"Zika Virus as a Possible Risk Factor for Autism"?

The paper by Vianna and colleagues [1] provides the blogging fodder today, discussing a potentially important topic around whether "the fetal brain infection caused by ZIKV [Zika virus] could predispose to ASD [autism spectrum disorder]."

Just before anyone gets the impression that a *link* has been found between Zika virus exposure and autism or autism spectrum disorder (ASD), I'm going to say no, not yet. The current peer-reviewed evidence at the time of writing this post has not identified autism as an outcome following exposure to the Zika virus. That's not to say that various other neurodevelopmental issues have not been associated with Zika virus exposure (see here for example) or that Zika virus exposure might not have biological consequences akin to that noted in some autism [2]. But as far as I'm aware, there is no peer-reviewed science yet suggesting that autism is for example, over-represented among those exposed to Zika virus in-utero. Not yet anyway.

Nonetheless, Vianna et al go through a number of lines of evidence suggesting how autism *could* be an outcome of Zika virus exposure. They start by talking about how Zika virus exposure during pregnancy can have a devastating effect on the developing child (see here) as a function of its teratogenic status. Severe microcephaly (small head size), a cardinal feature linked to infants' Zika virus exposure during pregnancy, has notable effects on the brain and its development, and is one of the more noticeable effects associated with Zika virus exposure in-utero. But further: "this phenotype is now considered only “the tip of the iceberg” and there is a spectrum of less severe abnormalities after congenital Zika infection."

Authors go on to talk about how pregnancy is a time of 'change' when it comes to maternal immune system functions, as a reprogrammed immune system has to become 'tolerant' of the developing foetus. Such reprogramming means that the foetus can survive and thrive. It also however means that the maternal immune system might be more susceptible to certain infections, where for example "opportunistic infections... take advantage." This is also the point where autism enters into the conversation, and the idea that exposure to various infections (viral and bacterial) occurring during pregnancy "can alter [offspring] brain development and are associated with alterations, such as brain calcifications, microcephaly, and neurodevelopmental disorders." I've covered a few possible examples on this blog with autism in mind (see here and see here).

Putting all this together, as well as talking about some of the immune system chemistry that *might* link Zika virus exposure and autism, and the authors come up with a model of 'neuroimmunomodulation' talking about "an ineffective anti-viral response" and increased levels of pro-inflammatory cytokines as being potentially important. I'll also provide another quote from Vianna which is similarly intriguing: "Moreover, in the case of ZIKV, previous infections with other flaviviruses, such as dengue virus and yellow fever, may trigger a secondary immune response of differential magnitude given the great molecular similarity of some immunogenic epitopes among these correlated viruses."

Reiterating that there is currently no link between Zika infection and risk of autism, I do find the Vianna paper interesting. It offers some testable hypotheses that could examined in the lab and beyond. It also provides some further support for the various surveillance and monitoring initiatives that remain in place with regards to Zika virus and the promise of further important data to come from them.

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[1] Vianna P. et al. Zika Virus as a Possible Risk Factor for Autism Spectrum Disorder: Neuroimmunological Aspects. Neuroimmunomodulation. 2019 Jan 10:1-8.

[2] Beys-da-Silva WO. et al. Zika Virus Infection of Human Mesenchymal Stem Cells Promotes Differential Expression of Proteins Linked to Several Neurological Diseases. Mol Neurobiol. 2018 Oct 30.

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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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Regressive vs. non-regressive autism: limited chemical differences noted

The paper published by Antonio Gomez-Fernandez and colleagues [1] examining whether or not there may be some potentially important biological differences as a function of reported regression vs. no regression in autism provides the blogging fodder today. Not for the first time has the immune system and 'regressive autism' been mentioned in the peer-reviewed science literature (see here and see here), but the current work focuses on the examination of various immune system and other related compounds: in a seemingly well-defined cohort: "Analyses of plasma molecules, such as cathepsin, IL1β, IL6, IL8, MPO, RANTES, MCP, BDNF, PAI NCAM, sICAM, sVCAM and NGF."

"Fifty-four children (45 males and nine females) aged 2-6, who were diagnosed with ASD [autism spectrum disorder], and a control group of 54 typically-developing children of similar ages were selected." Authors relied on quite an extensive battery of assessments looking at behaviour, alongside their use of the DSM-5 diagnostic criteria for autism (see here). Also accompanying physical examination "with a special emphasis on neurological and nutritional status", authors garnered blood samples from participants (overnight fasting) for their immune system and related functioning evaluations.

"The group of ASD children was further divided into two subgroups based on the presence or absence of neurodevelopmental regression during the first two years of life, which was assessed using a five-item questionnaire following the guidelines used by the Autism Diagnostic Interview-Revised (ADI-R) for the evaluation of this process." The ADI-R has been previously discussed on this blog in relation to regression in autism (see here). And just in case you might not be totally convinced that regression can be part of a pathway to autism, here's some more evidence for you (see here)...

Results: "there were 20 children included in the AMR [neurodevelopmental regression] subgroup and 32 in the ANMR [without neurodevelopmental regression] subgroup; two children could not be classified in these subgroups because they were adoptees, allocated by a national adoption agency." Bearing in mind that we cannot rule out any recruitment bias that might have leaned towards including those with regressive autism on the Gomez-Fernandez study, the figure of approaching 40% of their cohort showing such a regressive profile is notable. I'd also draw your attention to the finding that the behavioural profile for the regressive group (AMR) was also significantly different from the non-regressive group (ANMR) insofar as perhaps painting a picture of greater [group] autism severity...

Interestingly, the study did not show too many immune system and other compound differences between those diagnosed with autism and the asymptomatic (for autism) control group. So: "No differences were found between the two groups in terms of the cytokine and adhesion molecule levels studied, except for NGF [nerve growth factor], in which the group of ASD children was found to have twice the plasma levels compared to the control group." NGF is no stranger to autism research, and other studies have come to a similar conclusion [2].

When it came to examining results based on comparing the regressive (AMR) and non-regressive (ANMR) groupings, things got slightly more interesting but again no complicated pattern of difference was noted. So, for the ANMR (non regression) grouping: "lower plasma levels of the NCAM adhesion molecule were detected compared to the levels in the AMR subgroup and the control group. This ANMR group also exhibited higher NGF levels than the typically-developing children, which could indicate an alteration in neuronal development." Again, adhesion molecules have been mentioned in other autism research (see here).

"In conclusion, the results of this study show that there is not a typical profile for the expression of relevant plasma cytokines, adhesion molecules or growth factors in children with ASD compared with that in typically-developing children." OK, there are caveats to the phrasing used by the authors; not least that the participant numbers were quite small in the Gomez-Fernandez study and the idea that within the very heterogeneous autism spectrum, there may be smaller groupings (phenotypes) that perhaps show a tendency to greater immune system and related 'issues' (see here). But there are also some strengths attached to the Gomez-Fernandez study; not least the study "benefits from a careful selection of children of similar ages, as well as the complete diagnosis of ASD with multiple tests, clinical follow-up and associated complementary tests."

Questions still remain. Perhaps an important one is the question around why some children show a regressive pattern of behaviour as part of their path to a diagnosis of autism? Yes, issues such as infection do seem to be part-and-parcel of the clinical profile for some (see here and see here for examples) and perhaps more detailed focus is required in such areas. But much like a group showing the opposite of regressive autism - those who seemed to 'grow out' of autism - currently thought to include as many as one in ten (see here), a wider range of biological as well as psychometric measures are required to help pick out potentially important mechanisms pertinent to the idea that autism is not necessarily 'hard-wired' for all...

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[1] Gomez-Fernandez A. et al. Children With Autism Spectrum Disorder With Regression Exhibit a Different Profile in Plasma Cytokines and Adhesion Molecules Compared to Children Without Such Regression. Front. Pediatr. 2018. September 26.

[2] Dinçel N. et al. Serum nerve growth factor levels in autistic children in Turkish population: a preliminary study. Indian J Med Res. 2013 Dec;138(6):900-3.

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Endogenous Retroviruses (ERVs) and autism continued

It's been a while since I last mentioned Human Endogenous Retroviruses (HERVs) in the context of autism (see here) on this blog. I still however retain an interest in how these 'fossil viruses' - remnants of our ancient battle with various viruses down the ages that are still present in the genome - *might* impact on our health and well-being for all-manner of reasons.

One name in particular has been at the forefront of the work on HERVs with particular reference to autism (and ADHD and beyond) - Emanuela Balestrieri - and alongside, quite a significant peer-reviewed research record has been built up (see here). A new paper from the Balestrieri 'research group' (if I can call it that) continues their research journey in this area, from Chiara Cipriani and colleagues [1]. The aim of the research game this time around was to look at two popular mouse models of autism - "inbred BTBR T+tf/J mice and CD-1 outbred mice prenatally exposed to valproic acid (VPA)" - and examine the transcriptional activity of various ERVs (that's ERVs not HERVs 'cos these were mice) compared with control mouse strains ("C57BL/J and CD-1 untreated mice").

From what I gather, there were two main elements to the Cipriani study: (1) assessing intracisternal A-particle elements (IAPs) and Type II early transposons (ETns) in blood and brain samples of those autism-related mouse models, and (2) evaluating "the transcriptional activity of proinflammatory cytokines (IL-1β, IL-6, TNF-α) and Toll-like receptors (TLR3 and 4)... in whole embryos and, from the offspring at different time after birth, in blood and brain samples to investigate on the hypothesised link between the ERVs transcriptional activity and the immune system." If much of that sounds like a foreign language to you, well, you're not alone. From what I gather, intracisternal A-particle elements (IAPs) are "endogenous retroviral sequences" that can "induce genomic mutations and cell transformation by disrupting gene expression." Type II early transposons (ETns) are "mobile members of the repetitive early transposon family of mouse long terminal repeat (LTR) retroelements and have caused a number of mutations by inserting into genes" [2]. Clear as mud right?

Results: "In the two distinct mouse models analysed, the transcriptional activity of the ERV families was significant higher in comparison with corresponding controls, in whole embryos, blood and brain samples." Taking the BTBR mice first, in comparison to control mice (C57BL6/J), pretty much all of the expression levels of the ERV genes looked at were higher in embryos. In both blood and brain, over the course of 120 days post-natally, ERV expression was also higher than that in control mice. I say all this acknowledging that absolute number of 'mouse participants' in this study was relatively small.

Also, compared with 'vehicle treated' control mice, those offspring embryos exposed to maternal VPA ("VPA mice were produced by treating outbred pregnant CD-1 female mice with a single dose of VPA (500 mg/kg, sc) at gestational day (GD) 10.5") also saw higher transcriptional activity of 'most' ERVs. The data from blood and brain samples were similarly interesting; in brain samples in particular, those exposed to VPA always showed higher ERV transcriptional activity than non-exposed controls, and notably so. Insofar as the inflammatory cytokine look-see side of things, well in both autism-related mouse models, "the expression levels of the proinflammatory cytokines and TLRs [Toll-like receptors] were significantly higher than controls."

What does this all mean? Well, bearing in mind that mice are mice and not humans (see here), researchers conclude that "results are in agreement with our previous data showing a distinctive expression profile of some human HERV families in blood samples from two different cohorts of young autistic patients and support the hypothesis that ERVs could be implicated in ASD." The fact that authors used not one but two mouse models of autism - including something akin to an 'acquired' autism phenotype in that VPA mouse model - and found similar things adds something a little extra to their findings. The possibility of a tie-up between ERV expression and immune system 'activation' requires more investigation save any sweeping generalisations about linkage. I am wondering if autism research in this area might however benefit from looking at some initial research on myalgic encephalomyelitis (ME) (see here) and onward some chatter about a possible link between HERV proteins acting as 'superantigens' in immune system terms.

Hopefully further research will be forthcoming sooner rather than later...

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[1] Cipriani C. et al. High expression of Endogenous Retroviruses from intrauterine life to adulthood in two mouse models of Autism Spectrum Disorders. Sci Reports. 2018; 8: 629.

[2] Baust C. et al. Structure and Expression of Mobile ETnII Retroelements and Their Coding-Competent MusD Relatives in the Mouse. Journal of Virology. 2003; 77: 11448-11458.

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Cytokines and chemokines in depression point to immune system involvement: meta-analysed

Two papers are presented for your reading pleasure today, both covering a topic of growing importance on how at least some types of depression very much seem to show some immune system involvement whether causative or as part of the illness/condition course.

The first paper is by Leighton and colleagues [1] and provides results based on a systematic review and meta-analysis of the literature pertinent to the role of inflammation or chemicals involved in inflammatory processes ("chemotactic cytokines (chemokines)") in relation to depression. The authors begin their paper abstract with the words "Inflammatory illness is associated with depression" and concluded that their collected results "finds evidence linking abnormalities of blood chemokines with depression in humans."

The second paper is by Köhler and colleagues [2] and, although published slightly earlier this year (2017), similarly set about examining "measured cytokine and chemokine levels in individuals with major depressive disorder (MDD) compared to healthy controls (HCs)" also using a systematic review and meta-analysis. Their results similarly concluded that "a cytokine/chemokine profile [was] associated with MDD."

Combined these two review papers (and other research) provide a compelling case for immune system involvement in at least some cases or classes of cases of depression. Alongside other research [3] asking where the inflammation comes from in potential cases of 'inflammatory depression', there is still much to do in this promising area of interface between immune system/function and psychiatric/behavioural presentation (see here). Then also comes another potentially important implication from such studies: attend to the cytokine/chemokine profile and treat the depression? (see here). I say this with no medical advice given or intended and also acknowledging that the immune system is a mighty, mighty complex thing (see here)...

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[1] Leighton SP. et al. Chemokines in depression in health and in inflammatory illness: a systematic review and meta-analysis. Molecular Psychiatry. 2017. Nov 14.

[2] Köhler CA. et al. Peripheral cytokine and chemokine alterations in depression: a meta-analysis of 82 studies. Acta Psychiatr Scand. 2017 May;135(5):373-387.

[3] Berk M. et al. So depression is an inflammatory disease, but where does the inflammation come from? BMC Medicine. 2013;11:200.

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Pregnancy hypertension and offspring autism reloaded

"There is growing awareness that prenatal adversity may increase the risk of autism spectrum disorder (ASD)."

Go on.

"These findings indicate that HDP [hypertensive disorders of pregnancy] exposure may increase the risk of ASD in the offspring."

So said the findings reported by Eileen Curran and colleagues [1] whose results have previously appeared on this blog with hypertension in mind (see here). I'm not altogether sure if this latest publication from this group represents 'new data' or is the same as/similar to that previously published [2]. It doesn't really matter to be honest given that the latest publication also includes a research addition insofar as the examination of "cytokine expression in the serum of women with pre-eclampsia, which is the most common HDP, and whether exposure of foetal neurons to this serum could change patterns of neuronal growth."

Drawing on data from some 13,000 children whose details were included in the Millennium Cohort Study (MCS) researchers, after "adjusting for several potential confounders including maternal alcohol consumption, education, depression, age, and poverty status", observed "a significant association between HDP and a twofold increased risk of ASD." They also reported that: "exposure of foetal cortical neurons to 3% serum isolated from women with an established HDP increased neuronal growth and branching in vitro." The authors have reported similar things with attention-deficit/hyperactivity disorder (ADHD) in mind too [3]; something that might be particularly important in the context of autism and ADHD overlapping in quite a few people (see here).

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[1] Curran EA. et al. Exposure to Hypertensive Disorders of Pregnancy Increases the Risk of Autism Spectrum Disorder in Affected Offspring. Mol Neurobiol. 2017 Oct 3.

[2] Curran EA. et al. Hypertension in pregnancy and autism spectrum disorder in a British cohort: Long term consequences for mother and child. Pregnancy Hypertension: An International Journal of Women's Cardiovascular Health. 2016; 6: 153.

[3] Curran EA. et al. The effect of hypertensive disorders of pregnancy on the risk of attention-deficit/hyperactivity disorder in the offspring: Long term consequences for mother and child. Pregnancy Hypertension: An International Journal of Women's Cardiovascular Health. 2016; 6: 169–170.

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Hornig, Lipkin and chronic fatigue syndrome again

Drs Mady Hornig and Ian Lipkin once again provide some fodder for this blog, continuing one of their important research themes on how chronic fatigue syndrome (CFS) (sometimes also referred to as myalgic encephalomyelitis, ME) might show some important immune-related issues [1].

This research tag-team and the teams of dedicated scientists who surround them are making some real progress with regards to the idea that ME/CFS is a physical condition (not psychosomatic and not 'biopsychosocial') with some readily identifiable biological features potentially accompanying cases. Of course we're not there just yet when it comes to a biological test for ME/CFS but science has at least started down that particular research path...

With accompanying media attention in tow (see here), the focus of the most recent results were on how disease sub-types might be important to CFS and specifically, how: "Immune signatures in the central nervous system of ME/CFS patients with atypical features may be distinct from those with more typical clinical presentations."

Authors described how cerebrospinal fluid (CSF) samples from "32 ME/CFS cases with classical features and presentations and 27 ME/CFS cases with atypical features or clinical presentations" were included for analysis. On what basis was 'typical' and 'atypical' described? Well: "The ‘classical’ (C-ME/CFS) group had acute onset of disease marked by a prodrome consistent with infection; ‘atypical’ (A-ME/CFS) ME/CFS patients met full diagnostic criteria for ME/CFS at onset of their illness, but had a less standard onset of ME/CFS and/or developed other disorders after illness onset of ME/CFS." Interestingly one person included in the A-ME/CFS group was described as having Gulf-War Illness (another important condition talked about on this blog).

The results: various cytokines (chemical messengers of the immune system) were assayed for and with some nifty statistical 'corrections' authors reported some potentially important differences between the groups. So: "We found discrete differences in immune signatures of the CNS in ME/CFS subjects with atypical presentations that included sparse inter-cytokine networks and lower levels of two inflammatory mediators, the Th17 cytokine, IL17A, and the IFNγ- and TLR4-induced chemokine, CXCL9." All-in-all results suggested a "less robust CNS immune activation in A-ME/CFS."

Much more research is required in this area for sure. But these results are interesting and pertinent to the idea that within the heterogeneity (where have a I heard that before?) of CFS/ME, there may be quite a few phenotypes and subgroups that might be readily separable with a little biological research effort. Does this therefore mean when we talk about the pluralisation of lots of labels (the autisms, the schizophrenias, the depressions, etc), we might also one day called it 'the chronic fatigue syndromes'? Well, I've kinda speculated about this before in the peer-reviewed domain...

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[1] Hornig M. et al. Immune network analysis of cerebrospinal fluid in myalgic encephalomyelitis/chronic fatigue syndrome with atypical and classical presentations. Transl Psychiatry. 2017 Apr 4;7(4):e1080.

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Hornig M, Gottschalk CG, Eddy ML, Che X, Ukaigwe JE, Peterson DL, & Lipkin WI (2017). Immune network analysis of cerebrospinal fluid in myalgic encephalomyelitis/chronic fatigue syndrome with atypical and classical presentations. Translational psychiatry, 7 (4) PMID: 28375204

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

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.

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[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.

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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

A distinctive microbial signature in kids with autism and GI issues?

"Our findings identify distinctive mucosal microbial signatures in ASD [autism spectrum disorder] children with FGID [functional gastrointestinal disorders] that correlate with cytokine and tryptophan homeostasis."

So said the study results published by Ruth Ann Luna and colleagues [1] who "compared mucosa-associated microbial communities in children with ASD to previous reports characterizing stool in this population" among other things. If you are eating breakfast/lunch/dinner at the time of reading this post, maybe give it a few minutes before reading on...

So rectal biopsies and blood specimens were the samples under investigation and the focus was very much on those children on and off the autism spectrum who also presented with various functional bowel issues. Just before anyone starts to question the ethics of taking biopsies and the like, the authors expand by reporting that participants were "undergoing a lower endoscopy for one of the following symptoms: abdominal pain, altered stool patterns, or painless bright red blood per rectum." In these days of health inequality attached to the label of autism (see here for an example), these were children who were being investigated for their bowel issues and not being unnecessarily subjected to such invasive techniques just for the sake of science.

The various analyses undertaken on those blood and biopsy samples were pretty wide-ranging. Bacterial species present in mucosal samples and their supernatants were included but so researchers also looked at cytokines (various chemical markers linked to immune function among other things) in blood samples and supernatants and levels of "serotonergic metabolites" (chemicals related to the aromatic amino acid tryptophan) pertinent to their "microbiome-neuroimmune signatures" hypothesis testing. Bear in mind that when it comes to the neurotransmitter called serotonin (5-HT), the gut truly is the second brain.

Results: taking into account the relatively small participant numbers included for study - "ASD children with functional GI disorders (ASD-FGID, n=14), as compared to neurotypical (NT) children with (NT-FGID, n=15) and without abdominal pain (NT, n=6)" - there were some interesting, if not unexpected, results to be seen. "Principal component analysis showed clear separation between the ASD-FGID group and the NT-FGID and NT groups" on the basis of the bacterial communities present in those mucosal samples. In the autism group, several mucosa-associated Clostridiales species were predominant as per that noted in other independent findings (see here). Interestingly authors also observed "marked decreases in Dorea and Blautia, as well as Sutterella" species perhaps contrasting with other research in this area (see here). I'll let readers trawl through the other bacterial families talked about in the paper including those potentially linked to the presence of specific functional bowel states derived from questionnaire data from participants.

Looking at any potential associations between mucosal bacterial communities, cytokines and those tryptophan metabolites, researchers also reported some important, if preliminary, observations. So: "Group comparisons revealed that IL-6 [interleukin 6] and tryptophan release by mucosal biopsies was highest in ASD children with abdominal pain, whereas serotonergic metabolites were generally elevated in children with FGIDs." I'd like to see these findings replicated in larger groups before I make anymore of what their potential significance could be but it is intriguing that pain might play some hand in immune signalling and the production of amino acid metabolites. More so when one considers other related research [2].

On the back of a recent post talking about blood-based 'biomarkers' pertinent to more pathological bowel states occurring alongside cases of autism (see here) it is good to see that autism + GI issues is starting to receive a little more scientific attention. It shows that science has moved on from the question 'are bowel symptoms over-represented when it comes to autism?' (answer: yes) and actually started to look at the questions of 'why? and 'how?' The focus on gut bacteria is a worthy cause (see here) and if replicated and found to be important, opens up various intervention options derived from work in other areas of medicine (see here). Indeed, there is a 'watch this space' call for investigations looking at probiotics and autism for example (see here) with the promise of more to come [3]. Oh, and don't forget the good old 'gut-brain axis' when it comes to a possible tie-up between bowel and brain with at least some autism in mind.

But for now, autism, gut disorder, gut bacterial composition, mucosal immune function and little old tryptophan and its metabolites get some well deserved combined research attention...

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[1] Luna RA. et al. Distinct microbiome-neuroimmune signatures correlate with functional abdominal pain in children with autism spectrum disorder. CMGH Cellular and Molecular Gastroenterology and Hepatology. 2016. Dec 11.

[2] Ahmad SF. et al. Imbalance between the anti- and pro-inflammatory milieu in blood leukocytes of autistic children. Mol Immunol. 2016 Dec 24;82:57-65.

[3] Navarro F. et al. Can probiotics benefit children with autism spectrum disorders? World J Gastroenterol. 2016 Dec 14;22(46):10093-10102.

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Luna, R., Oezguen, N., Balderas, M., Venkatachalam, A., Runge, J., Versalovic, J., Veenstra-VanderWeele, J., Anderson, G., Savidge, T., & Williams, K. (2016). Distinct microbiome-neuroimmune signatures correlate with functional abdominal pain in children with autism spectrum disorder CMGH Cellular and Molecular Gastroenterology and Hepatology DOI: 10.1016/j.jcmgh.2016.11.008

Maternal immune activation (MIA) and Old World monkeys

Old World monkeys detailed in the title of this post, specifically refers to a type of animal called a rhesus macaque who were the 'participants' of choice as detailed in a recent study by Destanie Rose and colleagues [1] looking at a concept called maternal immune activation (MIA).

Those who followed this blog down the years will no doubt have seen me discuss MIA before in the context of autism and/or schizophrenia (see here for example). The basic theory is that whilst in-utero and enjoying approximately nine months in a warm and comfortable environment with a reprogrammed maternal immune system to stop a mother's body from 'rejecting' a developing foetus, infections encountered by the mother at critical periods of pregnancy might themselves or through their effects on the maternal immune system, have the ability to 'affect' offspring outcomes in a variety of ways. The majority of work on the concept of MIA has been in smaller animals such as rodents, so the inclusion of rhesus macaques is an important step as it was in other work with the immune system and autism in mind (see here).

So, take 21 pregnancy rhesus macaques and give them either "three injections over 72 hours of poly I:C-LC [an immune stimulant], a double stranded RNA analog (viral mimic), or saline as a control." Said injections were given either "near the end of the first trimester or near the end of the second trimester" to see whether timing of immune stimulation might be important. Macaque offspring were subsequently born and followed for about 4 years. Blood samples were collected from offspring "at the end of their first (year 1) and fourth (year 4) years to assess dynamic cellular immune function." At the same time, the behaviours of monkey offspring were also analysed to see if there were any effects from MIA exposure.

Results: behaviour did seem to be affected by MIA exposure, particularly stereotyped behaviours, noted to be a core feature of autism. Similarly, researchers reported some important immune system 'changes' associated with MIA exposure: "elevated production of innate inflammatory cytokines including: interleukin (IL)-1β, IL-6, IL-12p40, and tumor necrosis factor (TNF)α" at 1 year of age. Immune system changes were also noted longer-term at 4-years: "the MIA exposed offspring continued to display elevated IL-1β, and there was also a pattern of an increased production of T-cell helper type (TH)-2 cytokines, IL-4 and IL-13." Although being careful not to generalise too much when it comes to immune system markers and what they mean for pro- or anti-inflammatory signals, the leaning towards the production of Th-2 cytokines is typically linked to atopy and 'the promotion of IgE and eosinophilic responses in atopy.' The authors - including some notable names from the MIND Institute - conclude by suggesting that: "Data from this study suggests long-term behavioral and immune activation was present in offspring following MIA."

Accepting that animal models of something like MIA are not necessarily the same as human MIA and its responses, this is interesting work. If one however accepts the data on something like vaccine function being modelled in animals (see here for example) is akin to what happens in people, real people, there is some added strength to the information published by Rose and other groups on how MIA may indeed be a relevant factor when it comes to immune function potentially affecting offspring behaviour and development.

This research also intersects with quite a lot of other peer-reviewed science talking about how (human) pregnancy infection does seem to be related to offspring risk for conditions such as autism (see here). That various immune-related conditions such as asthma in mothers might also 'prime' for offspring neurodevelopmental issues is another important strand of research potentially pertinent to this area (see here). And then also there is the idea of an 'inflammatory autism subtype' (see here) also previously suggested continuing the important theme of immune function and behaviour/development being linked. There are, as you can see, quite a few potentially important connections that can be made between the Rose results and other data on MIA and offspring development.

Oh, and I'll be coming to the recent paper by Zerbo and colleagues all in good time...

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[1] Rose DR. et al. Long-term altered immune responses following fetal priming in a non-human primate model of maternal immune activation. Brain Behav Immun. 2016 Nov 19. pii: S0889-1591(16)30522-0.

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Rose, D., Careaga, M., Van de Water, J., McAllister, K., Bauman, M., & Ashwood, P. (2016). Long-term altered immune responses following fetal priming in a non-human primate model of maternal immune activation Brain, Behavior, and Immunity DOI: 10.1016/j.bbi.2016.11.020

On biological markers and "subphenotypes" of autism

I don't want to keep you today. Just long enough to draw your attention to the paper by Jones and colleagues [1] regarding "ongoing efforts toward identification of early biological markers specific to subphenotypes of ASD [autism spectrum disorder]."

The potential biomarkers in question this time around were the cytokines/chemokines - those various signalling molecules that seem to have more than a few connections to important processes like inflammation - and how their profile ("mid-gestational serum profiles") might show some interesting links to different phenotypes of autism. Similar sentiments are already present in the peer-reviewed literature (see here) I might add.

Comparing 22 different cytokines and chemokines from mothers' samples across various groupings (ASD, developmental delay, 'not-autism' controls) and importantly including a distinction between autism diagnosed with intellectual (learning) disability (ASD+ID) and autism on its own (ASD-noID), researchers reported some interesting trends. Not least was the suggestion of an "immunologic distinction between mothers of children with ASD+ID from mothers of children with ASD-noID" including elevations in something called interleukin-6 (IL-6) (see here).

Bearing in mind a continued focus on immune function in relation to [some] autism (see here) taking into account the very wide heterogeneity present across the spectrum (autisms people, autisms), the requirement for yet further study in this area is immense. With all that's starting to be known about the various presentations of immune function in autism (see here and see here for example) and beyond (see here), I find it surprising that so little research has so far been translated from 'bench-to-bedside' in terms of screening and intervention when immune-related issues are found. Yes, dogma about what autism(s) is and isn't still pervades the scientific literature and beyond (in many areas!), but surely routine immune panel screening could be looked at being rolled out for example?

Just one more thing to add about the Jones paper. The eagle-eyed among you might already have noted the involvement of the MIND Institute in the authorship group bearing in mind their long (long) history in autism research (see here). But did you also pick up a certain Robert Yolken from the 'Stanley Division of Developmental Neurovirology' as also being present? Yes, one and the same researcher, who along with various other members of the this facility, are making some real research waves when it comes to the interplay between behaviour, immune function and infection (see here and see here for examples).

Methinks this could be the start of a beautiful (and hopefully rewarding) research relationship [2]...

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[1] Jones KL. et al. Autism with intellectual disability is associated with increased levels of maternal cytokines and chemokines during gestation. Molecular Psychiatry. 2016. May 24.

[2] Grether JK. et al. Prenatal and Newborn Immunoglobulin Levels from Mother-Child Pairs and Risk of Autism Spectrum Disorders. Front Neurosci. 2016 May 18;10:218.

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Jones KL, Croen LA, Yoshida CK, Heuer L, Hansen R, Zerbo O, DeLorenze GN, Kharrazi M, Yolken R, Ashwood P, & Van de Water J (2016). Autism with intellectual disability is associated with increased levels of maternal cytokines and chemokines during gestation. Molecular psychiatry PMID: 27217154

Immunosuppression as a therapeutic pathway of clozapine?

"Our data suggest that the superior therapeutic effect of clozapine may be a result of its presently shown immunosuppressive action."

So said the findings reported by Markus Larsson and colleagues [1] (open-access available here) who set about investigating "the effects of chronic treatment with antipsychotic drugs on brain levels of cytokines and KYNA [kynurenic acid]" in a rat model. The rationale for the study came in part from the idea that schizophrenia (or least some schizophrenia) may show more than a passing connection to a state of inflammation (see here) as well as a link to the kynurenine pathway (see here). Whether or not antipsychotic medication might show some 'effect' on these processes is still a little up in the air.

Then: "Rats were treated daily by intraperitoneally administered haloperidol (1.5 mg/kg, n = 6), olanzapine (2 mg/kg, n = 6), and clozapine (20 mg/kg, n = 6) or saline (n = 6) for 30 days." Kynurenic acid (KYNA) in brain tissue and levels of various cytokines (chemical messengers of the immune system) in cerebrospinal fluid (CSF) were assayed for among the various groups in comparison to a control group of animals receiving saline.

"Clozapine, but not haloperidol or olanzapine-treated rats displayed significantly lower cerebrospinal fluid (CSF) levels of interleukin-8 compared to controls." This is an interesting finding that needs to be treated with some caution given that aside from IL-8 all other cytokines were reported as being below the limits of detection of the analytical methods employed. Interleukin-8 (IL-8) is normally considered to be a pro-inflammatory cytokine specifically associated with acute inflammation [2]. With schizophrenia in mind, IL-8 has been tied into the whole maternal immune activation (MIA) theory of schizophrenia as per data such as those reported by Alan Brown and colleagues [3]. Brown et al reported on: "a significant association between maternal IL-8 level during the second trimester and risk of schizophrenia spectrum disorders in the offspring." More directly, IL-8 levels in relation to 'some' schizophrenia [4] have been reported in the research literature.

That rats treated with clozapine showed a lower level of IL-8 than following use of the other antipsychotics has been translated by the authors as possible evidence that clozapine may have an immunosuppressive action. Granted we don't have 'before and after' data in the Larsson study so we have to be a little careful about generalisation but I do find this to be a tantalising prospect. Whilst it might not be new news to some readers that clozapine might be doing quite a bit more than we expected when it comes to its use in a condition like schizophrenia, the idea that immune function might be 'affected' by administration [5] is a new one for me. A quick survey of some of the other literature on the immuno-modulatory aspects to clozapine indeed reveals that there is something to see here. Chen and colleagues [6] for example, talked about the anti-inflammatory possibilities attached to something like clozapine.

Within the various discussions about how several psychiatric labels might have an important 'inflammatory' component behind them (see here) it's not outside of the realms of possibility that the other pharmacological actions of the drug might also be complemented by such immunological alterations too. This might be a bit of a double-edged sword insofar as working out a more targeted 'use' for the drug whilst at the same time realising how complex the immune system truly is and why we should be cautious about tinkering too much with it.

Music: Passenger - I'll Be Your Man.

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[1] Larsson MK. et al. Chronic Antipsychotic Treatment in the Rat - Effects on Brain Interleukin-8 and Kynurenic Acid. Int J Tryptophan Res. 2015 Sep 20;8:49-52.

[2] Harada A. et al. Essential involvement of interleukin-8 (IL-8) in acute inflammation. J Leukoc Biol. 1994 Nov;56(5):559-64.

[3] Brown AS. et al. Elevated maternal interleukin-8 levels and risk of schizophrenia in adult offspring. Am J Psychiatry. 2004 May;161(5):889-95.

[4] Zhang XY. et al. Elevated interleukin-2, interleukin-6 and interleukin-8 serum levels in neuroleptic-free schizophrenia: association with psychopathology. Schizophr Res. 2002 Oct 1;57(2-3):247-58.

[5] Røge R. et al. Immunomodulatory effects of clozapine and their clinical implications: what have we learned so far? Schizophr Res. 2012 Sep;140(1-3):204-13.

[6] Chen ML. et al. Regulation of macrophage immune responses by antipsychotic drugs. Immunopharmacol Immunotoxicol. 2013 Oct;35(5):573-80.

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Larsson MK, Schwieler L, Goiny M, Erhardt S, & Engberg G (2015). Chronic Antipsychotic Treatment in the Rat - Effects on Brain Interleukin-8 and Kynurenic Acid. International journal of tryptophan research : IJTR, 8, 49-52 PMID: 26448689

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