and reported on to further characterise those people with autism who such results are potentially relevant to. Whether gastrointestinal (GI) symptoms and dietary intervention might also be important factors [5] in such immune responses, also offers some potentially tantalising options for intervention...

Music to close: Rhapsody In Blue.

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[1] Jyonouchi H. et al. Cytokine profiles byperipheral blood monocytes are associated with changes in behavioral symptoms following immune insults in a subset of ASD subjects: an
inflammatory subtype? Journal of Neuroinflammation. 2014, 11:187

[2] Chang K. et al. Clinical Evaluation of Youth with Pediatric Acute Onset Neuropsychiatric Syndrome (PANS): Recommendations from the 2013 PANS Consensus Conference. J Child Adolesc Psychopharmacol. 2014 Oct 17.

[3] Jyonouchi H. et al. Immunological characterization and transcription profiling of peripheral blood (PB) monocytes in children with autism spectrum disorders (ASD) and specific polysaccharide antibody deficiency (SPAD): case study. J Neuroinflammation. 2012 Jan 7;9:4.

[4] Estes ML. & McAllister AK. Alterations in Immune Cells and Mediators in the Brain: It's Not Always Neuroinflammation! Brain Pathol. 2014 Nov;24(6):623-30.

[5] Jyonouchi H. et al. Dysregulated innate immune responses in young children with autism spectrum disorders: their relationship to gastrointestinal symptoms and dietary intervention. Neuropsychobiology. 2005;51(2):77-85.

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Jyonouchi H, Geng L, & Davidow AL (2014). Cytokine profiles by peripheral blood monocytes are associated with changes in behavioral symptoms following immune insults in a subset of ASD subjects: an inflammatory subtype? Journal of neuroinflammation, 11 (1) PMID: 25344730

Psychiatric effects of childhood inflammation?

"Higher levels of the systemic inflammatory marker IL-6 [interleukin 6] in childhood are associated with an increased risk of developing depression and psychosis in young adulthood". So said the paper by Golam Khandaker and colleagues [1] looking at the growing link between inflammation and psychiatry.

The bright light of Autumn @ Wikipedia 

The name Khandaker has appeared before on this blog (see here and see here), most recently with research looking at a possible link between the presence of a neurodevelopmental disorder and subsequent reports of psychotic experiences [2]. It's all rather fascinating research.

With the most recent investigation in mind...

• Two inflammatory markers, IL-6 and C-Reactive Protein (CRP), were the variables of choice as per their inflammatory link (see here and see here). Said inflammatory markers were analysed in blood samples from quite a nice cohort (~4500) of 9-year olds who took part in the ALSPAC initiative. 
• When their cohort were 18 years old, their mental health was assessed for things like depression and psychotic experiences (PEs) using various questionnaires and semi-structured interviews.
• Results: depending on whether participants fell into groups suggestive of low, medium or high inflammation on the basis of inflammatory markers seemed to have some effect on their mental health almost a decade later. So: "participants in the top third of IL-6 values compared with the bottom third at age 9 years were more likely to be depressed... at age 18 years". This finding was reported after correction for various potentially interfering variables.
• Additionally: "Risks of PEs and of psychotic disorder at age 18 years were also increased with higher IL-6 levels at baseline".
• The authors conclude: "Higher IL-6 levels in childhood were associated with subsequent risks of depression and PEs in a dose-dependent manner".

There is, as one might expect, some accompanying media interest in these results (see here). I was interested to see that Judy Van de Water commented on the Khandaker results. Regular readers of the autism research scene will probably already known about Dr Van de Water's interest in immune function (including inflammation) and autism previously talked about on this blog (see here for example). She is quoted talking about: "kids who get fevers more often and for longer periods of time may also have higher levels of inflammation". Mmm...

Whilst the Khandaker results are very interesting, as always, I do think there is more to do in this area. Aside from correlating spot analyses of inflammatory markers with events almost a decade later, IL-6 is portrayed as the bad guy in this scenario based on it's connection to systemic inflammation. But things are rarely so straight-forward as per the paper by Scheller and colleagues [3] (open-access) on the two faces of IL-6. To boil depression and PEs solely down to childhood inflammation also does little to say how complex such conditions are; something which Dr Khandaker's other research has also hinted at. I've also talked about other correlates and things like depression including vitamin D levels (see here), certain bacteria (see here) and possibly even something like dietary components (see here) showing potential involvement. It's complicated as I said.

Oh and headlines like 'Could aspirin and ibuprofen help fight depression?' are perhaps a little premature at this time... but at least they didn't suggest paracetamol.

Music to close. MAGIC! and Rude (Marry that Girl!).

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[1] Khandaker GM. et al. Association of Serum Interleukin 6 and C-Reactive Protein in Childhood With Depression and Psychosis in Young Adult Life. JAMA Psychiatry. 2014. August 13.

[2] Khandaker GM. et al. A population-based longitudinal study of childhood neurodevelopmental disorders, IQ and subsequent risk of psychotic experiences in adolescence. Psychol Med. 2014 Apr 25:1-10.

[3] Scheller J. et al. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2011; 1813: 878-888.

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Golam M. Khandaker, Rebecca M. Pearson, Stanley Zammit, Glyn Lewis, & Peter B. Jones (2014). Association of Serum Interleukin 6 and C-Reactive Protein in Childhood With Depression and Psychosis in Young Adult Life JAMA Psychiatry : doi:10.1001/jamapsychiatry.2014.1332

Ultrafine particulate matter air pollution, mice and autism

Reading the headline "Study links air pollution to autism, schizophrenia" in a media piece about the study by Joshua Allen and colleagues* (open-access here) made me want to delve a little more into this research. I've talked before about air pollution and autism (see here) on this blog. Although a healthy degree of scepticism is to be expected with any autism correlation, particularly when it comes to something as generalised as air pollution (or pesticide exposure) there is a growing research interest in how this aspect of the environment may have some bearing on autism risk.

Cloudy with a chance of... @ Wikipedia 

A few details about the Allen study might be useful:

• This was a study involving mice. I'll repeat that: this was a study involving mice. It involved exposing a particular strain of mouse, modelled to represent a particular age "during early postnatal development" to "human relevant levels" of air pollution in the form of ultrafine particulates (<100 nm).
• Mouse brains were analysed at different time periods following exposure (24 hours, 40 days and 270 days after) looking at brain morphology, neurotransmitter levels and those all important immune system chemicals involved in processes like inflammation: the cytokines.
• Results: bearing in mind some quite detailed control of the amount of air pollution exposure mimicking ambient doses near roadways, quite a few effects were noted. There was for example, "a persistent dilation of the lateral ventricles" induced by CAPS (concentrated ambient ultrafine particles) "preferentially in male mice". I believe this is called ventriculomegaly.
• "CAPS induces brain region- and sex-dependent alterations in cytokines and neurotransmitters in both males and females". So in male mice, "increased hippocampal glutamate" among other things was observed. In females, "CAPS reduced hippocampal GABA" and more.
• Of the various cytokines included for analysis, an old friend ranked up there when it came to some of the results obtained: IL-6. Again, there seemed to be region and sex specific alterations to this cytokine and some of them were "unanticipated" as per the lower levels of IL-6 and other relations in certain areas. IL-6 shares some features of a pro-inflammatory and anti-inflammatory cytokine [2] although more often than not, it is the pro-inflammatory effects which get the headlines [3]. 
• The word 'microglia' also crops up in the Allen results. "CAPS altered IBA-1 immunostaining in the anterior commissure and hippocampus only in males". IBA-1 is a protein expressed in microglia.
• The authors conclude: "Collectively these data show a dramatic susceptibility of male mice to environmentally relevant levels of early postnatal air pollution exposure, with effects that persist into adulthood and cause permanent neuropathology characterized by ventricular enlargement, a pathology not seen in females".

Reiterating again that this was a study of mice and that mice are mice not humans, these are some intriguing data presented by Allen and colleagues. The focus on male mice slots nicely into the [seemingly] over-representation of autism in boys and men. Elevations in glutamate - hippocampal glutamate [4] in male mice - might also overlap with the growing fascination that autism and schizophrenia research have with this neurotransmitter (see here). Some light reading around the finding of "CAPS-induced ventricular enlargement" observed in males leads down some interesting paths such as a possible relationship with agenesis of the corpus callosum [5] reported to be "a major risk factor for developing autism" according to some authors [6]. In short, there are plenty of correlations seemingly heading back to conditions like autism.

But... there are a few important points to bear in mind before we get too carried away. First and foremost, nothing is reported in the Allen paper around mouse behaviour and how that may or may not have overlapped with other mouse data trying to model autism. One should always be a little cautious when one hears the words 'autistic behaviour' when it comes to a mouse and whether for example, they vocalise or not, or decide to bury their marbles in a particular way as being representative of facets of the condition. It isn't but it's some of the best animal model behaviour that we currently have including the rat models. Allen et al on this occasion reported nothing about behaviour and how it may or may not link to their physiological findings. 

Second is a question already asked by someone in/on the Twittersphere: "Air pollution was so much worse many decades ago yet autism rates staggeringly higher today, not then" (thanks Jill). This is an important point which may have lots of different answers bearing in mind your acceptance that things were worse back in olden times (see here for more news from urban China). Perhaps one of the most relevant issues at the moment was the study by Heather Volk and colleagues [7] discussed in a previous post (see here) talking about gene x environment interactions. If one assumes that genes, gene expression, are being affected by air pollution and that some people might already be more 'at risk' than others, there could be something more to do in this area of investigation.

Finally, Allen and colleagues seemed to have focused all their attention on the brain of their brave mouse participants. They don't talk about whether other organs or biological systems were affected by air pollution. I know that I'm probably going to get some rolling of the eyes for this but harking back to other mouse models of autism, I note some interest in things like the gastrointestinal (GI) tract to be an upcoming area (see here for example on the VPA mouse model). Assuming that the GI tract will also an important exposure point for air pollution [8], could there be merit in looking at this and other organs too all in the name of the gut-brain axis? Also, not forgetting lungs (see here) and skin as important exposure sites too.

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[1] Allen JL. et al. Early Postnatal Exposure to Ultrafine Particulate Matter Air Pollution: Persistent Ventriculomegaly, Neurochemical Disruption, and Glial Activation Preferentially in Male Mice. Environ Health Perspect. 2014 Jun 5.

[2] Scheller J. et al. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2011; 1813: 878-888.

[3] Rincon M. Interleukin-6: from an inflammatory marker to a target for inflammatory diseases. Trends in Immunology. 2012; 33: 571-577.

[4] Kraguljac NV. et al. Increased Hippocampal Glutamate and Volumetric Deficits in Unmedicated Patients With Schizophrenia. JAMA Psychiatry. 2013; 70.

[5] Amato M. et al. Fetal ventriculomegaly, agenesis of the corpus callosum and chromosomal translocation--case report. J Perinat Med. 1986;14(4):271-4.

[6] Paul LK. et al. Agenesis of the corpus callosum and autism: a comprehensive comparison. Brain. 2014; April 25.

[7] Volk HE. et al. Autism spectrum disorder: interaction of air pollution with the MET receptor tyrosine kinase gene. Epidemiology. 2014 Jan;25(1):44-7.

[8] Kaplan G. Air pollution and the inflammatory bowel diseases. Inflamm Bowel Dis. 2011 May;17(5):1146-8.

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Allen JL, Liu X, Pelkowski S, Palmer B, Conrad K, Oberdörster G, Weston D, Mayer-Pröschel M, & Cory-Slechta DA (2014). Early Postnatal Exposure to Ultrafine Particulate Matter Air Pollution: Persistent Ventriculomegaly, Neurochemical Disruption, and Glial Activation Preferentially in Male Mice. Environmental health perspectives PMID: 24901756

Meta-analysing cytokine involvement in autism

A fairly brief post today to draw your attention to the "systematic review and meta-analysis" paper by Masi and colleagues [1] on all-things cytokine in relation to autism. They concluded that there was "strengthening evidence of an abnormal cytokine profile in ASD [autism spectrum disorder] where inflammatory signals dominate". I should point out that other authors have reached similar conclusions in previous reviews [2] and here also [3].

"The herring does not fry here" @ Wikipedia 

In case you didn't know, cytokines are the chemical messengers of the immune system (see here) and perform various important tasks in relation to processes such as inflammation (see here). Autism research has a growing respect for the role of cytokines in quite a few cases of autism as per the growth in the amount of research literature available in this area (see here). I've covered quite a few papers focused on cytokines and autism on this blog (see here and see here for example) down the years.

The Masi paper is an important one because it gathered quite a bit of the peer-reviewed research literature mentioning cytokines and autism together and for want of better words, 'statistically spat' out the sum total of the findings from the various studies. A couple of old friends "were significantly higher in the participants with ASD" compared with control populations including interleukin 1-beta (IL-1β), IL-6, interferon-gamma (IFNγ) and monocyte chemotactic protein-1 (MCP-1) potentially indicative of a more pro-inflammatory state. A general reduction in the levels of more 'anti-inflammatory' molecules such as transforming growth factor-beta 1 (TGF-β1) [4] added to proceedings.

It's perhaps slightly unfair to say that these and other cytokines noted to be generally elevated in relation to autism are solely pro-inflammatory cytokines (i.e. inducing or maintaining inflammation) because that's not necessarily the way they always work. IL-6 for example, is now realised as being both a pro-inflammatory and anti-inflammatory molecule [5]. That being said, the growing recognition that inflammation and inflammatory processes may have some bearing on brain and behaviour (see here) ties in well with the Masi findings and where science perhaps needs to start looking with greater vigour if one is going to understand the interplay between immune function and psychiatry. As the authors note: "A better understanding of the inflammatory biology of ASD and possible associations with behavioral impairments and non-diagnostic features warrants further investigation and may have significant therapeutic implications". I can't argue with those sentiments, although as per the recent paper by Careaga and colleagues [6] how science goes about looking at that relationship is going to be important.

Oh, and on the topic of MCP-1, the paper by Zerbo and colleagues [7] observing elevations in the levels of this cytokine in newborn blood spots from those subsequently diagnosed with autism is indeed timely... (and again reiterates the potentially usefulness of those drops of blood which many children give in their earliest days welcomed into the big, wide world).

Music now. Daft Punk, and before Get Lucky, they were already Around the World.

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[1] Masi A. et al. Cytokine aberrations in autism spectrum disorder: a systematic review and meta-analysis. Molecular Psychiatry. 2014. June 17.

[2] Goines PE. & Ashwood P. Cytokine dysregulation in autism spectrum disorders (ASD): possible role of the environment. Neurotoxicol Teratol. 2013 Mar-Apr;36:67-81.

[3] Onore C. et al. The role of immune dysfunction in the pathophysiology of autism. Brain Behav Immun. 2012 Mar;26(3):383-92.

[4] Qian L. et al. Potent anti-inflammatory and neuroprotective effects of TGF-beta1 are mediated through the inhibition of ERK and p47phox-Ser345 phosphorylation and translocation in microglia. J Immunol. 2008 Jul 1;181(1):660-8.

[5] Scheller J. et al. The pro- and anti-inflammatory properties of the cytokine interleukin-6. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2011; 1813: 878-888.

[6] Careaga M. et al. Inflammatory profiles in the BTBR mouse: How relevant are they to Autism Spectrum Disorders? Brain Behav Immun. 2014 Jun 14. pii: S0889-1591(14)00171-8.

[7] Zerbo O. et al. Neonatal cytokines and chemokines and risk of Autism Spectrum Disorder: the Early Markers for Autism (EMA) study: a case-control study. Journal of Neuroinflammation 2014, 11:113

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Masi, A., Quintana, D., Glozier, N., Lloyd, A., Hickie, I., & Guastella, A. (2014). Cytokine aberrations in autism spectrum disorder: a systematic review and meta-analysis Molecular Psychiatry DOI: 10.1038/mp.2014.59