[Neurosciences/Junk Sciences] Autopsy of a flawed study of aluminum and brain inflammation (Li et al., J Inorg Biochem 2017)

Note: This is a special blog post coauthored by The Mad Virologist and The Blood-Brain Barrier Scientist (this article will be co-published on both our blogs). Another post has already been published on this paper, but we wanted to take a deeper look at everything that is wrong with this paper.

[UPDATE2] The study in question got retracted according to RetractionWatch:
http://retractionwatch.com/2017/10/09/journal-retract-paper-called-anti-vaccine-pseudoscience/

[UPDATE] I would strongly recommend the reader to look at the comments on Pubpeer about this paper. It is terrifying to think how it percolated through peer-review.
https://pubpeer.com/publications/4AEB7C8F30015079E2611157CF8983#undefined

A recent paper by ophthalmologist Chris Shaw was published and immediately touted as being proof positive that the aluminum adjuvants found in some vaccines are responsible for causing autism. Before we get into the paper, I have a few choice things to say about Chris Shaw. Despite not being an immunologist, Shaw has ventured into studying how vaccines and vaccine adjuvants cause neurological disorders such as autism. Shaw made headlines in 2016 when a paper he co-authored that claimed to show a link between the HPV vaccine and neurological disorders was retracted after being accepted by the journal Vaccine. It turns out that the statistics used in the paper were completely inappropriate and there were undisclosed conflicts of interests for some of the authors, including Shaw.These issues should have prevented the paper from being accepted in the first place, but mistakes do happen and science tends  to be self correcting. More surprising is that Shaw claimed that he didn’t know why the paper was retracted and that the science was of the highest quality. Shaw’s previous work has also been described by the WHO as deeply flawed and rejected by that body. This isn’t being brought up to dismiss the paper out of hand but to help illustrate why Shaw’s work is deserving of additional scrutiny. Hopefully by the end of this post, the logic behind the need for additional scrutiny of anything Shaw publishes is abundantly clear. We’ll begin by examining the methods used by Shaw’s research group and point out some of the issues.

Background for experimental design flaws: PK and species issues

One problem that is recurrent with Shaw is his “vaccination schedule” tries to consider rodents, such as mice and rats, as humans in miniature. It is wrong to assume that rodent and human primate species are alike, they’re not and there are notable physiological differences between rodents and non-rodents. For example, there are a couple of studies by Terasaki and colleagues (http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2011.07208.x/abstract) that have shown differences in the expression of solute carriers and drug transporters at the blood-brain barrier. We cannot exclude that such differences may bias the outcome observed in his studies, but this bias applies intrinsically to any in vivo studies based on a rodent model.
There is also the issue of brain development and mapping the vaccination schedule and the brain maturation. In this study (as well in the previous ones), Shaw and colleagues consider that applying vaccines from post-natal day (PND) 3 to 12 is representative of a human infant vaccine schedule. There is some differences in the literature, as previous studies from Clancy and colleagues mapped the PND12 to the 7th gestational months in humans (https://blogs.cornell.edu/bfinlay/files/2015/06/ClancyNeurosci01-17kkli7.pdf), some more recent publications map PND21 to 6th month post natal in humans, making the PND12 around the 3rd month infancy following full-term birth (http://www.sciencedirect.com/science/article/pii/S2352154615001096). You can easily appreciate that by following Shaw flawed experimental design, the total amount of Al administered during a 2 year period has been indeed administered within 90 days of birth, whereas the vaccination schedule according to the CDC does not start before the 2nd month of infancy if we exclude the two injections of Hepatitis B vaccines at birth and after the first month respectively (https://www.cdc.gov/vaccines/schedules/hcp/imz/child-adolescent.html).

In addition to a flaw in the experimental design, we cannot exclude some differences in the pharmacokinetic profile of Al adjuvants between mice and humans. The data available is fairly limited but a recent study from Kim and colleagues (https://www.ncbi.nlm.nih.gov/pubmed/26437923) failed to show a significant brain uptake of Al compared to controls following the single oral administration of different Al oxide nanoparticles at a concentration of 10mg/kg. Furthermore, the approximation of Shaw in terms of total burden of Al from vaccines (550 microg/kg) is not an accurate metric as we have a dynamic process involving absorption, distribution and elimination to occur simultaneously. A daily burden of Al from vaccines is a much more reliable parameter to consider. Yokel and McNamara (https://www.ncbi.nlm.nih.gov/pubmed/11322172) established it about 1.4-8 microg/day for based on 20 injections spanning over a 6-year period in a 20kgs individual.
If we consider Shaw calculation, then the total burden at age 6 would be 1650 microg/kg or 33’000 microg for a 20kgs 6-year old child. That’s about 15 microg/day of daily Al burden from vaccines, a value that is 2 to 10 folds higher than applied to humans. It makes therefore very difficult to compare apples to oranges, as Shaw experimental paradigm is flawed and not representative of a clinical scenario.

Selection of genes to measure:

Selecting which genes to measure is a crucial step in a study like this. If care is not given to ensure that the correct genes are selected, then the study will be a wasted effort. Shaw stated in the paper that they selected genes that were previously published. However, not all of the genes that they measured came from this paper. Only 14 of the genes were from this paper (KLK1, NFKBIB, NFKBIE, SFTPB, C2, CCL2, CEBPB, IFNG, LTB, MMP9, TNFα, SELE, SERPINE1, and STAT4). This leaves 17 genes the were measured but not found in the paper. Two of these can be explained. One gene, ACHE, was mentioned as having been selected because of other work, so it is sourced. The second gene, is the internal control gene beta-actin. This is a housekeeping gene that is often used as an internal control to provide a relative expression from. This leaves 15 genes unaccounted for. We suspect that these genes were selected because they are involved in the innate immune response, but no reason is stated in the paper.

The way these genes were selected is problematic. Because half of the genes seemed to be selected for uncited reasons, this study is what is known in science as a “fishing expedition.” There’s nothing inherently wrong with this type of research and indeed it can lead to new discoveries that expand our understanding of the natural world (this study that increased the number of sequenced viral genomes by nearly tenfold is a good example of this). But what fishing expeditions can show is limited. These types of studies can lead to other studies but they do not show causality. Shaw is claiming causality with his fishing expedition here.

There is also the problem that they used old literature to select their gene targets when much more recent research has been done. By happenstance, they did measure some of these same genes in their study. However, their results do not match has has been measured in children that have been diagnosed with autism. For example, RANTES was shown to be decreased in children with autism. In Shaw’s work there was no statistical difference in RANTES expression between mice given the aluminum treatment and those receiving saline. Likewise, MIP1alpha  was shown to be decreased in developmentally delayed children but was shown to be increased in the aluminum treated mice. This was also the case for ILIb which was found to be elevated in children with moderate autism yet there was no statistical difference between the mice receiving the aluminum treatment and those receiving saline. In fact IL-4 was the only gene to follow an expression pattern similar to what was found in children with severe autism (elevated in both cases). However, there is something odd with the gel in this case. This was the image for figure 4 that was included in the online version of the paper (we have not altered the image in any way). Look closely at the top right panel at the IL-4 samples and the IL-6 samples. You’ll notice that the bands for the control and the aluminum treated mice have different color backgrounds (We enlarged the image to help highlight this but did not adjust the contrast). If these came from the same gel, there would not be a shift in color like this where the treated bands have a lighter color encircling them. The only way this could happen is if the gel was assembled in photoshop. The differences could be real; however, since this image was modified we do not know for sure and this is scientific misconduct. Papers get retracted for this all the time and people have lost their degrees for doing this in their dissertations. These gel results cannot be trusted and the paper hinges on them. The Western blots and issues with them will be discussed below.

22016544_10102969159317918_1868648690_n

The unaltered figure 4.

22052798_10102969159312928_192591888_n

A close up of the panel with the regions in question highlighted.

Semi-quantitative RT-PCR:

In order to quantify the gene expression levels of the genes that Shaw’s group selected, they used an older technique called semi-quantitative RT-PCR. This technique uses the exponential increase in PCR products in order to show differences between expression of a gene under different conditions. There’s nothing wrong with the technique provided one understands what the limitations are. Let’s say you have a large number of genes that you want to measure expression of, but you aren’t sure which genes are going to be responsive and you have limited funds. Semi-quantitative RT-PCR is a good method to screen for specific genes to be examined further by more precise techniques, such as Real-Time RT-PCR, but it’s not appropriate to use this technique and then make statements about precise quantification. Where semi-quantitative RT-PCR excels is with genes that are normally not expressed but can be expressed after some sort of stimulus, such as terpene biosynthesis genes that are induced by insect feeding.

To put it bluntly, semi-quantitative RT-PCR was not used properly in the paper by Shaw. The way that it was used implied that it would be quantitative when the technique is not that precise. Without verification by another method, ideally Real-Time PCR which can determine what the exact abundance of a given target is, these results should be taken with a grain of salt. This would still be the case if there weren’t irregularities in the gel images. With those irregularities, this is absolutely essential and should have prevented this paper from being accepted.

Western-blots and data manipulationPCR and Western-blots data: the owl is not what it seems
As The Mad Virologist mentioned, the semi-quantitative PCR is an old-fashioned RNA quantitation method, with the use of Real-Time quantitative PCR (that quantifies the amplification product at each cycle, using a fluorescent dye as an indicator) is a much more accepted method nowadays (see his section for more details). For Western-blots, the semi-quantitative approach is more accepted but it is important to show data that are consistent between what you show (qualitative) from what you count (quantitative). In Western-blot analysis, we measure the relative darkness of a protein band (the black lines that you see in papers) between treatments and controls. Because you cannot exclude some errors due to the amount of protein loading, we also measure the band intensity for proteins that are very abundant, usually referred as housekeeping proteins (because they play essential functions in cells). In this case, beta-actin (named ACT in the paper was used).
Once you normalize to beta-actin, you can compare the effect of a treatment by comparing the relative band intensity ratios. In both cases (semi-quantitative PCR and Western-blots), “what you see is what you measure” or you have to show a “representative Western-blot” alongside a quantitative data to demonstrate that your quantification matches with band densities. The common practice is the use of image acquisition software like ImageJ to determine band density. Showing Western-blot is nice, but not foolproof. Indeed, Western-blots data (with fluorescence images) is amongst the most common method by which some researchers can manipulate or even falsify data but also the most common type of data that spark a paper retraction. Someone notice something fuzzy on a Western-blot data, creating some questioning reaching to the editors and asking access to the full dataset (usually the X-ray film or the original full scan of the blot). Often, the author will use the excuse “the dog ate the flash drive” or “the hard drive containing the data crashed” if they cannot provide such data.
There are some methods to spot some image manipulation on Western-Blots and include playing with the brightness/contrast, requesting the presence of quantitative data in addition of a representative blot, samples must be coming from a same gel (you cannot use a cookie-cutter and build-your-own perfect gel). There is an excellent article that describe the pitfalls and cases of bad Western-blot data representation if not image manipulation. (https://www.elsevier.com/editors-update/story/publishing-ethics/the-art-of-detecting-data-and-image-manipulationThere are, at this time, different issues raised both in the Western-blots pictures and their subsequent analysis raising the reliability of the data presented in this study.

In this post, we have used the full-resolution pictures provided by the journal website (http://www.sciencedirect.com/science/article/pii/S0162013417300417), opened just pictures in ImageJ to convert such pictures into 8-bit format, invert the lookup tables (LUT) and adjusted the brightness and contrast. We have exported such pictures in Powerpoint to ease the annotation and comments. We recommend the reader to judge by himself/herself and download the full-resolution images as well.

The first concern is by looking at Figure 1C. First, this is the original Fig.1.

1-s2.0-S0162013417300417-gr1_lrg

Then, this is the close-up analysis for Fig.1C

Slide1

There are several issues. First there are some bands that appears as band splicings, in which the author create a custom blots by assembling different bands from different gels. This is a no-no in Western-blots: all bands showed in a blot should come from the same gel. This is why Western-blot is a torture for graduates students and postdocs, you need to show your best blot with all bands showing the same behavior for your quantitative analysis.
Second, the presence of a rectangular grey piece that was added on the top of control 3 TNF band. This is a possible data manipulation and fraud, as you are voluntary masking a band and hiding it. Thats a big red flag on the paper. The third issue of Fig.1C is the consistent feeling of seeing bands either cropped on a grey rectangle or what I call a “Photoshop brushing” in which you brush off using the brush function area of the gel you consider not looking good enough. You can clearly see it with actin as we have a clear line between the blurred blot and a sharp and uniform grey in the bottom half of the blot, compared to the wavy top of the blot. This a grey area that I am not familiar with Western-blot but this is a no-no for any immunofluorescence picture. Any image manipulation that goes beyond the brightness/contrast adjustment and involves alteration of the acquired picture is considered as data manipulation. If you analyze the data upon correcting for the inconsistency of Figure 1C, the graph looks much more different and failed to show any differences between Al-treated and control, when you restrict yourself in over-normalizing it and plot straight the protein/actin band density ratios.

What is also concerning and surprising is the conclusion from the authors that males, not females, showing an inflammatory response. Of course, the authors failed to show the same outcomes from female animals and expect us to trust them on this. The problem is that such conclusion is in direct contradiction with the literature. There is a solid literature supporting the presence of a sexual dimorphism in terms of inflammatory response, in particular in terms of neuroinflammation and autoimmune disorders such as multiple sclerosis (https://www.ncbi.nlm.nih.gov/pubmed/28647490; https://www.ncbi.nlm.nih.gov/pubmed/27870415). There is also a growing call to the scientific community to provide results for both sexes (males and females alike). Although Shaw reports the study was performed in both males and females, he gives us this explanation at the end of section 3.1: Taken together, a number of changes indicative of the activation of the immune-mediated NF-κB pathway were observed in both male and female mice brains as a result of Al-injection, although females seemed to be less susceptible than males as fewer genes were found altered in female brains.

Yet the interesting part comes when Shaw try to compare ikB phosphorylation between males and females following Al injection (Fig.3C). When you analyze the data, you are raising concerns very rapidly. First, we have a possible case of cookie-cutter band in which you just paste a band that seems nice enough in a blank space. This is a very suspicious activity as you can make up data as easy as this. Second, there is again this “Photoshopping brushing/erasing” taking place in that figure, in which I suspect a case of fraudulent activity. As you can see in female, it is as if someone tried to mask some bands that should not have been here. Remember when he said that males but not females showed an inflammatory response? Is it trying to dissimulate data that contradict his claims?

image

Again, lets bring up Figure 3 at its full resolution.
1-s2.0-S0162013417300417-gr3_lrg

Finally, the same issues are persistent and even more obvious in Fig.5A. Again, we have a mixture of different Western-blots image manipulations including bands splicing, Photoshop brushing, cookie-cutter bands……

First, the unedited picture:
1-s2.0-S0162013417300417-gr5_lrg

And below the close up of Fig.5A

Slide3

These are some serious concerns that raise the credbility of this study and can only be addressed by providing a full-resolution (300dpi) of the original blots (X-ray films or the original picture file generated by the gel acquisition camera).  There has been a lot of chatter on PubPeer discussing this paper and many duplicated bands and other irregularities have been identified by the users there. If anyone is unsure of how accurate the results are, we strongly suggest looking at what has been identified on PubPeer as it suggests that the results are not entirely accurate and until the original gels and Western blots have been provided, it looks like the results were manufactured in Photoshop.

 

Statistics:
Long time followers know that I tend to go right to the statistics that are used in papers to see if what they are claiming is reasonable or not. Poor use of statistics has been the downfall of many scientists, even if they are making honest mistakes. It’s a common problem that scientists have to be wary of. One easy solution is to consult with a statistician before submitting a paper for publication. These experts can help point out if the statistical tests that were run are the correct or not. The Shaw paper could have benefited from this expertise. They used a Student’s T Test for all of their statistics comparing the control to the aluminum treated. This is problematic for a couple of reasons. These aren’t independent tests and the data likely does not have a normal distribution, so a T Test isn’t appropriate. Better statistical tests would have been either Hotelling’s T-squared distribution or Tukey’s HSD.  Another issue is how the authors used standard error (SE) instead of standard deviation (SD). To understand why this matters, it helps to understand what the SE and what the SD measure and what these statistics show. The SD measures the variation in samples and how far the measurements are from the mean of the measurements. A smaller SD means that there is low variability in the measurements. The SE measures the likelihood that a measurement varies from the mean of the measurements within a population. Both the SE and SD can be used; however, using the SE is not always appropriate, especially if you are trying to use it as a descriptive statistic (in other words if you are trying to summarize data). Simply put, the SE is an estimation and only shows the variation between the sample mean and the population mean. If you are trying to show descriptive statistics, then you need to use the SD. The misuse of SE when the SD needs to be shown is a common mistake in many research publications. In fact, this is what the GraphPad manual has to say about when to use the SD and when to use the SE:

If you want to create persuasive propaganda:
If your goal is to emphasize small and unimportant differences in your data, show your error bars as SEM,  and hope that your readers think they are SD. If our goal is to cover-up large differences, show the error bars as the standard deviations for the groups, and hope that your readers think they are a standard errors.” This approach was advocated by Steve Simon in his excellent weblog. Of course he meant it as a joke. If you don’t understand the joke, review  the differences between SD and SEM.” The bottom line is that there is an appropriate time to use the SE but not when you are trying to summarize data.

Another issue is the number of animals used in the study. A consensus in published study is to provide a minimal number of animals (usually n=8) needed to achieve statistical significance but also maintain to a minimum to ensure proper welfare and humane consideration for lab animals. In this study, such number is half (n=5). Also the authors are bringing some confusion by blurring the lines between biological replicates (n=5) and technical replicates (n=3). By definition, biological replicates are different organisms that are measured and are essential for statistical analysis as these replicates are independent from each other. Technical replicates are dependent on each other as they come from the same biological samples and are repeated measurements. By considering the latter as statistical relevant, you are biasing yourself to consider a fluke as a biological phenomenon.

 

Conclusions:
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.

Maybe there’s a benign explanation for the irregularities that we’ve observed, but until these concerns are addressed this paper cannot be trusted.

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[BBB/Junk Sciences] Polysorbate 80 and the BBB or how to put anti-vaxxers into a blowing cognitive dissonance

Here we go again, anti-vaxxers keeping on moving the goalpost to fit their belief instead to change to adjust it to the facts. First it was mercury, then it was formaldehyde, then aluminum, today the “ingredient du jour” is polysorbate 80 and tomorrow they will blame it to PBS saline solution.

The latest fad as I have seen is to blame polysorbate 80 as a source of “vaccine-injury” with the bold claim that it breaks down the blood-brain barrier (BBB). Lets put the fact straight and debunk this one for all. But what is even better is the “what if” counter-argument. What if polysorbate 80 was indeed a good ingredient? I will come to that later.

Polysorbate (aka Tween 80) is a amphiphile compound   as you can see the molecular structure below (source Wikipedia):
1200px-polysorbate_80

You can see the structure made of a lipophilic (loves fat) tail and a series of hydrophilic  (loves water) tails, loaded with oxygen and hydroxyl groups. This is a typical structure of a detergent: one side will mix well with water, the other will mix very well with fat and oils. The result? You can form microspheres that can dissolve well in water and dissolve fat into water. This is how a detergent works, it helps to breakdown fats into small spheres and dissolve them in the drain water.
Polysorbate 80, due to this property, is very good to dissolve drugs and medicines that under normal condition would barely dissolve into biological fluids. This is why we have it in vaccines, but we also have it in medicines. Thats the job of biopharmaceutics: finding formulations to dissolve drugs into the body and allow them to reach a concentration high enough to display their therapeutic activity.

The use of polysorbate 80 in drug delivery of anti-cancerous drug is probably the first and foremost main driving factor on investigating its effect on the BBB. Brain tumors (primary and metastatic alike) are up until now one of the most dreaded and deadliest form of cancer. For instance, the average expected lifespan upon diagnosis of a grade IV glioma (aka glioblastoma multiforme) is grim: 18-months, with less than 5% survival after 5 years. The major issue is being able to deliver drugs and chemotherapy across the BBB. As reported by Pr. William Partridge (UCLA) the BBB remains the bottleneck in drug development for the treating neurological disorders (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC539316/?fref=gc&dti=873247819461536)

The first report of the investigation of polysorbate 80 on the BBB is probably by Spiegelman and colleagues in 1984 (http://thejns.org/doi/pdf/10.3171/jns.1984.61.4.0674), investigating the effect of the solvent used in etoposide solution for treating cancer. According to their  result, they noted a statistical difference in the BBB permeability  (using Evans Blue and 99mTc as tracers) following the injection of 1.125ml/kg. According to their paper, 5mL solution contained 400mg of polysorbate 80 or a concentration of 80mg/mL. Based on this, we can assume that the BBB effect was observed for a dose of 90mg/kg. Thats a very huge dose.
If we go back to the manure anti-vaxxers say, the amount injected via vaccines is enough to cause a barrier opening. According to John Hopkins University Institute of Vaccine Safety (http://www.vaccinesafety.edu/components-DTaP.htm), the expected concentration of polysorbate is lesser or equal to 100mcg or micrograms. Thats 0.1mg per dose. If we assume such dose is injected to a newborn (average weight ~3 kgs), then the amount injected is about 0.033mg/kg. Thats 2700 times less than what has been reported to induce a BBB disruption. Also you have to factor the bioavailability of polysorbate (that is 100% upon IV route) making this number a very optimistic number.
Now, the interesting twist about polsyorbate 80 is its use to enhance some drug carriers and its widely used for finding novel formulation to enhance the delivery of anti-cancerous drugs across the BBB. You can find a list of publications on Pubmed about that aspect (https://www.ncbi.nlm.nih.gov/pubmed/?term=polysorbate+80+blood-brain+barrier). What if polysorbate 80 not only will not injure your brain, but actually may help deliver drugs to help your brain fight disease?

 

Keep in mind that polysorbate 80 is good at dissolving lipid in water solutions but it is not good to let charged molecules accross the BBB, just in case someone comes with the claims that it conjugates with aluminum. Thats some high-school chemistry level.

 

 

[Sciences/Junk Sciences] How the recent AHA recommendation on coconut oil is making many getting nuts (and why coconut oil is not an healthy choice)!

Coconut oil. Coconut oil. Yep, that same coconut oil that (almost) nobody knew about a couple of years ago and suddenly became the next big thing in fad diets. Some claimed it is healthier than vegetable oil (http://pilatesnutritionist.com/why-coconut-oil-is-better-than-vegetable-oil/; which turned out is not true), other claimed it can help you loose weight (https://authoritynutrition.com/coconut-oil-and-weight-loss/; that is also hard to imagine how to loose fat by keeping an high-fat diet) or even use it as a natural sunscreen (http://thecoconutmama.com/coconut-oil-sunscreen/; which of course will more likely help you roast like a rotisserie chicken).
You see the fad went a bit crazy with the habitual “wellness” bloggers making miraculous claim. The fact is coconut oil is no better than any oil and indeed maybe as bad as any saturated fats.
The only thing that I would say coconut oil is good, is giving you some tasty and crunchy fries that are not too greasy. Any French household know the “Vegetaline” brand (basically solid coconut oil that you mix with half sunflower oil to get a frying oil).

What is (in terms of chemical composition) coconut oil?

Coconut oil is extracted from the inner side of the coconut. It is also called copra oil. Some coconut oil are referred as “organic coconut oil” and even some referring as GMO-free coconut oil (you know the GMO-free project sticker that have no sense except operating as a form of racketeering? There are been never any GM-coconuts that hit the market. http://www.zebraorganics.com/organic-virgin-raw-coconut-oil-1-gallon-tub-zebra-organics.html?gclid=Cj0KEQjwyZjKBRDu–WG9ayT_ZEBEiQApZBFuK3KbEfSPhyNyx9z9eNUIwAmd6OwcxTWJUYKADA_fhEaAnvd8P8HAQ). Therefore, we consider all coconut oil equals (maybe slight variations between cultivars but this should not affect much the overall composition to be considered significant).

Before we discuss about the composition of coconut oil, it is important to know what a fatty acid is. Fatty acids (FA) are hydrocarbon chains (made of carbons and hydrogens) that are very similar to molecules belonging to alkanes (these are the molecules such as propane, butane and octane that are present in your propane gas tank right now fueling your grill, fueling your gas stove or fueling your SUV).
In contrast to alkanes, FA have a carboxyl (-COOH) “head” denominated and seen below:

We have two type of FA: saturated FAs (fully loaded with hydrogens) and unsaturated FAs (that have one or several C=C double bounds). Saturated FAs are usually found in fat products from animal origin (lard, butter, ghee…) whereas unsaturated FAs are usually found in plants (olive, rapseed/canola, corn, sunflower…) and in fish and seafood (usually polyunsaturated fatty acids or PUFAs aka omega- fatty acids). Unsaturated FAs either show a cis-form (like the oleic acid depicted, in which the two carbon branches are in the same side) or a trans-form (in which the two pieces of the carbon branches are opposing each other). Trans unsaturated FAs (aka trans-fats) have been already a bad rep because of their detrimental effects on the cardiovascular system (they are suspected to increase LDL levels which are known to contribute in the atherosclerotic plaques formation). Saturated FAs are also having a bad rep because they are also associated with increased risk of cardiovascular diseases, whereas unsaturated FAs (commonly found in “the Mediterranean diet”) are considered healthier.
FAs composition are usually denominated as the following: Cn:m with n referring to the number of carbons (usually an even number), m referring to the number of C=C. In our cases, stearic and oleic acid share the same number of carbon (C18) but the former has no C=C bounds (C18:0) and the latter has a C=C bound (C18:1).
Based on this table, you can see how coconut oil fares to other oils (https://www.chempro.in/fattyacid.htm)
It contains 90% of saturated FAs and 10% unsaturated FAs, whereas most of other oils commonly used in Western countries have at least 50% or more of unsaturated FAs. To give you an idea lard, tallow (beef) and butter contains 40%, 37% and 41% respectively.  You can see how coconut oil is exploding the chart.

But, but this is coming from one study and science has been wrong all the time

If you stick to mainstream media, you will get this impression right. News outlets like to sell single studies as sold and irrefutable evidence and often oversell the claims of that study. Science is never settled, especially on a single study. Many things can go wrong that result in bias. Sometimes, scientists even cut the corners and publish fraudulent data to support their claims (thats what you see a lot with anti-vaccines, anti-GMO papers, climate-deniers, creationism……).
Science build a consensus on the amount of publications and their robustness in their experimental design. When you have an overwhelming majority of papers show you a same trend, arrive to same conclusion on a phenomenon using different approaches and different observations by different groups, you reach a conclusion and set a consensus.
A consensus is only broken once you have new studies that refute the existing claims with more robust and more precise data than the existing literature. This happens very rarely as you have to being in a weight of evidence bigger than the existing literature.

The science on FAs and their effect on cardiovascular diseases is not new, this have been known for over 50 years and keep refining. This consensus built on the detrimental effects of high-fat diet is well-known and served to establish guidelines and public health recommendations. The American Heart Association, the leading association worldwide gathering both basic and clinical scientists as well as any healthcare actors establish guidelines.

The AHA has a clear statement, visible here:
http://news.heart.org/advisory-replacing-saturated-fat-with-healthier-fat-could-lower-cardiovascular-risks/
Replacing saturated fats may help to reduce your risk of cardiovascular events, in addition to an healthy (balanced) diet and physical activity.

The study that made the uproar is available here and comes from the scientific board of the AHA. You can download it for free and you can see another fat composition of different oils:
http://circ.ahajournals.org/content/early/2017/06/15/CIR.0000000000000510

As you can see, coconut oil tops the list of saturated oils and fats, followed by butter and lard. Saturated fats consumption are clearly associated with increased risk of coronary heart diseases (CHD, aka heart attack), replacement with unsaturated fats reduce such risks. Replacement with PUFAs appears even more beneficial. Such effects is not limited to CHDs, but appears involved in other diseases as well (see Figure 4).

In conclusion, dont ditch your coconut oil yet. As small amount, coconut oil is fine. What is not fine was the fad diet that was basically pushing you to switch everything to coconut oil. In my personal opinion, I would say that butter (real unsalted butter like the French “President”, Irish “Kerrygold” or Danish “Lupak” butters; not the things called margarines that were at the basis of the trans-fat problem),  was even a better alternative  than coconut oil.

In conclusion, keep your peanut oil for your deep-frying cooking, keep your canola oil for your dressings and use olive oil for cooking instead of lard and coconut oil. If the taste of coconut oil is good, just add the minimal amount needed to taste.

 

[Sciences/Junk Sciences/BBB] Aluminum adjuvants in vaccines and the blood-brain barrier. Removing the facts from the fiction, the good science from the junk science.

Preface:

This post constituted a direct response I had to the article posted by VaccinePapers.org in February 10 2015 (http://vaccinepapers.org/vaccine-aluminum-travels-to-the-brain/). However, it recently got a recycling on the Collective-Evolution article (written by an author from VaccinesPapers) through this post:
http://www.collective-evolution.com/2017/04/14/groundbreaking-china-study-links-immune-activation-by-vaccination-autism/

I will not focus more on the first half of this article and I am just providing with some facts about it that I discussed earlier on Facebook.

1. First paper (i exclude the CalTech thing since it is not peer-reviewed): They observed that mothers experiencing inflammation during pregnancy had higher risk of having offsprings with autism. Thats legit science. The senior author (Patterson) was (died in 2014) was a legit and recognized neuroscientist in the field of autism. This inflammation is either the result of some autoimmune disorder or infection. Thus the need to have mothers infection-free or avoid infectious diseases by keeping an updated vaccine schedule.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2387067/
2. The second paper, again from the Patterson lab, show again the same conclusion, this time on rhesus monkey. It again emphasize the impact of infectious disorders on maternal gestation and the risk associated on the baby. Another good reasons for expecting mommies to keep their vaccines schedule up-to-date.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3322300/
3. Now the China study show that if you increase interleukin-6 levels in the brain (IL-6, a well-known pro-inflammatory molecule), you can induce behavioral outcomes in mice that are considered representative of the ASD. It goes in the same direction that what Patterson showed and further underline the danger of having an “overactive/boosted” immune system and its ability to cause neuroinflammation. This is a growing field as we speculate that some psychiatric (depression) and neurological diseases other than multiple sclerosis (Alzheimers, stroke) maybe aggravated or induced by an inflammation and overactive immune system.
http://www.sciencedirect.com/…/pii/S0925443912000234. Then we have the slippery slope in which CE drank the Kool-Aid of BS by “ergo post hoc” fallacy. The false association fallacy: If cars run on petrol and because cars kill pedestrians, therefore petrol kill pedestrians”. This is the BS they are doing. Since vaccination will induce a transient inflammation during the immune reaction and inflammation cause autism therefore vaccines cause autism”. Of course this was debunked but here they came back with the moving goalpost. Since thiomersal did not cause an increase in number of autism, then the anti-vaxxers moved to “then it should be formaldehyde. No? Then it should be aluminum….”

To better debunk the bogus claim brought by the VaccinePapers post, I have written a long but detailed description on what is wrong with that post. Since I had initially written down into a Word documents with elements embedded in it, this may have been some formatting issues in this post. My sincere apologizes.

In this blog post, the author primarily focuses on the vaccine aluminum nanoparticles to enter and accumulate in the brain. Using several peer-reviewed articles, the author tries to convince that aluminum nanoparticles in the vaccines are uptaken by macrophages, such macrophages are capable to enter the brain and trigger neuroinflammation.

Therefore, the message of this post is clear: vaccines contain aluminum nanoparticles, aluminum is neurotoxic, and therefore vaccines are neurotoxic.  If you travel in time, back in the early 2000s, the same blog post title would have talked about ethylmercury (formulated as thiomersal) contained in some vaccines and would have cited the famous “Wakefield paper” that was just published (and will be retracted a couple of years later due to gross scientific misconduct).

The blog website “Vaccine Papers” has the following slogan “an objective look at vaccine dangers”. Is it really an objective look or it is another anti-vaccine website distorting scientific studies to make fallacious claims in order to support an anti-vaccine agenda?

The goal of this article is to analyze, criticize and debunk claims made in this article and reveal the scientific fraud of this post and raise questions about the credibility of this website as a whole. In this post, we will explain why vaccines contain aluminum, which aluminum formulation has been used and is currently used in vaccines. Then we will refute the author’s arguments by directly citing passage of the post and provide a clear discussion about it.

  1. How do vaccines work?

To better understand the use of aluminum in vaccines, it is important for the reader to understand some fundamental concepts of immunology and how vaccines work. In mammals, we have a particular set of blood cells that are taking care of any foreign entity entering our body. It is called the immune system. To give an analogy, you can compare the immune system to the Department of Defense and the Department of Homeland Security, ensuring that anyone coming in to the United States is not posing a threat to the country.

Immune cells are all derived from a particular set of cells residing in the bone marrow: the hematopoietic stem cells (HSCs), such cells provide all the different cell types in your blood, whether we are talking about red blood cells, white blood cells or platelets.

In the figure depicted below (source: http://textbookofbacteriology.net/adaptive_2.html), HSCs will give two major cell lineages: the lymphoid stem cells (that will give the T and B lymphocytes) and the myeloid progenitors (including monocytes and macrophages).

When a foreign agent enters the system, it will induce two different types of response. In the case of a virus, it will infect some cells (depending on the type of virus) and will trigger the expression of molecules (usually viral proteins necessary for the spreading of the virus) on the cell surface. In the case of a bacteria or any entity that has a reasonable size (one thousandth of a millimeter), the bacteria will be swallowed by macrophages circulating nearby, digested and expose some fragment of this entity on its cells’ surface.

Finally circulating foreign agents can be recognized by a particular class of lymphocytes called B cells. B cells are like “keymasters”, they harbor millions of different types of keys capable to recognize any type of fragments coming from a foreign body. These tiny fragments are called in both cases, antigens. They are made of proteins, sugar or fatty acids, depending on the nature of the pathogen.

By exposing the antigen on the surface, it attracts the attention of certain immune cells. In the case of a virus, it will attract the attention of a set of T lymphocytes called CD8 T-cells that in turn will contact another type of lymphocyte called CD4 T-cells. In the case of a bacterium, macrophages and B-cells will recruit directly CD4 T-cells. CD4 T-cells acts a commander-in-chief, it will coordinate the immune response.

cellsindefenses75

It will train some immune cells to seek and destroy the immediate danger by having B-cells turning plasma cells capable to secrete antibodies. These antibodies are “keys” fitting exactly inside the antigen “key lock”. By this key-key lock interactions, plasma cells will trigger the immune response that will destroy the foreign agent and eliminate it from the body. Because of this threat, CD4 T-cells is training some naïve T- and B-cells as reservists to gain the military experience and that can be rapidly mobilized in case of a future threat.

The major caveat of this response is it takes time. It takes a couple of weeks to be up and running to fight off the infection and the immune system may forget it over time.

This is how vaccines work; we use decoys mimicking the foreign agent to train our immune system so they know the profile of this agent. In case of a real threat, they can rapidly mobilize and stop the threat. By having a immune system aware and ready to fight off the infection, it considerably limits the damage caused by an infection that for many of them lead to crippling conditions (e.g. polio, varicella….) or even death (e.g. measles, whooping cough….).

 In the paragraph below, you will see a chart from the Center of Disease Control (CDC) website describing of the number of crude death rate during the 20th century:

m829a1f1

Modern healthcare practice and medical breakthroughs (discovery of antibiotics, introduction of Salk vaccines….) introduced during the 20th century have considerably reduced the number of crude death rate in less than 100 years.

Yet, some people may argue this decrease in death rate may be inherent to the introduction of hygienist practices and refute such decrease in death rate is driven by massive vaccination campaign. To refute such claims, we are referring to a study published by the Vaccine-Preventable Disease Table Working Group published in JAMA in 2007 (http://jama.jamanetwork.com/article.aspx?articleid=209448).

Picture1

This table may appear confusing but four columns are in interests here: the pre-vaccine average number of cases and death prior the introduction of the first vaccine against the disease and the average number of cases and death as measured in 2006 and 2004 respectively. Note the dramatic decrease in number of cases and death for several of these diseases. For instance polio was a devastating disease over 70 years ago and we have zero number of cases reported.  These data clearly show the potential of vaccinations. This is why vaccines are life-saving type of therapeutics and have been able to considerably reduce the number of cases if not capable to eradicate certain diseases.

However, vaccines suffer from two important caveats: Firstly, the decoys are obtained from attenuated, dead, or small bits of such infectious agents. Secondly, such decoys are by themselves are weak to induce the “immune boost” needed to provide the biological effects necessary to create the “vaccine memory”.

Therefore, these vaccines are formulated with a solution called adjuvants, that act as a “booster” to improve the immune response, resulting in a fast and bold immune response but also help to stabilize the antigen in suspension (shelf life), as well as maintaining the sterility of such vaccines (http://www.roitt.com/elspdf/Newgen_Vaccines.pdf).

Historically, the adjuvant of choice was the Freund’s adjuvant (formulation can be found here:https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/f5881pis.pdf) but suffered from non-negligble side-effects.

Therefore a common consensus practiced in modern biomedical and clinical sciences is a gradual shift from an empirical (for instance mineral oil contained in Freund’s adjuvant has a chemical composition that is not fully known and controlled) into a synthetic (ingredients are all known and coming from primary materials with an extremely high purity) and controlled approach.

Adjuvant and formulations varies between manufacturers and between types of vaccines. The Cender of Diseases Control provides a public fact sheet detailling the composition of all vaccines currently sold in the U.S. (http://www.cdc.gov/vaccines/pubs/pinkbook/downloads/appendices/B/excipient-table-2.pdf)

  1. Why is there aluminum in my vaccines? Is aluminum safe?

Aluminum (symbol element “Al”) constitutes a particular potent class of adjuvant and have shown to have a potent stimulatory effect on the immune response for over 50 years with no particular side effects or increased risk observed (for review:http://www.nature.com/icb/journal/v82/n5/full/icb200476a.html#bib1).

Picture2

The mechanism of action of aluminum as an adjuvant remains complex and unclear. It is the third most abundant element on Earth. It is considered as a metalloid, with its ionized form being Al3+. In contact with water, aluminum undergoes a chemical reaction resulting in the formation of aluminum hydroxide (AH): 2Al + 6H2O -> Al2(OH)3 + 3H2 (dihydrogen gas).

AH is classically used in adjuvants as it does not precipitate in solution, although an alternative form called aluminum phosphate (AP, AlPO4) is also used in certain vaccines formulation. This criteria is very important as chemicals used in injectable solutions have to be in an homogenous suspension. AH can organize and align themselves into crystalline structures, forming particles

gibbsite-crystal-3d-balls

Depending on how many AH are involved in these crystalline structures, these particles will have a particular sizes. In vaccines, AH particles distribution ranging from 2 to 10 micrometers, with a median size of 3 micrometer. In this post, the author refers to a particular type of aluminum, the aluminum adjuvant nanoparticles (AANs) without ever giving a bibliographical source or a definition to describe the nature of such term:

Aluminum adjuvant nanoparticles (AANs) are transported through the body differently than ingested aluminum. Most vaccines contain aluminum adjuvant, an ingredient necessary for stimulating a strong immune response and immunity. The aluminum is in the form of Al hydroxide and/or Al phosphate nanoparticles.

A request on search engine failed to provide us with a link on Pubmed and Sciencedirect databases with the term “aluminum adjuvant nanoparticles”, instead refers to “aluminum hydroxide nanoparticles (AHNs)” or “aluminum oxide nanoparticles (AONs)”. The usage of an uncommon term by the author is questionable and bring confusions on exactly what the author is referring to. However, the author later appears to refer to AH or AP nanoparticles, thus we speculate that AANs maybe an umbrella terms to refer to AH and APs and therefore we will refer on AHNs as AANs for the rest of this article.

Then the authors states the following: “Aluminum has been used in vaccines since the 1920s. Despite this long history, aluminum adjuvant was not studied much beyond its effect of making vaccines more effective. The safety of injected Al adjuvant was assumed, largely because aluminum is a normal (if unhealthy) component of many foods. Its one of the most common elements of the Earth’s crust. Its everywhere. So consideration of Al adjuvant safety was entirely based upon studies of ingested aluminum.” The author creates confusions for the reader as such sentences introduce a lots of concepts with few explanations and sounds more like a “word salad” than anything else.

As we have previously stated, aluminum is the third element in abundance in Earth’s crust (http://www.sandatlas.org/composition-of-the-earths-crust/). We can reasonably speculate that living organisms have been growing in an aluminum-rich environment since LUCA (last unicellular common ancestor) and therefore have adopted evolutionary traits to cope with such exposure to aluminum on the surface of Earth’s crust.

 In this section, the author discussed and misused an important concept used in pharmacokinetics that will be discussed later: bioavailability. Bioavailability defines the amount of substance that reaches the systemic circulation (in other words the bloodstream) compared to the amount dispensed at the delivery site. It is a ratio of the amount measured in blood plasma following its delivery through an extravascular route (oral ingestion, intramuscular injection, dermal patch….) divided by the amount measured in blood plasma following a delivery through vascular route (most of the time an intravenous (IV) injection). When you inject a substance by IV route, this ratio is by definition 100%. Now if I re-use the example of the author, the bioavailability by oral route is 0.3%. If a patient swallows 100g of aluminum (Al), only 0.3g will make it inside the systemic circulation, thus giving us a bioavailability of 0.3%.

Although the author will cite an article by Flarend and colleagues (http://www.sciencedirect.com/science/article/pii/S0264410X97000418) later in his post, it failed to report the bioavailability reported by the same study. By courtesy, we introduce this study in this section to bring clarity. In this study, Flarend et al measured the pharmacokinetics of AH in rabbits, using radioactive aluminum (as radioactive-based analytical methods are the method of choice to quantify metals).   In this study, they injected two rabbits with an intramuscular injection of AH at a concentration of 13.24 mg/mL. In an equivalent dose, each rabbit got “vaccinated” with 0.85mg of AH. According to the FDA guidelines, the amount of AH present in vaccines sold in US have a maximal amount of 1.25mg/dose with a cumulative amount (total amount you will get from vaccines) estimated of 4.22 mg over the infant vaccination schedule (source: http://www.fda.gov/BiologicsBloodVaccines/ScienceResearch/ucm284520.htm). [UPDATE] Keep in mind that such amount does not accumulate over time and we have a clearance of aluminum taking place over time. Because an intramuscular injection is not a vascular route, the bioavailability is below 100%. The authors here estimated the amount absorbed over 28 days to be 17%. What does it means? The amount absorbed is the amount that was able to diffuse through the epithelial barrier or from the connective tissue into the bloodstream (systemic circulation).  It also means that 82% of the Al is still present at the site of injection (take note, AVers: if it does not absorbed, it is not absorbed). So why is that? Its all about solubility. This is the solubility equation:
Al(OH)3<-> Al3+ + 3OH-
It is an equilibrium equation, in which Al3+ is only soluble at its ionic form. Therefore, you need to deplete the Al3+ to dissolve the AH nanoparticles. At physiological pH, AH is practically insoluble. This drives the bioavailability of Al3+. If we consider the absorbed amount over 28 days, we can deduct that the bioavailability is about 0.6%, slightly higher than its absorption via GI tract.
Thus, AH releases its aluminum at a very slow pace but also at the very long time. This is why we have this biphasic curve after Cmax, with the second slope being more steady and explaining the long half-life of Al.

Picture3

At baseline values (before injection), the amount of Al detected in plasma was about 30ng/mL. Upon injection of these four doses simultaneously, AH (black triangles) show a tmax (a time by which the concentration of aluminum peaked) at 10 hours, bringing a Cmax (maximal concentration) of 32ng/mL. In other means, a 6% increase in the amount of aluminum. Thats a bit of extra noise over noise. Not much a dramatic peak that would double up your exposure to aluminum. You don’t face an aluminum storm upon vaccine injection, but more a slight added noise over time. This is not a problem for healthy patients unless those with a kidney condition (we will discuss that later). [END UPDATE].
In the body, the authors estimated that AH mostly accumulated in the kidney, followed by the spleen and liver. This high retention is understandable as these organs are highly perfused with blood and therefore may accumulate more aluminum than other organs. These organs (liver and kidneys) are always monitored when drugs are developed as they can have serious toxic effects. However the amount accumulating is very negligible. The authors report an amount of 0.00001mg/g of tissue after 28 days. Put back into the context, at the time of injection, this tissue concentration may have peaked at 0.000283mg/g. Brain tissue, in contrast, have shown 100 times less accumulation than kidney. After 28 days, 3% of the initial aluminum injected remains in kidney, we can therefore estimate only 0.03% of the initial amount is accumulating in the brain. Aluminum has a long half-life (time to eliminate 50% of a compound from your body), as it is estimated around 100 days.

Because it takes some times to eliminate it, we can reasonably raise the question: what about the acute and chronic toxicity? The acute toxicity is defined by the toxicity obtained by a single injection, whereas the chronic toxicity is obtained from a continuous exposure.

An important concept in toxicology has been established in the 17th century by Paracelsus, the father of modern pharmacology and toxicology: “Every substance is poison, no substance is no poison. The dose and only the dose makes the substance the poison”.

It is all about how much you get exposed over time and about how long it takes to get it eliminated. A poison with a short half-life can see its toxic effect cleared very fast, whereas a poison that has a long half-live will accumulate if exposed continuously and show its toxicity after weeks if not years. This is often the case observed in poisoning with heavy metals (like lead, silver, mercury, arsenic). An historical example is Napoleon Bonaparte’s death by poisoning during his exile on the island of St. Helena. Because the amount of arsenic ingested was  low and did not alter taste, it did not raise suspicion of poisoning. However because arsenic half-life is high (12 days), it kept building up in the body until it reached a toxic level.

In a review published in 2007 by Krewski and colleagues (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2782734/), non-occupational exposure to aluminum is mostly driven by food consumption, with a daily intake of 8.6 and 7.2mg/day for males and females respectively. The author of this post claim 0.3% oral bioavailabity in the following statement: “Ingested aluminum has a low absorption (about 0.3%), and when this low absorption is taken into account, there is good reason to expect vaccines to create aluminum toxicity. But that is not the subject of the present commentary. Commentary about the total amount of aluminum in vaccines can be found here: http://vaccinepapers.org/danger-aluminum-vaccines/

[UPDATE] For our demonstration, we will rely on the data provided by the review, citing 0.1% of bioavailability. Based on this number, we can estimate males and females are exposed daily to 0.0086 and 0.0072mg/day (or 86 and 72 micrograms/day) (values were corrected for bioavailability). If we use the study from Flarend and colleagues, we should expect to add 0.21mg (or 210 micrograms) of AH per injection. Considering the 6% increase in Cmax  concentration observed in rabbits following injection, we are expecting about an extra 12.6 micrograms of aluminum added to the systemic circulation. Another review by Yokel & McNamara (https://www.ncbi.nlm.nih.gov/pubmed/11322172) provides a more exhaustive comparison and sources for the amount of aluminum exposure summarized in the table below.

Yokel_Al.jpg

Now, we have more information available since I have written the first iteration of this post. We have an estimation of 0.07-0.4 micrograms/kg/day provided by vaccines. We will assume the more conservative number of 0.4 micrograms/kg/day. With the average weight of an infant (median weight at birth ~3.3kgs), we can assume a daily dose from vaccines of about 1.32 micrograms/day or about 237 micrograms over a 6-months period. These are plasma concentrations, therefore a better comparison would be to compare to the administration of aluminum via IV route as well.

As you can see in the chart, the most common medical procedure involving the IV infusion of aluminum is the total parenteral nutrition (TPN), or feeding directly via intravenous diffusion. The daily amount via TPN are much higher, we have values of 11-27micrograms/kg/day or for an infant getting a TPN at birth of about 36.3-89.1 micrograms/day. Therefore, the daily amount of aluminum obtained by vaccines is about 200 times less than the daily amount obtained by TPN route.
Now, this review came out before the FDA roundtable and risk assessment of aluminum in TPN bags. Following recommendation, the FDA consider an IV concentration of aluminum of 4-5micrograms/kg/day as possible neurotoxicity in premature and neonates connected to TPN bags.
Again, we have to compare to same scale. For an newborn, thats about 13.2 micrograms/day. Put the vaccine daily exposure, we are about 10% of this threshold value. Therefore, daily aluminum exposure in a vaccinated newborn infant is 10 times less the amount considered to have a possible neurotoxicity.
We have also to assume that the water and food intake does not apply to an infant and may bias our calculations. According to the Children Hospital of Philadelphia website (http://www.chop.edu/centers-programs/vaccine-education-center/vaccine-ingredients/aluminum), they estimate the total aluminum intake in infants from vaccines during the first 6 months 4.4 miligrams (thats about 4000 micrograms). This have to put into contrast from the 7 milligrams (about 7000 micrograms) from breast milk and 38 milligrams (38’000 milligrams) from formula-fed.  At this point, the author is simply hand waving about the danger of aluminum by ignoring the fundamental concepts of pharmacology, pharmacokinetics and toxicology. Therefore, the question I would like to raise is why the author failed to mention the study of Flarend in this section? It constitutes the right place to discuss about it.

Now, does aluminum is harmless? Well it is all about the dose and the patients condition. Again, the review by Krewski and colleagues can bring us useful information. Al is excreted primary via kidney route, with about 95% of renal clearance. For a patient with normal kidney function, this is not a problem. For infants that receive injections on a monthly basis, this is not a problem either. The problem arises if you have a patient or a premature that have to be on a constant IV infusion, like patients on TPN bags. There is a risk of aluminum accumulation as the intake amount is much more important than the amount excreted. Such case in patients that display either immature kidneys or signs of renal failure. However, in such cases, such patients will have aluminum-depleted IV bags to avoid such accumulation. [END UPDATE]

  1. Aluminum and neurotoxicity

If there is a concern about aluminum toxicity, it is its possible effect on the central nervous system (we refer to it as aluminum neurotoxicity). A rapid review on Pubmed using “neurotoxicity aluminum” bring us a total number of 387 articles, including 73 reviews. A classical model to assess aluminum neurotoxicity is the model of aluminum chloride (88 articles) in rodents.

In these models, aluminum chloride (Al(Cl)3) is administered by oral route with concentration varying from 5mg/kg/day (http://www.ncbi.nlm.nih.gov/pubmed/21543463), 50mg/kg/day (http://www.ncbi.nlm.nih.gov/pubmed/25940660), with a maximal values of 200mg/kg/day as reported by Prakash and colleagues (http://www.ncbi.nlm.nih.gov/pubmed/23315010). In all these studies, anatomical changes in the brain as well as motor and cognitive functions were reported. Now, it is important to relativise these amount to a 70kg body. At the minimal concentration of 5mg/kg of Al(Cl)3, the dose-equivalent needed to achieve these neurotoxic effects would be 350mg/kg/day. Thats about more than 43 times the daily dose of aluminum obtained with food intake. Every single day. With a chronic high exposure to aluminum, we would expect to reach such toxic level. However such model is not representative as Al(Cl)3 and AH (Al(OH)3) are distinct chemical entities and therefore do not share same physical and chemical features. But this did not stop the author from making false assumptions.

“Ingested aluminum enters the blood from the gut. In the blood, ingested aluminum is in a water-soluble ionic form, typically Al3+ or an aluminum complex*. This aluminum is separated into individual atoms, like ordinary salt dissolved in water. Ionic aluminum is toxic, but it is blocked from entering the brain by the blood-brain barrier (BBB), and it is rapidly filtered from the blood by the kidneys. Unless large amounts are consumed it does not cause a problem.”

A critic I have with his statement is how the author can exclude that Al3+ cannot cross the blood-brain barrier (BBB)? I will talk about the BBB later but I wanted to mention this logical fallacy. We just discussed about the neurotoxic effects of aluminum in the CNS, using Al(Cl)3.
To better understand the difference, we have to compare the solubility of Al(Cl3) and Al(OH)3 (AH). To be soluble, a compound has to interact with water molecules and breakdown its chemical bonds to become an ion. Some can easily break their bonds (example H-Cl breaks into H+ or Cl-), some less (like H-O-H or water). Water is a polar solvent. The oxygen atom attract the electrons of the shared bonds more towards it, it then becomes electronegative. In the other hand, hydrogen discretely loses its electron and becomes slightly electropositive. Ions will mix very well because they counter the charges around. If a compound can ionize, it will dissolve in water. If it cannot ionize (like hydrocarbon chains found in oil and fat, because the carbon atom is not greedy for electrons), then it will not mix with water. That’s why oil and water never mix.

According to Wikipedia, Al(Cl)3 solubility index is 43.9g/100 mL of water and AH is only 0.0001g/100mL. In other words, Al(Cl)3 in solution is under Al3+ and Cl- forms, whereas AH remains in its Al(OH)3 form. How can the author explain the experimental studies that show the neurotoxic effects observed in animals treated with Al(Cl)3 if he claims Al3+ cannot cross the BBB?

In a section of his article, the author cites one study to disagree with it, the study of Movsas  (http://www.ncbi.nlm.nih.gov/pubmed/23856981) published in JAMA Pediatrics.

The Movsas study (published in 2013) used human infants and obtained similar results. Movsas looked for aluminum in urine and blood before and after routine vaccination with 1200mcg aluminum at the 2-month date. No change in urine or blood levels was observed. Movsas states: “No significant change in levels of urinary or serum aluminum were seen after vaccination.“ Of course, these results contradict the claims by vaccine advocates that aluminum adjuvant dissolves and is removed by the kidneys.”

An important criteria when investigating journals is the impact factor. A high impact factor is usually associated with high-quality studies as the peer-review process in such journal has a higher expectation level. In the other hand of the spectrum, we have a new category of “predatory journals” (always based on fee for publications) that will publish studies with a weak or not peer-review process. JAMA Pediatrics impact factor is fairly good (7.13) to consider the study reliable. The authors investigated the levels of aluminum before vaccination, about 11.1+/-10.3 ng/mL. Note the extreme variability of these levels among 15 pre-term babies. The author reported no changes in aluminum after vaccination and estimated to increase the concentration to 1% following the publication by Flarend (see previously). You have to remember Flarend used radioactive Al to measure the kinetics, whereas in this study, we measure aluminum using another analytical technique that may have less sensitivity. It also indicates that the aluminum contained in vaccines injection is not giving higher values than the basal aluminum level, thus you cannot distinguish the aluminum from the vaccine from the aluminum contained in food. But this important point, the author failed to understand.

Indeed, there is another study that tried to reproduce a model of vaccines injection using mice that the author surprisingly failed to cite in his report: the study of Shaw CA and Tomljenovic L published in 2013 in Journal of Inorganic Chemistry (http://www.sciencedirect.com/science/article/pii/S0162013413001773). In this study the authors tried to develop a mouse model of newborn vaccine schedule and see the effect of such repetitive doses would impact on the neurological function.

Picture4

However, the experimental design is inconsistent and raises question about the validity of the data. The author never explain why they change the experimental paradigm in the low AI group. A common sense in science is when you want to show a biological effect you change one parameter at the time. For instance, the dosing schedule should stay the same, only the dose be different (as presented 50% of the normal dose) and have the control (injection with saline solution only).  A poorly designed experimental setup can only to poor results and poor interpretations.

Picture5

Based on this experimental setup, the authors observed an increase in weight in mice following the normal (or in this case, high) Al injections. No weight changes were observed in low AI or the control. Because of the flaw in the experimental design, we cannot tell if this effect is due to stress (remember the mice received more frequent injections than the other groups) or due to the treatment. Because of the poor experimental design, this result is worthless and resulted in an unfair use of animals to get this data. Now things become very interesting, the authors use behavioral tests for all the experiments and determined that high Al showed a decrease in the number of successful tasks. There is also a sexual dimorphism as males showed statistical differences but not females. Again, there is the experimental flaw that do not let us know if it stress related or if it is due to aluminum.  In a behavioral test, you are observing your animal and try to count how many times your animal displays a movement of interest. For instance, in neurosciences, we can put a mouse into a Y-shaped maze and put a bit of cheese in one branch of the maze. Each day, you put the mouse in the bottom of the Y-maze and let the mouse find the right branch (the one containing the cheese). After 10 seconds, you will take out the mouse and score if it succeeded (1) or not (0). After scoring, you will put the mouse again in the maze and score again the mouse for 9 times, this everyday. After a few days, the mouse will remember where is the cheese and will achieve a perfect score. If the mouse has some memory problems, it will perform poorly and will maintain a low score. Now the problem is if the mouse is stuck between the two branches, do you count that as a success or a failure? That’s the problem of subjectivity. I may consider it as a success because the mouse faced the right branch, another experimenter may consider it a success only if the mouse reaches and touches the cheese.

There is also some concerns about the authors’ affiliations. Both researchers were faculty members from the University of British Columbia, Vancouver, BC (Canada) (Dept. of Ophthalmology and Visual Sciences), (Program in Experimental Medicine; Program in Neuroscience) until 2013. Surprisingly one of the authors address displayed an unusual email for an academic researcher in a public institution (cashawlab@gmail.com) but furthermore later publications saw a change in the affiliation (Neural Dynamics Research Group, 828 W. 10th Ave, Vancouver, BC, V5Z 1L8, Canada). Firstly, I would question how a faculty researcher appointed in a department in which the mission is related to eye research has the expertise to study vaccines and toxicology.

Here is the website of Neural Dynamics and surprise, we find the same authors. What is interesting, is this page has been outdated for a while and it seems funding occurred for up to 2007. Are these authors still funded? Things become more and more murky when you see the name of Stephanie Seneff (a computer scientist at the MIT that claim autism is caused…..by glyphosate….in a journal called “Entropy”) as a co-author in one the publications (http://www.ncbi.nlm.nih.gov/pubmed/25349607)……in a predatory journal! Yes, that’s what I call entropic, sorry messy publication records. Orac from “Respectful Insolence” raised a red flag on this study published earlier on and it is worth a read (http://scienceblogs.com/insolence/2011/12/08/and-global-warming-is-caused-by-the-decr/). How reliable is a peer-review from a journal aiming to publish inorganic chemistry, in assessing the validity of scientific claims that are aimed for experts in vaccines and neurotoxicology? As reliable to publish my work on the blood-brain barrier in a journal that studies plankton biology.

[UPDATE] The lab of Shaw and colleagues came again in the spotlight recently for a retracted study on the effect of HPV vaccine on behavioral issues in mice, as mentioned by Retraction Watch (source: http://retractionwatch.com/2016/02/15/journal-temporarily-removes-paper-linking-hpv-vaccine-to-behavioral-issues/). The editor-in-chief of the journal (Vaccine) did not comment about the cause of the temporary retraction. It is also worth noting that the WHO called this study on aluminum adjuvants “seriously flawed”. The full report on the WHO related to the study on the effect of aluminum adjuvant can be found here: http://www.who.int/vaccine_safety/committee/reports/Jun_2012/en/

  1. Aluminum nanoparticles and macrophages

Later in the post, the author discussed about how aluminum nanoparticles (AANs) can enter into macrophages as citing the following: “This model is wrong because what actually happens is that a type of white blood cell called a macrophage (MF) engulfs or “eats” (process is called “phagocytosis”) the AANs before they can dissolve. Eating foreign material is normal behavior for MFs. When MFs detect bacteria or other pathogens, the MFs engulf the pathogens, and digest them with enzymes. They then tell other immune system cells about the pathogen and how to detect it. The problem with AANs is that they are not digested by the MF enzymes. And the AANs, once inside the MF, dissolve much more slowly. The AANs persist for a long time and cause the MFs to slowly leak aluminum. MFs that consume the AANs become highly contaminated with aluminum, and spread this aluminum around wherever they go.”

Again, the author never identifies the nature of these AANs, bringing confusion to the reader. Because the author focuses on the vaccines, we speculate that he is referring to Al(OH)3 particles (AH). The author continues his explanation on why macrophages are the main cause of “MFs that consume the AANs become highly contaminated with aluminum, and spread this aluminum around wherever they go. And they go everywhere in the body.”  Now the author claims these AH enter macrophages (MF), then these macrophages enter the brain and deliver aluminum across the BBB. Interestingly, after a search on Pubmed using the query “aluminum hydroxide AND macrophage”, I failed to find any relevant literature that demonstrated the inclusion of AH inside MF. Therefore the quote “Several studies show, with certainty, that MFs engulf AANs. In several studies, the AANs have been stained and photographed inside the MFs, and identified using several different methods. This is not surprising because it is well known that MFs will engulf nanoparticles just from being grown in a solution containing nanoparticles. The composition of the nanoparticles does not seem to matter“. This statement is not only exaggerated (the authors failed to provide citations to support that claim) but also provocative and fraudulent. In biological sciences, we rarely use a bold statement such as “certainty”, only when you have millions of individual records.

Only pseudoscientists would take a single study as the absolute truth.

The only study that can bring some information is the study cited by the author of this post (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155332/) using THP-1 cells.  What are THP-1 cells? THP-1 cells are monocytes derived from a patient that suffered from an acute monocytic leukemia. As any other cell line isolated from patients, these cells are readily available through cell collections such as ATCC (http://www.atcc.org/products/all/TIB-202.aspx). Technically, there are monocytes and not macrophages (see the schematics in chapter 1). Macrophages are derived from monocytes but in terms of biological identify these cells have distincts identity. It is like claiming my daughter is like my spouse. My daughter shares 50% of her DNA with my spouse and have the other 50% of mine, but she is different and unique. That simple concept appears not obvious for the author as at the end of his post cited: “Monocytes and macrophages are basically the same thing.”. Such statement is simply wrong and raises some skepticism on the rationale the author will use this study to establish his claims.

Furthermore, we have to remember that THP-1 cells are by essence a cancer cell line, they came from a patient suffering from a certain form of leukemia. Cancer cells are known to have a complete different biology than normal cells, because they are cancer cells. This is where we can start to discuss and question the validity of the authors claim: why did he not cited a study using macrophages isolated from healthy patients. The problem is there is no study that have investigated the uptake of AH by normal macrophages and we can reasonably speculate that THP-1 may have an abnormal uptake activity, resulting in an abnormal accumulation of AH.

However, there are several studies mentioning a condition called “myofasciitis”, all sharing the same co-author: Gherardi RK, the same Gherardi cited in this blog post. The author cited the following article to support the claim of AH-laden MFs: http://brain.oxfordjournals.org/content/124/9/1821. Myofasciitis (also referred as autoimmune/inflammatory syndrome induced by adjuvant) is a rare medical condition, as reported by Orphanet (http://www.orpha.net/consor/cgi-bin/OC_Exp.php?Lng=GB&Expert=592) but not listed in the Office of Rare Diseases (National Institutes of Health). The World Health Organization (WHO) provides a fact-sheet page available for information (http://www.who.int/vaccine_safety/committee/topics/aluminium/questions/en/). According to the website, it was identified in 1993 and most of the cases are located in France. Knowing that Gherardi is a French scientist currently affiliated with the Assistance Publique-Hopitaux de Paris Creteil (Department of Pathology, H. Mondor Hospital, Assistance Publique-Hôpitaux de Paris/Paris-Est University, Créteil, F-94010, France; Reference Center for Neuromuscular Disorders, H. Mondor Hospital, Assistance Publique-Hôpitaux de Paris, Créteil, F-94010, France; INSERM U955-Team 10, Créteil, F-94010, France.).  The WHO teaches us two important features about the diseases:

Q1. What is MMF and how is it related to aluminium in vaccines?

  1. Deltoid muscle biopsies performed in France in patients with a variety of complaints have revealed in a small number the presence of a minute inflammatory focus of macrophages, along with crystal inclusions and associated microscopic muscle necrosis. These localized lesions have been shown to contain aluminium salts. Since the location of the lesions in the deltoid muscle coincides with the usual site of injection for vaccines, it would appear that these microscopic lesions are likely to be related to immunization with vaccines that contain aluminium adjuvants.

Q4. Does everyone vaccinated with an aluminium-containing vaccine have the MMF lesion?

A. Since muscle biopsies have only been carried out in patients with myopathic symptoms, there is currently no information on whether the characteristic localized histological pattern would be found in the healthy population after vaccination. It has been suggested that there might be a predisposed subset of individuals with impaired ability to clear aluminium from the deltoid muscle. Whether this reflects macrophagic dysfunction, or the tail-end of a normal population distribution of aluminium clearance and local tissue response, has not been determined.

It seems the disease is related to vaccination in patients marked by macrophages infiltration and aluminum deposition at the site of vaccination,  that apparently harbor a genetic background predisposing these patients to elicit an autoimmune reaction following vaccination.

This article appears as legitimate as it shows the presence  of macrophages in the muscle biopsies by tissue imaging and by using other techniques such as atomic force microscopy to quantify the amount of aluminum in such tissue samples. However, the major flaw of this paper? The absence of proper controls. There are controls documented (1252 individuals) but they never show the tissue sections from these controls or the quantification in the amount of aluminum. I found it very disturbing that such blatant flaws in the experimental design had been overseen by the peer-reviewed process, since Brain has a fairly high impact factor (IF~10).

If you inject a vaccine, you will expect an immune reaction to take place (that’s the goal of a vaccine). This translates into an inflammation stage that everyone experience few hours after injection: hot, red, swollen and painful. Inflammation also recruits a lot of macrophages (thus you would expect to see them under the microscope in your tissue samples) and you also expect  to see an important amount of aluminum in the site of injection, an amount that will take time to disappear (remember the half-life of aluminum? 100 days, it takes some time to get rid of it).  There is no biopsy samples from patients that have shown no side effects.

Most patients are identified in France. So there may be a rare autoimmune disease, that has a genetic background and that may be triggered by vaccines adjuvants following vaccinations. Because this condition appears only after vaccination, we may be tempted to claim vaccines caused this autoimmune disease. This conclusion is wrong as correlation is not causation. If adjuvant was causing autoimmune diseases,  we should see the same condition occurring in patients not carrying the genetic mutation, with a number of cases high enough to raise some epidemiological alert. Again remember, 600 cases in France, a country that count 60 millions inhabitants.

In conclusion, although the authors theory of macrophages loaded with aluminum nanoparticles may have some scientific basis, it still remains unclear as we have almost no studies to support its claim and have a rare disease that is only documented to one country (France) and mostly by one single group (Gerhardi group). To make a claim valid, you need a high number of studies (20 and more) coming from independent laboratories and described into different parts of the world. Therefore the macrophage-loaded theory raises some skepticism and clearly contradicts the claim of “certainty” posed by the author.

  1. Aluminum, macrophages and the blood-brain barrier

The author claims the blood-brain barrier protects the entrance of ionized aluminum (Al3+) but completely ignored the studies showing the toxic effects using Al(Cl)3. Indeed, the author come with one esoteric theory to explain the claims: “And they go everywhere in the body. The MFs are able to travel across the blood brain barrier (BBB). The MFs, once loaded with AANs, act like a Trojan Horse and carry the AANs into the brain. This is very harmful, because the brain is very sensitive to aluminum.” Before we can talk about this theory, we have first to understand the blood-brain barrier (BBB) and how aluminum may cross the BBB.

The BBB is a blood-brain interface separating the blood flow from the brain tissue (source: http://medicalterms.info/img/uploads/anatomy/blood-brain-barrier.jpg). As other blood vessels, brain blood vessels are lined by endothelial cells separating the blood flow from the brain tissue. These endothelial cells (that we usually call brain endothelial cells) are unique: they are very tight, much tighter than other vessels. Such tightness is ensured by the presence of tight junctions between endothelial cells, that block exchange of molecules as small as water and ions between the blood and the brain. These tight junctions provide a “physical barrier”. In addition to such barrier, the BBB has another type of barrier: a “chemical barrier” .

Picture6

As we mentioned, if you are a small water-soluble compound (a, like sucrose (table sugar) or water), your entrance into the brain is very limited. Now if you are a chemical compound that dissolves in oil (b, lipophilic), then you can diffuse across the BBB because cells have membranes made of fat (think about biological oil droplets). However, many of the compounds are still pumped out the brain because of presence of efflux pumps. These pumps block the diffusion of toxins and also drugs (such as cyclosporin A or AZT). Indeed, these pumps are responsible in the blockade of 95% of known chemicals, either natural or synthetic. This is one of the reasons why we fail to have effective treatments for brain cancer, because our current chemotherapy drugs are blocked by the BBB and we don’t have techniques to open this barrier.

Picture7

If you have a bigger cargo to deliver (like protein), it is almost impossible to deliver it across unless you have a dedicated receptor (key lock) and you have the right key to unlock it (d, receptor-mediated transcytosis). We only know a few of them, among them are insulin (insulin receptor), transferrin (transferrin receptor) and low-density lipoprotein (LDL, LDL receptor). There is an important discussion about when the BBB appears during fetal development and when do we have a mature BBB that has the function of an adult. Up to ten years ago, we believed the BBB was immature in newborns based on studies using rat and mouse pups. However, with the recent development in modern biology techniques, it seems that indeed humans have a functional BBB that maybe are as mature as adults after full term pregnancy. This was firstly supported by Saunders and colleagues that demonstrated that BBB from the opossum, a small marsupial from the same family than kangaroos and koalas, have a tight barrier (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267212/). This observation was also observed on rat pups following stroke injury. During stroke, the BBB opens and lets water and ions enter inside the brain and causes brain damage by brain swelling. If the newborn BBB was more fragile than the adult one, the damage would be more devastating. Indeed it seems not and maybe the opposite. In a recent study, Vexler and colleagues demonstrated that rat pups better dealt with stroke injury than adult rats and showed lesser brain swelling (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539825/).

Thus, the concept of the newborn BBB being more sensitive to vaccines than adults’ BBB maybe completely wrong. If vaccines induced BBB disruption, we should have epidemiological data showing an increase in severe neurological disorders including cerebral palsy or epilepsies. As we mentioned in the previous section, aluminum is known to show neurotoxicity, based on the Al(Cl)3 model. In this model, we speculate that Al3+ is in free form. Al3+ can bind to transferrin (a protein that normally delivers iron to the brain), thus using the transferrin receptor as an entrance mechanism (http://www.ncbi.nlm.nih.gov/pubmed/7580055). Indeed, there is little or no studies that investigated if Al(OH)3 can enter the BBB. One possible mechanism is that aluminum may disrupt the BBB, which in turn induce a BBB leakage and brain damage. The only study found is from Wiesnieski and colleagues (http://www.ncbi.nlm.nih.gov/pubmed/3730864) that investigated the effect of Al(Cl)3 and Al(OH)3 on rat BBB using radioactive sucrose to follow changes in the barrier tightness. The authors noted an increase in BBB leakage 2 and 4 hours after administration but did not show any differences after 24 hours. More interestingly, Al(Cl)3 triggered such leakage whereas Al(OH)3 showed no difference on the BBB. This study therefore refutes the idea that AH cross the BBB and/or induces BBB breakdown.

However, the author believes in the AH-loaded macrophages theory and made the following claim: “The MFs are able to travel across the blood brain barrier (BBB). The MFs, once loaded with AANs, act like a Trojan Horse and carry the AANs into the brain. This is very harmful, because the brain is very sensitive to aluminum.”
This claim is fairly outrageous for the BBB expert that I am, for different reasons. Firstly, the author simply ignores the notion of “immune privilege” organ such as the brain (for review: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597357/). In the difference of other organs, the brain has no immune cells residing inside healthy patients. Only one type of cells, microglial cells (derived from monocytes), is the only immune cell residing inside the brain. Immune cells (including lymphocytes and macrophages) cannot enter the brain because they don’t have the right keys to open the BBB.  Immune cells only cross the BBB following certain neurological disorders such as stroke or multiple sclerosis. In such diseases, brain endothelial cells undergo a phenomenon called “endothelial cell activation”, resulting in the expression of cell adhesion molecules (for review: http://www.ncbi.nlm.nih.gov/pubmed/20946472).

Following this activation, now immune cells (also known as leukocytes), have anchor points to anchor at the surface of the BBB as displayed in the schematics below. Leukocytes get anchored, undergo a complicated tango dance with the activated endothelial cells, squeeze through the endothelial cells (by diapedesis) and finally enters inside the brain. Because the brain is an “immune-privileged system”, these immune cells identify antigens present in the brain as foreign agents and triggers an neuroinflammation. A poster child of such neuroinflammation is multiple sclerosis. Therefore macrophages can only enter the brain, if you have have an activation of the BBB that will allow these cells to bind to the endothelial cell surface.

To support the claim that Al-loaded MFs are causing a neuroinflammation, the author goes again with another study from Gherardi again (remember the previous article missing the proper controls?). This time, he uses another study looking at the effect of fluorescent latex beads surface-coated with AH and published in BMC Medicine (IF~7) (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3616851/). Before we further investigate this paper, it is important to note that BMC has been recently caught in a massive fraud scale involving fake peer-reviews and the subsequent retraction of 43 papers (http://retractionwatch.com/2015/03/26/biomed-central-retracting-43-papers-for-fake-peer-review/)

The author used the following image figure to claim the presence of aluminum inside the brain section. What appears disturbing is the inconsistency of the panel presented. On panel a, the author show a muscle biopsy tissue sample using hematoxylin-eosin staining, a common mixture of chemical dyes to observe tissue samples under a light microscope. Then for the spleen and the brain, the author show fluorescence tissue sections. Why did the authors switch from technique to another? Then we have the pseudo-colored pictures showing aluminum deposition. For the injected muscle, we can see some accumulation of aluminum consistent with the macrophages (deep purple) staining. But we cannot associate the aluminum deposition with the tissue sections of spleen and brain. Therefore nothing proves to me that the aluminum deposition observed in the spleen and brain are consistent with the presence of macrophages in that region. Also the aluminum analysis fails to show a proper scale bar. I cannot tell if panel b, c and d used the same magnification. For a manuscript of such caliber, it is unacceptable. Finally, the bar graph on the bottom right is not annotated properly. What tissue are we measuring aluminum levels over time? Muscle? Spleen? Brain?

This is the figure annotation: “Aluminum deposits in tissues following injection of alum-containing vaccine in the TA muscle. a) Granuloma composed of PAS+ cells is formed in the injected muscle envelope; b) PIXE mapping shows muscle Al deposits in pseudocolors, with confirmatory Al emission spectrum (d21); c) Section of spleen tissue (left panel) displays the large 500 × 500 μm and restricted 100 × 100 μm protonized fields corresponding to the PIXE maps (middle and right panel, respectively) enclosing eligible Al spots (d21); d) Section of brain tissue (left left panel) displays the restricted 100 × 100 μm protonized field corresponding to the PIXE map (middle panel) enclosing eligible Al spot (d21); the number of fields containing one or more Al spots was increased at all tested time points compared to unvaccinated (right panel) mice. (bars: 100 μm). d, day; PIXE, particle induced X-ray emission, TA tibialis anterior.

We have been just at figure 1 and we have already the same botched and neglected experimental paradigm that not only make the results inconclusive but also a complete waste of animals for the experiment. A complete opposition to the author’s claim as “In an impressive study in mice, AANs and other nanoparticles (e.g. latex) were injected intramuscularly into mice”. It is not impressive; it is a deeply flawed study that anyone holding a Ph.D. degree would be outraged to read. An important point to note: I never found information on how many mice have been used for the study. The minimum required to perform statistical analysis in animal models is 8 mice per group or treatment. Therefore, I call this study as “n=1” or a single mouse study. A single individual study has no scientific value unless it is a clinical case report (however, a case report remains the lowest level in the pyramid of scientific evidence).

In the next part of this study, things become even murkier. The authors now use fluorescent latex beads (FLB) to model alum agglomerates. This is something very important. Latex and AH are completely two different chemicals. Latex is a natural polymer made of number of cis-isoprene repeats (http://www.learnnc.org/lp/media/uploads/2008/09/polyisoprene1.png). It is basically a series of repeat of a molecule made of carbon and hydrogens atoms. These are two distinct structures and FLB cannot model AH. Furthermore, why did the authors not inject AH-loaded macrophages? even AH-loaded THP-1? So instead of investigating AH accumulation in the brain, we are now having a paper that investigates the accumulation of latex particles inside the brain. Latex is a natural product that is biodegradable. Such degradation is ensured by micro-organisms (http://www.wiley-vch.de/books/biopoly/pdf/v02_kap10.pdf). Animals do not have the enzymatic toolkit to degrade latex. Latex beads will accumulate in our body. The information in Figure 2 is basically telling us we are accumulating latex beads in the site of injection and because latex is not synthesized by our cells, it is recognized as a foreign agent and macrophages will try to clear these beads from the injection sites by swallowing them.

Picture8

we are accumulating latex beads in the site of injection and because latex is not synthesized by our cells, it is recognized as a foreign agent and macrophages will try to clear these beads from the injection sites by swallowing them.

In Figure 3, we are seeing the accumulation of FLB inside the brain. This is a natural consequence of injecting a substance that cannot be degraded. Because of the systemic circulation, these beads will reach the circulation from the site of injection and being spread in all areas perfused by the circulatory system. At the BBB, you will expect some non-specific uptake occurring. It is called pinocytosis, as endothelial cells will form some small sacks trapping liquid from the blood side and deliver this content to the other side. This event is rare but that happens. We can also speculate that because these animals are undergoing an important inflammation, such inflammation may be sufficient to activate brain endothelial cells and to allow FLB-loaded macrophages to enter the brain parenchyma.

Now, the onset of inflammatory response at the site of injection maybe what drives the opening at the BBB. If you look at figure 6, the intravenous injection of FLB did not trigger the infiltration of the BBB, simply because you need to have macrophages to swallow these beads in order to trigger an inflammatory signal. Macrophages are rarely circulating and mostly located in tissues. Following inflammation, macrophages will migrate to the inflammatory site. Thus explaining the absence of FLB infiltration inside the brain once injected by intravenous route. Now if you have a compromised BBB (like in mdx mice), you can see an important increase of FLB detected in brain sections from mdx mice.

To conclude, this story fails to directly demonstrate that AH triggers an inflammation in healthy mice, the authors using a complete different material (latex) to make a scientific claim that has no scientific values. You cannot claim that if I observe an allergic reaction following the ingestion of apples, it can be reproduced by ingesting oranges instead. I cannot show data using oranges and claim that these results reflect what happens when you have apples!

In addition to this study, the author also cited a study that used gold silica-loaded macrophages to target brain metastasis originated from metastatic breast cancer using a study from Clare and colleagues and published in Cancer Nanotechnologies (no impact factor as it is an open-access journal) (http://www.ncbi.nlm.nih.gov/pubmed/23205151). The authors showed they can load gold-silica nanoparticles inside macrophages directly obtained from whole blood samples of their institution Blood Center. Silica is a different entity than aluminum. It is formed by silicium (Si) that can ionize into Si2+. Like AH, silicium can bind hydroxyde and form Si(OH)2 that can crystallize and form particles. Why didn’t Gherardi and colleagues perform the same approach?

Again, this is a form of cherry-picking data, because the author tries to make the analogy between AH (Al(OH)3) and silica (Si(OH)2) – remember that these are completely two different chemical entities. Second Gherardi paper, second poor experimental designed study, second misleading conclusion. Not only did the author just drank the “Kool-Aid” without questioning it, he is also clueless on whether the Kool-Aid was genuine or tainted.

 

  1. Aluminum, autism and inflammation

Until now, we have been able to breakdown and debunk one piece at the time all these studies and showed that either their data were cherry-picked by the author to support its claim or the papers are of questionable quality that let us wonder how such papers went through the peer-review process without rejection. In the last piece, the author uses a study from Vargas and colleagues (http://www.ncbi.nlm.nih.gov/pubmed/15546155) published in Annals of Neurology, a journal with a good impact factor (IF=9.97).

It is important to note, this study is not investigating if aluminum or vaccines are causing autism. It is investigating what anatomical and biological changes can be observed between autistic brains and healthy brains. The authors used brain tissue samples from 11 autistic patients characterized by an IQ less than 70. Normal IQ score ranges from 90 to 110, so we can consider these patients as borderline. They have intelligence below average but not showing severe mental retardation. There is also an association of with epileptic seizure, with ~30% of autistic patients were epilepsy-positive. This study is legitimate and fairly well designed. We have proper controls and autistic patients. Interestingly, there is an increase in GFAP (activated astrocytes) astrocytes  and HLA-DR (a cellular marker expressed in antigen-presenting cells such as macrophages, dendritic cells or B-cells) in brain samples from autistic patients. This suggests the presence of brain inflammation in these patients compared to controls. The authors further investigated and measured changes in cytokines extracted from brain tissue homogenates and cerebrospinal fluid (CSF). The brain homogenates will tell us if cells produce (and maybe releases) these cytokines, whereas the CSF (a fluid in which our brain is soaked) will tell us if these cytokines are freely circulating. Cytokine measurements were done by antibody array using cell extract and patients CSF. It measures the cytokines by trapping them on a surface and then are detected by chemical reaction resulting in the formation of a dark spot. The darker and bigger the spot is, higher is the amount of cytokine. This method has the advantage to provide some information, although a more absolute method for quantification would have been a 2-D gel electrophoresis that directly identify these cytokines by their chemical structure and count the exact amount present.

You get interesting information from this table that compares the levels of various cytokines detected in the CSF from autistic versus controls.

Picture9

We can clearly see very high levels of cytokines known to promote inflammation (such as IFN-gamma, MCP-1) but there are also other cytokines known to promote BBB leakage (VEGF) and other cytokines with a biological function that remains unclear (TGF-beta2, FGF-9). This is some serious study that is supported by another research group, as published by Croen and colleagues (http://www.jneuroinflammation.com/content/11/1/113).

These studies tell us that there is an inflammatory component in a certain form of autism, with experimental data that are robust and reliable. Yet, these studies is not telling us autism is caused by inflammation or if autism causes brain inflammation. Furthermore, this study do not tell us if AH causes autism or if vaccines causes autism. However, by citing such articles at the end of this tortuous and fallacious pseudo demonstration, the authors want us to follow in the following fallacy: “Vaccines contains aluminum. Aluminum induces inflammation and get swallowed by macrophages. Macrophages causes brain inflammation. Inflammation is causing autism. Therefore vaccines causes autism”. You can see that such construction is invalid, flawed and completely irrational. Correlation IS NOT causation.

[ADDED SECTION] 7. Aluminum, the blood-brain barrier and neurotoxicity

As I have mentioned, the claim that macrophages loaded with aluminum leave the injection site and migrate to the brain via the lymphatic system and enter the brain via the BBB is unfounded and not back by science. Does it mean that aluminum does not cross the BBB?
The answer is no, aluminum can cross the BBB and therefore exert a neurotoxicity. This is why the presence of aluminum from external sources such as TPN bags can be problematic for patients that have either immature kidneys (premature newborns) or present some kidney malfunction (kidney failure, patients needing dialysis).
If I have to cite an expert in aluminum and neurotoxicity, the name of Pr. Robert Yokel (University of Kentucky) comes in mind (http://pharmacy.uky.edu/faculty/ryokel/Robert-Yokel). He has an impressive track-record in terms of peer-reviewed articles (145 publications) and other documentations. You can find his studies in Pubmed (https://www.ncbi.nlm.nih.gov/pubmed/?term=aluminum+yokel)
Aluminum can enter the BBB via various mechanisms. Because aluminum is an ion, it cannot diffuse through the BBB per se, it requires the use of carriers to piggyback on. According to a study by Yokel and colleagues, the monocarboxylate transporter 1 (MCT1) might be one of these carrier (https://www.ncbi.nlm.nih.gov/pubmed/9699794).
Another study speculate the possible use of zinc-transporter (ZnT) as Song and colleagues (https://www.ncbi.nlm.nih.gov/pubmed/18786610) described a protective effect of zinc on aluminum-induced disruption of the BBB following co-administration with zinc. We can speculate a possible competition between Zn and Al for the uptake at the BBB or we can speculate a competition for the site of action. Keep in mind that such study noted a disruption of the BBB following intraperitoneal (IP route, considered similar to IV route in terms of pharmakonetics) injection that were 5 and 10mg/kg, a value 1000x and 2000x higher than the value considered by the FDA to display neurotoxic effects (5microg/kg/day).
Another name that comes in the aluminum and neurotoxicity is the name of Chris Exley from Keele University (Staffordshire, UK, you can see his profile here: https://www.keele.ac.uk/aluminium/groupmembers/chrisexley/).  He has a legitimate expertise in alumium research in chemistry, but his positions and claims on aluminum neurotoxicity are not very accurate (the link between Al and Alzheimer’s Disease is mostly based on a case report from occupational exposure: https://www.ncbi.nlm.nih.gov/pubmed/24513181), fallacious (calling on the risk of aluminum as adjuvants in vaccines without providing direct evidence for the claims: http://www.sciencedirect.com/science/article/pii/S0264410X11013089?via%3Dihub), if not simply borderline conspiracy theory (https://www.ncbi.nlm.nih.gov/pubmed/25386158).
One of the most fascinating yet skeptical claim is the ability of silicon-rich water to “clear out the brain from aluminum”, according to his clinical trial  published in 2013 (http://content.iospress.com/articles/journal-of-alzheimers-disease/jad121231) and promoted by “woo” website and UK tabloid (in his case, The Daily Mail) as here (http://www.dailymail.co.uk/health/article-4320406/Silicon-rich-mineral-water-new-healthy-have.html), citing brands like Fiji water and Volvic (French mineral water)
I don’t know what he calls “silicon-rich water” (in other words, silicium dioxide (SiO2) the same chemical that make sand and your electronic chips) but I am highly skeptical how this highly polarized molecule can cross the GI barrier and the BBB, trap the Al inside the brain and clear it out.
To refute his claim, I would use the data from a study by Che and colleagues that looked at the effect of chronic exposure (13-weeks) to various dioxide via oral administration in rats (http://onlinelibrary.wiley.com/doi/10.1002/jat.3125/abstract;jsessionid=57E87335BDC127B06DAB5E9DAE7BF815.f04t01).
One figure worth a thousand words, the one about brain distribution of SiO2 in the brain of males and females. Brain_SiO2

Note the dose given (mg/kg) and the dose measured in the brain (ug/g). Even by literary eating silicon (245mg/kg/day), only a bare fraction make it inside the brain (0.28% to be precise). But foremost, even at the highest amount (almost 1g/L), there is no difference between the background noise (see the control values). I would be curious how does the silicon contained in Fiji water (95mg/L) and Volvic (32mg/L) will show higher SiO2 levels when concentration 2 to 7 fold higher cannot do better than background noise. Should we assume Exley is profiteering from Big Mineral Water?

[ADDED SECTION] 8. Aluminum and infants: my comments on the “Dorea” papers

I have some comments claiming the toxicity of aluminum from vaccines, using the publications of Jose G. Dorea (Department of Nutritional Sciences, Universidade de Brasilia, Brazil). You can find his publication on that topic on Pubmed (https://www.ncbi.nlm.nih.gov/pubmed/?term=aluminum+dorea).
His research can be interesting because of the issue of soil contamination of Amazonian soil with heavy metals (lead, methylmercury, aluminum….), but also is flagged by his claims that aluminum and mercury contained in vaccines is responsible for such effects.
For example, I refute his claims that aluminum and mercury contained in vaccines are providing much higher levels than breastmilk from his 2009 paper because his claims are based on a flaw in data interpretations (http://www.nature.com/jes/journal/v20/n7/full/jes200964a.html?foxtrotcallback=true).
First, his claims are based on values published, not from experimental data. Therefore, he never provide experimental data for his claims and we are just speculating. Second,  in the only figure of his paper, he assumes the bioavailability factor for the absorption of breastmilk but completely went south on the bioavailability of vaccines following IM or SC injection (see Figure below). If we assume the calculated bioavailability of the Flarend paper (~0.5%), then the graph looks much less impressive and completely destroy his claims. There is a big difference between 100% and 0.5% bioavailability, 200x  difference.
Thats enough to bring aluminum from detectable range to potential toxic range.

jes200964f1

We can go the same way with his other papers, they suffer the same methodological flaws: they all assume the toxic effect of Al and Hg based on hypothetical values and poor correlation, comments to studies that have nothing to do with the data presented in that study (the classical “whataboutism” and gaining one citation in Pubmed on the way, https://www.ncbi.nlm.nih.gov/pubmed/21664460). When the few times he published a paper with experimental data, he dismiss such data because it simply does not fit to his narrative. One of them is about measuring Al in infants hair, with the idea that infants who got immunized will show much higher accumulated Al levels than those not-vaccinated. Of course, the amount of Al from vaccines is marginal from Al from dietary exposure and resulted in no statistical differences. He was unhappy enough to conclude that this technique is not reliable enough. It is like trying to find patterns and hidden images in the static of an old TV (https://www.ncbi.nlm.nih.gov/pubmed/24183841).
In conclusion, I would definitely recommend not to take the Dorea’s papers as the gospel and strongly recommend anyone that has enough critical thinking and statistic literacy  to not suffice with the abstract and dig in to re-analyze the data to sort the scientifically correct from the misinterpretations. There are some nuggets of interesting data in his publications but also a lot of speculations and wrong conclusions.

 

  1. Concluding remarks

In this article, we investigated, analyzed and criticized the blog post that questioned the safety of aluminum in vaccines, with an ending clearly pointed to associate autism with vaccines. The same logic can be applied to thiomersal, an adjuvant containing mercury and initially cited as a causative agent of autism following MMR study in the Wakefield study. [Correction: One reader noted the inaccuracy of this claim and I make an apology for this mistake. The retracted Wakefield paper made an association between children displaying autistic traits with MMR vaccinations (see Table 2). However, the origin (manufacturer) of such MMR vaccines was not reported and therefore such study could not pinpoint which agent contained in those vaccines was the cause of such condition. Neither such study mentioned which MMR vaccines contained thiomersal. End of correction]. Of course, the Wakefield study scientific fraud has been raised and resulted in the retraction of it. Thiomersal has been removed but anti-vaccinationists now turn to another component: aluminum hydroxide, despite the clear evidence of no association between autism and vaccines (http://www.sciencedirect.com/science/article/pii/S0264410X14006367).

Early on, we demonstrated the confusion brought by the authors. The author creates confusion by introducing the terms “aluminum adjuvants nanoparticles”. There is two class of aluminum adjuvants used in vaccines: aluminum hydroxide (AH) and aluminum phosphate.  For the clarity of this article we focused on the AH nanoparticles. Unlike some websites making claims without providing any source for primary literature to support their claim, we indeed observed a smart strategic move from the author to use primary literature as sources but never showing the real data or discussing the main information coming from these studies. I call such move as a “hijack” method in which a legitimate study is used as a decoy making the claims supported by scientific evidence. A neophyte will accept this claim for granted but a more scientifically alert person will access the primary source to ensure what have been claimed on the website is in agreement with the original study. This strategy has been applied by pseudoscience websites such as “Natural-News” or “Collective-Evolution” that will have legitimate references listed to make their claims appear credible.

Another fraud observed in this post is the obvious copyright infringement by hosting and embedding PDF files of manuscripts published under conventional editorial processes. Unlike open-access journals, conventional journals requires the subscription to access their article databases. As a scientist, once my manuscript is accepted by a journal for publication, I have to secede my author’s rights to the journal. I can share the link to the abstract of my studies. If I put the PDF of the journal article on my website, freely available, I am violating the journal copyrights (you can see the copyrights information here: http://www.elsevier.com/about/company-information/policies/copyright).

However, if my studies have been funded with federal research grants (from the National Institute of Health), then my papers will be freely available through Pubmed Central after 12 months of publication embargo (I have linked to the PMC versions whenever it was possible).

Finally, we also found the few articles that were directly supporting the author claim were also the ones with questionable origin, flawed experimental setup if not suspicion of blatant scientific misconduct, even after undergoing a peer-review process.

The peer-review process has been initially designed to use the expertise of scientists to filter studies with an adequate methodology and results from those with a poor or questionable methodology. However such method is not perfect and have been subject to criticism. Even a fair and genuine peer-review in journals with the highest impact factor (Nature, Science) may fail to detect the presence of data manipulation and fabrication. If you are interested to follow on paper retraction, an excellent website to consult is Retraction Watch:http://retractionwatch.com.

We rightfully questioned several publications, all coming from two groups. An important criteria for robust scientific discovery is the ability to reproduce similar results in different laboratories, but also having such publications being reviewed in journals harboring an editorial board capable to assess the quality of your research. As a BBB researcher, trying to publish my research inside a plant biology journal may not give me the best peer-review.

Finally, statistics and certainty in science. Scientific method starts with a question (hypothesis), the design of an experiment and the subsequent outcome of such results and eventually the share of such results by publication into peer-reviewed journals. Once peer-reviewed anyone can ask to reproduce your data in their lab settings. A single study is not sufficient to qualify a phenomenon as a fact. When you have the same phenomenon that has been published a number of times by different groups and most of them show a similar outcome, we can call about a consensus. If this consensus rely on thousands of experiments that predict the same outcome each time, we can consider it robust enough to set as a theory.

In our study, we presented evidence that the additional aluminum due to vaccines injection is negligible and non-distinct from our daily exposure (food), an allergic reaction to vaccines is possible but affect so far only 1 individual per 100’000. Therefore, these individual will have a greater chance to develop such condition following massive public health campaign (for instance, France had a massive Hepatitis B vaccine campaign in 1994, which was marked by  the first report of myofasciitis).

Based on the current literature, there is no evidence that AH incorporate into macrophages, such macrophages cannot enter the healthy BBB and therefore the neurotoxic effects of aluminum due to vaccines is unproven. The house of cards built by the authors just crumbled under an intense and scientific scrutiny.

In my final words, I would say to anyone trying to argue with conspiracy theorists is the same than with wrestling with a pig: you will end up exhausted, covered with mud and the pig will feel happy.

[Pharmacology/Junk Sciences] About the FDA recall on Dr. Hyland’s homeopathic tooth relief….

You may have heard about the recent FDA recall for Dr. Hyland’s homeopathic teeth relief and any products similar to it as listed in their report below (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm523468.htm).

There are 10 children deaths and 400 adverse events reported with the association of this homeopathic product. Homeopathy has always lauded itself as “natural, effective and harmless”. Even when it was faced with the lack of efficacy, proponents of homeopathy always use the “what’s the harm?” as a defense. Seems that not only it will not treat your ailment. Worse it can kill you. Why? Simple pharmacology.

A tenet of pharmacology is known since Paracelsus with his famous citation “Every substance is poison. No substance is no poison. The dose and only the dose makes a substance a poison”. Even the natural products are poison. You see, Mother Nature in all its grandeur has developed a certain affinity for developing some of the most toxic substances designed to kill living species. Because of evolutionary constraints, plants have been very good as being “chemical plants” and use chemical warfare as a method of invasion and defense.

Now, you can use this poison effect to treat certain conditions. If you find the right dose, you may treat a condition. This is how pharmacology works. Under a certain dose, there is no effect. We only start to see some biological activity after a certain dose. This is what we call the Minimum Efficacy Concentration (MEC). On the other hand, if you reach concentrations high enough, then you start to hit on off-targets and develop the minimum toxic concentration (MTC). This is where we start to see side effects and if the doses are high enough you will see adverse and toxic effects.

Homeopathy is by definition an unproven therapy because its principles defy common laws of physics and chemistry. Because it is relying on extreme dilutions to explain its activity, the amount of active substance is so low that it can be compared to dilute one single molecule into a swimming pool.

In this case, we don’t exactly know what compounds and what amount of each alkaloids are extracted. We know the total amount is 0.0000000000003% or 0.3 pico-grams per 100mL of solution or 100g tablets.

belladonna

That amount is simply ridiculously low. First, we have to assume that its PO administration will result in a 100% bioavailability (that is probably false and surely below). Second, you should be able to detect the compound in plasma samples. At that level (pg), the odds of detection are very low if not non-different from background. Therefore the odds of having this single molecule finds its target inside a body is almost zero (and the probability is something like 0.0000000000000000000000000000001%).

Yet, homeopathic treatment are all starting from raw extract (usually an hydro-alcoholic solution as extracting solvent) called “tintura matter”. Thats a concentrate and it contains indeed active substances in a high concentration. The nature and quantity of active substance in it are not known and not even reported since homeopathic products are not falling under the FDA supervision (you can thanks the DHSEA provision for that). Basically, no one (even the manufacturer) will tell exactly what and how much of active compounds are inside this tintura matter.

This raises a problem because Mother Nature has been excelling in producing poisons, what only matter is the dose. In the case of the teething relief product, the main culprit appears Atropa belladonna, a common poisonous plant that get easily confused with edible berries.  One of the classical substances purified from Atropa is atropine, an acetylcholine muscarinic receptor blocker. Because Atropa belladonna is known for its poisonous activity, we had some disaster in waiting that was cooking here. Anyone working with drugs will tell you how dangerous it is to overdose on an active substance.

Acetylcholine is an important neurotransmitter in mammalian cells, with peripheral (heart, muscles, lungs….) and central actions. Acetylcholine is a key target of organophosphates and sarin gas, as these agents block its degradation by acetylcholine esterases (this is why you have atropine administered as a side treatment for poisoning).

However, atropine can and will have severe side effects if ingested in a poisoning event (see this case report of accidental ingestion of Belladona: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361210/)

So whats exactly happened in this situation? We dont know and only now the FDA can provide clues. The FDA is toothless and cannot execute any order unless there are fatality cases involved. On their page, they are aware of the anticholinergic side effects of it but minimized the risk (that logically make sense since the amount is ridiculously low)

belladonna2

At this point, I speculate that something wrong likely happened with these homeopathic products. My speculation is something went wrong with the preparations, resulting in inconsistant extraction and effective concentrations in tintura matter. Because homeopathy are not ruled by FDA, they do not have to follow the stringent quality control and good manufacturing practices (GMP) imposed on pharmaceutical companies. So we have here a production that was on a free ride without any oversight on the QC. Now you can easily imagine that we may have ended up on with overdosed preparations.

Considering the special population (pediatrics), this risk of overdosing was even more amplified. One thing we consider when we develop a drug and prescribe a drug is the benefits/risk ratio. If the risk or adverse effects are too high, the drug cannot be used because it overcomes the benefits.

In that case, not only the benefits were quasi-null, we provided access to a product that was deemed “safe because natural” but indeed has a risk of adverse effect. Now we have likely some precedent in directly demonstrating the danger of poisoning with homeopathic products. This is already the case with essential oils (there are cases of children poisonings with Eos) and now we have the same with homeopathic products.

[Junk Sciences] Thanks, Michael Phelps, for glamorizing cupping quackery!

Olympic games are a great time to see the accomplishment of athletes, years of hard work and sacrifice to achieve their best athletic performance. Yet, not everything is all shiny and pristine with Olympic games. Between corruption scandals, doping, abuse from coaches over their proteges, misogyny to female athletes, I guess we can add pseudosciences and quackery as the plight on our athletes.
Proof? The recent cupping fad that have been glamorized by some athletes including Michael Phelps.
Oral from Respectful insolence has been, as usual, debunking that quackery that will give you as much superpowers as a chicken soup!

Source: Thanks, Michael Phelps, for glamorizing cupping quackery!

[Sciences] Blood-brain barrier and EMF: the good (science), the bad (science) and the ugly (tinfoil hats)

A friend on social media recently has been debating about some concerns of electromagnetic fields (in particular radiofrequencies and microwaves) on the brain function, in particular their impact on the blood-brain barrier and on brain tumors. I guess this has to also do with some comments of a hopeful presidential candidate making some dubious claims on scientific facts such as vaccines, GMOs and Wifi (!) (https://youtu.be/IGQjaSJP2Xg) with a certain sense of flip-flopping to pander to a particular fringe of the electorate. Some even found indeed a juicy business by selling overpriced cheap caps as EMF shielded caps.

I will not dive into the studies on EMFs on brain tumors becasue I want to keep this post focused on the BBB, but until now there is no evidence of increased incidence of brain tumors in rats being continously exposed to 3x the signal strength of an active call (the study in question has not been peer-reviewed but available on BioRxiv here: http://biorxiv.org/content/early/2016/06/23/055699), as well as the most recent epidemeliogical study from Japan (a country with one of the highest wireless coverage and use) that failed to find any correlation (http://www.ncbi.nlm.nih.gov/pubmed/27197787)

1. What makes a EM wave harmful?

To understand EM radiations, you have to understand the physics behind it. EM waves stands for electromagnetic waves. These are waves formed by two oscillating waves: an electrical wave and a magnetic wave. I will not enter deep into it because I am honestly lame in explaining physics but two things are important for the explanation.
First, oscillating waves have a sinusoidal pattern. Think about a roller coster pattern, in which in a succession your wagon goes up and down in a regular basis. However, this wagon always reach the same height at regular distance: this distance is refered as the wavelength (also symobilized by the Greek letter lambda). The wavelength (a distance unit), usually in the metric system) is inverse to another parameter called frequency (a time unit, expressed in Hertz), by the following formula: Wavelength=1/Frequency. Frequency is usually represented by the Greek letter “nu”
Second, the frequency is associated with energy (E in joules) by the equation of Planck as Energy (E) = Frequency x h, with h being the Planck constant.
Energy is never lost, it is only transformed. A mechanical energy can become thermal energy, that in turn can be transferred into chemical energy and so on.
This brings on the idea of non-ionizing and ionizing energy as depicted below.
ionizingvsnon-ionizing

If your EM wave can provide enough energy to kick out an electron from an atom, it will create an unstable ion that will do all the best to get back its missing electron, even if it means stealing from its neighbors. This is how free radicals are formed. Free radicals are fairly nasty because they are lacking an electron and will do their best to steal from its neighbor, which in turn will also become an electron thief. This is why ionizing radiations (UV, X-rays, gamma rays from certain radioactive isotopes, cosmic rays) are so dangerous and are known cause of cancer.
In the other hand, common EM waves we are constantly bombarded from our electronic devices such as the car FM radio, the satellite dish setup, the wireless signals from our phones and from the Wifi and even the microwaves from your microwave oven (that had some fearmonger at its launch) are simply too weak to ionize an atom and create the dreaded free radicals chain reaction.

2. Does EM waves make your BBB leaky? 

This is some of the claims that some people use to justify their fear of EMFs, since the demonstration of EMFs to cause brain tumors failed to reach a conclusive level. To support of debunk this claim, we will use “Pubmed”, the gold-standard of scientific article database, listing every peer-reviewed studies. However one of the caveat of Pubmed is to publish studies from reputable and less reputable (aka predatory) journals making for a profane hard to distinguish the good from the bad science. However, a graduate student with a BS in Sciences (preferentially with a major in Biology) should have the knowledge to read a paper and assess the quality.
If we use the terms “blood-brain barrier AND electromagnetic field“, we are ending with 68 studies. We will also limit our search to 2005, as we in science consider obsolete any literature that is 10 years and over. We have in total 47 hits with this criteria. I am also narrowing to studies that specifically looked at the EMFs within the 900-5000Mhz, a band spectrum covering the GSM signal from wireless phones up to the Wifi signal (2400Mhz for the 802.11a/b/g norm and 5000Mhz for the 802.11n/ac norm). After removing local (non-translated studies) journals, reviews and non-related studies (one study on EMP was removed as it was not fitting the criteria defined by our analysis), we end up with 17 studies over a 10-year period (11 done in vivo and 5 done in vitro). I have summarized the finding in an easy to read graph

Data 1

Two things to note.

First, the overwhelming majority (>80% of published studies) show no or even a positive effect  of EMFs in drug delivery of nano-particles loaded with HIV antiviral drugs as published here here and  here.

Second, the relative poor publication record on that particular niche (less than 5 studies and review published a year) that makes the argument in favor of harmful effects of EMFs either under investigated or overblown. I would stand with the latter as the odds of publishing negative results (in this case, no effects of EMFs on the BBB function) is much smaller than publishing positive results. Therefore we may indeed overblown the number of studies showing the effect of EMFs on the BBB function, as this number maybe much smaller if all studies left in a drawer were published.

Now let see these few outsiders that have shown a negative effects of EMFs on the barrier function. I have identified four studies: Zhu et al., Brain Res 2015Kumar et al., Electromag Biol Med 2012; Demir et al., Bioelectromagnetics 2010 and Sirav et al., Electromag Biol Med 2009. With the exception of the latest study (IF2014~2.78), all the three other studies were published in journals with a very poor impact factor (IF<2), with Bioelectromagnetics and Electromagnetics Biology Medicine having a displayed IF of 1.20 and 1.5 respectively.

The second caveat is the lack of explicit definition of the intensity of exposure, especially the specific absorbance rate (or SAR) expressed in W/kg. This is the average exposure rate deemed safe. For wireless communications, the SAR in the US set by the FDA is 1.6W/kg, whereas it can go up to 3.0W/kg (with focal acute head exposure) if you are subjected to an MRI head scan. The only study that specifically provide such information is the study from Zhu and colleagues with a SAR of 2W/kg.

Now the SAR expressed a mass unit has a caveat as it does not take into account the physiological difference between a rodent brain and a human brain, both in terms of volume and surface. A rat brain has an average weight of 2g, a human brain has an average weight of 1400-1600g (source: https://faculty.washington.edu/chudler/facts.html). However, a human brain has a thickness much more important than a rat brain, especially the human cortex is twice larger than a rat cortex, so the penetration of EMF waves is twice less in humans than rats. Indeed, we should be consider the surface area that is exposed to such EMFs, in the same manner we are describing the pressure in physics as a unit of force over a surface area (Pressure (N/m2) = Force (N)/ surface area (m2)).
A rat has a surface area of 6cm2, a human has a surface area of 2500cm2.

If you apply the same force into two difference surface area, the pressure will be completely different. The force (N) is the product of the mass (kg) by the Earth gravity acceleration (9.81 m/s, for this example we will ease the calculation by rounding it to 10 m/s). If I apply one ton (1000kg) mass, it has a force of 10’000N.

Now the pressure of this one ton mass will be different if I apply to 1m2 surface (P=10’000N/m2) or if I apply it to 1000m2 surface (P=1’000N/m2). The pressure is ten-times less for the same force applied.

Now, if we replace the force by the EMF exposure limit recommended by the FDA for non-medical application (wireless communications), we have a SAR of 1.6W/kg. If we do the maths using the pressure formula, then the “EMF pressure” a rat exposed to a 2W/kg EMF has a EMF pressure of 2/6 = 0.33W/kg/cm2 or 33.3W/kg/m2. A human exposed to a 2W/kg EMF has a EMF pressure of 2/2500 = 0.0008 W/kg/cm2 or 0.08W/kg/m2.
This gives us a ratio of 33.3/0.08=41.65. So a rat in a experiment exposed to EMF radiations was exposed to a level that was over 40 times the maximal recommended dose. You have to have your head exposed to 40 wireless phones in active call next to your ear constantly to experience some of the issues reported. Or you have to stick your head next to a wireless antenna relay continuously or put your head inside a working microwave to get radiations that are at least as much as reported.

This is how bad and poorly experimental design leads to poor conclusions and fuel bad studies. You see now that not only the numbers of studies pointing to the dangers of EMFs are insufficient, but they are also so poorly designed that their translation to a real-life situation is almost impossible unless you are competing for a Darwin Award.

In conclusion, there is no evidence to support claims made by some about the dangers of the EMFs on the blood-brain barrier, the data supporting their claims are indeed so poorly designed that the only concerns someone may have are those exposed to strong EMFs as their occupational work (MRI technicians….) but this is part of OSHA regulations.

You can however play the precocious card, by limiting the time your cell phone is on your ear, if you are an avid phone person then consider using a bluetooth headset. Don’t hack your Wifi into a WiMax to send signals miles away from your router and please please please put down that tinfoil hat :p. Not only it is worthless but you just making a fool of yourself as well.