[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 First paper (i exclude the CalTech thing since it is not peer-reviewed): They observed that mothers experiencing inflammation during pregnancy had higher risk of having offsprings with autism.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 mercury (formulated as thiomersal) contained in 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 (source: http://www.fda.gov/BiologicsBloodVaccines/ScienceResearch/ucm284520.htm).

Because an intramuscular injection is not a vascular route, the bioavailability is below 100%. The authors here estimated the bioavailability to be 17%, in other means the amount reaching the blood compartment will be only 0.45mg out of the 2.64mg injection. In the study, the authors estimated the aluminum plasma concentration at 30ng/mL before vaccination.

Picture3

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. 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/

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 0.86 and 0.72mg/day. If we use the study from Flarend and colleagues, we should expect to add 0.21mg of AH per injection/day. Thats 25% and 29% acute exposure. The problem would arise if you inject such vaccines everyday for months as you will keep accumulating aluminum in your body.

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.

  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.

  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.

[Sciences/Junk Sciences] Essential Oils: The good, the bad and the ugly science behind some claims

Essential Oils (EOs for simplicity). These little bottles are almost everywhere. Advertised as “natural”, “pure”. Some even are trying to sell them as the next big fad. as the next “miracle cures all” remedy.

Everybody swears by EOs, giving them some curative properties despite the lack of evidence backing such claims. Their therapeutic activity is far from being demonstrated, but their ability to siphon wallets and fill bank accounts of those selling them is as efficient as  the Bernouilli’s principle.

The problem with EOs is to sort the good, the bad and the ugly science behind them.

@MommyPhD recently pointed this out in a nice chart taken from a company making a living on EO.

What I can tell, as the pharmacologist that I am, I was not only perplexed but mind-blown by this chart. It was not a nice mind-blowing effect. It was more like a trigger that turned into a “ballistic mode”.

Just see by yourself the chart below:

17634361_1898070473737773_8852685320786733500_n

What is wrong? Well, all the claims from the original infographic (left) are wrong! It just simply no sense if you have any basic in pharmacokinetics. Its time for the BBB scientist to deflect such woo with some science deflector shields.

First, in order to understand EOs, you have to understand their origin. To understand their origins, you have to have some basic understanding of pharmacognosy.

Pharmacognosy and EOs:

Pharmacognosy is the science that studies the chemical and biological properties of substances produced by plants and fungi. They are seasoned experts in botany, plant biology and analytical chemistry. Their main interest is to extract chemicals from different part of the plant (stem, sap, roots, leaves, flowers, fruit….), identify the substances present in such extract and identify their possible biological properties (this is often linked with ethnopharmacology, in which scientists are trying to identify the potential of some medicinal plants with their use from healers and shamans).

Plants and fungi synthesize two major classes of molecules: those involved in the primary metabolism and those involved in secondary metabolism.

Primary metabolism mostly aimed to ensure growth plant and reproduction. You can consider it as the core chemical plant. These are chemicals important for the plant function.

The second metabolism is on its own very interesting. At first, these compounds have no role in plant growth and therefore may appear useless. Indeed compounds produced from secondary metabolism are very important for the plant because these are essential for its survival. Plants evolved to have limited mobility and therefore are easy target for predators. But what plants traded out for limited mobility have indeed traded in one of the most sophisticated chemical warfare. Plants have evolutionary developed one of the most advanced and versatile chemical warfare aimed to control and deter any dangerous entities that may compete for limited resources (water, minerals, oxygen, light, CO2…).

Here are some examples of chemicals synthesized by plants secondary metabolism: Caffeine, atropine, cocaine, morphine, tetrahydrocannabinol, strychnine, nicotine, digoxin, ouabain, terpenes, cyanide, colchicine, vinblastine, paclitaxel, acetylsalicylic acid, phalloidin, forskolin, turmeric acid…….these are all products from the secondary metabolism. Many of them sounds like “poisons” and they are rightly called poisons because they can kill you at the right dose. But if you use these compounds at the right dose, these compounds can also be used to treat cancer, heart failure, glaucoma……..considering the dose makes the poison.

EOs are a particular class of chemicals, because they harbor particular chemical features. They are volatile (they belong to the superclass of volatile organic compounds or VOCs), lipophilic (soluble in fat and oils) and are odorant (this is why we can smell them). They are also capable of some biological activity.

These EOs have to be extracted from the plants via the use of organic solvent. One of the most common solvent is ethyl alcohol or ethanol (CH2OH), that is convenient organic solvent. Ethanol can help dissolve both lipophilic and hydrophilic substances. It is also has a low evaporation temperature (78ºC), allowing it to dissipate fast once on skin contact.
Another property of these compounds contained in EOs is their ability to become volatile. We can refer these compounds as volatile organic compounds (VOCs). This is why we use them as fragrance. Because they are volatile, these compounds are spread in the air and can be caught by our olfactory receptor neurons (ORNs), making what we refer a smell a smell. Smells are very powerful stimuli, even for humans. This is why we are all fond of “eau de toilette”, “eau de parfum” and all these molecular cues that can turn our reptilian brain upside-down.

EOs are very diverse by their origin and their composition. For the simplicity of this article, I will focus on the major source of EOs by their production: Citrus sirensis (sweet orange) and Mentha arvensis (mint) EOs. These are the two most prevalent sources of EOs worldwide.

Two studies that I have found listed the EO composition from these two plants.  C. sirens is  has about 50 different compounds identified, mostly classified as terpenes. M. arvensis  have about 30-40 compounds including terpenes and other organic compounds (http://www.ifrj.upm.edu.my/18%20(04)%202011/(10)IFRJ-2011-062.pdf and http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.927.8806&rep=rep1&type=pdf)

EO composition vary between cultivars, between crops, between extraction procedures. …..It means that EO by nature are anything but “pure”. EOs are therefore impure because you have a mixture of different compounds at different concentrations. It also means  that such EOs are setting the perfect storm for some drug interactions and some toxicity due to photo activation (some terpenes like limonene are know to become phototoxic following exposure to light)  or induction of an allergic reaction.

Pharmacokinetics and EOs:

In this second part, we will refute the claim brought about the penetration and tissue targeting mentioned in the infographic. The infographic have it wrong at so many different levels but two are striking: firstly, the sequence of events followed the extent of these events.

In order to understand the rebuttal, we have to understand some basic aspect of pharmacokinetics (PK). PK is the science that will tell you the fate of a chemical in your body. It will tell you how it is absorbed, how much reach the bloodstream and the tissue, how it gets detoxified and finally how it gets eliminated.
PK focuses on the fate of drugs inside our body, whereas toxicokinetics (TK) focuses on the fate of poisons and toxins inside our body.

Both follow the ADME acronym: Absorption (tegumentary/skin, intestinal/gut….), Distribution (bloodstream, tissues and brain), Metabolism (“detoxification” via chemical transformation and inactivation by the liver) and Elimination (via the liver and kidneys).

This is where the infographic has it completely wrong. It makesthe assumption that the EOs enter the brain, then the bloodstream and finally cells is just what I call “bullshit” and simply a reflection of a sheer ignorance of human anatomy and physiology. I dragged a small sketch to described the EOs ADME profile.

EssentialOilFirst, EOs have to pass the skin (or gut) barrier and diffuse all the way through the bloodstream. This operate through a passive gradient that result in the progressive loss of EOs compounds during the diffusion (depicted by the yellow arrow) into the bloodstream. This phenomenon is called “bioavailability” and investigate how much of a compound can reach the bloodstream following an administration route that is not obtained via direct injection into the bloodstream (intravenous or intra-arterial).

At the end of the day, this is the appropriate order of sequence: skin->bloodstream->brain (if you are lucky enough).

The amount of compounds contained in the EOs reaching the blood circulation remains unclear and poorly understood. But we can use some analogies with known chemicals. We will discuss the case of hydrocortisone (a topical steroid) and nicotine (a known compound capable to cross the BBB and act on the central nervous system).

Hydrocortisone is commonly used by practitioners to treat skin rashes and other irritations with a cream. The good thing about it is that such cream act topically. A thesis has documented previous studies that estimate about less than 5% of the amount of hydrocortisone applied to the skin was able to get a full ride into the kidneys (https://www.unispital-basel.ch/fileadmin/unispitalbaselch/Bereiche/Querschnittsfunktionen/Spital-Pharmazie/Diss_Pellanda.pdf). It is also telling you that a topical administration is probably not the best option for administration of a drug.

Now, there are other cases of topical administration that result in brain delivery. This is the case for nicotine and nicotine patches. These delivery systems are good in giving a good bioavailability, but yet these compounds will take some time to reach the brain. Considering the tmax (time by which a compound reaches a maximum plasma concentration), such delivery systems can only deliver nicotine with a tmax of 5-6 hours following patch application (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1364642/?page=4). You can understand that the probability of compounds contained in EOs to reach the brain within 30 seconds is impossible, unless you perforate the skull and perform an “intraventricular injection”. This is a very invasive procedure requiring a brain perforation and the insertion of a canule deep inside the brain.

Now we can argue that some drugs can reach the brain within minutes following injection. This is true for anesthetics like propofol. However propofol administration route and chemical properties are very different from EOs: they are injected via IV infusion and propofol penetration across the blood-brain barrier (BBB) is known and documented. Yet, it still takes about 4 mins for a IV infusion of propofol to achieve tmax , making the alleged claims of 22 seconds in the infographic completely bogus (https://www.fda.gov/ohrms/dockets/ac/08/briefing/2008-4354b1-01-FDA.pdf).

Even if your compounds can diffuse the skin barrier at the speed of light (100% absorption and bioavailibity) and have no metabolism (0% loss in EO compounds), you still have to demonstrate that such compounds can cross the blood-brain barrier (BBB). The BBB  blocks about 95% of chemical compounds known by humans. Therefore it is very unlikely that all these EO compounds magically fall within the 5% range.

In addition, analyzing the fate of every single chemical compound present in one EO can be an analytical nightmare even for the most seasoned analytical chemist. @SciBabe can explain you that in more details.

In conclusion, the ability of EOs to exert their biological activity beyond their skin application is simply “dead in water” and subsequently the claims posted in the infographic.

EOs and their “therapeutic claims”: the FDA warning letters
EOs may smell good but they have no scientific basis to support their claims of therapeutic use as depicted on their website. This is why the FDA has decided to enforce its authority via warning letters to two companies.

In 2014, the Food and Drug Administration (FDA) sent two warning letters to Young Living (https://www.fda.gov/iceci/enforcementactions/warningletters/2014/ucm416023.htm) and DoTerra (https://www.fda.gov/iceci/enforcementactions/warningletters/2014/ucm415809.htm) noticing them of the violation of the Food, Drug and Cosmetic Act by advertising therapeutic claims that have not been asserted. Unless you file an application in which you document with a great care the safety and the efficacy of a therapeutic agent, you have no rights to make a claim that compound X will prevent or cure cancer or other illnesses. What was true for these two companies also applies for any companies selling EOs.

Do not use EOs to treat or cure any illnesses because their therapeutic activity have not been proven by scientific methods. Worse, if misused these EOs can become dangerous poisons if swallowed  (http://www.poison.org/articles/2014-jun/essential-oils). You have been warned.

In conclusion,  EOs make great scents and fragrances to make your house smell nicely. But that should be their only application. Use them as personal fragrances with extreme precautions and avoid their swallowing and use as medicines.

[Sciences/Stroke] PCSK9-FOURIER trial and why newer drug is not always better

You may have heard about the FOURIER trial making the news…..or maybe not. What you have maybe heard was this new published clinical trial that has created a divide in the news: In one hand it sounds evolocumab (a PCSK9 inhibitor) is the next cholesterol lowering blockbuster that would put statins into the category of “old drugs”, in the other hand it was raised how this new treatment provide little benefits for a hefty added price ($14’100/year price tag according to this article) compared to statins ($1’409/year price tag for a branded form).

So what is about this study, how does it fare and does it really have such a beneficial value that worth paying the extra $$$? Lets go through together. For this post, I will base my comments on two articles: the FOURIER clinical trial  (Ray, Landmesser et al. 2017) and a recent meta-analysis on the benefits of statins on cardiovascular events (Chou, Dana et al. 2016) .

How does statins and PCSK9 work?

You have heard about statins as a cholesterol-lowering drug, heard about the LDL/HDL ratio with one being “bad” (LDL) and the other being “good” (HDL). Cholesterol is part of our normal functioning. We need cholesterol for our cells and our tissues. Cholesterol is an important component of our cell membrane allowing it to have a certain “fluidity”. Cholesterol is also the precursor of any steroid hormones and it is the source for essential groups of steroid hormones such as glucocorticoids (anti-inflammatory), mineralocorticoids (blood pressure), androgens (male sexual hormones) or estrogens (female sexual hormones).

Cholesterol can come from two sources: diet (by consuming animal products) and by de novo synthesis by the liver from acetyl-coenzyme A (acetyl-coA, through a very long and complex biochemical pathway).

346px-hmg-coa_reductase_pathway

Our liver can produce the amount of cholesterol we need daily, therefore the cholesterol obtained from the diet is an extra amount that is pooled with our endogenous cholesterol. Cholesterol is a fatty acid, it is a lipophilic compound (like fat) that is poorly dissolving in water. One biological adaptation in our evolution was to combine these cholesterol on some proteins called “lipoproteins”, allowing cholesterol to piggy-back and therefore be “soluble” in blood and in interstitial fluids. You have different flavors of these “lipoproteins” based on their physico-chemical properties as observed by ultra-centrifugation. We have the light form called “low-density lipoproteins” (LDL) and the heavier form called “high-density lipoproteins” (HDL).

ng0208-129-f1

LDLs are taken up by cells through a particular receptor called “LDL receptors” (LDLR). These receptors are expressed on the cell surface, in particular by liver cells and endothelial cells (cell lining the inner side of blood vessels). LDLs are the one found mostly in atherosclerotic plaques that are responsible for myocardial infarction (MI or heart attack) and cerebral ischemia (ischemic stroke).

Therefore, it is important to keep these LDL levels within normal range. You have two possible actions: either you reduce your dietary intake and/or use medication aimed to block the endogenous cholesterol production.

This is where statins and PCSK9 inhibitors come into action.
Statins target the enzyme called HMG-coA reductase that is involved in the early step of cholesterol biosynthesis.

Proprotein convertase substilisin/kexin 9 (PSCK9) inhibitors in the other hand block the interaction of PSCK9 with LDLRs. PSCK9 is produced by most of our cells and have an affinity to LDLRs. Therefore PCSK9 can compete with LDL for binding to LDLRs and therefore increase the amount of circulating LDL. Worse, once bound to its receptor, PCSK9 forces the internalization of LDLR and block its recycling, further accentuating the amount of circulating LDLs. By blocking PCSK9 ability to bind to LDLR, you can free more LDLR to bind circulating LDLs and therefore decrease your LDL levels.


What is evolocumab?

One compound have been approved by the FDA in this matter, its name is evolocumab (Repatha®, Amgen). It is a biologic, an antibody that will bind to PCSK9 and impeach its binding to LDLR. Because it is a biologic, it is very expensive to produce because it is a high molecular weight protein that cannot be synthesized by chemistry. We have genetically-modified yeasts or mammalian cells that know how to synthesize it if you give them the right gene. Yet, synthesis is only one problem solved, the other problem is protein folding.
It is like an origami folding, a very tortuous and complicated series of step to achieve a desired shape. Like an origami, fail to properly fold and you will have a far cry of a desired finished product.
Yeast and mammalian cells may not fully be efficient at this task and therefore you have to deal with small amount of biologically active product at the end of the manufacturing process. Evolocumab has been approved by the FDA in 2015 for the use in people that cannot use statins and that failed to control their cholesterol levels by dietary intervention. Because it showed interesting outcomes, it was tempting to consider the added benefit of this biologic over statins. We have a good idea on statins risks and benefits and one major advantage of statins are their price. We have generic forms that are very cheap, much more cheaper than evolocumab (at least 10 times cheaper, considering the full price before coupons and insurance plans).

The FOURIER trial
This is the clinical trial that is at the epicenter of the news. In this trial, they assess how evolocumab (EVO) stood against statins and possibility outperformed statins. Statins have been shown their added benefits for patients with high cholesterol compared to placebo treated.

Because it is unethical to assess EVO versus non-treated individuals, you have to compare groups taking their statins with EVO versus groups taking their statins without (refered here as placebo). This is also a method to show that EVO outperforms statins.

We have two groups of reasonable sample sizes: EVO (N=13’784) and placebo (N=13’780) with matched parameters (age, sex, ethnicity, condition, medication……) to ensure the effect you observe are mostly due to EVO and nothing else.
The striking data here is the lowering of the LDL. In placebo, this level is about 90 mg/dL. In EVO group, we are going down to 30mg/L. That’s fairly impressive. But at the end, you want to see the outcome right?

In our case, our outcomes are how many cases of heart attack and stroke we are dealing with.

This is were the results are becoming more contrasted and less exciting.
If we look at secondary endpoint (death by cardiovascular event, heart attack or stroke), EVO fared a bit better than placebo (5.9% versus 7.4%).

For 2000 persons suffering, EVO saved the life of one person death by cardiovascular death. For 200 patients, it saved one person from an heart attack and one person to stroke injury compared to placebo (statins) group.

Considering the possible side effects reported to the medication, the lack of pharmacovigilance and foremost the price of the drug, you have to weight the benefits over risk and costs. This is where things are less shiny. This study fails to show us how EVO will replace statins.

Statins are the devil we know, they are doing their job well, we know their risks and associated side effects fairly well and they are very affordable. EVO is certainly great for patients that cannot rely on statins to control their cholesterol. But the benefit of EVO over statin is marginal and not worth the risk and costs.

References:
Chou, R., T. Dana, I. Blazina, M. Daeges and T. L. Jeanne (2016). “Statins for Prevention of Cardiovascular Disease in Adults: Evidence Report and Systematic Review for the US Preventive Services Task Force.” JAMA 316(19): 2008-2024.

Ray, K. K., U. Landmesser, L. A. Leiter, D. Kallend, R. Dufour, M. Karakas, T. Hall, R. P. Troquay, T. Turner, F. L. Visseren, P. Wijngaard, R. S. Wright and J. J. Kastelein (2017). “Inclisiran in Patients at High Cardiovascular Risk with Elevated LDL Cholesterol.” N Engl J Med.

 

[Science/Stem Cells] Stem Cell Therapy and Age-related Macular Degeneration – A Tale of Two Cases Reports

Yesterday, a breaking news in the stem cell field came from a case report published in the New England Journal of Medicine (a respected journal in medical research) (http://www.nejm.org/doi/full/10.1056/NEJMoa1609583?query=featured_home).
What is probably the most intriguing about this case report from a stem cell clinic, that had a clinical trial submitted to the FDA and subsequently withdrawn in 2015 (https://clinicaltrials.gov/ct2/show/NCT02024269?term=NCT02024269&rank=1).
This case report focuses on three patients, elderly patients (70+) and all three suffering from age-related macular degeneration (AMD).
They all three being injected with mesenchymal stem cells. For those not familiar with stem cells, stem cells comes into different flavors depending on their origin and potency abilities. The most pluripotent type of stem cells are the embryonic stem cells (hESCs) that are derived from embryos. Following in their pluripotency comes in the induced pluripotent stem cells (iPSCs). Unlike hESCs, iPSCs are reprogrammed somatic cells (usually skin fibroblasts). Then we have bone marrow stem cells (from bone marrow) that have a narrower pluripotency but proven efficacious for treating patients with leukemia. Finally we have the mesenchymal stem cells (MSCs) obtained from the stream fraction of adipose tissue. Usually these cells are obtained by liposuction and isolated from the fat tissue via defined protocols.
Now this last type of cells has a certain classification that allows to use a loophole. In order to conduct a clinical trial, you have to submit an investigative new drug (IND) application to the Food & Drug Administration to show evidence of safety and efficacy from pre-clinical studies. These are safeguards that ensure patients and medical researchers enrolled in such trials.
Now, when you are dealing with MSCs, these stem cell “clinics” can exploit some loopholes and apply for a non-IND application if your trial follows two criteria: it is an autologous procedure (you are injecting cells from yourself back into your body) and there is no procedures that modify the material used (in that case, the purification steps are not altering the MSCs identity and function).  In addition, these trials have to be done without any financial link. In academic institutions, you neither ask the patient to pay for the clinical trial nor  provide the patient with a financial compensation (only a possible therapeutic outcome, if the treatment work). In this case reports, all patients paid $5000 to that clinic. it is also important to note a certain level of deception from the stem cell clinic as this registration at the ClinicalTrials.gov website appeared to these patients as a clinical trial were it was not. It is even mentioned by the authors of this case report that none of the consent form signed by these patients displayed “clinical trial”.
There is also report in the consent form that the patient were informed of the risk of blindness and were requesting to have the procedure done in both eyes.
These patients rapidly developed post-operative complication including retinal detachment, increased ocular pressure and hemorrhagic events, such complications were not taken care by the clinic involved in the stem cell procedure and were done in eye clinics in patients domicile.

By coincidence, NEJM also published another case report of the use of stem cells in AMD in a clinical trial in Japan (http://www.nejm.org/doi/full/10.1056/NEJMoa1608368?query=featured_home). This procedure was done using iPSCs from patients and derived into retinal pigmented epithelial cells (RPEs). These cells are lining the outer side of the retina and serve as a barrier for protecting the retinal neurons involved in vision. This is the famous “iPSC clinical trial” from Takahashi and colleagues that had to be halted due to some safety issues that were corrected (https://ipscell.com/2016/06/good-stem-cell-news-as-takahashi-ips-cell-trial-to-resume/).
So far, the encouraging part is the absence of post-operative complication and graft rejection and the patient showed an improvement in her vision.Yet, this is a single report and the authors were also very cautious about to make overstating conclusion.

These two cases report should inform into two things: we are making little steps but confident steps in stem cell based therapies but it is also important to raise awareness and vigilance about the methods of some stem cell clinics making unrealistic claims of stem cell therapies or posing as clinical trial center.

My recommendation is if you decide to jump into a clinical trial is to verify the affiliation of the center (avoid any private firms and check the credentials of any institution supporting this trial), ask your doctor to help you read through the consent form and never ever be asked to pay for the procedure. The trial is funded by research grants, if it is requested that you cover the expenses of such procedures run away!

 

[Neurosciences/Alzheimer] Structural variation in amyloid-β fibrils from Alzheimer’s disease clinical subtypes

Interesting study published in Nature about how differences in Abeta fibrils have an implication on the clinical symptoms.
A quick refresher for those not much familiar (it is also a bit of a headache as I just dive in the Alzheimer’s research). Alzheimer’s disease is characterized by the formation of senile plaques and tangles, such structures have been considered as the causative agents in neuronal cell death.
These plaques are clumps of a peptide called Abeta (for amyloid beta) peptides. We know how these peptides are formed (by cleavage of the amyloid precursor protein or APP) and we know there are different “flavors” of Abeta that have been described: Abeta 1-40, Abeta 1-42…..
We also know that these peptides are “sticky”. They are released as monomers (single peptide) and because they are hydrophobic (hates water, like oil hates water) they will try to bind together and form oligomers (think about little sticky balls). These oligomers then can form fibrils.
It is a very “dry” study because it is heavy on structural biology and computational biology but in the same time very interesting.
It shows us that plaque formation is not a linear processes, there are different combinations possible (imagine like comparing snowflakes) that have a direct impact on the clinical presentation and outcomes in patients.
That’s maybe providing another way of thinking in targeting Abeta that can help us learn from the failure of previous clinical trials.

Abstract and link to the original paper

Aggregation of amyloid-β peptides into fibrils or other self-assembled states is central to the pathogenesis of Alzheimer’s disease. Fibrils formed in vitro by 40- and 42-residue amyloid-β peptides (Aβ40 and Aβ42) are polymorphic, with variations in molecular structure that depend on fibril growth conditions. Recent experiments suggest that variations in amyloid-β fibril structure in vivo may correlate with variations in Alzheimer’s disease phenotype, in analogy to distinct prion strains that are associated with different clinical and pathological phenotypes. Here we investigate correlations between structural variation and Alzheimer’s disease phenotype using solid-state nuclear magnetic resonance (ssNMR) measurements on Aβ40 and Aβ42 fibrils prepared by seeded growth from extracts of Alzheimer’s disease brain cortex. We compared two atypical Alzheimer’s disease clinical subtypes—the rapidly progressive form (r-AD) and the posterior cortical atrophy variant (PCA-AD)—with a typical prolonged-duration form (t-AD). On the basis of ssNMR data from 37 cortical tissue samples from 18 individuals, we find that a single Aβ40 fibril structure is most abundant in samples from patients with t-AD and PCA-AD, whereas Aβ40 fibrils from r-AD samples exhibit a significantly greater proportion of additional structures. Data for Aβ42 fibrils indicate structural heterogeneity in most samples from all patient categories, with at least two prevalent structures. These results demonstrate the existence of a specific predominant Aβ40 fibril structure in t-AD and PCA-AD, suggest that r-AD may relate to additional fibril structures and indicate that there is a qualitative difference between Aβ40 and Aβ42 aggregates in the brain tissue of patients with Alzheimer’s disease.

Source: Structural variation in amyloid-β fibrils from Alzheimer’s disease clinical subtypes : Nature : Nature Research

[Sciences/BBB] Histamine-induced blood-brain barrier disruption in teething children: a “post hoc ergo” on glucocorticoids.

Recently, I have been aware about some parents concerned about the impact of teething on the blood-brain barrier integrity, such claims was wrapped through one of the most bizarre “ergo post hoc” fallacy following that sequence:
1. Teething induces histamine release
2. Histamine is a vascular hyperpermeable vascular factor
3. Blood-brain barrier in babies is leak
4. Therefore Teething induces a blood-brain barrier breakdown in children.

You have to agree that is one of the most bizarre fallacious association, but it has been repeated and spread enough to have parents concerned about the impact of teething on the blood-brain barrier. To dispel that myth and beat that dead horse once for all, I think it is important to demonstrate why this information is fallacious.

 

  1. Teething and histamine release: understanding the mechanism of inflammation.

First, in order to understand the physiological response of teething, you have to understand the mechanism of inflammation. Everyone can provide a clinical presentation of an inflammation: it is red, it is swollen and it is hot.
Inflammation is triggered by lesion or a wound due to internal or external stimuli, in our case teething. Teething involves a mechanical stress (due to teeth growth) and eventually cells and tissue laceration. Such laceration release intracellular contents into the extracellular space that turns on resident immune cells. These cells in turn release what we refer to “pro-inflammatory factors”, a cocktail of different chemicals that triggers the inflammatory bomb on:

We have amino-acids derivatives and peptides (bradykinin, histamine, inflammatory chemokines and cytokines) and arachidonic acid (AA)-derived molecules. The production of AA-derived molecules (also known as prostanoids) are driven by cyclo-oxygenases (COXs). There are two types of COXs: COX1 that is constantly produced at small level and COX2 that is increased during inflammation.  COX1 produce the “good prostanoids” and COX2 the “bad prostanoids”, the latter being the driving force of the inflammation. COXs are the classical targets of the classical NSAIDs found in OTC products including acetaminophen (Tylenol), ibuprofen (Advil) and naproxen (Aleve). All these small molecule target COXs and stop prostanoids production. In the case of teething, inflammation is mostly driven by the release of prostanoids (such as prostaglandin PGE2) and interleukins (IL-1beta) (Blakey, White et al. 1996). Aside of two obscure studies published 40 years ago in obscure medical journals (Cotias, de Medeiros et al. 1968, Soliman, Abdel Wahed et al. 1977) there is no evidence of histamine release following teething. This therefore nullify claim 1.

  1. Histamine and the blood-brain barrier

Histamine is not the major mediator of inflammation, but it is indeed the major mediator in allergic reaction. During an allergic reaction, the immune system respond to the allergen by the production of certain types of antibodies called IgE.

IgE are produced by B-cells that positively responded to the allergen, recognizing it as a foreign body. IgE binds to a certain type of immune cells called mast cells. Mast cells are about less than 1% of the total population of immune cells. These cells are super-loaded with histamine, ready to puff it upon signal. Once IgE binds to its appropriate receptor, mast cells puff and release vast amount of histamine. Histamine in turns triggers the anaphylactic response such as “asthma”, “runny nose” and in the worst case an anaphylactic shock. The main treatment for mild allergic reaction is solved by taking anti-histaminic drugs such as Claritin D or Benadryl.
Because the histamine released in teething is much more negligible than the amount of prostanoids produced, the use of anti-histaminic is worthless because you only address a minor component of the inflammation and omit to block the major component. So the histamine relationship with teething is also refuted at this point. But does histamine can cause the BBB disruption and its leakiness? Yes, but only at high doses and only in very specific cases. Studies that have investigated the biological effects of histamine at the BBB are very old (20+ years) and were achieved with high concentrations (10-100 micromol/L) (Gross, Teasdale et al. 1981, Domer, Boertje et al. 1983, Watanabe and Rosenblum 1987, Butt and Jones 1992, Mayhan 1996). If we consider that histamine is produced during teething, we can conservatively assume that such level would not be over plasma levels found during a severe allergic reaction such as an anaphylactic shock. Reported values for an anaphylactic shock are about 6.35 nmol/L (Laroche, Gomis et al. 2014). Even at that high level, that’s put us about 1000X to 16000X less than values reported to have an activity on the BBB. So claim 2 is also refuted.

  1. Is the BBB leaky in newborns and babies?

TL; DR the short answer is NO. If you want to understand why and what is the science behind my statement, please check my previous post about it: https://scientistabe.wordpress.com/2016/05/21/neurosciencesbbb-thiomersal-and-the-blood-brain-barrier-where-does-the-science-stand/)

 

  1. Conclusions

By now, we should agree that the reason of delaying vaccines in children due to histamine-induced barrier disruption does not stand to science. There is no scientific rationale to support the hypothesis of a massive release of histamine during teething, such release being well below reported values for achieving a BBB disruption and leakage. If you have your baby teething right when he/she is due for immunization, consult with your AAP-accredited pediatrician for what is best for baby.

    5. References

Blakey, G. H., R. P. White, Jr., S. Offenbacher, C. Phillips, E. O. Delano and G. Maynor (1996). “Clinical/biological outcomes of treatment for pericoronitis.” J Oral Maxillofac Surg 54(10): 1150-1160.

Butt, A. M. and H. C. Jones (1992). “Effect of histamine and antagonists on electrical resistance across the blood-brain barrier in rat brain-surface microvessels.” Brain Res 569(1): 100-105.

Cotias, C. T., E. C. de Medeiros, U. V. Lima and C. F. de Santana (1968). “[Determination of histamine release in the blood serum of children during deciduous tooth eruption].” Rev Fac Odontol Pernambuco 1(2): 95-100.

Domer, F. R., S. B. Boertje and S. A. Sweeney (1983). “Blockade of the acetylcholine-and histamine-induced changes in the permeability of the blood-brain barrier of normotensive and spontaneously hypertensive rats by atropine and pyrilamine.” Res Commun Chem Pathol Pharmacol 42(1): 157-160.

Gross, P. M., G. M. Teasdale, W. J. Angerson and A. M. Harper (1981). “H2-Receptors mediate increases in permeability of the blood-brain barrier during arterial histamine infusion.” Brain Res 210(1-2): 396-400.

Laroche, D., P. Gomis, E. Gallimidi, J. M. Malinovsky and P. M. Mertes (2014). “Diagnostic value of histamine and tryptase concentrations in severe anaphylaxis with shock or cardiac arrest during anesthesia.” Anesthesiology 121(2): 272-279.

Mayhan, W. G. (1996). “Role of nitric oxide in histamine-induced increases in permeability of the blood-brain barrier.” Brain Res 743(1-2): 70-76.

Soliman, N. A., S. Abdel Wahed, A. M. Abul Hassan, G. el-Asheiry and A. K. Abdallah (1977). “Systemic disturbances accompanying primary teething: a clinical and pharmacological study.” Egypt Dent J 23(1): 1-8.

Watanabe, M. and W. I. Rosenblum (1987). “In vivo studies of pial vascular permeability to sodium fluorescein: absence of alterations by bradykinin, histamine, serotonin, or arachidonic acid.” Stroke 18(6): 1157-1159.

[Neurosciences/BBB/Stroke] Breaking The Wall: Brain pericytes impairs the BBB integrity following ischemic stroke injury. 

Interesting study, reminds me one of my earlier study. Brain pericytes are a particular cell type with a disputed origin (some think they come from neural crest, some think they come from mesoderm and some think they are mesenchymal stem-cell like cells) that play an important role in the brain and retinal vascular integrity.
Pericytes play an important function by stabilizing vessels and tightening the blood-brain barrier and the blood-retina barrier. Loss of pericytes have been associated with disruption of the retinal barrier in diabetic retinopathy whereas it was associated with leaky vessels and impaired amyloid beta clearance.
Interestingly, pericytes have been also associated with an impaired barrier recovery following stroke injury acceding to this study by Shih and colleagues (http://www.jneurosci.org/…/2016/11/14/JNEUROSCI.2891-16.2016). The mechanism of action is something known as it involves matrix metalloproteinases (MMPs). MMPs are enzymes that cleaves matrix proteins into fragments as well as tight junction proteins. The increase in MMP activity following ischemic stroke injury was known but until now it was mostly associated with astrocytes and endothelial cells. In my previous work, I have demonstrated that pericytes were deleterious to the barrier function following hypoxic stress.

Source: Rogue Cells Leave the BBB Defenseless During Stroke Trending | Labroots | Virtual Events, Webinars and Videos