A central thesis of this blog is that aluminum adjuvant causes brain injury and autism by an immune activation mechanism. Specifically, the hypothesis is that aluminum adjuvant causes autism by inducing interleukin-6 (IL-6) production in the brain. Elevated IL-6 in the brain causes autism.

There is strong evidence for this hypothesis, but we do not yet have conclusive experimental results showing that Al adjuvant induces IL-6 (and/or IL-17a) in the brain. Instead we have experiments showing:

1) water-soluble aluminum induces IL-6 in the brain and other tissues, and

2) Al adjuvant causes inflammation and microglial activation in the brain. Some of these studies use aluminum adjuvant dosages essentially the same as dosages infants receive according to the CDC schedule.

A Special Relationship Between Aluminum and IL-6?

Aluminum strongly induces IL-6 production, and there are good scientific reasons why. Aluminum causes oxidation, and IL-6 is produced in response to oxidative stress (e.g., low glutathione, elevated free radicals). IL-6 stimulates tissue repair and healing of oxidation injury.

One way aluminum likely induces oxidation is by displacing iron from a protein (transferrin) where iron is safely stored. Transferrin is specifically designed to bind iron and prevent it from causing oxidation. Free (unbound) iron floating around is very toxic, because its a potent catalyst for oxidation reactions. So, one way aluminum induces oxidation is by unleashing free iron. In living cells, free iron creates an explosion of destructive oxidation.

The Viezeliene et al. paper (described below) states:

“It has been suggested that…IL-6 is involved in the repair process responding to oxidative stress and depletion of reduced glutathione. The protective effect of IL-6 against oxidative stress and mitochondrial dysfunction has been proved by the increased toxicity of reactive oxygen species in IL-6 deficient mice. These studies demonstrated specific induction of IL-6 is a response to the disturbed redox status.”

AND

“The mechanism by which Al causes tissue damage by an oxidation-mediated process is probably caused by an increase in the iron content due to the competitive binding of Al to transferrin, an iron binding and transport protein. It was shown previously that upon exposure to Al the iron content in several tissues, especially in the liver, increased substantially. Since iron is the most probable initiator of oxidative stress reactions in tissues, the Al-induced increase in oxidative stress could be mediated by iron.“

However, aluminum likely causes oxidation by other mechanisms as well. It is well proven that aluminum strongly induces oxidation, but the importance of the various proposed mechanisms are debated. Here is a paper describing several ways that Al can cause oxidation, including the iron-release theory: Exley: The pro-oxidant activity of aluminum

Regardless of the mechanism, oxidation explains why aluminum may have a special relationship with IL-6. IL-6 is part of the body’s natural response to oxidative stress.

Al Dosage from Vaccines

According to the US vaccination schedule recommended by the CDC, infants may receive the following (maximum) dosages of Al adjuvant:

Birth (Hep B): 74 mcg/kg (250 mcg for 3.4 kg infant)

2 month: 245 mcg/kg (1225 mcg for 5 kg infant)

4 month: 150 mcg/kg (975 mcg for 6.5 kg infant)

6 month: 153 mcg/kg (1225 mcg for 8 kg infant)

Because these are dosages of injected Al adjuvant particles with low solubility, they cannot be directly compared to the dosages used in the feeding studies below. Relating ingested aluminum and injected Al adjuvant is covered here:FDA’s Flawed Study Of Al Adjuvant Toxicity

Ingested dosages of 3.4 and 4 mg/kg/day (used in studies below) are relevant to exposure from Al adjuvant human infants receive from vaccines because ingested Al has a low absorption of about 0.3%. By comparison, 100% of injected Al adjuvant enters the body. So for example, if 1000 mg of water soluble aluminum is ingested, only about 3mg enters the body, and 997 mg is eliminated in feces. If 4mg is injected, then obviously all 4mg enters the body.

However, comparing water soluble Al with Al adjuvant is misleading and oversimplified, even if the low oral absorption is considered. This is because Al adjuvant is made of solid particles that slowly dissolve in body fluids. Hence, Al adjuvant produces exposure to two different substances: 1) water soluble Al3+ ions, and 2) solid particles of Al adjuvant materials (Al hydroxide or Al phosphate).

Both water-soluble Al and Al adjuvant are toxic, but in different ways, in different places in the body, and with different dynamics over time. Ingested Al3+ (the absorbed portion) is almost completely eliminated in urine within a few days, and small amounts persist in bone, brain and other tissues. Al adjuvant particles are not eliminated except by dissolution, and they dissolve very slowly. Solid particles of Al adjuvant have been observed years later. So, with Al adjuvant, exposure to the particles and water soluble Al3+ is chronic and continuous.

Despite these differences, studies of water soluble Al are relevant to Al adjuvant toxicity and show that aluminum induces IL-6 in the brain and other tissues.

Studies of Water-Soluble Aluminum and IL-6

Experiments using water-soluble aluminum (not Al adjuvant) such as aluminum chloride (AlCl3) show that it increases IL-6 in the blood (Viezeliene et al 2013, Khafaga 2017, Borai 2017), kidney (Dera 2016), and brain (Cao et al 2016, Alawdi et al 2016).

below are summaries of the IL-6 findings in these studies:

Alawdi et al., 2016

Dosage: 3.4 mg/kg/day Al (from 17mg/kg/day AlCl3)

Route: Ingestion

Dosing duration: 6 weeks

Form: AlCl3

Animal: Adult rats

Neuroprotective Effect of Nanodiamond in Alzheimer’s Disease Rat Model: a Pivotal Role for Modulating NF-κB and STAT3 Signaling.

In this experiment, adult rats were fed 3.4 mg/kg/day soluble aluminum (from 17 mg/kg/day AlCl3) for 6 weeks. This is a vaccine-relevant dosage, because it is much lower than the oral dosage (26 mg/kg/day) used to argue that aluminum in vaccines is safe (by Mitkus 2011, see this article: The Foundation for Al Adjuvant Safety Is False). The aluminum increased IL-6 about 4-fold in the brain (hippocampus).



Above: Ingestion of 3.4 mg/kg/day soluble aluminum caused a 4-fold increase in IL-6 in the hippocampus, a part of the brain. The aluminum also elevated other signs of brain inflammation, and caused adverse effects on learning, memory, and behavior. “ND” and “MEM” refer to nanodiamond and memantine, which reduced some of the adverse effects of aluminum. Memantine is an FDA-approved drug for alzhiemers disease, which, like autism, is caused by brain inflammation. From Alawdi et al 2016.

Borai et al., 2017 (NEW! As of Dec 2017)

Dosage: 3.4 mg/kg/day Al (from 17mg/kg/day AlCl3)

Route: Ingestion

Dosing duration: 4 weeks

Form: AlCl3

Animal: Adult rats (12-15 weeks)

Therapeutic impact of grape leaves polyphenols on certain biochemical and neurological markers in AlCl3-induced Alzheimer’s disease

In this experiment, adult rats were fed 3.4 mg/kg/day soluble aluminum (from 17 mg/kg/day AlCl3) for 4 weeks. This is a vaccine-relevant dosage, because it is much lower than the oral dosage (26 mg/kg/day) used to argue that aluminum in vaccines is safe (by Mitkus 2011, see this article: The Foundation for Al Adjuvant Safety Is False). The aluminum increased IL-6 about 4.5-fold in the serum (blood). Also, Borai et al. reported that the cerebellum and purkinje cells were damaged by the aluminum. Purkinje cell and cerebellum damage is a consistent (practically universal) finding in human autism. Borai states: “Al-treated rats showed marked histological changes and showed decreased number of cells in the granular and Purkinje layers, indicating cellular degeneration and atrophy.”



Above: Ingestion of 3.4 mg/kg/day soluble aluminum caused a 4.5-fold increase in IL-6 in the serum (blood). From Borai et al 2017.

Viezeliene et al 2013

Dosage: 25 mg/kg Al (from AlCl3)

Route: Injection into body cavity

Dosing duration: single dose

Form: AlCl3

Animal: Mice

Selective induction of IL-6 by aluminum induced oxidative stress can be prevented by selenium

In this experiment mice received a single injection of 25mg/kg aluminum (AlCl3), a very high dosage compared to vaccines. The mice were sacrificed and analyzed for acute effects 16 hours later. The aluminum caused a large increase in IL-6 in the blood (serum). Note that selenium caused a smaller increase in IL-6. Selenium is an essential nutrient, but it was given at very high (possibly toxic) dosage. Selenium provides antioxidant protection, which likely explains why it reduced the IL-6 surge caused by the aluminum.



Above: A single injection of 25 mg/kg soluble aluminum (in the form of AlCl3) caused a large increase in IL-6 in the blood (serum) 16 hours later. TNF-a was unaffected, which suggests that Al has a particular connection to IL-6. The connection between aluminum and IL-6 may be because IL-6 is induced by oxidation, and aluminum strongly induces oxidation. This result does not prove that aluminum adjuvant increases IL-6 in the brain, but it is suggestive. From Viezeliene 2013.

Dera 2016

Dosage: 4 mg/kg/day Al (from 20mg/kg/day AlCl3)

Route: Ingestion

Dosing duration: 40 days

Form: AlCl3

Animal: Adult rats

Protective effect of resveratrol against aluminum chloride induced nephrotoxicity in rats

The Dera study looked for effects on the kidneys. The aluminum caused many adverse effects on kidney function. Aluminum ingestion caused a nearly 3-fold increase in IL-6 in the kidneys.



Above: Ingestion of 4mg/kg/day aluminum for 40 days caused a nearly 3-fold increase in IL-6 in the kidneys of rats. The antioxidant resveratrol (RES) reduced the adverse effects of aluminum. From Dera 2016.

Cao et al 2016

Dosage: 10, 30 or 90 mg/kg/day Al (from AlCl3)

Route: Ingestion

Dosing duration: 90 days

Form: AlCl3

Animal: Adult rats

Aluminum chloride induces neuroinflammation, loss of neuronal dendritic spine and cognition impairment in developing rat.

Brains of the rats (hippocampus, specifically) were analyzed for gene expression of inflammatory cytokines. Gene expression of IL-6 was elevated (in fact, gene expression for all inflammatory cytokines was elevated).



Above: Ingestion of soluble aluminum (AlCl3) increased IL-6 gene expression in the brain. Gene expression of other inflammatory cytokines (IL-1B, TNF-a) also increased. The combination of IL-6 + IL1B causes epilepsy (seizure disorders). Autism is strongly associated with elevated TNF-a. From Cao et al. 2016.

Khafaga, 2017

Dosage: 20 mg/kg/day Al (from 100 mg/kg/day AlCl3)

Route: Ingestion

Dosing duration: 8 weeks

Form: AlCl3

Animal: Adult rats

Exogenous phosphatidylcholine supplementation retrieve aluminum-induced toxicity in male albino rats

The aluminum increased IL-6 in the serum (blood) 3-fold. Lecithin (phosphatidylcholine) prevented some of the adverse effects of aluminum.





Above: Ingestion of 20 mg/kg/day aluminum increased IL-6 in serum (blood) 3-fold. Lecithin (phosphatidylcholine) reduced the inflammation cause by the ingested aluminum. From Khafaga 2017.

Aluminum Adjuvant and IL-6

I found only one study reporting IL-6 measurements after Al adjuvant injection: Kool et al.

Kool et al., 2008: Cutting Edge: Alum Adjuvant Stimulates Inflammatory Dendritic Cells through Activation of the NALP3 Inflammasome

In this experiment, 1000mcg Al adjuvant (a very large dosage for mice) was injected into the body cavity (“intraperitoneal”, or i.p.) of mice. This was compared to an injection of ovalbumin (OVA), an egg protein commonly used in allergy and immunology research. The Al adjuvant+OVA caused a much larger induction of IL-6 than OVA alone. IL-6 was measured in the body cavity in this experiment. This study has limited applicability for vaccines because it used a very high dosage and i.p. injection, and because it did not measure IL-6 in the brain. But it does show that Al adjuvant induces IL-6, so it is suggestive.



Above: Injection of Al adjuvant+OVA (ovalbumin) caused a much larger increase in IL-6 compared to OVA injection alone. IL-6 was measured in the body cavity (“peritoneum”) 2 hours after injection. From Kool et al, 2008.

Aluminum Adjuvant and Brain Inflammation

Several studies show that injection of Al adjuvant causes inflammation and microglial activation in the central nervous system. None of these studies measured IL-6, unfortunately. But microglial activation suggests that IL-6 is elevated, since microglia can produce IL-6, and often do produce IL-6 when activated and/or exposed to oxidative stress.

Shaw et al, 2009: Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration

In this experiment, 300mcg/kg Al adjuvant was injected into adult mice in 6 doses of 50mcg/kg, over a period of two weeks. Behavioral and other tests were performed, but here we are interested in the test for activated microglia. Microglia are the immune cells of the brain. Microglial activation indicates inflammation and cytokine production. Microglia are capable of producing IL-6. Activation per se is not proof that microglia are producing IL-6, but it is suggestive.

Microglial activation is indicated by the protein iba-1. A normal, healthy brain has a low level of activated microglia producing iba-1. 300 mcg/kg Al adjuvant caused a significant increase in activated microglia. In this study, the activated microglia were located in the spinal cord. Other regions of the central nervous system (e.g. the brain) were not analyzed for activated microglia.



Above: Injection of 300 mcg/kg Al adjuvant caused a large, significant increase in activated microglia, as indicated by iba-1 production. This measurement was performed on the spinal cord, not the brain. Elevated iba-1 indicates the microglia are in an activated state, and likely producing cytokines. From Shaw et al 2009.

Crepeaux et al. 2016: Non-linear dose-response of aluminium hydroxide adjuvant particles: Selective low dose neurotoxicity

In this experiment, mice injected with 200 mcg/kg (in 3 doses of 66mcg/kg) were analyzed for microglial activation in the brain. This dosage caused a significant increase in activated microglia, as indicated by iba-1 production. Measurement of iba-1 was performed 180 days after injection, so the brain inflammation appears to be chronic, and perhaps permanent. This study is essentially a replication of the Shaw et al 2009 finding that 300mcg/kg caused microglial activation in the spinal cord.



Above: Injection of 200 mcg/kg Al adjuvant caused a significant increase in activated microglia, as indicated by iba-1 production. This measurement was performed on a part of the brain (the “ventral forebrain”), not the spinal cord. Elevated iba-1 indicates the microglia are in an activated state, and likely producing cytokines. From Crepeaux et al, 2017.

Crepeaux 2017 also reported that the 200mcg/kg dosage caused behavioral abnormalities and a 50-fold increase in brain aluminum content. This study is described here: Al Adjuvant Causes Brain Inflammation and Behavioral Abnormalities; Low Dose Is More Harmful

Microglia and Autism

Microglia are definitely involved in autism. Human autistics have chronic microglial activation throughout the brain. A recent review paper (Takano 2015) on the topic of microglia and autism states:

“…any factors that alter the number or activation state of microglia either in utero or during the early postnatal period can profoundly affect neural development, thus resulting in neurodevelopmental disorders, including autism.“

Paper (Takano 2015): Role of Microglia in Autism: Recent Advances

Obviously, the finding that aluminum adjuvant induces chronic microglial activation in the brain, at vaccine-relevant dosages, strongly implicates this vaccine ingredient as a cause of autism.

Li et al. 2015: Neonatal vaccination with bacillus Calmette-Guérin and hepatitis B vaccines modulates hippocampal synaptic plasticity in rats.

This very important study is explained in detail here: http://vaccinepapers.org/two-vaccines-opposite-effects-brain/

In this experiment, baby rats were injected with the bacillus calmette-guerin (BCG) vaccine (for tuberculosis), or the hepatitis B vaccine, with timing that imitates human exposures (dosage was higher than in humans, however). The rats received one dose of BCG or 3 doses of Hep B vaccine. The hepatitis B vaccine contains aluminum adjuvant, and BCG vaccine does not. BCG vaccine does not contain any adjuvant, because it is a “live” vaccine, with live bacteria. At ages 2, 4, and 8 weeks, the brains were analyzed for inflammation and other things.

IL-6 level was measured in the brain (in the hippocampus, a part of the brain). The hepatitis B vaccine increased IL-6, and the BCG vaccine had no effect on IL-6. Results (at 8 weeks of age) are shown below.

Note that IL-6 was normal (in the Hep B vaccinated group) at ages 2 and 4 weeks. Elevated IL-6 was therefore a delayed response, starting sometime between 4 and 8 weeks.



Above: Hepatitis B vaccination caused an increase in IL-6 in rat brain (in the hippocampus) at age 8 weeks. BCG vaccine had no effect on IL-6. Hep B vaccine contains Al adjuvant, and BCG vaccine does not. Elevated IL-6 in the brain causes autism. From Li et al. 2015 (Fig. 5F).

Why do BCG and Hep B vaccines have different effects on IL-6 in the brain? Is it the aluminum adjuvant in the hep B vaccine, or something else? While its not certain the Al adjuvant is responsible, its the only ingredient that is linked to IL-6 and known to be transported into the brain. All available evidence suggests the Al adjuvant is responsible for increasing IL-6 in the brain.

Ingredients in the BCG and Hep B vaccines are shown below.



Above: Ingredients in the BCG and Hep B vaccines. Ingredients present in the Hep B and not present in the BCG vaccine include aluminum adjuvant, yeast protein, soy peptone, formaldehyde, and of course the Hep B virus antigen. Of these ingredients, only aluminum is known to induce IL-6. The mineral salts and other ingredients (potassium phosphate, sodium chloride, magnesium sulfate, citric acid, glycerin, dextrose (another name for glucose)) are naturally present in body fluids and are harmless. It is not plausible they are responsible for the IL-6 increase.

Conclusion

The elevated brain cytokines indicate that aluminum adjuvant can cause brain injuries by the same mechanisms as immune activation. Aluminum adjuvant causes long term, chronic immune activation/inflammation in the brain, and such inflammation is a proven cause of neurodevelopmental disorders, including autism.