Magnesium and ADHD meta-analysed

The study results published by Mohammad Effatpanah and colleagues [1] provided some food for thought recently on the topic of whether there may be an *association* between "serum magnesium levels and the diagnosis of attention deficit hyperactivity disorder (ADHD)."

The name of the research game was meta-analysis, that well used 'boiling down' of the published (hopefully peer-reviewed) science literature into something like a coherent 'conclusion'. The starting point for Effatpanah was that: "Current research suggests conflicting evidence surrounding the association between serum magnesium levels and the diagnosis of attention deficit hyperactivity disorder (ADHD)." It's interesting that this isn't the first time that magnesium and ADHD has been put under the meta-analysis microscope [2] and that particular meta-analysis didn't suggest such conflict.

Never mind. Seven studies made the grade for Effatpanah, together revealing that "subjects with ADHD had 0.105 mmol/l (95% CI: -0.188, -0.022; P < 0.013) lower serum magnesium levels compared with to their healthy controls." Researchers did also talk about 'high heterogeneity' across the studies analysed. This indicates that whilst there may well be "an inverse relationship between serum magnesium deficiency and ADHD" overall, the individual studies included in their meta-analysis weren't always in agreement with one and another.

So what conclusions can we take from the Effatpanah and other (meta-analysis) studies in this area? Well, more investigation is required on the suggestion of a *link* between magnesium and ADHD. We need to know more about the biology of why reduced biological levels of magnesium might be important to ADHD or ADHD-type behaviours [3] and whether something as simple as supplementing with magnesium *might* make a difference for some people [4] (minus any medical or clinical advice from me on this or any other topic). Indeed, on that last issue, I might refer you back to some other occasions where magnesium has been mentioned in the context of nutritional intervention for ADHD (see here). I'm also inclined to mention that there may be other 'labels' where magnesium might require a little more study (see here), some of which might 'overlap' with a diagnosis of ADHD. And of course, we should remember that magnesium 'issues' in the context of autism might not be the end of the story when it comes to trace metals and ADHD (see here)...

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[1] Effatpanah M. et al. Magnesium status and attention deficit hyperactivity disorder (ADHD): A meta-analysis. Psychiatry Res. 2019 Feb 19;274:228-234.

[2] Huang YH. et al. Significantly lower serum and hair magnesium levels in children with attention deficit hyperactivity disorder than controls: A systematic review and meta-analysis. Prog Neuropsychopharmacol Biol Psychiatry. 2019 Mar 2;90:134-141.

[3] Black LJ. et al. Low dietary intake of magnesium is associated with increased externalising behaviours in adolescents. Public Health Nutr. 2015 Jul;18(10):1824-30.

[4] Ghanizadeh A. A systematic review of magnesium therapy for treating attention deficit hyperactivity disorder. Arch Iran Med. 2013 Jul;16(7):412-7.

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Nicotine exposure and offspring ADHD (yet again)

The study findings reported by Andre Sourander and colleagues [1] talking about "an association with and a dose-response relationship between nicotine exposure during pregnancy and offspring ADHD [attention-deficit hyperactivity disorder]" continue an important research theme (see here and see here).

What was different about the Sourander results compared with some of the other studies in this area was their focus on the measurement of cotinine levels - cotinine being a biomarker for exposure to tobacco smoke - in mums-to-be as "measured by using quantitative immunoassays from maternal serum specimens collected during the first and second trimesters of pregnancy and archived in the national biobank." Indeed, such a biological marker measurement protocol mimics other research from members of this authorship group when looking at maternal nicotine exposure and offspring risk of schizophrenia for example (see here).

Based on the analyses of samples from over a thousand participants born in the late 1990s and diagnosed with ADHD compared with samples from a similar number of non-ADHD control participants, researchers came to their possible *link* observation. They mention how the relationship between maternal cotinine levels and offspring ADHD diagnosis was statistically significant even when other important, potentially confounding, variables were taken into account. When categorising their maternal cotinine results into bands approximating light to heavy nicotine exposure and the possibility of a link with offspring ADHD diagnosis, researchers also reported something that looked like a dose-response relationship. Ergo, a biomarker of nicotine exposure during pregnancy *looked* to be potentially linked to offspring risk of ADHD.

Although important work, my first thought when reading this research was about how these results are 'set' within the context that historically, smoking rates or tobacco exposure rates during pregnancy were so much larger decades ago than they are now (see here), but ADHD is seemingly showing only quite a recent rise in numbers (see here). Although no expert on pregnancy tobacco consumption during the 20th century, I'm assuming that all those adverts about smoking being 'healthy' in the 1940s and beyond (see here) probably meant that quite a few women smoked during their pregnancy in the belief that it was 'healthy'. At the very least, it probably meant that they were exposed to a lot more second-hand tobacco smoke as a result of smoking being allowed in various public places and also more likely to be observed in the home environment. Surely then we would have seen an explosion of ADHD diagnoses at that point in time if the link was so simple? That is, assuming that the tobacco of today is the same as the tobacco of yesteryear.

I'm also intrigued that within the various potentially confounding variables which Sourander and colleagues adjusted for - "maternal socioeconomic status, maternal age, maternal psychopathology, paternal age, paternal psychopathology, and child’s birth weight for gestational age" - there's another variable that could exert an effect on ADHD risk: relative age (see here and see here). Relative age refers to the observation that the youngest children in the school classroom compared to their older classmates, are more likely to be diagnosed with ADHD. It strikes me that alongside something like tobacco or nicotine exposure, so age and other effects could be important.

I'm not trying to poo-poo the link that Sourander and various other research teams have independently observed. I'm also not trying to downplay the harms that tobacco (nicotine) exposure can have for the unborn child. I merely suggest that with typically falling rates of (reported) tobacco exposure during pregnancy in many countries (see here) and increasing levels of childhood (and adulthood) ADHD being reported, there must be other factors at work in any such relationship (see here for example).

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[1] Sourander A. et al. Prenatal Cotinine Levels and ADHD Among Offspring. Pediatrics. 2019. Feb 25.

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Serum zonulin testing via ELISA: be very careful

I appreciate that the findings reported by Mary Ajamian and colleagues [1] probably aren't going to set many research hearts racing. Their observations that "current commercial zonulin assays are not detecting the actual protein as prehaptoglobin-2" is not exactly 'change the world' science, but that doesn't mean that they aren't important findings.

So, zonulin is the name of the research game. A protein described with properties "capable of reversible tight junction disassembly and, therefore, is implicated in the regulation of mucosal permeability" means that zonulin and dysregulation of the zonulin pathway has found a home in the science of 'gut permeability' a.k.a leaky gut. And it is with mention of the misnomer called 'leaky gut' and it's *association* with some autism (see here) that I gravitated towards the Ajamian findings. Indeed, zonulin has already made a mark in autism research too (see here). We'll come back to this shortly.

Researchers zoomed in on some of the commercially available methods currently available to 'test for zonulin' - "commercially-available ELISA assays" - and whether they are cutting the scientific mustard. And before I go on I should mention that Ajamian et al aren't the only ones who have looked at this issue [2]. Two ELISA assays were examined: "from CUSABIO (Wuhan, China) and Immundiagnostik AG (Bensheim, Germany)" and pitted against each other and various other analytical techniques to assess "whether the assays are reliably detecting zonulin as prehaptoglobin-2 and if not, what they may be detecting instead." I note the words 'mass spectrometric analysis' are also used in the Ajamian paper, which is music to my analytical ears.

Results: "Serum samples were collected from well-characterised patients and healthy individuals between the ages of 16 and 70 years living in Melbourne, Australia." Those 'well-characterised' participants included those diagnosed with non-coeliac wheat sensitivity (NCWS), coeliac disease, and ulcerative colitis (N=93) and their results were compared with nearly 50 asymptomatic controls. "The majority of study participants were zonulin-producers" as haptoglobin phenotype (see here) was also described in the Ajamian study.

Then to the serum [purported] zonulin levels as measured by those commercial assays: "Compared with the cohort of healthy individuals with a median (IQR) of 0.00 (0.00) ng/mL, patient median (IQR) values for purported zonulin were elevated (all p<0.0001) at levels of 0.032 (0.90) ng/mL in NCWS, 0.07 (1.27) ng/mL in coeliac disease, and 1.73 (2.17) ng/mL in ulcerative colitis" using the CUSABIO assay. Unfortunately, when compared with the other commercial assay (the Immundiagnostik assay), there was apparently little relationship observed between the two when it came to [purported] zonulin levels. And things didn't get any better when for example we are told that "2 of 19 participants who were zonulin non-producers had levels detected by CUSABIO assay."

Various other experiments were carried out and reported on in the Ajamian paper. These included attempts to find out what else might be being picked up by those ELISA assays. Unfortunately, even with the notable analytical prowess of something like mass spectrometry, no definitive compound(s) emerged. Something called complement C3 is discussed, as are other potential matches: "haptoglobin, and albumin." But again unfortunately: "neither complement C3 nor haptoglobin, despite both being candidate target proteins as determined by mass spectrometry, was detected by the CUSABIO assay." So we're not really any further forward when it comes to what might be being detected by such assays.

"In conclusion, the current commercial zonulin ELISA assays investigated in this study detect different proteins, neither of which was zonulin. Therefore, there can be no value of circulating concentrations in assessing intestinal mucosal barrier dysfunction and permeability until the target proteins are indeed identified." A harsh conclusion but faithful to the results observed. What this means is that the literature already published talking about zonulin levels in this, that and t'other label/diagnosis/condition (see here) need to be treated with some caution. And yes, that includes studies that have looked at zonulin levels in autism (see here) and related labels like attention-deficit hyperactivity disorder (ADHD) (see here).

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[1] Ajamian M. et al. Serum zonulin as a marker of intestinal mucosal barrier function: May not be what it seems. PLoS One. 2019;14(1):e0210728. Published 2019 Jan 14.

[2] Scheffler L. et al. Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family. Front Endocrinol (Lausanne). 2018;9:22.

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T. gondii infection might be "a contributing causal factor for schizophrenia"

T. gondii mentioned in the title of this post refers to Toxoplasma gondii, a parasite with something of a rather interesting profile and history (see here and see here). I've talked quite a bit on this blog about T. gondii and it's various 'associations', but of particular interest has been the suggestion of a 'connection' between T. gondii exposure and risk of psychiatric diagnoses like schizophrenia (see here).

The findings reported by Kristoffer Sølvsten Burgdorf and colleagues [1] (open-access available here) add further evidence to such a 'psychiatric' connection with their conclusion that: "exposure to T. gondii might be a contributing causal factor for developing schizophrenia." Researchers arrived at their conclusion following the examination of an intriguing initiative called the Danish Blood Donor Study (DBDS). Started in 2010, the DBDS includes records for over 100,000 patients and "contains DNA and EDTA plasma samples, consecutive for all donors returning for blood donation after enrolment." That's a lot of data. So: authors "identified all individuals in the DBDS cohort registered with psychiatric disorders, suicidal behavior, or traffic accidents (N=5,953)." Said participants were matched with 'suitable' controls (N=7,101) and stored samples were analysed for "immunoglobulin (IgG) class antibodies against T. gondii and CMV." CMV by the way, refers to cytomegalovirus. Contact with (congenital) CMV has also been talked about on this blog (see here). CMV (exposure) also shares a potential *link*  with "psychiatric disorders, cognitive deficits, suicidal behavior, and traffic accidents."

Results: "Of the 11,546 studied individuals, 2,990 and 7,020 individuals, respectively, tested positive for IgG class antibodies against T. gondii (25·9%) or CMV (60·8%)." Onward: "We found that individuals with a T. gondii infection had increased odds of being diagnosed with schizophrenia disorders compared to those without infection." Because researchers were also able to access other national databases containing details on outcomes like diagnosis of a psychiatric disorder and 'attempting suicide' and cross-reference them with their participants, they were also able to look at "temporality, with pathogen exposure preceding outcome" as a factor. And when they did, that T. gondii exposure - schizophrenia association was described as "even stronger." The other data on T. gondii or CMV exposure in relation to traffic accidents or suicide attempts was not as statistically strong, and indeed nothing showed significance when temporality was taken into consideration in relation to causation. On that basis, I'm gonna leave that part of the results without further comment.

This was a good study. It drew on data from a well-defined group (those Scandinavian databases 'do it' yet again) and was able to take into account the important issue of temporality. It wasn't a perfect study - "We cannot rule out that socio-economic factors could potentially account for part or all of the observed causal effect" - and said nothing about possible mechanism(s) of effect however. That being said, I'm willing to go along with the conclusions made and the need for a lot more investigation in this area linking T. gondii exposure and subsequent risk of mental illness. In particular whether new or existing treatment methods for T. gondii *might* hold the promise of much more...

And whilst on the topic of T.gondii and the specific input from cats on the spread of T. gondii (see here and see here), I'll state here and now that I am not a great believer in the idea of 'cat eradication' as mentioned by some researchers recently [2]. That being said, a toxoplasmosis vaccines for cats (see here) sounds like a really good idea...

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[1] Sølvsten Burgdorf K. et al. Large-scale study of Toxoplasma and Cytomegalovirus shows an association between infection and serious psychiatric disorders. Brain Behav Immun. 2019 Jan 24. pii: S0889-1591(18)30699-8.

[2] de Wit LA. et al. Potential public health benefits from cat eradications on islands. PLoS Negl Trop Dis 13(2): e0007040

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"serum levels of certain endocannabinoids are substantially decreased in people with ASD"

The quote titling this post - "serum levels of certain endocannabinoids are substantially decreased in people with ASD [autism spectrum disorder]" - comes from the paper published by Adi Aran and colleagues [1]. It adds to previous study on this topic (see here) and continues a research theme from members of this authorship team where the word 'cannabis' is being discussed - in the peer-reviewed science domain - in the context of [some] autism (see here).

Distinct from the last time authors' research appeared on this blog talking about the feasibility of "Cannabidiol-Rich Cannabis" 'for autism' [2], the name of the research game this time around was to assess "the circulating levels of several endocannabinoids and delineate the correlations between their levels and disease characteristics in a large group of children with ASD and their matched controls with typical development." Researchers mention how previous studies in this area "were not designed to comprehensively characterize the involvement of the ECS [endocannabinoid system] in the pathogenesis of ASD" in quite a sweeping blow to some of the other research in this area.

So, endocannabinoids are part of a system that is involved in various important biological processes [3]. As the name suggests there's an overlap between 'endogenous cannabinoids' and some of the chemical components seen in cannabis that provides as good an answer as any as to why cannabis use/misuse is the continuing issue that it is in a population sense. Authors talk about their study focusing on various endocannabinoids: AEA (anandamide), 2-AG (2-arachidonoil-glycerol), AA (arachidonic acid), PEA (N-palmitoylethanolamine), and OEA (N-oleoylethanolamine). They report how said compounds in serum samples were "analyzed by liquid chromatography/tandem mass spectrometry in 93 children with ASD... and 93 age- and gender-matched neurotypical children." Please don't however get me started on the nonsense that is the word 'neurotypical' (see here). Various other behavioural, psychometric and demographic data were also collected and thrown into the statistical mix.

Results: "Serum levels of the main endocannabinoid AEA and its structurally related compounds OEA and PEA were lower in children with ASD versus age-, gender-, and BMI [body mass index]-matched control group of typically developed children." Nothing particularly new there, as the lower levels of anandamide for example, mimic those reported by Karhson and colleagues [4]. Researchers also mentioned how their findings *might* also have some other potential: "circulating AEA, OEA, and PEA might be used to identify a biologically homogeneous subgroup of ASD, predict response to treatments and adverse reactions to medications, and assist in the development of novel drugs that target specific core symptoms of ASD." Interestingly, some of these 'options' have already been explored [5] in humans and also some animal models [6] with autism in mind.

As to the biochemical *links* between the Aran findings and indeed, the ECS more generally with autism, well, there's still a way to go to decipher them all yet. There are clues emerging [7]; clues that intersect with other important autism-relevant concepts like inflammation among other things. I note also the authors mention how their findings "support the rationale in the ongoing and emerging clinical trials of CBD [cannabidiol] in ASD" (see here) and some results to come.

I'm well and truly [cautiously] interested...

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[1] Aran A. et al. Lower circulating endocannabinoid levels in children with autism spectrum disorder. Molecular Autism. 2019; 10:2.

[2] Aran A. et al. Brief Report: Cannabidiol-Rich Cannabis in Children with Autism Spectrum Disorder and Severe Behavioral Problems-A Retrospective Feasibility Study. J Autism Dev Disord. 2018 Oct 31.

[3] Lu HC. & Mackie K. An Introduction to the Endogenous Cannabinoid System. Biol Psychiatry. 2015;79(7):516-25.

[4] Karhson DS. et al. Plasma anandamide concentrations are lower in children with autism spectrum disorder. Mol Autism. 2018 Mar 12;9:18.

[5] Antonucci N. et al. Beneficial Effects of Palmitoylethanolamide on Expressive Language, Cognition, and Behaviors in Autism: A Report of Two Cases. Case Rep Psychiatry. 2015;2015:325061.

[6] Servadio M. et al. Targeting anandamide metabolism rescues core and associated autistic-like symptoms in rats prenatally exposed to valproic acid. Transl Psychiatry. 2016 Sep 27;6(9):e902.

[7] Brigida AL. et al. Endocannabinod Signal Dysregulation in Autism Spectrum Disorders: A Correlation Link between Inflammatory State and Neuro-Immune Alterations. Int J Mol Sci. 2017;18(7):1425. Published 2017 Jul 3.

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Elevated zonulin levels in ADHD = more hyperactivity and "impairment of social functioning"

"Children with ADHD [attention-deficit hyperactivity disorder] had higher serum zonulin levels and were more impaired in social functioning compared to controls."

So said the findings reported by Gonca Özyurt and colleagues [1] exploring a topic quite close to my research heart, zonulin and the assumption that "the level of zonulin increases when intestinal permeability is impaired."

Before heading further into the Özyurt findings, I'll perhaps refer you to some of my previous musings on the topic of zonulin (see here) and the hows-and-whys of this potentially important compound. It's rooted in the idea that intestinal permeability is perhaps rather more than it should be in some people with some labels (see here) and this *could* have some important implications for biochemistry and beyond; particularly the notion of a 'gut-brain' relationship (see here).

Özyurt et al examined zonulin in the context of attention deficit hyperactivity disorder (ADHD) based on the idea that: "Zonulin has been shown to be associated with social impairment in children with autism spectrum disorder" but such functions (and other attention-related behaviours) have not yet been looked at with ADHD in mind. Based on the examination of serum zonulin levels in some 40 kids diagnosed with ADHD and a similar number of not-ADHD controls, analysed via "enzyme-linked immunosorbent assay", researchers reported that: "Children with ADHD had higher serum zonulin levels and were more impaired in social functioning compared to controls." Also: "The level of zonulin was independently predicted with hyperactivity symptoms and SRS [Social Responsiveness Scale] scores in regression analysis."

Bearing in mind that the Özyurt study was a fairly small scale study that utilised a methodology that has its critics (see here), I'm cautiously interested in the presented findings. I don't want to say anything further about this at the present time; aside that is, from the need for quite a bit more data on this potentially interesting relationship...

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[1] Özyurt G. et al. Increased zonulin is associated with hyperactivity and social dysfunctions in children with attention deficit hyperactivity disorder. Compr Psychiatry. 2018 Oct 29;87:138-142.

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Folate receptor autoantibodies and autism... replicated (yet again)

Ah yes, scientific replication. A cornerstone of good science, when findings are independently reproduced and confidence increases that A is linked to B or Y affects Z (insert other letter from the alphabet as appropriate). No, it doesn't prove anything - proof is not something that sits well with the scientific method - but it does imply that a particular relationship is much more likely not to be just due to chance given a similar answer being found across different investigations and hopefully, different cohorts of people.

The findings reported by Jiaxiu Zhou and colleagues [1] represent scientific replication in action. Not only that, they represent scientific replication covering an increasingly important issue in relation to [some] autism: a possible role for folate receptor autoantibodies (FRAA).

I don't really want to re-type everything describing FRAAs, what they and what they mean, because I've covered such descriptions before on this blog (see here and see here). Suffice to say these are autoantibodies - where the body mounts an immune response against 'self' - that target a particular protein called folate receptor protein alpha which plays an important role in transporting something called 5-methyltetrahydrofolate (5-MTHF) into the brain. 5-MTHF is a biologically active form of folate, a pretty important nutrient by all accounts; and something with some 'significant' autism research history (see here).

Building on various other reports suggesting that FRAAs might be *over-represented* in relation to the diagnosis of autism [2], Zhou et al examined serum samples provided by 40 children diagnosed with an autism spectrum disorder (ASD) and some 42 matched not-autism controls. They were specifically looking for FRAAs as "measured by enzyme-linked immunosorbent assay" bearing in mind there are blocking FRAAs and binding FRAAs.

They reported more frequently finding FRAAs in the serum samples from those with autism compared with controls (77% vs. 54%). The difference was significant and led researchers to conclude that "children with ASDs may have defects in folic acid absorption that play a role in the onset of ASDs."

As you can see, whilst the rates of detection of FRAAs in the serum samples of those with autism are quite frequent, the presence of FRAAs are not something 'autism-specific'. I say that bearing in mind that FRAAs have been reported in various other conditions/states/diseases and are also seemingly influenced by the presence of certain dietary components too, such as milk consumption [3]. But that doesn't mean that they aren't potentially important to [some] autism...

Then to the next question: intervention. What can be done as and when FRAAs are detected? Well, I've talked before about some of the the scientific evidence on the use of folinic acid (leucovorin) in the context of autism and FRAAs (see here), investigated under double-blind, placebo-controlled conditions. Folinic acid represents an alternative way of getting a biological active folate into circulation in the context of FRAAs being detected. It needs quite a bit more investigation with autism in mind, but could be a useful intervention (minus any medical or clinical advice given or intended).

Also, a milk-free diet. I know some people don't like the idea that [some] dietary elements might 'affect' [some] autism, but again, there is some initial peer-reviewed evidence to suggest that a milk-free diet might be able to dampen down things like folate receptor autoimmunity [4]. This added to the already quite voluminous peer-reviewed science suggesting a possible 'diet-related phenotype' in relation to autism [5] that mentions milk (casein) as well as other dietary components (see here).

In short, folate receptor autoantibodies are probably important to at least some autism.

Oh, and while we're on the topic of folate, I see that someone recently has been talking about why a 'one-size-fits-all' model of folic acid use during pregnancy isn't going to cover all the biological bases (see here). The MTHFR (methylenetetrahydrofolate) gene that is mentioned, has also got quite a bit of peer-reviewed research history with autism in mind (see here)...

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[1] Zhou J. et al. High prevalence of serum folate receptor autoantibodies in children with autism spectrum disorders. Biomarkers. 2018 Mar 26:1-9.

[2] Quadros EV. et al. Folate receptor autoantibodies are prevalent in children diagnosed with autism spectrum disorder, their normal siblings and parents. Autism Res. 2018 Feb 2.

[3] Berrocal-Zaragoza MI. et al. High milk consumers have an increased risk of folate receptor blocking autoantibody production but this does not affect folate status in Spanish men and women. J Nutr. 2009 May;139(5):1037-41.

[4] Ramaekers VT. et al. A milk-free diet downregulates folate receptor autoimmunity in cerebral folate deficiency syndrome. Dev Med Child Neurol. 2008 May;50(5):346-52.

[5] Whiteley P. Nutritional management of (some) autism: a case for gluten- and casein-free diets? Proc Nutr Soc. 2015 Aug;74(3):202-7.

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Plasma anandamide concentrations are lower in children with autism

The findings reported by Debra Karhson and colleagues [1] piqued my interest for two primary reasons. First, they detail "the first empirical human data to translate preclinical rodent findings to confirm a link between plasma anandamide concentrations in children with ASD [autism spectrum disorder]." Second, authors also report on the use of a gold-standard technique when it came to their analyses: the development and use of "a LC-MS/MS [liquid chromatography-tandem mass spectrometry] method to quantitatively analyze AEA [anandamide] concentrations in small volumes of banked plasma with short sample preparation time and high sample repeatability."

OK, the basis for the Karhson study was the 'increasing interest for ASD' examining the endogenous cannabinoid or endocannabinoid system. This is a system, an internal system, that comprises of quite a few compounds, enzymes and receptors that play "important roles in central nervous system (CNS) development, synaptic plasticity, and the response to endogenous and environmental insults" [2]. Yes, as the name suggests, there is an 'overlap' between some of the workings of the endocannabinoid system (ECS) and components of a certain drug of abuse but that doesn't insinuate anything at the present time.

Anandamide (AEA) is a sort of messenger molecule that is part of the ECS. It shares some chemical characteristics with the active compound found in cannabis, leading quite a few commentators to talk about AEA in terms of being a 'pleasure' or 'bliss' molecule. It does not however, have the 'staying power' of its molecular companion; chemically-speaking being fairly readily degraded in the body. Indeed, of the many biological roles and functions linked to AEA and the ECS more generally, I'd in particular, like to direct your attention to some of the science-so-far literature in relation to pregnancy (see here). And, no, that does NOT mean that smoking marijuana during pregnancy is a good thing...

The authors highlight how the ECS is a research area rising in relation to autism (and associated diagnoses) based, quite extensively, on animal models of autism and all the associated 'issues' that this carries (see here). So: "despite the promise of these preclinical data, no studies to date have investigated AEA concentrations in humans with ASD." They sought to remedy that situation.

Results are reported based on the LC-MS/MS analysis of plasma samples provided by some 59 children with autism and 53 not-autism controls. As per my continued interest in all-things mass spec when specifically applied to autism research, I was encouraged by the use of a "commercially available stable isotope-labeled AEA-d8" being used as an internal standard, and the fact that the lower limit of detection for AEA was in the femtogram range. In short, authors were able to train their system to specifically look for AEA and were able to get down to some really quite low levels of detection.

"Two significant findings were observed: (1) plasma AEA concentrations significantly differentiated ASD cases from controls, such that children with lower AEA concentrations were more likely to have ASD, and (2) AEA concentrations were significantly lower in ASD compared to control children." I don't really need to say much more than that, aside from adding in another quote: "These results, although preliminary, corroborate preclinical evidence that AEA signaling may be impaired in patients with ASD."

The question of what these findings actually mean is still unanswered. It should for example, be noted that the Karhson results in real people were based on the analysis of plasma samples, where previous animal work has tended to be a little more 'invasive' in terms of the tissue types looked at (e.g. in the brain). The authors also mention that further investigations need to give due credit to the idea that autism rarely exists in some sort of diagnostic vacuum (see here), and some of these 'comorbidities' could very well influence the results obtained [3].

Further studies on this topic are very much required.

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[1] Karhson DS. et al. Plasma anandamide concentrations are lower in children with autism spectrum disorder. Molecular Autism. 2018; 9: 18.

[2] Lu H-C. & Mackie K. An introduction to the endogenous cannabinoid system. Biological Psychiatry. 2016;79(7):516-525.

[3] Romigi A. et al. Cerebrospinal fluid levels of the endocannabinoid anandamide are reduced in patients with untreated newly diagnosed temporal lobe epilepsy. Epilepsia. 2010 May;51(5):768-72.

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Vitamin D and autism... another double-take?

A quick-ish post today as I bring the results published by Dong and colleagues [1] to your attention and the [translated] observation that: "Serum 25-hydroxyvitamin D level in children with ASD [autism spectrum disorder] is obviously lower than that in the healthy control group, and there are negative correlations between vitamin D levels and core symptoms of ASD." 'Obviously' eh?

It's another study from China (see here) and yet again I find myself correcting language as per the authors' use of the term 'healthy control group' when describing not-autism. In much the same way that the word 'neurotypical' is a bit of a nonsense, so the insinuation that a diagnosis of of autism automatically means 'not healthy' is far too broad a sweeping generalisation.

Anyhow, vitamin D and autism was the name of the research game for these authors; something not altogether new and novel for at least some of the authorship group (see here and see here for examples). Indeed, my use of the term 'another double-take' in the title of this post refers to the observation that this group have really gone to town with their clinical trial registered research project in this area (see here for example).

"Serum vitamin D level in ASD children was significantly lower than that of the control group... and the between-group percentage difference of normal, insufficient and deficient levels of vitamin D was statistically significant." Bearing in mind that vitamin D levels were checked using a gold-standard technique (liquid chromatography-mass spectrometry, LC-MS), I'm inclined to accept these results as they stand. I'm not saying that other methods of vitamin D analysis are all bunk, but rather that LC-MS is a mighty powerful method for sample analysis with vitamin D in mind.

Further: "There were negative correlations between serum vitamin D level in ASD children and total ABC [Autism Behavior Checklist] score or ABC subscale scores (body behavior, self-care, language and social interaction). There were negative correlations between serum vitamin D level in ASD children and total CARS [Childhood Autism Rating Scale] score and CARS subscale scores (imitation, nonverbal communication and general impression). There were negative correlations between serum vitamin D level in ASD children and SRS [Social Responsiveness Scale] behavior subscale or ATEC [Autism Treatment Evaluation Checklist] social interaction subscale." In short, vitamin D  levels seemed to *correlate* with quite a few behavioural results, although I'm slightly less inclined to read too much into such findings given the relatively small participant group included for study and the 'snaphot' study methodology.

But yet again, this is another example illustrating that vitamin D metabolism should very much be a part of any assessment when it comes to autism (see here and see here)...

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[1] Dong HY. et al. Correlation between serum 25-hydroxyvitamin D level and core symptoms of autism spectrum disorder in children. Zhonghua Er Ke Za Zhi. 2017 Dec 2;55(12):916-919.

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On iron and vitamin D and autism

"This study suggests that deficiency of iron and Vitamin D as well as anemia were more common in autistic compared to control children."

'This study' refers to the findings reported by Abdulbari Bener and colleagues [1] (open-access available here) who set out to "investigate iron deficiency anemia and Vitamin D deficiency among autism children" in Qatar, a part of the world not renowned for its 'lack of sunshine' (a source material for the production of vitamin D).

Looking at some 300 children diagnosed with an autism spectrum disorder (ASD) and an equal number of controls, not-autism controls "who visited the primary health-care centers", researchers concluded that as a group, those with autism were more likely to present with low serum iron levels (and various related measures) and further that: "Vitamin D deficiency was considerably more common among autistic children." The authors provide some background details on what constitutes vitamin D deficiency and other 'levels': "Participants were classified into four categories: (1) severe Vitamin D deficiency, 25OHD <10 ng/ml; (2) moderate deficiency, 25OHD 10–19 ng/ml; (3) mild deficiency, 25OHD 20–29 ng/ml; and normal/optimal level is between 30 and 80 ng/ml."

When attempting to ascertain what factors might be important to the autism vs. not-autism participants, researchers reported that: "serum iron deficiency, serum calcium levels, serum Vitamin D levels; ferritin, reduced physical activity; child order, body mass index percentiles, and parental consanguinity can all be considered strong predictors and major factors associated with autism spectrum disorders." I might add that consanguinity defined as "unions between couples who share at least one common ancestor" is perhaps something more 'culturally-relevant' to autism in certain countries and societies [2] but not necessarily widely applicable...

What's more to say about the Bener findings? Well, given that issues with iron (see here) and issues with vitamin D (see here) are no strangers to the autism research landscape, there is little novelty in the conclusions reached even if being "the first report on an establishing level of iron deficiency in children with autism in Qatar and in Arabian Gulf Countries." The implication is again that preferential screening and treatment of such issues should be offered when a diagnosis of autism is received, save any further health inequalities arising. Whether or not treating something like iron deficiency and/or vitamin D issues will impact on behavioural presentation (see here) is perhaps an issue for another day. I say this bearing in mind the sentiments expressed in the recent paper by Philippe Autier and colleagues [3] examining the collected data on vitamin D supplementation "on non-skeletal disorders" and results seemingly "strengthening the hypothesis that low vitamin D status is a consequence of ill health, rather than its cause."

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[1] Bener A. et al. Iron and vitamin D levels among autism spectrum disorders children. Ann Afr Med. 2017 Oct-Dec;16(4):186-191.

[2] Mahajnah M. et al. Clinical characteristics of autism spectrum disorder in Israel: impact of ethnic and social diversities. Biomed Res Int. 2015;2015:962093.

[3] Autier P. et al. Effect of vitamin D supplementation on non-skeletal disorders: a systematic review of meta-analyses and randomised trials. Lancet Diabetes Endocrinol. 2017 Oct 25. pii: S2213-8587(17)30357-1.

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Zonulin (testing): "its clinical utility questionable"

The quote making up part of the the title of today's post is taken from the paper by Aristo Vojdani and colleagues [1] (open-access available here) providing some well needed analysis of a compound of some interest for various clinical areas: zonulin.

Just in case you weren't familiar with all-things zonulin, this is a compound that has found some scientific favour when it comes to the concept of intestinal barrier function being perturbed in several diagnoses [2]. I must admit to being pretty interested in some quite recent research talking about zonulin in the context of 'some' autism (see here) based on the idea that intestinal barrier function might not be 'optimal' for some people diagnosed with an autism spectrum disorder (ASD) and what implications that might have (see here and see here).

Vojdani - who is also no stranger to autism research - cautions that the inevitable testing 'free for all' that has ensued as zonulin has risen up the scientific ranks might not be all good, as they pitted the direct measurement of serum zonulin levels against "antibodies against zonulin" to see which measure might provide the most accurate results. Antibodies against zonulin by the way, meant IgA and IgG antibodies against zonulin and was carried out "using enzyme-linked immunosorbent assay methodology."

Results: based on the analysis of over 70 blood samples from "18 volunteers at intervals of 0, 6, 24, and 30 h[ours]" authors noted that a third of participants (6/18) had low levels of serum zonulin "very close to the detection limit of the assay." We are told over the course of the hours, levels of serum zonulin "did not significantly fluctuate" in their trace amounts in this group. For the other 12 participants, it was a slightly different story as "significant fluctuation in zonulin levels was observed in almost all 12 of these subjects at the 6-, 24- or 30-h blood draws." When it came to those antibodies against zonulin, the clinical picture appeared to be slightly more calm as data showed that "both IgG and IgA antibody levels from blood obtained at 0, 6, 24, and 30 h were highly stable with variations of less than 10%." On that basis, the authors recommend that a single measurement of zonulin itself may not be a suitable indicator "for assessment of intestinal barrier integrity."

There was also another part to the Vojdani study looking at serum zonulin levels in "30 healthy controls along with 30 patients with known celiac disease." Coeliac or celiac disease (CD) is the archetypal gluten-related autoimmune condition and has some connection to zonulin. Results for this part of the study indicated a significant group difference between CD and non-CD groups where serum zonulin levels were higher in those with CD. When comparing serum zonulin levels against those antibodies to zonulin in the CS vs no-CD groups, authors reported "detection of antibodies against zonulin in 67% of patients with CD while zonulin level elevations were detected in only 33%." They suggested that these results could be due to "zonulin fluctuation in the blood and its removal by the immune system."

These types of results are interesting and help to add some 'detail' to big, sometimes sweeping, scientific findings with an emphasis on the technology and techniques used to measure such compounds. In the context of the Esnafoglu paper [3] that was the source material for my blogpost on zonulin and autism, there may be lessons to be learned as per their use of an enzyme-linked immunosorbent assay to analyse for serum zonulin levels in that particular cohort. That being said, a comparison of the range of zonulin levels reported in their autism cohort "(ASD (122.3 ± 98.46 ng/mL) compared with the healthy controls (41.89 ± 45.83 ng/mL)" compared with the Vojdani results (CD mean = 8.5 ng/mL vs. controls mean = 3.7 ng/mL ) shows that there may be quite a bit more to see when it comes to zonulin and [some] autism outside of just testing factors.

Just before I go, I do have one possible suggestion which might help matters in the area of zonulin measurement. Being quite a big fan of techniques such as mass spectrometry over other analytical methods and bringing in other recent data suggesting that *some* immunoassay kits purposed for zonulin analysis might be missing the mark [4], I'm minded to suggest that a more direct analysis of something like serum zonulin in various groups could be warranted based on mass spec and related techniques including those diagnosed with CD and autism (or even both)...

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[1] Vojdani A. et al. Fluctuation of zonulin levels in blood vs stability of antibodies. World J Gastroenterol. 2017 Aug 21;23(31):5669-5679.

[2] Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Annals of the New York Academy of Sciences. 2012;1258(1):25-33.

[3] Esnafoglu E. et al. Increased Serum Zonulin Levels as an Intestinal Permeability Marker in Autistic Subjects. J Pediatrics. 2017. May 11.

[4] Scheffler L. et al. Widely used commercial ELISA for human Zonulin reacts with Complement C3 rather than preHaptoglobin2. bioRxiv preprint. 2017. Jun 30.

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On genotype and environmental exposure patterns

I was rather interested to read the paper by Michela Traglia and colleagues [1] (open-access available here) concluding that: "maternal and fetal genetic make-up are important determinants of mid-gestational maternal circulating levels of some environmental organohalogens." Interested because, in these days of gene-environment interactions being applied to just about everything, the detail that is missing - which genes might potentially be linked to which environmental factors - has not yet been suitably addressed in the peer-reviewed science literature.

So, based on data - "serum levels of a set of 21 organohalogens in a subset of 790 genotyped women and 764 children" - derived from participants included in the Early Markers for Autism (EMA) Project, researchers set about assessing how genetics might impact on environmental pollutant exposure profiles. Maternal blood samples were collected at around 15-20 weeks pregnancy. Children provided blood samples via the fabulous resource that is the newborn screening program, where: "Newborn blood spots were collected on filter paper 1-2 days after birth." Maternal samples were analysed for various environmental pollutants and both sets of samples were analysed for the genetic material they contained pertinent to whether "circulating mid-gestational levels of organohalogens would be driven by common maternal genetic determinants, and that these results could shed light on the observed associations between the organohalogens and ASD [autism spectrum disorder]."

Results: yes, the authors "found evidence that a large proportion of maternal circulating levels of BB-153, BDE-47, -100, -153 [polybrominated congeners] and their sum was significantly controlled by common genetic factors." Those 'common genetic factors' typically referred to the presence of point mutations (SNPs) that litter everyone's genome and on occasion, can affect the function/production of specific biological processes. So: "Genome-wide association analyses identified significant maternal loci for p,p'-DDE... in the CYP2B6 gene and for BDE-28... near the SH3GL2 gene, both involved in xenobiotic and lipid metabolism." In other words, although the environmental pollutants measured are not great products in the first place (in terms of safety), a person's genetic make-up can influence how such products are eventually dealt with by the body and potentially onwards, what subsequent effects they might have.

Additionally: "results suggest that the maternal circulating levels of some compounds were more highly influenced by fetal genetic factors than maternal genetics." This leads into another aspect of the current study whereby foetal genetic factors might also play a part in "controlling the toxicant disposition between mother and fetus." Specifically, authors noted that aspects of the individual genetics of a foetus (distinct from its mother) "contributed to the levels of BDE-100... and PCB187... near the potential metabolic genes LOXHD1 and PTPRD, previously implicated in neurodevelopment."

And finally: "We confirmed that the serum levels of BDE-100, -153 and the total sum of PBDEs were significantly lower in mothers of ASD-affected children compared to mothers of control children." This is interesting in light of other discussions about PBDEs and autism in particular (see here). The authors do discuss various scenarios to account for their results not least that "transplacental transfer of organohalogens during pregnancy may be driven by the fetal genome expressed in placenta." Further analyses of the 'placentome' might therefore be indicated.

To reiterate, this is interesting research. It tells us that many [adverse] environmental exposures, whilst typically to be avoided, don't act on the body in a uniform way as a function of differing genomes and differences in the ways that the body 'handles' such exposures. With autism in mind, this is not necessarily new news (remember paraoxonase gene variants and organophosphate metabolism [2] and air pollution and offspring autism?) but is a useful reminder. Such work also provides a template for looking at the myriad of other environmental factors put forward to influence autism risk and whether individual product safety is necessarily the only or most important factor when it comes to assessing relative risk profiles.

I might finally also draw your attention to a recent interesting meta-analysis of the various environmental risk factors potentially linked to autism [3] (open-access) and another article talking about similar things [4] (open-access) (thanks Annabelle). Genes and environment, genes and environment...

Music: Petula Clark sings the Beatles? Personally, I think it's better than the original...

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[1] Traglia M. et al. Independent Maternal and Fetal Genetic Effects on Mid-gestational Circulating Levels of Environmental Pollutants. G3 (Bethesda). 2017 Feb 24. pii: g3.117.039784.

[2] D'Amelio M. et al. Paraoxonase gene variants are associated with autism in North America, but not in Italy: possible regional specificity in gene-environment interactions. Mol Psychiatry. 2005 Nov;10(11):1006-16.

[3] Modabbernia A. et al. Environmental risk factors for autism: an evidence-based review of systematic reviews and meta-analyses. Molecular Autism. 2017; 8: 13.

[4] Parker W. et al. The role of oxidative stress, inflammation and acetaminophen exposure from birth to early childhood in the induction of autism. Journal of International Medical Research. 2017. Jan 20.

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Traglia M, Croen LA, Lyall K, Windham GC, Kharrazi M, DeLorenze GN, Torres AR, & Weiss LA (2017). Independent Maternal and Fetal Genetic Effects on Mid-gestational Circulating Levels of Environmental Pollutants. G3 (Bethesda, Md.) PMID: 28235828

"Autoimmune epilepsy is an underrecognized condition..."

"Among adult patients with epilepsy of unknown etiology, a significant minority had detectable serum Abs [autoantibodies] suggesting an autoimmune etiology."

So said the findings reported by Divyanshu Dubey and colleagues [1] continuing a research theme previously discussed on this blog (see here) on how epilepsy / seizure-type disorder(s) for some might have more to do with immune function than many people might think.

OK, a brief bit of background: epilepsy is a blanket term covering a wide variety of different presentations that affect the brain and specifically, 'the electrics' of the brain. Seizures are the most common symptom. Treatment typically comes in the form of anti-epileptic medicines (although other options are being considered for some). It's been known for a while that outside of the 'brain' focus of epilepsy, other biological systems might also play a role in the development/maintenance of the condition(s); specifically the immune system and quite often in cases where traditional anti-epileptic medicines don't seem to be able to control seizures effectively. The details are still a little sketchy but studies like the one from Dubey et al are trying to put some scientific flesh on to the bones of what facets of the immune system are potentially involved, specifically under 'autoimmune' conditions where the body fails to recognise 'self' as self and mounts an immune response against the body's own tissue(s).

Dubey and colleagues looked at a group of participants "presenting to neurology services with new-onset epilepsy or established epilepsy of unknown etiology" and tested donated serum samples "for Abs reported to be associated with autoimmune epilepsy (NMDAR-Ab, VGKCc-Ab, leucine-rich glioma-inactivated protein 1 [LGI1] Ab, GAD65-Ab, γ-aminobutyric acid type B receptor [GABAB] Ab, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor [AMPAR] Ab, antineuronal nuclear antibody type 1 [ANNA-1 or anti-Hu] Ab, Purkinje cell cytoplasmic antibody type 2 [PCA-2] Ab, amphiphysin Ab, collapsin-response mediator protein 5 [CRMP-5] Ab, and thyroperoxidase [TPO] Ab)." Quite a lot of those autoantibodies probably sound like gibberish to the lay reader but some of them have been discussed in other contexts on this blog (see here and see here for examples).

Results: some (15) of the 127 participants initially enrolled in the study were "subsequently excluded after identification of an alternative diagnosis." This in itself is interesting, as diagnoses such as "hypoxic or anoxic injury following cardiac arrest" and "ischemic stroke" are mentioned, illustrating how several different roads can lead to epilepsy and/or the presentation of seizures.

Then: "Serum Abs suggesting a potential autoimmune etiology were detected in 39 (34.8%) cases." Over a third of the cohort showed serological evidence of autoantibodies and some presented with more than one type of autoantibody as being present. Breaking down those serologically positive participants, we are told that: "19 patients (48.7%) had new-onset epilepsy and 20 patients (51.3%) had established epilepsy." The authors did also subsequently limit their findings to those cases excluding TPO-Ab and low-titer GAD65-Ab (autoantibodies where a specific role to epilepsy is unclear or not specific) but even then reported that: "23 patients (20.5%) with unexplained epilepsy had positive serologic findings strongly suggestive of an autoimmune cause of epilepsy." There is also a final part to the Dubey paper which also merits mention: "Among the 23 patients who were seropositive, 15 (65.2%) received some sort of immunotherapy. Better seizure outcome was associated with use of immunomodulatory therapy... especially with use of intravenous methylprednisolone... or plasmapheresis."

Alongside other (independent) studies in this area, the peer-reviewed evidence does seem to growing to suggest that within the wide (and heterogeneous) 'spectrum' that is epilepsy, at least some of that epilepsy might have an important immune component to it. To quote again from Dubey: "The data presented here suggest that autoimmune encephalitis may explain at least 20% of adult-onset epilepsies of unknown etiology." Aside from the importance of screening for said autoantibodies when certain cases of epilepsy appear at clinic, there are a few other potentially important points that could be raised about such data. Autism is area that I would be interested to see some further investigations carried out on with the Dubey findings in mind. Epilepsy is an important comorbidity 'over-represented' when it comes to autism (see here) and given the suggestions down the years that immune function (specifically autoimmunity) might be a facet of 'some' autism (see here for example) it's not beyond the realms of possibility that comorbid epilepsy might be a further facet of any autoimmune processes. Birds of an autoimmune feather tend to stick together and all that (see here). Add in the findings specifically talking about 'anti-NMDA-receptor encephalitis "mimicking an autistic regression"' (see here) and how methlyprednisolone might not be an uncommon medicine for some types of (autoimmune-related autistic presentation) and the hypotheses to be tested are laid out in front of you. By saying that, I don't want to take anything away from the more typical forms of epilepsy that can present (either alone or alongside autism) but rather point to the expanding knowledge base suggesting that immune functions may extend much further than just protecting the host from infection et al...

To close, slightly related to some of the content included in this post, the trailer for the film Brain on Fire (from the book of the same name) is out and looking like required viewing.

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[1] Dubey D. et al. Neurological Autoantibody Prevalence in Epilepsy of Unknown Etiology. JAMA Neurol. 2017 Feb 6.

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Dubey D, Alqallaf A, Hays R, Freeman M, Chen K, Ding K, Agostini M, & Vernino S (2017). Neurological Autoantibody Prevalence in Epilepsy of Unknown Etiology. JAMA neurology PMID: 28166327

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