If there’s one thing about antivaccine activists, it’s that they rely on bad science (and the shameless misinterpretation of valid science) to give the impression that there is solid scientific evidence behind their pseudoscientific claims. I’ve discussed examples—and deconstructed them, explaining why they didn’t show what the investigators claim they showed—more times than I can remember, most recently when Christopher Shaw and Lucija Tomljenovic tortured yet more mice in the name of autism pseudoscience to produce a paper that was ultimately retracted because of readers on PubPeer who found images that reeked of manipulation. Of course, most antivaccine “research” is never revealed to have suffered from scientific fraud, but it’s basically all bad. I long ago lost track of the number of truly atrocious studies I’ve evaluated over the years published by antivaccine “scientists” dating all the way back to Andrew Wakefield and the father-son duo of pseudoscientists, Mark and David Geier.

Speaking of Mark and David Geier and bad antivaccine autism science, I encountered yet another example yesterday when I came across an article written by our old friend J.B. Handley for Medium entitled New study: Massive Aluminum levels in Autism brains, is this the smoking gun for vaccines? Spoilers: The answer is no. However, in this case, the voyage to that answer is important; so I had to look through the study. First, however, let’s take a look at how JB does what JB does best: Gloat prematurely whenever he thinks he’s found “smoking gun” evidence supporting his belief in the discredited idea that vaccines cause autism and win friends and influence people. Of course, J.B. Handley and his wife founded Generation Rescue, a group originally devoted to the belief that mercury due to the thimerosal preservative in vaccines is the primary cause of the “autism epidemic.” However, moving with the times and the shifting milieu in the antivaccine movement in which aluminum (atomic symbol: Al) is the new mercury, a milieu that shifted because of how, 15 years after mercury was removed from childhood vaccines, autism prevalence hasn’t fallen, Handley has now latched on to aluminum in vaccines as the cause of autism. It’s hip, it’s happening, and, above all, to Handley it’s always about the vaccines, as it is for all antivaccine zealots.

After citing the usual litany of bad antivaccine science about aluminum, Handley gloats:

The one thing missing from all the work done to date about aluminum and its possible role in autism? Actual brain tissue of people with autism. All the studies published that appeared to be demonstrating strong biological certainty of how the aluminum in vaccines could trigger autism were done with MICE, and Professor Exley and his colleagues’ new research studied the actual brains of people with autism. The conclusions should make you gasp.

The aluminium content of brain tissues from donors with a diagnosis of ASD [Autism] was extremely high…the mean aluminium content for each lobe across all 5 individuals was towards the higher end of all previous (historical) measurements of brain aluminium content, including iatrogenic disorders such as dialysis encephalopathy …We recorded some of the highest values for brain aluminium content ever measured in healthy or diseased tissues in these male ASD donors…Why, for example would a 15 year old boy have such a high content of aluminium in their brain tissues?

Actually, the only thing that makes me gasp is the unjustified conclusions being made from this paper, Aluminium in brain tissue in autism, coauthored by Matthew Mold, Dorcas Umar, Andrew King, and Christopher Exley and published in the Journal of Trace Elements in Medicine and Biology. We’ve met Exley before on this blog twice, and his track record is not—shall we say?—encouraging. The first time I dealt with Exley, he had published a truly execrable paper in which he measured aluminum content in mastectomy specimens taken from 17 women with breast cancer in which he tried to link aluminum from antiperspirants to breast cancer. He also wrote a review article trying to make the same argument. In both cases, he failed to make anything resembling a compelling scientific case for aluminum from antiperspirants as a cause of breast cancer. The second time, I noted that he’s one of a group of scientists funded by the Child Medical Safety Research Institute (CMSRI), which is a group funded by Claire and Al Dwoskin, who are as rabidly antivaccine as anyone I’ve seen, including even Mike Adams. Among that group of antivaccine “scientists” funded by CMSRI? Anthony Mawson, Christopher Shaw, Lucija Tomljenovic, and Yehuda Shoenfeld, antivaccine crank “scientists” all. And guess what? This study was funded by CMSRI, too. Fair’s fair. If antivaxers can go wild when a study is funded by a pharmaceutical company and reject it out of hand, I can point out that a study funded by an antivaccine “foundation” is deserving of more scrutiny and skepticism.

A study must rise or fall on its own, however, regardless of who funded it or what obnoxious antivaxer is promoting it. So let’s dig in. One thing that stood out to me reading the manuscript is just how fast it was accepted for publication. It was submitted on October 26, resubmitted in revised form on November 21, and accepted on November 23. It was then published online on November 26. That’s amazingly fast for a paper to be reviewed, revised, accepted, and published. This sends up a number of red flags, implying either that the review was rushed (and thus suboptimal, to put it kindly). The journal is not known to be a predatory one or a “pay to play one.” It has a halfway decent impact factor (3.225). So what’s going on here? Well, Exley is on the editorial board of the journal. Whenever I see a paper by someone on the editorial board of a journal published in that journal I tend to raise an eyebrow and wonder about how objective the peer review was.

The introduction also gives away the game:

Autism spectrum disorder (ASD) is a group of neurodevelopmental conditions of unknown cause. It is highly likely that both genetic [1] and environmental [2] factors are associated with the onset and progress of ASD while the mechanisms underlying its aetiology are expected to be multifactorial [3-6]. Human exposure to aluminium has been implicated in ASD with conclusions being equivocal [7-10]. To-date the majority of studies have used hair as their indicator of human exposure to aluminium while aluminium in blood and urine have also been used to a much more limited extent. Paediatric vaccines that include an aluminium adjuvant are an indirect measure of infant exposure to aluminium and their burgeoning use has been directly correlated with increasing prevalence of ASD [11]. Animal models of ASD continue to support a connection with aluminium and to aluminium adjuvants used in human vaccinations in particular [12]. Hitherto there are no previous reports of aluminium in brain tissue from donors who died with a diagnosis of ASD. We have measured aluminium in brain tissue in autism and identified the location of aluminium in these tissues.

First off, no, aluminum has only been “linked” with ASD by antivaccine cranks. There is really no good evidence that aluminum in vaccines causes ASDs, try as as antivaxers might to try to “prove” that there is. Particularly telling is that Exley cites papers by Tomljenovic and Shaw, who are well known for their proclivity to publish papers chock full of bad (and fraudulent) science purporting to link aluminum adjuvants to autism and other conditions. Any citation of one of their papers is a good indication of bad science. Heck, I even deconstructed one of the papers cited.

So, right off the bat, the scientific justification for this study is highly dubious, but it was done anyway; so we have to deal with the results. What did Exley’s team do? They obtained brain tissue samples from autistic people from the Oxford Brain Bank, which is hosted in the Department of Neuropathology of the Oxford University Hospitals NHS Trust and the Academic Unit of Neuropathology of the Nuffield Department of Clinical Neurosciences of Oxford University. The bank stores sample and clinical data relevant for research into neurological diseases. From this bank, it appears that Exley received five specimens of frozen brain tissue (4 males, 1 female, age range 15-50), consisting of samples from tissue from temporal, frontal, parietal and occipital lobes and hippocampus of each individual. Aluminum levels were measured using transversely heated atomic absorption spectroscopy.

Also, the authors received frontal, parietal, occipital, temporal and hippocampal tissue from 10 donors ( 3 females and 7 males) with a diagnosis of ASD was supplied by the Oxford Brain Bank as three 5 μm thick serial paraffin-embedded brain tissue sections per lobe for each donor. These were subjected to staining with the fluorescent probe lumogallion, which is purported to be a selective stain for aluminum in cells and was first used to localize aluminum in plant roots. It’s sold, however, by Santa Cruz Biotechnology as a stain for aluminum, gallium, and other metals. The stained sections were then subjected to fluorescence microscopy to visualize where the lumogallion binds to aluminum in the tissue.

I could see a number of problems with the methods used. First, there are no controls. Certainly, if Exley were accessing the Oxford Brain Bank to obtain tissue from autistic people, then why couldn’t he have also obtained brain tissue from age-matched normal controls? If the bank doesn’t have such tissue, then it should have been justified. Either way, the relevance of these measurements are suspect, given that we have nothing to compare them to. There’s also an extreme paucity of clinical information about the samples used. There are no dietary histories, no medical histories, and all with a very small sample size. While dietary history might be hard to come by in a typical database associated with a tissue bank, there’s no excuse for not including medical histories, given that the Oxford Brain Bank definitely includes clinical information about the patient from which each sample came from.

There’s another thing. The raw aluminum levels measured by atomic absorption spectroscopy are reported as ranging from 0.01 (the limit of quantitation) to 22.11 μg/g dry wt. Now here’s an interesting thing. The authors go further to report that the aluminium content of all tissues ranged from 0.01 (the limit of quantitation) to 22.11μg/g dry wt. and that the aluminium content for whole brains (n=4 or 5 depending upon the availability of hippocampus tissue) ranged from 1.20±1.06) μg/g dry wt.to 4.77±4.79 μg/g dry wt. Exley then cites a previous study of his, which he refers to as “our 60 brain study,” that has “allowed us to define loose categories of brain aluminium content beginning with ≤1.00 μg/g dry wt. as pathologically benign (as opposed to ‘normal’).” I looked up the study, reference 15, and was puzzled. In it Exley reported measuring aluminum levels in the brain of one man, a 69-year-old who had died of Alzheimer’s disease. Either Exley screwed up the reference, or he hoped that no one noticed that he had referred to a reference that doesn’t describe what he claims it described. The first would just be sloppy. The second would be dishonest. Fortunately for Exley, in this case, it looks as though he was just sloppy.

I suspect that Exley meant this study, although I’m not sure. In that study, he found that the median aluminum content in 713 samples was 1 µg/g dry tissue and that 75% of the measurements were less than 2 µg/g dry tissue. Some things to note. First, these were all brains from elderly people. Second, Exley basically says that 75% of all values were less than 2 µg/g dry tissue. Looking at Table 1 of the present paper, I note that the vast majority of aluminum concentrations were below 2 μg/g dry weight, too. I also notice some rather extreme variability in measurements. For instance, the sample replicates for one specimen were 1.71, 1,64, and 17.10 μg/g dry weight. For those not familiar with scientific terminology, “replicate” means repeated measurements performed on the same sample, although in this case the replicates the samples were divided into three, and each portion subjected to the same procedure to measure aluminum. The replicates were then averaged and a standard deviation calculated. There were other replicates as questionable. For example, for another sample the replicates measured 2.44, 1.66, and 22.11 μg/g dry weight. For still another sample, replicates were 0.01, 0.64, and 18.57 μg/g dry weight. Remember, again, these are replicate measurements of the same samples. True, there could be “clustering” of aluminum in different parts of the sample, but even allowing for that possibility, these values do not give me a lot of confidence in the reproducibility of Exley’s methodology. So what does Exley do with such widely variable results? He reports the average of the three values for these specimens, which is—surprise! surprise!—hugely dominated by the spuriously high value in each case, thus falsely inflating the value for each of those specimens. Of course, even in Exley’s “60 brain study,” he shows no evidence that aluminum content greater than 2.0 or 2.0 μg/g dry weight is even pathological! He even notes “how difficult it might be to use statistical measures of brain Al content as reliable indicators of potential neurotoxicity.”

Also, if only we had an idea of the aluminum concentration in normal brain tissue. If only…

Oh, wait, we do. It’s between 1.4 to 2.5 µg/g dry tissue, as measured in a study that compared aluminum concentration in the brains of patients with Alzheimer’s disease. Oops! That’s not very different from the values that Exley found, and if you take into account the huge variability in some of his sample replicates, I have a hard time thinking there’s anything particularly alarming in these values. Heck, even compared to Exley’s “60 brain paper” I have a hard time being alarmed by the numbers in this new study.

When it comes to fluorescent staining of tissue samples, I must admit that I’m not an expert, although I have done some fluorescence microscopy work in the past, including some confocal microscopy. So I’m not completely without knowledge here. I know some of the pitfalls, and I know that certain controls are very, very important. I also know that counterstains are usually necessary if you’re going to make assertions about which cells whatever it is that you’re looking for is localizing in. In this paper, Exley makes a number of conclusions, such as that aluminum located in inflammatory cells associated with the vasculature, in round and amoeboid glial cell bodies, neurones and glia-like cells, and other areas. I looked at the figures for which these claims were made, and my reaction was: WTF? Maybe. Maybe not. I can’t tell. Here’s the thing, though. There are specific stains for such cells and structures. If you really want to know where the aluminum is localized, you stain with both the stain for aluminum and a different stain with a different colored fluorescent molecule attached, and then you can tell. Yes, I know it’s more complex than that, and that there are a lot more pitfalls of dual fluorescence staining with different probes, but, seriously. Scientists are getting good at this. It should be doable.

Fortunately, when it comes to the fluorescence microscopy, The Blood-Brain Barrier Scientist knows a lot more than I do, and blogged about this study. This analysis is a lot more technical than mine, particularly for the fluorescence microcopy. The discussion gets into such minutiae as how different sections are superimposed, and a lot of it overlaps what I found troubling as well, but it’s certainly worth reading if you’re more technically inclined. Bottom line: The fluorescence microscopy is dodgy, and it is vastly overinterpreted. That doesn’t even take into account the nonexistent to weak statistics in the paper.

Nowhere is this overinterpretation more evident than in the discussion:

This implies that aluminium somehow had crossed the blood-brain barrier and was taken up by a native cell namely the microglial cell. Interestingly, the presence of occasional aluminum-laden inflammatory cells in the vasculature and the leptomeninges opens the possibility of a separate mode of entry of aluminium into the brain i.e. intracellularly. However, to allow this second scenario to be of significance one would expect some type of intracerebral insult to occur to allow egress of lymphocytes and monocytes from the vasculature. The identification herein of non-neuronal cells including inflammatory cells, glial cells and microglia loaded with aluminium is a standout observation for ASD. For example, the majority of aluminium deposits identified in brain tissue in fAD were extracellular and nearly always associated with grey matter [19]. Aluminium is cytotoxic [21] and its association herein with inflammatory cells in the vasculature, meninges and central nervous system is unlikely to be benign. Microglia heavily loaded with aluminium while potentially remaining viable, at least for some time, will inevitably be compromised and dysfunctional microglia are thought to be involved in the aetiology of ASD [22], for example in disrupting synaptic pruning [23]. In addition the suggestion from the data herein that aluminium entry into the brain via immune cells circulating in the blood and lymph is expedited in ASD might begin to explain the earlier posed question of why there was so much aluminium in the brain of a 15 year old boy with an ASD.

This is speculation on steroids. More important, though, is the subtext. Where, I wonder, does Exley think this evil aluminum is coming from? He specifically mentions immune cells bringing aluminum into the brain as an explanation for “so much aluminum” in the brain of a 15 year old boy. Hmmm. He couldn’t mean…vaccines…could he? Oh, let’s cut the crap. Of course he means vaccines. Exley thinks aluminum adjuvants cause autism, and this study was looking for a way to “prove” that they do. This is, like most studies funded and carried out by antivaxers, horrible science. No wonder J.B. Handley is so impressed by it.

Finally, there’s aspect of this study that angers me greatly. Brain tissue is a precious resource. Tissue from autistic brains is an even more precious resource. This poorly designed, poorly executed, poorly analyzed study squandered some of that precious resource in the service of a study that tells us nothing scientifically useful about autism that could lead to a deeper understanding of the abnormalities of the autistic brain and the pathogenesis of this neurodevelopmental disorder. Such a waste of precious tissue borders on the criminal.