Aluminum causes autism? Anti-vaccine Shaw and Tomljenovic UPDATED

Every morning I wake up with the vain hope that the vaccine deniers will give up on the thoroughly debunked vaccines-cause-autism tropes. And every morning I’m disappointed. Today’s trope is that vaccine aluminum causes autism. Despite the claims, there still is no evidence.

Two of our favorite anti-vaccine shills, Christopher Shaw and Lucija Tomljenovic, have been the subject of scathing reviews numerous times by the feathery dinosaur. And they’re back again, using bad science, trying to convince the world that aluminum in vaccines is somehow linked to autism.

Well, let’s take a look at research, but don’t expect a different result when I last looked at a Shaw and Tomljenovic article about aluminum in vaccines. Which was retracted.

Aluminum causes autism – the paper

A new paper was published in the Journal of Inorganic Chemistry (we’ll get back to the choice of journal later in this article) by Shaw and Tomljenovic, from the University of British Columbia, Department of Ophthalmology. Digesting the article down to its basics, it attempted to show that injecting aluminum is related to autism.

In case you are new to the vaccine world, aluminum salts, in microgram quantities, are used as an immunologic adjuvant, that is, a chemical that enhances the immune response to the antigens in the vaccine. There are claims that aluminum is dangerous in vaccines, but there is simply no evidence that supports that claim. In fact, the amount of aluminum that is inhaled and ingested far surpasses the amount of aluminum injected via vaccines. And there’s simply no physiological reason why injected aluminum salts would be more dangerous than ingested aluminum.

But back to the paper. The authors concluded that,

Based on the data we have obtained to date, we propose a tentative working hypothesis of a molecular cascade that may serve to explain a causal link between Al and the innate immune response in the brain. In this proposed scheme, Al may be carried by the macrophages via a Trojan horse mechanism similar to that described for the human immunodeficiency virus (HIV) and hepatitis C viruses, travelling across the blood-brain-barrier to invade the CNS. Once inside the CNS, Al activates various proinflammatory factors and inhibits NF-κB inhibitors, the latter leading to activation of the NF-κB signaling pathway and the release of additional immune factors. Alternatively, the activation of the brain’s immune system by Al may also occur without Al traversing the blood-brain barrier, via neuroimmuno-endocrine signaling. Either way, it appears evident that the innate immune response in the brain can be activated as a result of peripheral immune stimuli. The ultimate consequence of innate immune over-stimulation in the CNS is the disruption of normal neurodevelopmental pathways resulting in autistic behavior.

Yes, that’s some heavy sounding science, but what does it say in simple terms. Well, Shaw and Tomljenovic attempted to provide evidence to support their preordained conclusion that aluminum adjuvants in vaccines can be linked to autism. The paper reports measurements of higher levels cytokines in the brains of mice injected with the aluminum adjuvant. And then they claim these cytokines are elevated in human autism.

I want to critique this paper in two ways. First, at the meta level, without actually looking at the data. Then, second, at the scientific level, examining their methods, results, and statistical analysis. Maybe we can answer the question whether this study actually provides any sort of evidence that aluminum causes autism.

A meta critique

Although the scientific critique is going to be more telling about the limited quality of this article, let’s take a look at some serious issues I have with this whole aluminum causes autism claim.

  1. Let’s be clear once again, there is simply no evidence whatsoever that shows a link between vaccines, with or without aluminum, having any link to autism. There are literally over 100 papers published in high quality peer-reviewed journals, looking at millions of vaccinated children, and they have found no links. None.
  2. There is no evidence that aluminum is associated with any neurodegenerative disease, including Alzheimer’s disease. In fact, the Alzheimer’s Society does not mention aluminum once in their discussion of risk factors for dementia. Maybe Big Aluminum is in control of the Society. In a meta review (the peak of the hierarchy of scientific evidence), no correlation was found between environmental aluminum and neurological diseases. None. 
  3. The claim that cytokines, which are small proteins produced by immune system cells to signal other cells to do their job, cause autism is weak at best. It is possible, though evidence is really not overwhelming, that increased levels of cytokines are indicative of a inflammatory response in the brain as a result of autism. But there’s no real evidence that cytokines cause autism, even if there was robust evidence that aluminum adjuvants increased cytokine levels.
  4. Publishing this article in the Journal of Inorganic Chemistry is curious. Inorganic biochemistry is the study of organometallic compounds. Aluminum is most definitely an inorganic chemical, but 99% of the paper discusses organic biochemistry, biology, genetics and immunology. As someone said online in a discussion of this paper, “what do the editors of this journal know of a DNA assay?” Well, they wouldn’t, it’s not part of the training of an inorganic chemist. And that becomes more obvious when we look at the data.
  5. Shaw and Tomljenovic are not immunologists. They are not epidemiologists. They have no training in any area related to vaccines. In fact, Shaw is located in the Department of Ophthalmology in the University of British Columbia. Shaw does claim he’s a neuroscientist, but his research focus is in  on amyotrophic lateral sclerosis (ALS) and the ALS-parkinsonism dementia complex, not autism.
  6. This research was paid by “the Dwoskin Family Foundation, the Katlyn Fox Foundation, and the Luther Allyn Shourds Dean estate.” The Dwoskin Family Foundation is one of the most profoundly anti-vaccine sponsors of research in the world. Claire Dwoskin is a board member of the anti-vaccine group, the National Vaccination Information Center. In 2011, the Dwoskins also underwrote the anti-vaccine “safety” conference in Jamaica, which included as speakers, Shaw and Tomljenovic.The other two groups, the Katlyn Fox Foundation and the Luther Allyn Shourds Dean estate, are also strongly anti-vaccine. These funding groups are just so anti-vaccine, it’s difficult for me to overlook the funding of this research in this case. I have found no indication that they give grants to any studies that are not supportive of any of the important tropes of the anti-vaccine movement.  I do think that the funding source should be given some weight in determining the bias of research. Even the anti-vaccine crowd agrees, because read just about anything they write in which they automatically dismiss anyone or any research that even has a tenuous tie to Big Pharma.

These six points alone, or together, are significant enough to dismiss this research out of hand. In fact, I was going to end my article here. Why bother with the scientific research when there’s much better research that shows no link between neurodegenerative diseases and aluminum, and we have not a shred of evidence that vaccines, with or without aluminum, are linked to autism.

But, let’s look at the study, because that’s what we do.

Vaccine aluminum causes autism – more critiques

So, let’s dig down into the science. Does this article actually given us any evidence that vaccines, and the aluminum adjuvants, are linked to autism? Not really. Here are some specific points:

  • The researchers used a mouse model. That in of itself is not a reason to dismiss this research, but let’s remember a key point – only about 1% of mouse research ever ends up being clinically significant. Frankly, most animal model research cannot be repeated. The insolent Orac is a skeptic of mouse research – “Again, this is a very artificial system basically designed to show something.” And that’s what Shaw and Tomljenovic did – they kept squeezing results until they got something liked.
  • I am troubled that they injected the aluminum adjuvant subcutaneously, that is, under the skin. Vaccines are almost always injected intramuscularly (into the muscle), as that is the best way to invoke an immune response. The authors rejected doing an intramuscular injection because they wanted to follow previous protocols. That’s unacceptable. The metabolism of the aluminum is simply different in subcutaneous delivery of the adjuvant, and no way compares to what is done in real world vaccinations.
  • The equipment they used to do PCR’s (polymerase chain reaction), a technique to amplify the amount of a focused segment of DNA is ancient, was last built 20 years ago. There are more modern, and not expensive, PCR equipment that are the standards for current literature. This is suspicious, but not necessarily a reason to dismiss the article out of hand.
  • The authors used an atypical methodology for determining gene expression (which is the whole point of the article). They used an older PCR method (possibly because of the ancient equipment) that does not provide quantitative measurements.  Furthermore, they did not confirm or repeat their results with more precise techniques. In summary, they used old equipment and old techniques (yes, PCR has improved over 20 years), without verification of the results. Shaw and Tomljenovic, looking to get answers that support their a priori conclusions, are trying to push results that are probably unsupported by the poor quality or overstatement of their results. (See Note 1)
  • The authors then used a Student’s t-test for statistical analysis. The problem with this type of statistical analysis is that the researcher can set the level of significance of the data, thereby rejecting or accepting the hypothesis, in this case that aluminum is associated with autism, simply by changing the significance level. There are better statistical tests that prevent manipulation of the significance levels of the results, providing confidence intervals that help determine the significance.
  • They didn’t test for expression of genes that are actually linked to autism. Let’s be clear, if aluminum supposedly is linked to autism, then there should be some up regulation of these autism-related genes that even their ancient technology and techniques could uncover. If they showed that, maybe, just maybe I’d be convinced that they had some evidence, but they didn’t show me anything except cytokines, which may or may not be an indicator of autism.
  • A cursory review of 179 citations highlights some troubling issues. Many of the articles simply do not support or even contradict the hypothesis presented by the authors. As Zared Schwartz stated online in a review of this article, the authors “spammed the paper with references.” Amusingly, 12 of the references were from Shaw and/or Tomljenovic. The problem is that their prior research has never stood up to scientific critiques. In fact, their research has been discredited by the World Health Organization. I wonder how often that has happened in scientific history.

Update – 26 September 2017

I am not a PCR expert by any means. My graduate degree and subsequent research never involved DNA testing, and it’s too complicated to even pretend to be an expert. I suspect that Shaw and Tomljenovic, based on some other analyses I’ve read, are trying to pretend to be DNA experts, and they’re failing miserably.

PubPeer, a website that runs like a journal club in grad school, where we would pick a paper and critically analyze it, took on the paper by Shaw and Tomljenovic. A lot of the analysis is highly technical, but there are a few takeaways that the reader should know:

  1. The same exact figure is used in two different papers (the other paper isn’t even indexed in PubMed), which in itself not a problem. However, the description of how the means and standard errors were derived were completely different. One is wrong.
  2. Some of the manipulation of the photos of the gels by the authors seemed to be non-standard or even attempted to bias the results. Of course.

There isn’t one positive comment about their results by real scientists examining their work.

The Mad Virologist, who is also an expert on DNA analysis, and the Blood Brain Barrier Scientist, who writes about the blood-brain barrier and heavy metal music, jointly analyzed the paper by Shaw and Tomljenovic. They did a masterful job in looking at some of the technical errors and other issues with the paper. I won’t pretend to summarize what they write, so I’ll use their own summary:

Based on the methods that were used in this paper, Shaw et al. went too far in declaring that aluminum adjuvants cause autism. But there are six other key points that limit what conclusions can be drawn from this paper:
1) They selected genes based on old literature and ignored newer publications.
2) The method for PCR quantification is imprecise and cannot be used as an absolute quantification of expression of the selected genes.
3) They used inappropriate statistical tests that are more prone to giving significant results which is possibly why they were selected.
4) Their dosing regime for the mice makes assumptions on the development of mice that are not correct.
5) They gave the mice far more aluminum sooner than the vaccine schedule exposes children to.
6) There are irregularities in both the semi-quantitative RT-PCR and Western blot data that strongly suggests that these images were fabricated. This is probably the most damning thing about the paper. If the data were manipulated and images fabricated, then the paper needs to be retracted and UBC needs to do an investigation into research misconduct by the Shaw lab.
Taken together, we cannot trust Shaw’s work here and if we were the people funding this work, we’d be incredibly ticked off because they just threw away money that could have done some good but was instead wasted frivolously. Maybe there’s a benign explanation for the irregularities that we’ve observed, but until these concerns are addressed this paper cannot be trusted.
Since this paper was funded by money from Big Anti-vaccine, they didn’t waste it. They had a pre-ordained conclusion, and they found evidence, manipulated by the way they analyzed the non-quantitative methods of DNA analysis, that supported that a priori conclusion.

Summary

Of course, the usual suspects of the vaccine denial world are all over this research. Frankly, all of the real epidemiological and clinical research show that there’s no link between vaccines and autism, so these science deniers have to jump on a weak and easily discredited study from Christopher Shaw and Lucija Tomljenovic.

So let’s remember the key points:

  1. There is no link between vaccines, or aluminum in vaccines, with autism. None. The vast bulk of really high quality science outweighs this study by orders of magnitude that are beyond description.
  2. Shaw and Tomljenovic are not credible researchers. They are essentially paid shills of the anti-vaccine industry. They have had papers retracted, and their research was described by WHO as “seriously flawed.
  3. This research was poorly executed using outdated equipment and outdated techniques for DNA analysis. If we are to accept research that changes our fundamental policies about life saving vaccines, then we need powerful evidence to make that change. This isn’t it.

It is becoming clear that this paper tell us nothing about whether aluminum causes autism – in fact, there seems to be evidence that this paper should be retracted. I think a lot of smart people have reviewed this paper, and there are a lot of substantial number of errors, intentional or not, that can only lead a reasonable person to reject this paper.

Since well designed scientific studies reject any link between vaccines and autism, to contradict that conclusion, we need well-done, strong papers. This isn’t it. This paper isn’t even close.

I am really tired of Shaw and Tomljenovic – but they give me fodder for articles.

Notes

  1. Alma Glenn Laney, a real scientist, wrote a more detailed analysis of the DNA techniques which I digested down to its essentials. This is what he wrote in full:

    They use a technique called semi-quantitative RT-PCR to try and quantify expression of the genes. However, they did not digest the extracted RNA with DNase to remove any contaminating DNA (they claim they used a technique to avoid this, but this isn’t proper procedure for doing this type of test) and they try and quantify the results from this assay. The assay is good to see if a gene is expressed or not, but they ran it for longer than is normal (30 cycles instead of 20-25 cycles which is a huge difference as PCR is exponential; typically endpoint PCR is 30-35 cycles, so this is pushing the semi-quantitative RT-PCR beyond what it meant to do) and this method is not not meant to be quantitative (and far more precise techniques exist to measure gene transcription levels). They also did the experiment in a very old thermocycler that hasn’t been made by the company they cite in almost 20 years (it got bought by another company), so I don’t trust the PCRs to be as accurate as they claim. They only show cut out portions of the gel images and not the entire gel, so we don’t know if weak bands are due to low template abundance or because of off target amplification reducing the amount of resources available (this is crucial for this type of experiment). Another problem is that they do not confirm these results with a more precise technique like realtime PCR or RNASeq.

Citations

The Original Skeptical Raptor
Chief Executive Officer at SkepticalRaptor
Lifetime lover of science, especially biomedical research. Spent years in academics, business development, research, and traveling the world shilling for Big Pharma. I love sports, mostly college basketball and football, hockey, and baseball. I enjoy great food and intelligent conversation. And a delicious morning coffee!