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Pharmacology Sciences

[Sciences/Pharmacokinetics] Do nano-particles of the Pfizer COVID-19 vaccine cross the blood-brain barrier and infect your brain with mRNA (or will fritz your gonads)?

1. Introduction
[EDIT: Updated the article on 07/05/2021 to reflect some updates on my analysis]

I have recently seen some claims I considered moot resurfacing on social media: first that COVID-19 vaccines render women infertile; second that mRNA vaccines cross the blood-brain barrier and therefore lead to neurological diseases.
These claims have been rebutted by various science communicators including Edward from Deplatform Disease and myself on Skeptical Raptor few months ago, as the Pfizer and Moderna vaccines were rolling out in the US.

Thing is, with anti-vaxxers, claims are never completely dead and keep rising up like some zombies straight out of a Walking Dead episode.

This time, it seems to be materialized through this screenshot, that appear to spread virally on social media over the weekend, especially in various iterations of that screenshot, with a yellow highlight in a table with the following tissue: “ovaries” and total lipid concentrations as only information.

Screenshot depicting estimated aminolipid contents in rats following injection of the Pfizer COVID-19 nanoparticle formulation (source: Facebook).

2. What is the screenshot coming from?

As always, getting back to the source of a document is essential to put this information back into the context. This screenshot appeared to be coming from a leaked document (if I have to judge on the “Pfizer – Confidential” footers) that I was able to find the source. Unfortunately the document is in Japanese but I can speculate this document likely came from an application packet submitted to the Japanese equivalent of the FDA to seek authorization of sale of the vaccine on the Japanese market. 3. What is the document about?
It seems the document provides us with some pharmacokinetics data on the mRNA vaccine done in rats (Wistar Han strain, both males and females) to assess the pharmacokinetics of the nanoparticles inside these rodents to assess the pharmacokinetics of both the lipid nanoparticles and the mRNA (using the luciferase as reporter of mRNA transcription, I will explain it later).
For the majority of the experiments, we have the following situation been used (according to Table 1):

Nanoparticles were used using two aminolipids (ALC-0315 and ALC-1059) at concentrations of 15.3 and 1.96mg/kg respectively. mRNA was encapsulated in these nanoparticles at 1mg/kg (to give you an idea, the actual dose of mRNA in a Pfizer shot is 30ug or 0.03mg from patients ranging of 12 years and older)

Table 1 provides us with some pertinent PK parameters including the half-life (time to eliminate 50% of a drug), the AUC (to compare the relative bioavailability, distribution and calculate the clearance of a drug) and finally the Kanji translated by Google Translate (sorry but that poor Gaijin is illiterate to Japanese despite decades of anime) as “Distribution ratio to the liver“, with 60% of ALC-0315 found in the liver, 20% of ALC-0159 respectively. The number of animals also appear to be N=3/group (male, female as groups).

We have therefore extensive data on the aminolipids metabolism and the metabolites obtained both in vivo (plasma samples mostly), in vitro (using liver microsomes homogenates, a classic in PK/PD studies); distribution of LNPs in tissues and organs using a non-metabolized radio-tracer ([3H]-08-A01-C0 which I quote the document “[3H]-08-A01-C0 = An aqueous dispersion of LNPs, including ALC-0315, ALC-0159,distearoylphosphatidylcholine, cholesterol, mRNA encoding luciferase and trace amounts of radiolabeled [Cholesteryl-1,2-3H(N)]-Cholesteryl Hexadecyl Ether, a non-exchangeable, non-metabolizable lipid marker used to monitor the disposition of the LNPs“, which was given at a dose of 50ug in animals) and finally bio-luminescence assays in which it consisted of injecting 2ug of RNA encapsulated in the LNP formulation in the hind-limbs of rats (we can assume these were adult rats, therefore a weight of 200-250g is not unheard of), followed by live imaging of the animals to track the luciferase activity (following injection of coelenterazine, the conversion of this substrate by luciferase results in bio-luminescence at close proximity which can be detected through a special camera, as Figure 2).

4. What the data is telling us?


The first thing I would tell is that the person behind the yellow highlight not only have absolutely no idea of what to look for in Table 3 but also went into a cherry-picking expedition to use numbers in scaring people with numbers. That person is providing us with amount of the radiolabeled tracer detected in the tissue (e.g. ug/g tissue), with the approximation of total lipids amount in tissue. This assumes that the nanoparticles made it through the tissue complete, but we cannot exclude that we are maybe measuring only the 08-A01-C0 compound accumulation.
In practice, we usually focus our attention on the percentage of injected dose (% ID) when it comes to appreciate the distribution and the delivery of a drug into an organ/tissue. In some fields, like the BBB, such value is usually not sufficient, and we further correct these values to sort the amount that diffused across the blood-brain barrier (BBB) against the amount that is retained in the cerebral vasculature by the time of euthanasia.
Therefore, we have to put our attention on the right-half of the table. I have plotted these values into a plotting software (Graphpad Prism 9) to have a graphical representation.

What we can see is that the LNPs reach a Cmax value of 52.9% of the ID by 1 hour following IM injection and see a biphasic phase of distribution and elimination (which I suspect the drug would follow a 2-model compartment). Liver is the organ with the highest uptake (we know that 60% of the LNPs are uptaken by the liver) with a Cmax of about 18% of the injected dose by 8 hours. This is expected as liver has a formidable blood flow compared to other organ (Q=1500mL/min). Spleen (very important lymphoid organs) comes in as a good second with a Cmax~1%ID by 8 hours. Kidneys in the other hand sees a much lower uptake despite being an organ with a decent blood flow (GFR=~120mL/min) with a Cmax`0.2%ID, suggesting these LNPs maybe eliminated mostly via hepatic clearance route (including metabolism).

[EDIT: I have performed an area-under-the curve analysis, just for the fun of it. We are lacking data, so we will use for informational purpose. The use of the AUC trapezoidal method can allow to guesstimate how much of that radiotracer accumulated in the tissue/organs over the 48 hours period.
If we look at the AUC values of these from 0 to 48 hours, about 57% of the injected dose is found in the liver, 3% in the spleen, 0.25% in the kidneys, 0.17-0.18% in the gonads and finally 0.04% of the injected dose is found in the brain). ]

What about ovaries? Well we are in the same ballpark than kidneys and indeed nothing really much about (0.1%ID after 48 hours). Interestingly, the author hyper focused on female gonads and occulted to show that male gonads (testes) were getting the same %ID (0.07%). I don’t think it was an accident from the author, just a sign of a deliberate attempt to manipulate the narrative by spinning the numbers.
And last, brain, my favorite organ. The amount entering the brain is maybe the lowest of our organ of interest as we measured a meager 0.02% ID there. Keep in mind, we have to be careful on this number as we may have an overestimation here. In the field, when you do brain perfusion and you are about to collect your last plasma timepoint before sacrificing the animal, you have to be sure to perform a “flushing” of your cerebral blood vessels with PBS, to remove any residual blood volume that can contain your drug. Unless you can correct for the vascular volume (which is not as simple), you have to perform this procedure as we did in a paper I collaborated on. Failure to do so can can lead to overestimation of your brain uptake. Until I have evidence of such flushing occurred, we can hypothesize that the investigators sacrificed their animals at the time points, extracted and weighted all organs and proceeded with the radioactive counts. Therefore, that 0.02% ID should be considered as a grand maximum, likely overestimating the real concentration.

Taken together, we can see that aside of the liver and spleen, the uptake of the radiolabeled tracer (and by extension nanoparticles) remains very low in gonads and in the brain, with amounts of 0.1% and 0.01% respectively at 48 hours.

The second set of data we have to look at is the bio-luminescence data (see Page 5). The lab injected 1ug of mRNA in each hind leg, totaling 2ug mRNA in each rat. Considering an average weight of 200g per rat, we can approximate a dose of 10ug/kg for the luciferase assay. As a control (to remove the background noise), control animals were injected with saline buffer. The average bio-luminescence signals were given, and I personally added 10% of this average as an estimated standard deviation to have an error margin, which a value commonly accepted in biological sciences (10% variation around average is considered pretty good data variability).
[Added: The bio-luminescence is also set to a mininum of 10E6 AU, which is important for the rest of the analysis.]

We can see that the luciferase activity at the injection site (which we can refer as our reference tissue) is significantly high within hours of injection (2 hours being the first reported timepoint) and decreases over time. [Added: What is important to note is how does the %ID actually compares to the bioluminescence. The common sense would be the more of the lipids are biodistributing in the tissue, the more mRNA (and therefore luciferease activity) we should detect, no? Well it is more complicated than this. Let’s plot the %ID in the tissue versus the bio-luminescence.

As you can see, an increase of lipid tracer in the tissue does not correlate with an increase in mRNA activity (as seen by Luc activity). It can be meaning two different things:
* The accumulation of the radiotracer present in the LNPs does accumulate in the tissue because of its non-metabolization and therefore may overestimate the half-life of the LNPs.
* Lets assume the LNPs found a way in the tissues, does not mean they made it safely with their cargo. They may accumulate as residues, or may come as empty shells with little or no mRNA left.]


We can assume that the luciferase expression at the injection site last for up to 10 days before being no different of background noise (we also have to be careful to not extrapolate as-is for the spike S protein, as the mRNA and protein kinetics of luciferase enzyme may greatly differ from the recombinant spike protein). However, the risk of off-target effect and having the mRNA expressed outside the injection site seems to be quite dim. Luciferase activity in the liver (which apparently uptake 60% of the injected dose) is down to background level by 48 hours post-injection. [Added: If we look at the profile, we can guess there is some metabolism in the liver that makes the clearance of LNPs and/or mRNA faster than the muscle tissue. From the data of the muscle bio-luminescence, we can see the decay of the bio-luminescence follows a first-order kinetics and puts with a half-life of ~0.75 days].
Ovaries luciferase activity was basically in the range of the saline group (and would be barely detected over noise, if we refer to the expected min. The penetration of the dye emission wavelength should be enough to be caught by the camera, even through solid tissue. If we don’t see any luminescence, it is likely because it is below or same intensity than background in saline) and brain luciferase activity in the brain was basically noise from the beginning to start (remember we have no access on the standard deviation but the numbers being that close from saline suggest we are scrapping background noise).
In conclusion, the risk of having the mRNA expression outside the injection is very unlikely and meaningless when it comes to biological activity.

5. The perils of dismissing the dose and the allometric scale in assessing the risk
So, we have evidence that the LNPs are pretty safe by barely accumulating in gonads and in the brain, that the mRNA activity is mostly not being found to have off-target, but what about the dose and how does it correlate to clinical situation?
This is where important concept of doses and allometric scale have to be introduced.
First, the dose used for the PK study. It was 1mg/kg of mRNA given in rats. As a comparison, the regular dose of the Pfizer vaccine is 30 ug (0.03mg) given to any patient of 12 years and older.  An average 12-years old girl would be 40 kgs per the CDC chart (rounded up to the lower value and for the ease of calculation). This would indicate a dose of 0.00075mg/kg. That’s already a difference of 1333-fold between what we gave to these rats and what we gave to humans, but there is more!
We also have to account to the allometric factor, because rats are not small human. [EDIT: For adjusting to the allometric scale, we will use this calculator ]. The allometric scale tells us that 1mg/kg dose in rats results in a human-equivalent dose (HED) of 68mg/kg if your patient is a 70-kgs adult; 45mg/kg if you are a 40-kgs teenager (~12 year old girl falling in the 50th percentile of the CDC growth chart).

Therefore, we have to multiply it by 45 (40-kgs patient) or 68 (70-kgs), which means if we want to transpose the PK findings as done in the rats, we would need to inject about 60’000 doses of the Pfizer vaccine in ONE girl (91’000 doses if you are a 70-kgs adult). That’s about half one-fourth of all doses distributed to Amarillo until now given to only ONE person [EDIT: One 12-year old teenage girl that is in the 50th percentile], ALL AT ONCE! You see where we going? The very extreme implausibility of the claims that COVID19 vaccines affect ovaries and the brain.
To finish it up, we can also look at the actual mRNA and luciferase.
We know that 8microg/kg was sufficient to see some liver activity, but no activity in gonads and brain. How does it translate to humans? First, lets apply the allometric scale (68x). We would need 544microg/kg for the HED, and translated to a 12-years old girl that would be 21760microg of mRNA delivered, which is about 725 doses of Pfizer given in ONE person at once! You can see that since we cannot detect notable activity if I give 725 doses at once, chance are I will not detect any activity when given a single dose or even two doses of Pfizer.

6. Concluding remarks

In conclusion, we can take the following messages:
– This is a document leaked on the PK of nanoparticles as found in the Pfizer vaccine, showing animal studies have been done before or during the clinical trials and we have the documentation.
– It helps clarify an ambiguous statement made by Pfizer in their summary submitted to the European Medicine Agency a couple of weeks ago about the distribution of the mRNA vaccine.
– The studies were done in a very conservative fashion at doses exceptionally high and impossible to reach in humans
– At such doses, it was shown that aside from the liver and spleen, the distribution of LNPs was minimal in gonads and the brain.
– The amount of mRNA required to be present in the tissue to appreciate an off-target effect is ridiculously low and impossible to achieve in real life and was transient in the liver.
– When accounting for the clinical dose and the allometric scale, this study shows that the Pfizer vaccine is very safe with a very low incidence of the off-target effect. To achieve the same result in humans, it would take a ridiculously high amount and a sheer incompetent healthcare practice to have the probability of having any issues of off-target effect occur in humans.

Categories
Pharmacology Sciences

[Sciences/Pharmacology] A tale of death by licorice poisoning

I guess you have heard in the last couple of days about this poor man death from a severe cardiac condition triggered by a severe case of hypokalemia (low potassium levels) as reported by the APNews here. The culprit in this death? Overconsumption of black licorice (one pack a day for few weeks).

So, how can it be possible? Well, to understand how licorice can be dangerous, and how this case is another validation of Paracelsus axiom “the dose makes the substance poison”, we have to go back into some biology and chemistry, all wrapped up in what we call “Pharmacology” and especially a sub-genre of it we call “Toxicology”.

Licorice (Glycyrrhiza galabra, remember that name for later) is an herb commonly found in Europe and West Asia, with edible roots. It can be consumed as-is by chewing and sucking the dry roots (I used to buy them from my pharmacist as a treat) or primarily used for extracting licorice, a black and bittersweet substance.

Like any plants, Glycyrrhiza produce various phytochemicals including chemicals falling into what we call “secondary metabolism”. One of them, glycyrrhizin, is the major chemical sought from these plant, it gives this bittersweet taste that some enjoy and some get repelled (Gosh, I hate processed licorice candies and I would throw my Haribo mixed bags once I ate everything but the licorice ones). Now comes the fun: Here is the structure of licorice:

If you are a chemistry nerd, you will note the two glucuronic acid on the left, but you will find more interest into the polycyclic saturated chain that looks a lot like cholesterol (see below)….

…..or similar to digoxin (a cardiac glycoside that is a potent poison extracted from foxgloves, but also a potent cardiotonic we give to patient suffering from heart failure).

But what is even more interesting is that glycyrrhizin share a lot of structural similarities with steroid hormones including aldosterone (mineralocorticoids, left) and cortisol (glucocorticoids, right):

Glycyrrhizin (or glycyrrhizic acid) is poorly present in the blood and urine, usually found at less than 2% of the injected dose. In the other hand, glycyrrhetic acid (GA), the degradation byproduct is considered the major form that is absorbed and distribute into the body. GA is mostly eliminated via liver metabolism (GA-3-glucuronide) but interestingly can get salvaged by the gut microbiota back into GA and re-enter the body as GA, hence resulting in a pretty long elimination half-life (between 6-10 hours, which would mean it would take 1 to 2 days to clear out a single dose of licorice).

You can appreciate that we are getting closer when it comes to chemical structure to aldosterone and cortisol. Here comes the interesting part. Cortisol can bind to its cognate receptor (glucocorticoid receptor), but also bind to other steroid receptors like the mineralocorticoid receptors (MR). MR target genes are various, but several of them are encoding for sodium (Na+) channels which will work on kidney epithelial cells to induce reabsorption of sodium in the nephrons. This is turn will change the dynamics of electrolytes, as the reabsorption of sodium (Na+) will result in an increased elimination of potassium (K+) by renal excretion. In turn, we will end up in a hypernatremia/hypokalemia situation which will manifest in any excitable cells, in particular in the heart tissue. Both Na+ and K+ play an important role in the heart electrical activity. Mess around with the extracellular concentration of one of these two and you are setting yourself into serious cardiac issues (arrhythmia, fibrillation, conduction block, impaired or asynchronic muscle contractions….weird EKG patterns ahead).

It would be tempting to assume GA would compete with cortisol, or mimic it for binding to the MR. Turns out, GA does not really fit to MR, but fits quite well into the catalytic site of an enzyme called 11-beta-hydroxysteroid dehydrogenase (11-bHSD). This enzyme will convert the hydroxyl group present in the carbon 11 position (see that OH group pointing on the left in the cortisol and aldosterone molecule?) into a keto group (=O). This is enough to kill the ability of cortisol (which now became cortisone) MR activity. What basically happens is that GA will compete with cortisol for 11-bHSD binding, has better affinity for the enzyme and block the transformation of cortisol into cortisone. Result? You create a buildup of cortisol, which means you have an increased activation of MR, increased expression of its target genes, increased Na+ channels and transporters in the kidneys that will increase its resorption during the renal filtration process…..and the resulting hypernatremia and hypokalemia.

It is very unlikely you will an issue in an acute exposure (the FDA recommends to people over 40 to not eat licorice for more than two weeks, keep it below 2 ounces) but likely to occur if ingested chronically. Plus having an history of cardiac events makes you worse.

So please remember the axiom of Paracelsus: “The dose makes the poison”. Limit your licorice as a once-in-a-week treat, limit your intake and avoid it if you have heart issues.

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Junk Sciences Junk Sciences Sciences Uncategorized

[Sciences/Junk Sciences] Anti-influenza activity of elderberry (Sambucus nigra)

First blog post of 2020, after taking a holiday break to reinvigorate and prepare myself for the second half of the academic semester, a semester with a lot of items on the menu.
It is also, for those living in the Northern Hemisphere, time for the flu season. Unfortunately this year, it is not looking good and seems to be a very bad year for Winter 2019-2020 as we have already an early spike in the number of cases about flu and already a certain number of fatalities.
For some reasons, the flu shot drag a very bad reputation amongst the population. We blame it gives you the flu (which is impossible since you are injecting a dead virus into your deltoid muscle, not even in your nasal cavity) and some prefer downplaying the danger of influenza (“It is just a cold!”) or claiming you can prevent it by using some grandma remedies such as elderberry syrup. If you wander around some “wellness” websites, you will see these website touting about the benefits of elderberry syrup, claiming it as a “cure-it-all” remedy and even will give you how to make your own.
To sugarcoat it with a layer of “scientific literacy”, they will cite studies showing its antiviral properties and therefore giving it credentials to other treatments. Back off Tamiflu(R), we have a serious challenger right here, and it is natural!
One study that I have seen becoming viral is this one:
Anti-influenza activity of elderberry (Sambucus nigra)” by Dehghani and colleagues, published last year in Journal of Functional Foods, a journal that aims on publishing any studies on nutraceuticals (studies showing the biological activity of food and food-derived products). It is a journal published by Elsevier with an okay impact factor (3.19 as of 2019).

About the conflict of interest:
The first thing I am checking when reading papers is the affiliation of the authors and the possible conflict of interests in the funding of the study. The authors are all affiliated with the University of Sydney (NSW, Australia) with the exception of Qayyum Adil that has an affiliation with PharmaCare Laboratories. This is the first item of interest. If you lookup PharmaCare Laboratories, you will find out that one of their product is Sambucol(R) (elderberry extract). Sambucol(R) is sold at any grocery chains like Wal-Mart as a dietary supplement (which means it was not approved by the FDA to diagnose, treat or prevent any illnesses) and natural remedy to “stay healthy through the year”. It is sold as syrup, or tablets, or gels with some packages claiming “homeopathic cold and flu relief”. There is also the disclosure of financial support by PharmaCare to the study, which accounts for a conflict of interest. A conflict of interest is not inherently bad and evil. But it is important to disclose it, as the authors may have a possible bias to present their study in a too much favorable and positive way that is supported by the data.

About the study:
This study aims to look at the antiviral property of elderberry extract (here mentioned as juice) as a potential antiviral against influenza virus (in this case H1N1 strain, and only the H1N1).  To address this activity, the authors have solely conducted an in vitro (aka in a Petri dish) study using two cell lines to assess the antiviral activity (A549 cell line which derives from a lung cancer) and MDCK (Mavin-Darby Canine Kidney cells, a cell line derived from a dog kidney). It is important to note that the A549 is only good at assessing adhesion and internationalization of the influenza virus (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692684/), but is not a great at allowing the virus to spread. In the other hand, MDCK is one of the cell line commonly used to grow the influenza virus for industrial production, as these cells are perfectly suited for large-scale culture in bio-reactors, and provide source of viral material including for vaccines production.

Like any plant-derived products, elderberry extract is reach in plant chemical products originating from the plant primary metabolism and secondary metabolism. Amongst them, polyphenols represent a major class of phytochemical found in elderberry. Polyphenols share the same chemical structure revolving around a benzopyrane structure (see below).
1280px-4H-Chromen.svg

Elderberry extract is indeed rich of a certain class of polyphenols called “anthocyanins”.
Examples-of-anthocyanins-found-in-different-foods.ppm

These anthocyanin share a common structure, with the presence of an “oxonium” (positively charged oxygen atom) in their structures. They differ from each other by the nature of the radical groups surrounding the aromatic rings (hydrogen, hydroxyl, or methyl groups) and being present either as their aglycone (no sugar moiety) or glycoside form (with a sugar moiety, quite often a glucose).
As I have mentioned, this “oxonium” is very unstable yet very useful. It gives these polyphenols a pigmented color usually in the blue-purple range, which gives the colorful blue-purple of red grades, roses and berries. The maintenance of the structure is indeed pH dependent and remains stable only under acidic condition (pH<5). At higher pH, these compounds quickly disintegrate into a chalcone.
Structure-change-of-anthocyanin-with-pH-in-aqueous-solution_W640

I know this to happen personally very fast, as I have been working on measuring levels of delphinidin in cell culture medium during my Master’s degree……and failed. Upon aqueous solution at physiological pH (7.4), it degraded into a chalcone, making it near impossible to detect on my HPLC-UV instrument. Without an internal standard, I could establish a validation method for its analysis. I lost half of my Master thesis on that issue.

About the method:
For the study, the authors extracted 200g of elderberries and obtained a juice at undocumented concentration, which they diluted via serial dilution for the study. A quick lookup of some data in Figure 1 allows us to estimate that 1:5 of elderberry dilution maybe representing 100mg of anthocyanin/100ml juice.
That would conclude that the stock (1X) solution would contain 500mg anthocyanin/100mL juice or a concentration of 5000mg/L (5g/L) of anthocyanins.
A lookup on the Sambucol(R) datasheet suggests that 10mL of serving (2 teaspoons) contains 3.8g of elderberry extract (assuming that his extract is 100% anthocyanins). This would correspond to about 0.38g/mL or 380g/L solution (which is roughly the same of a 76X concentrated juice). In parallel, the authors also used cyanidin-3-glucoside (C3G) as a standard. The authors used two cell lines (A546 and MDCK), measured cell viability via WST-1 assay, assayed the antiviral activity by measuring the inhibition of hemagglutinin (the H in H1N1) using red blood cells, H1N1 infectious level by measure plaque formation assay (which denotes cell death from viral infection, leaving the area empty), measurement of H1N1 infection in MDCK cells by flow cytometry (and using a FITC-labelled anti-H1N1) and measurement of cytokines using a cytometric bead array (CBA). This last experiment is a bit weird as usually an ELISA (direct measurement of cytokines release) being a more accurate choice.

About the results:
The first result is the presentation of the cytotoxicity. A common trope that flies over anti-vaccines and alternative medicine is the good old axiom of Paracelsus known as “the dose makes the poison”. Figure 1 is about that, assessing which dilution of elderberry juie makes the poison.1-s2.0-S1756464619300313-gr1

A caveat here is the absence of control (untreated cells) that would have helped set the baseline and determine which amount is toxic. If we take the highest dilution as control,  we can guess which dose is toxic. Usually if you achieve over 30% decrease, you can expect the difference is statistically meaningful. If we look at the values extrGapolated from the absorbance and normalized to the 1/20th dilution,  we have about 75% and 50% viability at 1/15th and 1/10th dilution for MDCK; 80% and 50% viability for A549 respectively. Similar outcome for panel b. Interestingly, is the absence of statistical significance (as reported as “*” which is indicative of a P-value lesser than 0.05), which is bizarre that no reviewers asked for.  One thing is certain, by 1:5th dilution, we have 0% viability. Both cell lines are dead. The dose makes the poison. It can be due to the extreme acidity (elderberry juice is pH 4.4, 1000x more acidic than the physiological pH).
If we look at the dilution (1/5th) and the amount of anthocyanin (~100mg/100mL or 1000mg/L juice or 1g/L juice), we can estimate that the average concentration of this elderberry juice is about 5g/L. I would assume dilution of 1/15th and higher can be considered not toxic.
Then comes trouble. And trouble came with the form of the IC50. In pharmacology, the IC50 is the concentration of a drug by which you obtain 50% of inhibition. From the IC50, you can deduct that you should achieve a very good inhibition at 10x to 100x this value.
In pharmacology this concentration has to remain under a certain level, usually below 1micromoles/L to avoid off-target effects (lack of selectivity, which is the basis behind drug side effects). Usually, we cap the maximum concentration to 100micromoles/L. If you cannot achieve a significant inhibition, you can toss your drug candidate in the trash or bring it back to the bench and let medicinal chemists tinker it.
The authors give us two values: A for during and after infection, B for during infection only. The average values provided are 6mg/100mL and 17mg/100mL.

Screen Shot 2020-01-09 at 2.20.43 PM

These equate to 60mg/L and 170mg/L respectively. If we assume that all this elderberry juice is 100% made of cyanidin-3-glucoside (C3G, which is the compound they consider bioactive) and harbors a molecular weight ~450g/mol, we can estimate IC50 values of 0.133-0.377mmoles/L or 133-377micromoles/L. We are talking here IC50 values, which means we have to factor in we will likely need 10x these concentrations to have an antiviral activity. These put us about 1.33-3.77mmoles/L. This assuming we have a concentration in tissues equal to the concentration in plasma (blood). Assuming a blood volume of about 5L in humans, we are talking about 6.65-18.8mmoles to enter the body.
The oral bioavailability of anthocyanins is indeed very small and reported to occur between 0.26-1.8% (based on comparative PK to IV administration) according to this review. If we assume a 2% bioavailability, this puts us to the consumption of at least 332.5-940 mmoles of anthocyanins. Assuming we are talking about C3G, that would represent a mininum intake of  150’000mg or 150g of C3G. If we had to put into perspective, that would be about 400mL of Sambucol to swallow, or 40 teaspoons or about 3 bottles at 4oz each. This would require to repeat it 4 times a day, which brings us to 1.6L of Sambucol(R) swallowed by an adult every day to achieve the same results than obtained in a Petri dish. With a price tag of $12.99/0.12L ($108.25/L) on Sambucol(R) website, it would cost you almost $200/day/person to have a chance in reducing your flu symptoms (if it works). Assuming a week-long treatment? About $1400 per week per person!
And BigPharma is here for their money, one $15 to get a flu shot you get once per season! The maths is here.
Now let’s reverse it. The company tells you to take 10mL four times a day. Thats 3.8g per serving. Out of it, 2% is bioavailable and ends in the circulation. Thats 0.076g circulating in your blood, with about 0.0152g/L(15.2mg/L) of average plasma concentration. Assuming it is 100% C3G, thats a plasma concentration 0.0337mmoles/L or 33.7 micromoles/L.  Or 25% of the IC50 values. In other words, you cannot achieve any antiviral effects with the dose recommended by the company. In both ways you lose.
A) The posology written on the insert for Sambucol(R) is too low to be efficient
B) To achieve the efficacy as reported in a Petri dish, you will have to drink gallons of it, with a price tag exceeding far more the cost of a flu shot.

Should we continue? Sure let’s continue. In the later stage of the study, the author use C3G as a comparator as they mention it being “the primary anthocyanin in elderberry,showed a strong anti-influenza effect with the IC50“. The IC50 reported is 0.069mg/mL or 69mg/L (or 153micromoles/L).  What is bizzare is the nature of the contradictory of the data shown.
One of them is the flow data hidden in the supplementary figures (which you have to download separately). First, the design of this figure is very convoluted and needed to rearrange the panels to become more readable. Putting aside the concern that the number of events are not equal between the different histograms (the authors should have presented as % max for the y-axis), you can see something that is not looking good for the authors.
If we compare between the two infected groups, we can see without hard numbers that elderberry syrup (at 1/20th dilution) was not changing the outcome of infected population. In the opposite, treatment with anti-H1N1 antibody completely blocked the infection. Guess what makes you produce anti-H1N1 antibodies? A $15 flu shot!

Slide1
What is even more bizarre is the cytokine expression profile. I personally not much confident in this method, and I would have much more preferred a good old ELISA, which gives you absolute values. But lets go through the figure 6.1-s2.0-S1756464619300313-gr6

Cytokines are important molecules that serve as communication methods for immune cells. Some are pro-inflammatory, some are anti-inflammatory. In this case, the cytokines measured are most pro-inflammatory (IL-1beta, IL-6, IL-8, TNF), with IL-10 being anti-inflammatory and IL-12p70 being involved in T cells differentiation into Th1 cells.
Under normal conditions, cells do not produce pro-inflammatory cytokines.
LPS is the acronym for lipopolysaccharide, a biomolecule present in Gram-negative bacteria. LPS is considered as the most potent bacterial stimulator of immune response during sepsis, and commonly refered as endotoxin which can induce a septic shock.
We can see that LPS alone induce the production of IL-6 by 50 fold and in lesser extent IL-8 (15-20 fold).
Interestingly, elderberry juice (1/15th dilution) triggered the expression of TNF-alpha by 50-fold and IL-6 by 200-fold. IL-8 by contrast was increased by 30-fold. Here is the problem. If C3G is the main anthocyanin in elderberry juice, why he could not achieve the same profile then elderry juice (which is about 5g/L anthocyanins) at a concentration half of it (C3G was given at 2.5g/L)?
The second issue is the extent of the release of pro-inflammatory molecules. This release can be high enough to cause what we call a “cytokine storm”, the equivalent of the nuclear option for the immune system by inducing a major immune response leading to the death of the patient. Cytokine storm is accepted now as the worst outcome of the flu pathphysiology (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4711683/), with current goals is to save patients with flu by trying to avoid the nuclear option using immunomodulators.
As I said, the last thing you want is to worsen the immune response. With such an increase in pro-inflammatory cytokines, you may indeed worsen the outcomes of flu rather than treating the flu.

Conclusion:
This study albeit interesting suffers from major flaws that unfortunately will not deter woo peddlers. But lets resume it here:
1. It has some skin in the game as one author is affiliated with a company making a living of elderberry syrup as dietary supplement.
2. The amount used to show some effects requires a ridiculous amount to see any effects.
3. Inversingly, the dosing regimen of the Sambucol as written on the package is way below the dose needed to see a semblant of biological effect.
4. The data is overall of poor quality and contradicts each other making the paper pretty unreliable.
5. And if you think you can save money by making your own, think twice. You can poison yourself to death with elderberry juice. I guess flu will have no chance…if you are dead.
https://www.cdc.gov/mmwr/preview/mmwrhtml/00000311.htm

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Junk Sciences Sciences Uncategorized

[Sciences/Junk Sciences] Contre-lettre au billet d’Adrien Senecat « Les évidences relatives de la tribune de No Fake Science sur l’information scientifique” (Le Monde – 07/26/2019)

Avertissement :L’article suivant est un article d’opinion servant à une contre-réponse à un article publie en tant que “article d’opinion” par un journaliste du quotidien “Le Monde”. Je suis scientifique français immigre aux États-Unis, enseignant-chercheur en pharmacologie et neurosciences. Pardonnez d’avance certaines coquilles et l’usage abusif de termes anglo-saxons en lieu et place de termes francises.

Il est rare que je prenne ma plume pour écrire un article sur mon blog en Français, ceci pour plusieurs raisons. Le fait de vivre aux États-Unis depuis 10 ans, d’avoir un contenu principalement international et puis surtout discuter d’un contenu scientifique. Cependant, un article (ou plutôt un billet d’opinion) signe par Adrien Senecat  publie dans le quotidien « Le Monde » (https://www.lemonde.fr/les-decodeurs/article/2019/07/26/les-evidences-relatives-de-la-tribune-de-no-fake-science-sur-l-information-scientifique_5493749_4355770.html) et partage sur le réseau social a servi d’une discussion vive entre moi et un ancien camarade de fac (pour la petite histoire, on a été ensemble sur les bancs de la Fac de Médecine il y a 20 ans. Il a réussi le concours de P1 et devenu médecin, j’ai raté mon concours de P1 et je suis devenu enseignant-chercheur aux États-Unis. Comme quoi il y a une vie après la P1).
Cette pièce d’opinion m’a surpris, et en même temps m’a donné du grain à moudre durant mon weekend. Pourquoi je reste sceptique et même absolument pas impressionne par cet article ? Je suis sceptique par les arguments avancés par l’auteur, par les qualifications du journaliste et en même temps exprimer ma lassitude de voir les sciences maltraites et caricatures par le journaliste de base. Venant d’un quotidien comme le Monde, je m’attends à une qualité journalistique digne d’un journalisme d’investigation, non de titres racoleurs et d’un nivèlement journalistique par le bas.

Mais allons au cœur du sujet et vidons le cahier de doléances envers cette pièce.

1. L’auteur en question :

Qui est l’auteur de cet article d’opinion e et quels sont les mérites qui lui donnent une qualification pour discuter d’un sujet pointu qu’est la science ?
Adrien Senecat (selon son profile LinkedIn) a un diplôme de journalisme de l’EFAP Lyon. Après un rapide passade dans l’hebdomadaire “Le Pays Roannais” et sur la radio “RCF Lyon Fourvière”, il s’est spécialisé dans le journalise “high-tech/web” pour L’Express d’Octobre 2011 à Avril 2015. Il quitta l’express pour Buzzfeed pour une période d’un an, avant d’obtenir son poste de journaliste d’Avril 2016 jusqu’à maintenant dans l’équipe “Les Décodeurs”, avec comme intitule “factchecking”. En conclusion, on peut supposer que l’éducation scientifique se limite à l’enseignement du lycée. Considérant que les personnes entrant une carrière de journalisme le font majoritairement à partir de filières générales débouchant vers un Bac L ou ES, on peut vraisemblablement considère que son éducation scientifique est au mieux anémique et absolument pas préparé pour ce sujet et encore moins donner le niveau requis pour un « factchecking » scientifique.

Je précise ici que c’est mon premier article lu écrit par Mr. Senecat. Donc, je suis à ce niveau « blind » et seulement lu avec mon propre niveau. Je ne peux juger de sa qualité sur ces autres « factchecking » et de ce fait ma contre-lettre s’applique qu’à ce billet écrit pour le quotidien.

2. Qu’est-ce la méthode scientifique et pourquoi elle est importante quand on parle de « Fake Science » ?

La méthode scientifique est à base de toute les sciences dure moderne et s’appuie sur l’expérimentation scientifique de Claude Bernard, un physiologiste Français du 19emesiècle. La pierre angulaire de la méthode scientifique est le scepticisme.
Toute nouvelle étude est passe au peigne fin, pour s’assurer que les résultats sont de hauteur a la rigueur scientifique. En science, les découvertes scientifiques les plus robustes se font dans l’intimité d’un journal de peer-reviewde haut vol tel que “Science” ou “Nature” et présente dans de grandes conférences scientifiques en tant que “keynote speakers”. J’ai l’habitude de comparer le monde de la recherche scientifique avec le monde de la musique métal. On a nos propres “Rockstar”, on a notre propre “Hellfest”, on a le même défi de vivre de notre travail par un système de mécénat (les demandes de financement de recherche restent assez proche de soumission d’une maquette d’album a un producteur de musique). Pour réussir dans le métier, il faut exceller dans la qualité du travail et dans l’innovation scientifique. Mais elle se fait généralement discrète, présenté rarement dans les médias conventionnels. Les scientifiques aiment rester des personnes discrète, détaché du « spotlight » des plateaux télés. C’est un peu comme le slogan d’une marque de frite surgelés, ceux qui paradent le moins dans les écrans télés en font le plus de découvertes scientifiques. Malheureusement, les scientifiques ont négligé d’adresser le public de leurs découvertes, laissant la porte ouverte à la « Fake Science » qui compense son incapacité scientifique par une esbroufe devant les plateaux télé et radio.
En science on a une deux issues possibles à une hypothèse : ou bien elle est validée par les résultats expérimentaux (reproduits par d’autres laboratoires et vérifies par des résultats convergents obtenues par d’autres approches) ou bien elle n’est pas. Quand la masse et la qualité des résultats et d’études atteint un niveau critique et que la réfutation de ces données devient difficile, on atteint un consensus. Un consensus n’est pas inscrit dans la pierre et s’adaptera en fonction de nouvelles informations et découvertes.
L’article en question publie par le collectif “No Fake Science” dans le quotidien « La Tribune » (https://www.lopinion.fr/edition/politique/science-ne-saurait-avoir-parti-pris-l-appel-250-scientifiques-aux-192812) et signée par plus de 250 signataires (médecins, scientifiques, pharmaciens, ingénieurs….) met en alerte sur la progression rampante de la « Fake Science » dans la sphère publique.
A titre personnel, l’utilisation du terme « Fake Science » est maladroit. Le mot « Fake » est utilisé en anglais Nord-Américain pour désigner un faux, une pâle copie, une escroquerie, une tromperie. Ce terme a une importance légale, car on peut avoir une étude complètement bâclée (par exemple, l’étude rétractée faite sur des rats nourris au maïs OGM. Cependant, le laboratoire a gagné un procès en diffamation car l’accusation a été faite que les résultats présentes dans l’étude rétracté étaient « Fake ». En réalité, les résultats aussi mauvais et bâclés (ayant de ce fait aucune valeur scientifique) étaient bien existants, avec une preuve physique de leurs existence (cahier de laboratoires sous forme physique ou électronique).
A moins qu’il y ait démonstration qu’une étude a été montée de toute poil, je préfère l’utilisation de « Junk Science » (science poubelle) quand je m’adresse au sujet de l’antiscience.

L’antiscience est la face oppose de la méthode scientifique. Que l’on parle des anti-vaccins, des médecines alternatives (homéopathie, acupuncture, reiki, cristaux…), des créationnistes, des négationnistes du changement climatique anthropocène, des anti-nucléaires ou des anti-OGMs, on retrouve souvent le même fil rouge qui font que leur approche est biaisée et leurs évidences de faible qualité scientifique (en particulier, un fil rouge que je vois quasiment tout le temps dans n’importe quel étude anti-vaccin) :

  1. On part sur une conclusion prédéfinie, et l’on réalise les expériences qui confirment le résultat.
  2. Généralement, les résultats obtenus s’alignent rarement avec la conclusion initiale donc on va éliminer l’utilisation de groupe contrôles, on va exagérer les doses administrer en utilisant des quantités faramineuses, ou bien un compose qui n’est communément utilise ou bien une approche exotique utilisant uniquement une seule technique.
  3. Si on n’obtient toujours pas le résultat voulu, on a sélectionné le résultat qui s’aligne à note conclusion et ignorer les autres communément appelé « cherry picking » (« cueillette des cerises ») ou bien couper les coins de la rigueur statistique par l’utilisation du « p-hacking » pour trouver une signifiance statistique là où il n’a point.
  4. Publier le tout dans un journal de basse qualité (car aucun journal de qualité accepte un torchon sans passer par un « peer-review » rigoureux), au pire un journal « Open-Access » a comportement prédateur (dans lequel le journal acceptera de publier n’importe quel torchon moyennant la somme coquette de $2000-3000 en frais de publications).
  5. Présenter ce torchon comme la preuve ultime d’une contre-étude fiable questionnant le consensus dans les « echo-chambers » sur les réseaux sociaux et par certains journalistes à l’éthique journalistique discutable sinon malhonnête. Jouer les cartes du martyr et du « whistleblower » (lanceur d’alerte) sur les plateaux télés, dénonçant une cabale et une censure qui a pour but de cacher la « vérité® » au public. Le but est de semer le doute dans l’esprit du public.
  6. Accuser les détracteurs et critiques comme « shills » (agent paye par un groupe d’intérêt), tout en cachant les conflits d’intérêts financier dont vous êtes vous-même coupables (par exemple, plusieurs scientifiques anti-vaccins siègent dans le directoire de fondations ayant un agenda anti-vaccins et bénéficie d’une manne financière de ces mêmes organisations à travers le financement de leurs propres recherches).

Une antiscience a aucune chance de soutenir ses thèses erronées devant ses pairs lors d’un congres scientifique international. Leur seule chance pour disséminer leur « Junk science » se fait par le biais de la « fausse-équivalence » et de trouver un journaliste assez naïf (comme est le cas avec cet article de Mr. Senecat) pour se dire qu’il y a deux camps dans chaque débat, y compris scientifique et que chaque camp a le droit à la parole sans peser le poids des « facts » de chaque camp. Ce que fait notre auteur avec ce paragraphe d’introduction :

« Le débat public autour de ces thèmes ne saurait être considéré comme “scientifiquement clos”, reconnaissent les auteurs. Pour autant, les points précis retenus en exemple sont consensuels parmi les spécialistes et doivent être présentés comme tels », assurent-ils. A y regarder de plus près, ces six « consensus scientifiques » n’en sont pourtant pas tous. Revue de détail.»

3. Les vaccins :

Dans sa globalité, la section est correcte de manière scientifique :

« Il est donc tout à fait juste de parler de consensus scientifique sur ce point. On peut cependant noter qu’un tel consensus n’éteint pas automatiquement tout questionnement sur la politique de vaccination. Ce n’était certes pas le propos de la tribune de No Fake Science, mais des questions demeurent ouvertes sur l’âge auquel vacciner, le nombre d’injections à pratiquer, la composition des produits utilisés. ».

L’auteur questionne, a juste titre la politique de vaccination. Mais l’auteur oublie de préciser que la politique de vaccination, bien que se basant sur la même littérature scientifique, reste à la discrétion de chaque gouvernement base sur son propre corpus scientifique et de la géographie. C’est un point récurrent que je vois utilise de manière sotte par les anti-vaccins anglophones qui considère que la politique vaccinale du CDC (Center of Diseases Control, organisme fédéral de sante publique) est en place non seulement aux États-Unis, mais aussi au Canada, au Royaume-Uni, en Australie ou en Nouvelle-Zélande !

Premier carton jaune a l’auteur pour le zeste de doute sur les vaccins, en pleine explosion d’épidémie de rougeole (les États-Unis ont déjà dépassé le record de cas de rougeole cette année compare à la plus grande crise d’épidémie depuis son éradication du territoire depuis 2000 (précédent l’effet vague de l’étude Wakefield).

4. L’homéopathie :

L’homéopathie, ou ce que j’appelle la poudre de perlimpinpin qui se base sur un principe développé par Samuel Hahnemann il y a 200 ans.

Ses deux principes violent les lois de la biologie et de la chimie :
* Que l’on puisse soigner “un mal par un mal” sans aucune évidence de causalité (par quelle mécanismes biologique un extrait de foie/cœur de canard ait capable de soigner un état grippal ? Aucune réponse).
* Comment expliquer que les produits homéopathiques puissent expliquer une activité pharmacologique malgré une dilution ridicule qu’il est statistiquement impossible de détecter une molécule de substance active dans une préparation diluée pour usage thérapeutique ?
* Comment expliquer “la mémoire de l’eau”, un argument souvent utilise par les homéopathes pour réfuter argument #2 ? Ça va faire plus de 200 ans que Lavoisier a établi les bases de la chimie modern et 400 ans que Paracelse a défini les bases de la pharmacologie. 200 ans et toujours aucune évidence des principes de Hahnemann démontré par la science moderne.
Le problème est que bien que ce sont des préparations homéopathiques, une etape-cle reste la préparation de concentre communément appelée “teinture mère” (Tinctura mater). Cette préparation (souvent hydro-alcoolique) reste un concentre de composes extrait de plantes avec une activité pharmacologique documentée. Une erreur de dosage peut avoir un risque important de surdosage qui peut être mortelle. Ce fut le cas avec un extrait d’Atropa belladonnautilise comme remède “Hyland teething tablets” antidouleurs pour les éruptions dentaires chez le nourrisson et le petit enfant. Le principe actif est l’atropine, un puissant antagoniste des récepteurs muscarinique de l’acétylcholine. A fortes doses, ce compose peut entrainer la mort du patient. Ce produit a été reitre par le FDA (https://www.fda.gov/news-events/press-announcements/fda-warns-against-use-homeopathic-teething-tablets-and-gels) après le rapport de cas fatal (on estime 10 décès lies a l’utilisation du produit. Pour rappel, tout supplément sur le marché US n’est pas régulé par le FDA, le FDA enquête qu’après cas d’effets secondaires sérieux ou fatal reporte par les médecins traitants).

« Là aussi, cependant, le fait qu’un consensus scientifique existe ne veut pas dire qu’une seule politique publique est possible. »

Si l’on réalise une expérience 100 fois pour valider une hypothèse et que l’on 100% un taux d’échec pour cette hypothèse, il est fort probable que cette hypothèse est invalide et se doit d’être abandonne. Les antis sont généralement têtus et pense que la 101eme expérience confirmera ce que 100 expériences ont échoué à vérifier. Avec l’homéopathie se posent deux problèmes, éthique et financier :
* Est-il éthique pour un médecin de prescrire un remède inerte juste pour satisfaire un effet placebo chez le patient sachant que le remède inerte a une probabilité quasi-nulle de traiter la condition du patient ?
* Dans un climat ou les dépenses de sante augmentent, est-il raisonnable to dévier des fonds dans une intervention thérapeutique qui a pratiquement zéro effet thérapeutique pour un produit qui est couteux ? On crie beaucoup à la chasse au gaspillage, le déremboursement des produits homéopathiques est un moyen de recentrer les dépenses vers des approches supporte par les faits scientifiques.
Un deuxième carton jaune pour l’auteur et l’on peut sentir le refrain “Je ne suis pas anti-X, mais…”, une autre tactique que je vois utilise par les trolls anti-vaccins quand je les débats sur les réseaux sociaux.

5. Le réchauffement climatique :

Là, je suis en alignement avec l’auteur. Le changement climatique est réel, que le réchauffement climatique est anthropogène (due par l’activité humaine) qui a contribué l’augmentation du CO2dans l’atmosphère, un gaz à effet de serre connue depuis au moins 100 ans. Les modèles mathématiques développés il y a 20-30 ans se sont révélés assez proches des valeurs expérimentales mesures sur le terrain. Que cela plaise ou non au gouvernement US, on a un effet sérieux dont on sent les premiers signes alarmant. En tant que climatologue et communicateur scientifique, je recommande de suivre les travaux de Michael Mann (https://www.michaelmann.net) et Katharine Hayhoe (http://katharinehayhoe.com/wp2016/) qui sont tous deux climatologues et excellent communicateur scientifique.

6. Le glyphosate :

Le glyphosate. Un sujet très a cœur des Français jusqu’en dans les plus hautes sphères, associe avec Monsanto comme un croquemitaine. Mais là encore beaucoup d’erreurs de jugements, de stéréotypes et d’exagération des faits que l’auteur, bien que habitue au « factchecking » selon ses dires, se laisse badigeonner dans la saumure.
« Inclure le glyphosate dans une liste de sujets qui font l’objet d’un consensus scientifique est discutable. Cet herbicide massivement utilisé dans le monde est, en réalité, au cœur d’une controverse scientifique, où chaque mot a son importance. »
Pour être honnête, la controverse n’existe que dans la tête des des politiciens, des « écologistes-bobos » et d’autres adeptes des délires conspirationnistes que même certains scientifique critique du glyphosate appellent à mettre de cote (donc je ne citerai point que selon Stefanie Seneff, informaticienne du MIT, que le glyphosate serait selon elle responsable des causes du spectre d’autisme chez les enfants, ou bien que le glyphosate change notre microbiome intestinal car une étude publie dans PNAS montre que le microbiome intestinal est fortement changée par la présence de glyphosate à haute dose) (https://www.ncbi.nlm.nih.gov/pubmed/29226121).

« Ici, les auteurs de la tribune mentionnent les « différentes instances chargées d’évaluer le risque ». Et il est vrai que celles-ci jugent « limités » les risques du glyphosate pour la santé humaine. Le problème, c’est que ces agences sanitaires ne sont pas les seules à explorer le sujet. Le Centre international de recherche sur le cancer (CIRC), une agence de l’Organisation mondiale de la santé (OMS), a ainsi classé le glyphosate comme « cancérogène probable » en 2015. Cette décision n’a pas de valeur réglementaire, mais elle est le fruit d’un travail scientifique. Plusieurs études sérieuses ont également pointé de possibles risques pour les agriculteurs qui utilisent des produits à base de glyphosate. »

Disséquons les points ici et commençons par la classification du CIRC du glyphosate 2A. Ce que Senecat a oublié de manière involontaire ou non est de pointer du doigt l’écran de fumée opaque délivré par Chris Portier (président du CIRC) dont ses relations étroites avec les firmes d’avocat qui ont pignon sur rue pour entrainer des poursuites pénales, résumé par Risk-Monger dans son enquête « Portier Papers » https://risk-monger.com/2017/10/13/greed-lies-and-glyphosate-the-portier-papers/

Cette suspicion d’intégrité a été confirme par une investigation journalistique par Kate Kelland de Reuters (ce n’est pas Buzzfeed, hein. On tape quand même dans du très haut de gamme quand on considère la qualité de l’information). Dans cet article, la journaliste a montré que certaines personnes ayant accès au brouillon final du monographie du CIRC a modifié de tel sorte pour faire passer le glyphosate d’être plus cancérigène que les études ont conclu. https://www.reuters.com/investigates/special-report/who-iarc-glyphosate/

Étonnant que notre « factchecking » ait ignore ces articles compromettants dans son analyse, j’assume que c’est un oubli involontaire de notre auteur. Si je peux souligner ce fait a l’auteur, ceci considère ce que l’on appelle un « conflit d’intérêts ». On a une technique assez rode que d’autres scientifiques à une intégrité scientifique douteuse comme Andrew Wakefield on utilise : une firme d’avocats cherche un moyen d’argent facile, trouve un scientifique comme « mercenaire » pour publier une étude qui compromet un produit chimique ou une procédure médicale populaire. Ce scientifique publie une étude suggérant un lien entre une maladie et le produit chimique en question. Avec une telle étude en poche, les firmes d’avocats sont prêtes pour envoyer des procédures de poursuites pénales et en même décrocher un sacre pactole.
Aussi étonnant est le silence de plomb de la position isole du CIRC dans sa décision de classifier le glyphosate en tant que « cancérigène probable »  seule contre plus de 17 organismes de sécurité sanitaire nationales, résumé dans une illustration infographique par « Toughtscapism » (une scientifique environnementale, qui blogue comme moi durant son temps libre) ici (https://thoughtscapism.com/gmos/#jp-carousel-41330).
Je suis curieux de savoir quelles sont ces études qui ont donné l’idée de Portier de classifier le glyphosate en catégorie 2A (pour comparaison, l’alcool est classifie 1, bon à se souvenir lors de la prochaine étude trouvant des traces de glyphosate dans le vin, la bière ou le schnaps).
« Ces éléments font que bon nombre de spécialistes se montrent beaucoup moins catégoriques que les auteurs de la tribune No Fake Science. « C’est un sujet difficile avec pas mal d’incertitudes et il est nécessaire d’approfondir nos connaissances »expliquait ainsi récemment au Monde Robert Barouki, médecin, toxicologue et directeur de recherche à l’Inserm. »
Je salue le langages mesure du Dr. Barouki, co-auteur d’une publication majeure qui démontre l’absence d’effets biologique longue durée du maïs OGM chez les rats (https://www.ncbi.nlm.nih.gov/pubmed/?term=barouki+R+glyphosate). Malheureusement, il semble que ce soit la seule étude auquel il approcha d’une manière indirecte la toxicologie du glyphosate et j’aurais préfère que d’autres chercheurs dont la toxicologie du glyphosate est le pain quotidien (avec une expertise adéquate base sur leur publications) soit également intéressé.

Mr. Senecat questionne la position du collectif par rapport au glyphosate : « Outre la santé des personnes, l’usage massif du glyphosate dans le monde pose également des problèmes environnementaux, qui sont documentés par des études scientifiques. Si bien que, en résumant, le glyphosate à un simple « improbable » risque cancérogène pour l’homme, le collectif No Fake Science semble s’écarter de sa propre recommandation de ne pas « choisir ce qui nous convient et laisser en rayon ce qui contredit nos opinions ».

Le problème du glyphosate est le même que n’importe quel pesticide (qui sont utilisé aussi bien dans l’agriculture classique et Bio, à bon entendeur), celle de déterminer les bénéfices/risques sur le rendement et sur la santé humaine et environnementale. Il faut en particulier comparer aux précédentes générations de pesticides. Depuis 30 ans, le glyphosate a été adopte par son profil faible de toxicité aiguë (de l’ordre de 500mg/kg et plus, on parle plutôt d’une DL50 autour de 5’000-10’000mg/kg), de faible usage (une canette de concentre est suffisante pour l’épandage d’un terrain de football américain. On est très loin de cette fausse idée que les champs sont trempes de glyphosate). Le problème d’écotoxicité bien que moindre compare aux anciennes générations, reste quand même assez longtemps (demi-vie estime de quelques jours à 90 jours, source : http://npic.orst.edu/factsheets/archive/glyphotech.html#ecotox).

Il y a également un risqué de résistance qui s’applique à n’importe quel pesticide et reste qu’une solution temporaire jusqu’à le développent d’une nouvelle génération de pesticides plus efficace et plus sure. Comme chaque chose dans la vie, le 100% efficace ou le 0% risque n’existe pas car c’est un paramètre impossible à achever dans la réalité. Donc quelle alternative on nous laisse ? Trouver des pesticides moins toxiques et plus efficaces dans le futur, mais en même temps le glyphosate reste un pesticide malgré son âge qui a le meilleur ratio bénéfices/risques de l’arsenal contemporain.

Troisième carton jaune pour Mr. Senecat, donc j’appelle cela une expulsion de terrains pour trois fautes journalistiques sérieuses. Si de telles fautes aurait été faite par un journaliste de « l’Écho des Savanes », j’aurais laisse passe. Mais de la part d’un journaliste qui se glousse d’être dans le « factchecking », cela est inacceptable.

7. Les OGMs :

Deuxième hystérie collective de la population Française de base, et un « cash-flow » profitable pour n’importe quel marchand de peur. Je m’y connais, moi aussi était jeune, con et anti-OGM. Les anti-OGM (et comme chaque antiscience) c’est comme une certaine marque de frites surgelé : « Ceux qui en connaissent le moins (biotechnologie) en parlent le plus », étude a l’appui (https://www.nature.com/articles/s41562-018-0520-3).

Pour marquer son scepticisme, l’auteur écrit « Le fait qu’un organisme soit génétiquement modifié (OGM) ne présente pas, en soi, de risque pour la santé. » Ici encore, la formulation retenue par les auteurs est contestable. La référence utilisée (un article de l’OMS sur les questions fréquentes sur les OGM) n’est, en effet, pas aussi catégorique. ».

Mais que dit l’OMS ? En fait pas grand-chose, et renvoie la patate chaude aux autorités nationales « En revanche, la plupart des autorités nationales estiment que les aliments génétiquement modifiés nécessitent des évaluations spécifiques. Des systèmes de circonstance ont été mis sur pied afin d’évaluer avec rigueur les organismes et les aliments génétiquement modifiés du point de vue de la santé humaine et de l’environnement. Les aliments traditionnels ne font généralement pas l’objet d’évaluations similaires. Il existe donc aujourd’hui une différence importante dans le processus d’évaluation qui précède la commercialisation de ces deux groupes d’aliments. »

Cette phrase explique bien le problème qu’on rencontre la science quand il vient aux décisions politique. La science n’a que faire de la politique, malheureusement la politique a souvent un problème avec la science, surtout quand celle-ci déraille des slogans politiques, surtout dans certaines populations électorales. Vaccins, avortement, changement climatique, mesures de sante publique, contraception, orientation sexuelle et identité sexuelle…on a souvent un antagonisme émanant de la classe politique refusant d’écouter ce que la science a de dire.

Malheureusement, par la nature même des autorités de sante, il peut être difficile de recommander une initiative impopulaire en disant que les OGMs sont aucun risque car Madame Michu donne plus de crédit a d‘anciens 68-ards devenu eux-mêmes un membre de la “nomenklatura” qu’a un panel d’experts de l’INRA quand a la question des OGMs. Les OGMs sont devenu un sujet tellement tabou qu’il a fallu la mobilisation de 107 Prix Nobel dénonçant la politique calamiteuse de Greenpeace par rapport aux OGMs (https://www.washingtonpost.com/news/speaking-of-science/wp/2016/06/29/more-than-100-nobel-laureates-take-on-greenpeace-over-gmo-stance/?utm_term=.c154b3d3f8b9).
Nous avons (en temps qu’Homo sapiens sapiens) modifie génétiquement toute notre agriculture et notre élevage depuis le Néolithique. Nous avons joué “au sorcier” maintes fois utilisant les lois de la génétique de manière aléatoire en croisant des variétés et sélectionnes des mutants ayant des traits d’intérêts que ce soit esthétique, nutritionnelle ou de rendement. On a joué ainsi plus de 9800 ans a “l’apprenti-sorcier à l’aveugle” jusqu’au expériences des petits pois de Gregor Mendel dans son cloitre.
Les OGMs reste jusqu’à présent une technique qui a montré son efficacité et sa sécurité quand on parle de temps, d’argent et de traits recherche. N’est-il pas hypocrite que l’on interdise une méthode qui permet d’éditer un génome de manière chirurgical (OGMs y compris CRISPR/Cas9) dans l’industrie Agricole, mais en même temps laisse le champ libre à la formations d’OGMs de manière complètement aléatoire (mutagenèse force) car considère de manière “naturelle” (http://www.info-nbt.fr/la-mutagenese-une-nbt-deja-ancienne.html)? Ou bien pourquoi les autorités sont si frileuses à certain types d’OGMs (agriculture) mais en même accepte sans ronchonner d’autres OGMs (produits pharmaceutique obtenue par génie génétique).
L’hypocrisie est encore pire lors ce que l’on interdit la culture de plantes transgéniques mais on autorise allègrement leurs importations (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592980/). Là est le manquement de l’auteur qui gratte que de manière superficielle ignorant ces détails et se contente que de trouver information qui confirme ses biais.

En es temps de changement climatiques, on a point le luxe d’attendre 50 ans pour trouver une variété résistante aux aléas climatiques ou à l’apparition de nouveaux pathogènes dans nos latitudes.

Oui les OGMs ont leurs problèmes, mais pas les problèmes imaginent et fantasmes par la population et maintenue par un journalisme malhonnête. On a l’issue de l’accès des ressources en biotechnologie pour les pays en voie de développent, afin qu’il puisse trouver une solution à leur problèmes spécifiques ; ou bien les financements servant à développer des brevets par des centre de recherche publique de protéger leurs inventions (eh oui, la propriété intellectuelle existe aussi pour les découvertes scientifiques et aide au financement de Nouvelles découvertes. L’histoire de la warfarine (un anticoagulant) développé par Dr. Paul Link at l’Universite de Wisconsin via le Wisconsin Alumni Research Foundation qui se finance grâce aux licences de brevet), mais également la mise en place d’un système de sécurité sanitaire pour s’assurer de l’innocuité de nouveaux produits OGMs.

Apparemment pour Senecat, vivre de ses brevets est le mal absolu:
« Au-delà des questions de santé évoquées par la tribune, les OGM posent néanmoins d’autres enjeux, notamment en termes de brevetabilité du vivant et de dépendance des agriculteurs aux sociétés qui en commercialisent les semences. Autant de réserves politiques qui ne relèvent pas forcément de l’obscurantisme ou de la mauvaise foi. ».

On accepte bien que le piratage d’œuvres artistiques (y compris films, séries TV et musiques) est une violation des droits d’auteurs, mais en temps on demande aux scientifiques de renoncer à une protection de leurs inventions. Le terme brevetabilité du vivant est souvent utilise comme argument de “straw man fallacy” (“fallacieux d’homme de paille”) pour discréditer le parti adverse par une exagérations des points argumentaires discute. On a un “straw man” aussi bien sur la “brevetabilité” (je me souviens de mes jeunes années ou l’on brandissait l’épouvantail Monsanto et ses grains OGMs avec le gène “Terminator”), que sur l’idée de voir les agriculteurs redevenus serfs sous le joug des multinationaux. Ce que Senecat n’a pas dû apprendre durant son passage sur Buzzfeed et sur les réseaux sociaux est ce que j’appelle les méthodes d’investigations.

Cette idée que Monsanto tient les fermiers comme je tiens mon chien par la laisse est le cas “Bowman vs. Monsanto” qui remonta jusqu’à la cour suprême des EU, donnant raison à Monsanto (https://www.npr.org/sections/thetwo-way/2013/05/13/183603368/supreme-court-rules-for-monsanto-in-case-against-farmer). Les agriculteurs sont libres d’acheter leurs semences ou leur plaisent et rarement gardent les semences pour l’année suivante. Les raisons sont multiples mais surtout pour s’assurer de la qualité des semences chaque année (surtout en termes de rendements). Le cas Bowman se base sur l’achat de graines de soja OGM “Roundup Ready” génétiquement modifie pour être résistant au glyphosate. Cela donne une certaine aisance a l’agriculteur avec un produit prêt a l’emploi, sans se soucier d’une perte de rendement ou le choix du pesticide. Comme chaque produit, on se doit de lire le CLUF (généralement on clique “OK” sans lire les clauses du contrat). Dans ce cas, l’agriculteur en question donna son accord écrit pour utiliser les semences pour les planter durant la saison et de ne pas les réutiliser l’année suivante”. Que l’on soit d’accord ou non avec cette clause reste à la discrétion du client. Mr. Bowman, en signant le contrat, accepta cette clause et accepta d’acheter le produit de Monsanto. Si Mr. Bowman n’était pas d’accord sur cette clause, rien ne l’empêchait d’acheter ses graines chez un autre pépiniériste. Le problème survenu quand Bowman décida de garder quelques graines de Soja “RR” pour un plantage hors-saison, violant les clauses du contrat. Monsanto eu vent de cette violation et entama une procédure juridique.
On est donc bien loin de cette image fantasme du pauvre fermier sous le joug de Monsanto que Senecat nous peints dans sa tribune. J’ai le droit d’acheter un CD, de le convertir en fichier audio AAC sur mon Mac pour écoute personnel. Mais je n’ai point droit de poster ces fichiers en téléchargement libre sur Internet. On a le même problème ici.

Quand on a des saccageurs fauchant des champs expérimentaux de cultures OGMs qui se passent pour des “héros et martyrs” dans les réseaux sociaux et sur le PAF, détruisant le fruit de plusieurs années de travaux de scientifiques de l’INRA tout en demandant les études complémentaires sur l’absence de risqué sanitaires, n’est-il pas indicatif de l’hypocrisie ambient à ce sujet ? Malheureusement, Senecat joue à l’apprenti-sorcier jetant de l’huile sur le feu en jouant sur la peur et l’appel au “naturel”.
Que différencie un faucheur d’un “sauvageon” incendiant une voiture lors de la veille du Nouvel An ? On a destruction et saccage de propriété d’autrui.

8. Le Nucléaire :
Le dernier parti du billet se focalise sur le nucléaire. A l’heure du changement climatique et de la série “Chernobyl” sur HBO, on a la discussion du nucléaire qui revient. Et à son habitude, l’auteur reste suspect des points aborde par la tribune des “No Fake Science”:
« Mais, de nouveau, la mise en exergue d’une seule affirmation, au détriment d’autres enjeux essentiels du sujet, peut donner l’impression que No Fake Science a fait son choix dans le « supermarché » de l’information scientifique. « Le point que nous voulions mettre en avant était la faible émission de CO2 de ce moyen de production électrique, pouvant participer à la lutte contre le réchauffement climatique. Le propos n’avait pas l’objectif d’aller au-delà », répond le collectif. ».
Le nucléaire en lui-même n’est pas la solution miracle à elle seule. Chaque source d’énergie a ses avantages et inconvénients. Les énergies fossiles ont dominé le 19eme jusqu’à maintenant au détriment du réchauffement climatique (gros producteurs de CO2), mais également de la pollution atmosphérique (dont les particules de gaz d’échappement des moteurs diesels ou bien des industries). Les énergies renouvelables sont une alternative intéressante, mais aussi ont leur limitation. Beaucoup de chemin reste à parcourir quant au rendement et a l’approvisionnement continue et stable en énergie. L’Allemagne qui a pourtant été le fer de lance “Gruene” (vert en allemand), n’a pu trouver une alternative aux centrales nucléaires pour une énergie propre (CO2) par la réouverture des centrales au charbon (http://www.leparisien.fr/societe/urgence-climatique-l-allemagne-doit-fermer-ses-centrales-a-charbon-28-07-2019-8124954.php).
L’urgence a court-terme reste à diminuer la production de CO2pour mitiger le réchauffement climatique. A ce point l’énergie nucléaire reste la méthode alternative accessible immédiatement. Oui il y a le problème des déchets mais on a des solutions. On a des méthodes pour recycler certains déchets et l’on a une expertise nationale à ce niveau. Le montant de déchets reste bien moindre que le montant de déchets et matériaux utilise pour la production de machines utiliser pour produire les énergies alternatives (solaire, éoliennes) tels représenté dans un diagramme par Toughtscapism (https://thoughtscapism.com/2017/11/04/nuclear-waste-ideas-vs-reality/).
L’autre alternative ? On retourne à l’Age de pierre pour couper net notre production de CO2avec zéro production de déchets et un licenciement sec pour Senecat : plus d’électricité, plus d’internet, plus de réseaux sociaux, plus de “buzz”, plus de “factchecking” et peut-être on réécoutera les sages paroles du vieux chef du village.

9. En conclusion :
Ma conclusion est la même et en phase avec ce que “Risk-Monger” appel le “poison du précaution” (https://risk-monger.com/2019/06/11/the-poison-of-precaution-iupac-keynote/). On vit dans une époque ou les réseaux sociaux ont une place prépondérante dans notre société. On vit à coup de “likes” sur Facebook, sur Instagram, sur Twitter. On a un changement de paysage ou soudainement on a donné une importance a des “motivateurs”, des “coach” ou bien “des experts” tout en regardant d’un mauvais œil les experts “classiques” comme dépassé, ou bien à la solde de groups d’intérêts dans nos délires conspirationnistes.

On vit dans un monde où l’on questionne les experts qui ont un Bac+8 et une réputation scientifique par leurs pairs par la qualité de ses publications.
Revenons à mon ancien camarade de fac et discutons comment cette mentalité peut être délétère.

Imaginons que je vais chez mon ORL pour un maux de gorge. Mon ORL diagnostique ce mal de gorge en tant qu’infection par streptocoque et me prescrit des antibiotiques.
En attendant mon tour pour récupérer ma prescription, je navigue sur le groupe Facebook « Crunchy Mommies », parlant de ma visite chez le médecin.

Karen, agent de caisse le jour et vendeuse « ceinture noire » des huiles essentielles (HE) Pomme Déterre la nuit (m’assurant que son insistance à vendre ses 5mL d’HE coutant $50 pièces, m’assurant que ce n’est pas un « Ponzi scheme ») commente sur mon poste : « Ne prends pas ces antibiotiques, tu vas bousiller sa flore intestinale ! Prends donc un flacon d’HE d’origan pour ton angine ! Ton docteur ne connaît rien et il est sous la solde de Big Pharma ! ». Voilà comment Karen, caissière s’improvise ORL.

Cette histoire peut paraître rocambolesque mais est assez proche des histoires que je rencontre avec des mamans « On The Fence » (hésitante à vacciner). Les réseaux sociaux ont paradoxalement ouvert les portes à n’importe qui sur Internet de se parader comme « expert » sans démontrer aucune qualifications et diplômes. SI l’on veut diminuer l’effet des « Fake Sciences/Junk Sciences », il faut une alliance entre les experts scientifiques et des journalistes scientifiques qui ont un bagage intellectuel et idéalement une formation scientifique de base pour pouvoir décoder un article de « peer-review ».
Malheureusement, Adrien Senecat est le symptôme plutôt que la solution dans le combat des « Fake Sciences ». De gré ou de force, Senecat via ce billet d’opinion a démontré son inaptitude et d’immaturité de « factchecking » quand on parle de questions scientifiques. Senecat est comme l’un de ces journalistes d’une planche de « Tintin au Pays des Soviets », auquel un commissaire Soviet montre avec opulence sous les yeux ébahis de journalistes occidentaux son « Village Potemkine ». Senecat est tel un étudiant qui pense plus connaître que le professeur. Ce symptôme a un nom, on l’appelle « l’effet Dunning-Kruger » et Senecat nous a démontré par cet article être plein dedans.

Categories
glut1 deficiency syndrome Sciences

[Sciences/Rare Diseases] Summary of the GLUT1 Deficiency Conference 2019 – Washington, DC

Last week occurred the GLUT1 Deficiency Conference 2019 that was hosted in the Hilton Crystal City. It was nicely located near the Ronald Reagan National airport (DCA) making it easy access to the conference and conveniently located in the 2ndfloor of the hotel, with different ballrooms used. Before I go into details, I have to disclose that I have attended half of the first day and attended mostly sessions that I considered relevant as a scientist. If anyone else wants to write a summary on the other session, please feel free to contact me, I will add to this post.

Wednesday Afternoon Session:

The first session I have attended was a research plenary session hosted by Dr. Juan Pascual (UT Southwestern) giving us some update on the current NIH clinical trial ongoing when it comes to the Triheptanoine Clinical Trial. Nutrigenix was running a parallel clinical trial and after initial results came in decided to discontinue such trial.

Dr. Pascual initial results reported a reduction in seizures in about 7 patients out of 12 on the TH. The treatment was defined in a time period (if I remember well it was 6 months), and stopped. Some patients continued to improve, some showed a drop in the improvement. An interesting feature was the report of changes in the EEG suggesting a possible interplay between excitatory circuits and inhibitory circuits.

The second session was provided by Dr. McKenzie Cervenka (John Hopkins Univeristy) that has been surveying patients that are adults. An interestin feature is that all children initially diagnosed with GLUT1DS in the 1990s are now all adults and we are learning about the disease as they age.

An interesting observation is that we have a glut of GLUT1DS occuring in adults, in particular a review of medical history suggest that mild symptoms reported in their childhood maybe indicative of GLUT1DS but went under the radar due to the lack of proper diagnosis or clinical symptoms.
In adults, it seems to characterize as a mild and chronic encephalopathy/cognitive impairment with infrequent seizures. Some varying spasticity and ataxia were reported, as the paroxysmal exercise-induced dyskinesia.

Although the recommended level of ketosis in children is about 5mmol/L, such level seems not reachable in adult patients.
There is no signs of sex or gender differences when it comes to occurrence (50%), the most common symptoms are: ataxia (63%), cognitive difficulty (66%), and speech difficulties.
About 82% reported some triggers (excitation, stress or anticipation, warm, hunger, fatigue….), and 67% reported changes in the symptoms when reaching puberty.

61% of the patients surveyed are on the ketogenic diet (with 52% on the classic one and 33% on the modified Atkins Diet). About 41% on the keto diet were seizure-free in childhood.
Thanks to the recent awareness, the age of seizure-free patients is getting younger.
About 46% of the patients were on AEDs, in particular acetazolamide, levetiracetam or lamotrigine.
Notably, 91% of respondent found physical activity reduced symptoms seizures,100% were capable of basic daily activities and 36% able to drive. Notably, 19% started families and have children with GLUT1DS.  This brought some discussion about how pregnant patients should handle the KD and the urgent need for more studies to assess the effect of KD on pregnancy. There is some speculation that such KD maybe protective in these patients and the fetus.
A recent update in the guidelines were published recently in Epilepsia and available as open-access here: https://onlinelibrary.wiley.com/doi/10.1002/epi4.12225

Other recommendations were to follow calcium/creatinine ration in urine, increase hydration, and consider the risk of carnitine deficiency induced by use of multiple AEDs. Carnitine levels should be monitored 1 and 6 months after initiation of KD.

Finally, a presentation from a Sanofi scientist about gene therapy, this is in particular in light of recent success and FDA approval of two gene therapies for RPE65 and SMA diseases.

The rest of the day was a poster session, that was pretty succinct (less than 10 posters) but really was an enjoying experience to have patients and caretaker asking about my science and explaining them. It was some challenging but enriching experience, as well as some data aligning with other studies.

Thursday morning session:

There was a series of different panels mostly driven for patients and about the ketogenic diet. The first speaker was Dr. Eric Kossoff (John Hopkins University) highlighting the interesting times for the keto diet. There is over 3000 publications as of today, and 7 randomized controlled trials. It becomes interesting enough to have the American Epilepsy Society to have a satellite session (or a session within the plenary session) about the keto diet in general. There are some indication that the ketogenic diet in mice is altering the gut microbiota, based on a recent study cited in Cell (https://www.cell.com/cell/retrieve/pii/S0092867418305208?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867418305208%3Fshowall%3Dtrue)

The authors reported a decrease in two types of bacteria (Akkermansia muciniphilia and Parabacteroides spp) in patients with refractory epilepsy. The keto diet was able to alter 4 days in mice. Interestingly, restoring these bacteria restored seizure protection. There is yet any clinical studies to support these claims. There are also no reasons to fluid or calorie restriction.

All diets seems to be valid, you choose which one can stick. However, it is recommended to adhere to KD for <2 years and to the modified Atkins for >12 years. About 80% of patients have over >90% seizure reduction on KD and MAD and 64% no longer required anticonvulsants. The keto diet seems highly effective for cognition and dyskinesia.

Although the discontinuation of KD in epilepsy patients can be considered about 2 years after seizure-free, the consensus is to keep on in GLUT1DS patients.

The keto diet is really entered a craze in the last two years, with some good information and also a lot of bad information. It has been recommended to operate precaution on some products labeled as keto-friendly.

Another discussion was on the long-term effect of ketogenic diet. The current data suggest that although an elevation was observed for total cholesterol and LDL were reported at 3 months, a normalization as a decrease seems to occur. More longitudinal studies are currently performed.

There is not conclusive risk of birth defects on patients following the KD.
Finally a quick information on CBD oil. Some patients admitted the use of CBD oil as adjuvants, found no additional benefits of it. Restriction of CBD oil restrains it the use of clinical trials.

 

Thursday afternoon session:

I have lesser notes taken because it was mostly general but there were some research highlights including from Dr. Umrao Momani (Columbia University) by selectively knocking-down of SLC2A1. It seems that knockdown the gene in infancy (P2) and early years (P28) mice fared worse on motor function compared to late stage silencing. This was also reported by worsened seizures and decrease capillary density in the brain of these two groups. Dr. DeVivo also reported about the issues of patients that are classified as GLUT1DS-like patients due to the presence of symptoms as encountered in GLUT1DS patients but showing no mutations known in SLC2A1 and also no known mutations in SLC2A3 genes.

These are some of the notes taken at the G1D conference, see you in San Diego in 2021!

 

Categories
Neurosciences Sciences Stem Cells Uncategorized

[Sciences/Neurosciences] Propionic Acid Induces Gliosis and Neuro-inflammation through Modulation of PTEN/AKT Pathway in Autism Spectrum Disorder (Abdelli et al., Sci. Rep. 2019)

Once a wise man said: “Be always wary of scientific studies trumpeted by mainstream news outlets as groundbreaking. Once the smoke settles down, the study in question is rarely groundbreaking, but rather limited with a lot of caveats”. If I have to summarize this study, that would be within the lines of the wise man. Despite what news outlets have been selling, this is a study that has its own merits, but its methodological limitations and caveats outweighs the novelty and significance. Especially considering the publication occurred in Scientific Reports, the response of Nature Publishing Group to the open-access model, this is an extra layer of concern as Scientific Reports prestige and quality has suffered major setbacks in the last few years due to papers retracted for blatant scientific misconducts that should have been spotted by reviewers.

About the authors: We have three authors. The first author seems to be a postdoc, as she apparently graduated from the same program from another lab. The second author maybe an undergrad, although a faculty with the same family name is listed. Finally the senior author is a faculty with an expertise (based on the publication record) on gastrointestinal (GI) tract physiology and pathophysiology. However, none of them seems to have a history of publication in any field of neurosciences. This is an important point, because it explains a lot of methodological flaws that anyone with neural stem cell biology (and brain development) could pick easily.

1. Introduction and hypothesis: The authors are basically using the rationale of changes in metabolomics observed in ASD patients and reported by several studies. In these studies, there are some indications that certain patients on the spectrum (especially those qualified as severely disabled) display an impaired GI function, in particular something we could qualify as something similar to inflammatory bowel diseases. There are studies suggesting that such GI condition is associated in a changes in the gut microbiota, yet with a fairly low resolution (we are able to document changes in a family of bacteria, but not able to pinpoint to the level of Genus species yet). In particular, there is a study that was recently published in Cell Stem Cells (very high impact factor journal) that highlighted changes in mice behavior and gene expression profile of several genes associated to autism following the fecal transplant from patient on the spectrum considered severe (https://www.cell.com/cell/fulltext/S0092-8674(19)30502-1).

In this study, the authors speculate that certain metabolites biosynthesized and/or bio transformed by these class of bacteria are contribution to the symptoms. In particular, the authors consider acetate (AC, CH3-COOH) , propionate (PPA, CH3-CH2-COOH) and butyrate (BA, CH3-CH2-CH2-COOH) as potential culprits, citing studies showing an elevated levels of these small chain fatty acids (SCFA) in fecal cultures of ASD patients compared to control patients. The authors also cite two rare genetic diseases such as neonatal propionic acidemia (PA) and propionyl coA carboxylase (PCC). PA will be very useful to us, because it will help us set what we would consider a pathological level of propionic acid (PPA) in blood. Yet comes the most speculative, and I would say the “jumping the shark” moment of the paper. The authors assume that since processed foods are rich in PPA, such amount of PPA can lead to the development of ASD in the fetal brain during pregnancy. That’s a lot of speculations with little or no layers of evidence. We have here the authors trying to make a statement four to five steps too far from the existing literature for several reasons (and also lakcing the literature backing them up) that be identified as the following:

1.   Where is the literature providing evidence that these SCFA cross the GI tract, at which extent (bioavailability studies)?
2.   What are the levels of PPA (and other SCFA) in the plasma/serum levels of neurotypic patients versus patients on the spectrum? For patients suffering from PA, I have found an old study referring to serum PPA as high as 0.337-1.35mM, with a normal level about 0.00337mM (https://www.jpeds.com/article/S0022-3476(81)80004-2/pdf)
3.   How much of PPA can cross the blood-brain barrier? This is an important question to answer. We can try to build on the analogy of the SCFA to ketone bodies (acetoacetate, beta-hydroxybutyrate) that are formed when someone is fasting or forced into a ketogenic diet. Considering that patients suffering from GLUT1 Deficiency Syndrome are showing improvement when put on a ketogenic diet (with BHB levels around 2-3mM), we can speculate these compounds can cross the BBB readily.
4.   How much of PPA can cross the placental barrier? I don’t have any clue either.

These talking points are important because it will determine if the experimental design of a study is sound or deeply flawed, which eventually will set the quality of the paper and the robustness of the conclusion made. That’s something I should mention by now, is that both the authors and the news outlets have been very fond of superlatives and trying to sell that paper at much higher level that it is meant to. Not only it is scientifically inadequate to make extraordinary conclusions without highly robust data to support them, but also it is nowadays dangerous to do so as such studies will be used by scammers, charlatans and other snakeoil salesmen to promote their supplements claimed to “cure autism” and use these type of studies to claim their products is supported by science.

2. Materials and methods: The authors used neural stem cells (NSCs) derived from fetal tissues (obtained from Life Technologies/Thermofisher) and maintained in a classical medium formulation aimed to maintain these NSCs in their pluripotency stage. The cells were passaged no more than three times, according to the author. This is important, as NSCs/NPCs passaging over time will coax them towards the astrocytes lineage compared to neurons (in terms of development, neurons appear earlier and mature earlier than astrocytes).
Now there is something intriguing. The authors claimed they looked at PPA and BA at concentrations ranging from 0.1mM (that would be physiological), 0.5, 1 and 2mM concentration. Considering such treatment would reproduce the fetal brain, we have to factor in what is the amount capable to cross the three barriers: GI barrier of the mother, the placental barrier and finally the BBB. However, the authors never showed what happened to these concentrations except the 2mM which is limit deadly (remember? This is the one that is detected in newborns having the rare genetic mutation). Are the authors trying to model the effect of PPA to model such diseases or are they assuming that the amount of PPA in processed found will be high enough to put the pregnant women into a severe metabolic acidosis? I don’t know but that like a red flag.
The differentiation of these NSCs into neurons/astrocytes were left to occur in a fairly random fashion, as the authors use the same medium used for NSCs maintenance but without growth factors. I think this is an important issue here as we may have a significant variability in terms of yield between passages in particular when it comes to neurons/astrocytes ratio (personal communication with Clive Svendsen).
Otherwise, nothing else really fancy and classical techniques found in any neurosciences studies: Immunocytochemistry, neurite outgrowth, qPCR…

3. Results:
3.1. Figure 1: I am a bit perplexed from what I see and from what I get when it comes to quantifications. The first issue I have is the lack of scale bar. A scale bar tells you how many pixels equals to a length. For example, a 512×512 pixel image may indicate you that 100 pixels equals to 50 micrometers. Here you have to trust that the experimenter did not fudge the data, crop the pictures and really show you a 10x magnification.
For simplicity, I will focus on the Day 10. My concern is about the health of the neurospheres in some of the groups, in particular the BHB-treated group. You can see in controls; we have nice rosette-shaped neurospheres with a dark core reflecting a dense. In contrast, look at the BHB treated group. These neurospheres are small, frail and lack the morphology observed in control. I wonder if BHB at this concentration is showing signs of toxicity? If yes, the authors were not concerned at all by this issue. And this is something concerning. If BHB is neurotoxic, so how can we make a conclusion to BHB as inhibitor. There are ways to show the viability of these neurospheres: Hoechst staining, propidium iodide staining, Fluorojade staining……..Because of this important issue, I will not consider the BHB treatment as valid.

Slide1

If you compare the data shown in Fig.1B and 1C compared to the quantification made in Fig.1A (and shown in the bottom), you can see we have a certain discrepancy here. I would skip the issue in the y-axis labelling (the correct symbol for micrometers is µ (mu) not n(nu)), but compare the 10 days timepoint to the data we actually obtain from the Fig.1A. In scientific publications, you have to be sure that what your representative blot/micrograph picture shows matches your quantitative analysis. In other words, what I see in the micrograph pictures in Fig.1A should be reflected in Fig.1B and 1C.
Then explain me why the differences in diameter reported is not as different between my quantitation (using ImageJ internal functions) is different from the one displayed. How do the authors justify the use of SEM instead of SD, except for making the graph look nicer (you can see the data actually suggest a much more variability that likely undermine the statistical difference)? How does the authors explain the 3x difference in neurosphere counts between me and them? Did they crop the pictures? If so they should have accounted for, and highlights the importance of having a scale bar in micrographs pictures and normalizing such data to a surface area (e.g. pixels2, µm2, mm2….)

3.2. Figure2: These are immunofluorescence pictures of plated neurospheres allowed to differentiate by their own on Matrigel-coated plates (Cultrex). The pictures are okay, although not very convincing for some and certainly not suitable for quantification. The use of flow cytometer is definitively a go-to when it comes to assessing cell populations.

Slide2

We are also here having a mixed results and missing important cell markers. First, the authors should have performed a nestin staining, as nestin is one of the markers present in NSCs/NPCs. Second, the use of GFAP as astrocytes marker has to be taken with a big grain of salt. I am not sure experts in the field would have let this fly with just one marker. GFAP can be expressed by NSCs and NPCs. Showing at least two markers per cell lineages (NeuN/bIII tubulin for neurons, S100B/GLAST1 for astrocytes) would have been much more convincing. bIII tubulin antibody (in particular the one used in this paper) is known to have a very strong non-specific staining. A good bIII tubulin would show nice neurites. I have attached a picture of an iPSC line developed by Sigma-Aldrich. You can use it to compare so you can see what a good bIII tubulin and a good GFAP staining should look like in NPC-derived astrocytes. Here we have just some blobs (that indicates a possible non-specific immunoreactivity) that dangerously overlap with GFAP. Technically, you cannot have a neuron that express both GFAP or bIII tubulin. It is either or but not both. R&D Systems has a nice interactive map that shows you the different cell markers expressed by the neural lineage as its differentiate into neurons and astrocytes here: https://www.rndsystems.com/pathways/neural-stem-cell-differentiation-pathways-lineage-specific-markers

What I am supposed to do with that?

3.3. Figure 3: The authors looked at both GFAP and bIII tubulin at mRNA levels (PCR) and protein levels (by ELISA). I would have personally put the PCR data first, followed by the ELISA data. The PCR data was normalized to GAPDH and the DeltaDeltaCt method was used, which is good, the authors also have represented the apparent bIII tubulin or GFAP/GAPDH ratio, which is good.  However, I am more skeptical on the ELISA data. The reason why? The data is represented as micrograms of protein/microliters of cell extract. I am skeptical why the authors did not run a Western-blot analysis for these two housekeeping genes, since you expect a lot of proteins being expressed. The authors also forgot to mention if they have diluted the samples or just added the crude extract at is. This is important because you can easily blunt the accuracy of your ELISA. LSBio is honestly a cheapskate when it comes to showing a standard curve, unlike more established ELISA kits manufacturer such as R&D Systems or Abcam, that will show you their standard curves and tell you the coefficient of variation in them. The maximum concentration of the standard curve is 1000pg/mL or 1ng/mL, with a detection range of 15.63-1000pg/mL and a sensitivity of 9.38pg/mL. The authors reported concentrations for GFAP was 0.8-3 pg/ml according to their graphs. Something is wrong here, and we have at least 2 reviewers that completely miss that. Are the authors telling me that they were able to detect GFAP and bIII tubulin below the sensitivity level (9.38 and 313pg/mL respectively)? Give me a break! I would also have advised the authors to normalize their concentrations into something meaningful like mg of proteins. It is easy to take a fraction of the cell lysate and measure the total protein concentration by BCA. I ask my students whenever they use an ELISA for quantifying a cellular protein to normalize their amount detected (pg/mL) to a total protein concentration (mg/mL) which allows us to normalize the data. Failure to do this normalization is like showing a Western-blot without a proper loading control (e.g. actin, GAPDH….).

3.4. Figure 4: In this figure, the authors are trying to show the expression of GPR41 (aka free fatty acid receptor 3 or FFAR3) in those cells. Honestly, this is my breaking point of tolerance. First, the authors underwent some cherry-picking of the data, showing you only the PPA treatment in astrocytes (where is the BA treatment? Where are the BHB treatments?) and only the BA treatment in neurons (where is PPA? Where are BHB?).
I am also very skeptical that what the authors call astrocytes are really astrocytes looking. What we see in Fig.4A looks very similar to 4B: very thin cytoplasmic projections looking like neurites. Only neurons form neurites in cultures. Astrocytes have more a flat-shaped feature, sometimes a bit fusiform like shape. Again, the GPR41 protein expression is really up when you have tons of PPA given (mM and more). How come this went through peer-review unabated and have at least 2 reviewers did not notice this gross conundrum in the data?

4. Rest of the figures and conclusion: I can go further with this paper. It was looking very interesting and promising, but the lack of expertise from the authors quickly percolated into loose and inconclusive data. This is the kind of paper you wish the authors would seek feedback from across the street, from some faculty with a neuroscience background and give them an honest feedback to make this paper good and scientifically sound. What we have indeed is a half-baked study, served as the next big thing since sliced bread. Not only the data is far from convincing of the claims made my authors (I would probably accept as a possible model for modelling PA or PCC, but this paper IS ABSOLUTELY NOT SHOWING THAT PPA IS CAUSING AUTISM for several reasons below:

1.   It does not account for the PPA levels found in normal persons, even less provide a study showing PPA levels in people eating processed foods (if such dietary habit even lead to such outcome).
2.   It does not consider that in order to be valid the authors have to show that you have a 100% bioavailability of PPA across the GI barrier, the placental barrier and the BBB, which are not reported or cited by the authors in any credible form.
3.   It does not account that the levels used as so ridiculously high that a pregnant mother would deal with a possibly deadly metabolic acidosis.
4.   It also ignored that BHB was showing signs of neurotoxicity.
5.   There is a worrisome pattern of data cherry-picking, with groups popping in and out intermittently, sometimes even in a complacent manner. This is a no-no and an unacceptable behavior that has no place in any respected peer-reviewed journals. Why did the reviewers overlooked that issue?
6.   There are several inconsistencies in the data, especially whether the axis labels are botched or if the authors really provided measurements that were nornally impossible to reach (below detection limit).

This paper should at least had a “major revision” to fill the gaps. Yet, it went through at least 2 reviewers and none of them were able to see the obvious methodological flaws. As a reviewer for Sci Rep on a seldom basis, I am very concerned about the quality of review provided by the journal in the recent years, especially in light of series of retraction. Conclusions? The news outlets have been trying to sell an overhyped paper that does not live much under scrutiny. This is just “same old, same old” when it comes to journalistic reporting on science (trying to fudge it as groundbreaking), but also opens a dangerous precedent. I will bet that within 12 months, there will be some quack doctors and snake oilsalesmen that will claim they can cure autism by selling you supplements aimed to reduce the PPA or by selling you a dietary fad book, claiming it will cure your child autism by dietary restriction. I guess the keto diet will soon join the casein-free/gluten-free diet as outdated and have another fad being served as dietary torture to children on the spectrum.

Categories
Neurosciences Sciences Stroke Uncategorized

[Sciences] Restoration of brain circulation and cellular functions hours post-mortem (Vrselja et al., Nature 2019)

You may have heard about that groundbreaking story last on “pig brains being revived” sounding almost like a scenario of a zombie movie. Let’s say science journalism love to use superlatives and sensationalistic headlines to grab few more clicks and views.
As usual, my skepticism was to first look at the paper and see how the claims hold on. The publication behind that “pig zombie paper” is the study from Vrselja and colleagues published in Nature last week and available here: https://www.nature.com/articles/s41586-019-1099-1
So what is about this story? First it is published in Nature, a top-tier peer-review journal. Second it is a huge paper, coming from Yale University. The paper was initially submitted on February 22, 2018 and got accepted March 1st. You can say a bit more than a year and that suggest that this paper likely went at least two rounds of review and probably more than three peer-reviews (three were named as well as other anonymous). One of the peer-reviewer was Pr. Constantino Iadeccola (Cornell University, NY), a “rock star” in the field of cerebral blood flow (which nicely match for the paper).
Overall, it is a very good paper with some reservations on the greater impact that I will explain later.
To understand the paper, you need to understand first that as until now we consider the brain highly dependent on continuous cerebral perfusion with blood flow to survive. The brain is highly dependent on oxygen and glucose (at least 20% of our daily uptake is taken by this tissue that only represent less than 2% of the human body weight).
We assume that if you stop flowing the brain with blood (e.g. cardiac arrest), you will die within minutes from massive and irreversible brain damage. The whole idea of this paper was: “what if we could maintain a blood flow for 24 hours, can we maintain some neurological function?”. In particular, the authors have developed a kind of artificial blood, cell-free, called BEx. It contains a hemoglobin carrier called Hemopure(R), glucose/pyruvate, as well as a cocktail of neuroprotective agents, antibiotics and some echogenic agents (to measure blood flow). The caveat is that as a control the authors used a simple saline solution without glucose and pyruvate (see supplementary tables below). Considering the importance of glucose for the brain tissue, and the absence of glycogen storage in that tissue, I would argue that this is a non-negligible flaw in the experimental design, giving a serious advantage to the BEx and maybe even overestimating the BEx activity.
Screen Shot 2019-04-17 at 2.06.26 PM

Nethertheless, let’s continue the discussion. Pig brains were not obtained from pigs euthanized for the sole purpose of the experiments, but rather obtained as waste from the slaughterhouse. Thats ethically much more acceptable, even 3R-friendly (as it valorize animal tissues considered as waste) and much more easy for obtaining an IACUC approval. About 300 post-mortem brains were used, I guess mostly for the development and optimization of the technique. The sample size (N) appears to be 32 pigs/group, which is very good for statistical power of analysis.
The surgical procedure (to connect these brain to the system) was about 10 minutes of warm anoxia, which would probably represent a severe cardiac arrest in which CPR is not performed immediately. They exposed these brain to either 1 hour or 10 hours post-mortem interval (PMI) without flow, with control perfusate or with BEx. Note that the perfusion to occur happened about 3 hours since the initial brain flush, the surgical preparation appears indeed tidious, but reproduce a pulsate blood flow similar to what would happen in animals. They also cooled down the brain to 25ºC, which is known that cooler temperature improve the chance of reducing brain damage (the common sense is that drowning in an hypothermic environment (frozen lake) increase your chance of resuscitation compared to drawing in a normothermic environment (swimming pool). The experiment lasted 10 hours for all groups, except the 1 hour PMI group.
The first results shown demonstrated the presence of a functional flow inside the brain tissue, and some vascular reactivity, using nimodipine infusion (a Ca channel blocker commonly used to reduce blood flow) and showing changes in blood flows. In other words, there is a proof of principle that it works.
The second result looked at changes in cerebral edema as a crude estimation of the blood-brain barrier (BBB) function. The control perfusate showed an increased water content, which is not surprising as some of our in-house data (and other studies) suggesting that the BBB function has a greater dependence to glucose than to oxygen when it comes to maintaining the barrier integrity. Since the CP is glucose-free, that is not surprising. The BEx group of course fare better (same level than 1 hour PMI group) but wonder how it would have fared if the CP contained the same amount of glucose and pyruvate. My personal thought is why did not the authors performed a gadolinium imaging of these two brains? They provide some T1 scans, which are nice but confirming changes in the BBB leakiness using gadolinium as contrasting agent would have been better.
Figure 3 show us a series of tissue staining of these brain, in particular from the hippocampal region. As expected, the Nissl staining worsened in the PMI, the presence of CP partially improved the situation and the BEx was similar to the 1 hour PMI and has the lowest cell death (as imaged by active caspase-3). When you look between CP and BEx, the difference is not that dramatic and makes me wonder that if we had the right CP formulation (with same glucose/lactate content), we would unlikely have a difference between CP and BEx, suggesting that perfusion with a saline solution oxygenated and with the correct amount of glucose can do as well than a more complex one.
Figure 4 show that the perfusion with BEx help to maintain astrocytes and microglial cells alive and functional (as measured by the secretion of pro-inflammatory cytokines following treatment with LPS).
Figure 5 show that there are some functional neurons present in BEx, capable to show electrophysiological activity. Small activity (not enough to be detected by EEG) but measurable by patch-clamp analysis.
Overall, it is a nice paper, considering it got published in Nature. There are some interesting stuff, but there are also some questionable limitations and caveats that I would have pointed as a reviewer and expected reviewers from Nature to have pointed it before letting it accepted. It shows that no matter what, never blindly trust a paper even if published in Nature.
The idea is very interesting and can help us better understand the post-mortem brain. It also raises the importance of CPR or any procedure aimed to keep a steady flow in the brain after injury or cardiac arrest and maybe worth considering it.

Categories
Blood-Brain Barrier Junk Sciences Junk Sciences Neurosciences Sciences Uncategorized

[Sciences/Junk Sciences] Zeolites, blood-brain barrier and “Autism Detox” scam.

Recently, it came to my attention of another scam popped up on social media. This scam came in form of the “Autism Detox” page on Facebook coming with the following description:
IMG_2005

“Zeolite”, “blood-brain barrier”, ‘detoxify toxins and heavy metals” and “cellular level”.
Incredible how much amount of BS claims can be packed in such a small vaporizer. Not only this was enough BS, the owner of this page went the extra mile and claims it is an “autism detox” as well.
I call this an utter amount of BS and since I am a scientist, I will explain why it is an utter amount of BS.

1. What are zeolites?
Zeolites are crystalline structure made of aluminum, silicium and oxygen. These crystals are formed by the aggregation of 4 oxygen atoms around aluminum Al3+ and Silicium Si4+ (notice how Avers that yells “shark” on aluminum in vaccines are fine absorbing aluminum from zeolites).
These frameworks of AlO4 and SiO4 can form 3-D geometrical structures harboring charges and possibly acting as a caging structure as shown below (Moshoeshoe et al., Am J Mat Sci 2017):

As you can see different structures exist. Now, which zeolites are used in the product described in this “detox”? According to the vendor website (https://www.coseva.com/toxin-removal/advanced-trs/), clinoptilolite (CLI) (amongst water and a proprietary formula). According to Mosheoshoe and colleagues, CLI harbors the following chemical composition ((Na,K)6(Si30Al6O72) •20H2O)) and harbor the following crystalline structure:
Clinoptilolite

Notably, CLI also display one of the lowest cation exchange capacity (CEC) of 2-2.6 mEq/gram. In summary, CLI is a small zeolite crystalline structure with limited cation exchange (against Ca2+, K+ and Na+). First, it shows that these compounds have a molecular weight exceeding the size recommended for small molecules (~832 Da>500 Da), a ring size bigger than the tight junction pore (5.6 Angstroms>4 Angstroms) and an non-negligeable amount of molecular charges. All these features make CLI very unlikely to cross the blood-brain barrier and no studies have provided a direct experimental evidence that CLI crosses the BBB.

2. Does zeolites even cross the GI tract?

Good question! The only paper that I found discussing about zeolites is a paper from Cefali and colleagues (Cefali et al., Pharm Res 1995). Unfortunately I cannot access the paper but the abstract provides two important parameters: Cmax and AUC. In particularly, it also provides the value of aluminum hydroxide (yep, that stuff found in vaccines).
Cmax is indicative of the maximal concentration reached upon administration via extravascular route (IM, PO or SC). The AUC is representative of the total amount that reached the circulation from the time of administration until the time the drug becomes undetectable in blood. From the abstract we have the following information The mean plasma silicon AUC values (+/- S.D.) were 9.5 +/- 4.5 [Note: Zeolite A], 7.7 +/- 1.6, 8.8 +/- 3.0, 6.1 +/- 1.9 mg.hr/L [Note: Aluminum Hydroxide] and the mean plasma silicon Cmax values (+/- S.D.) were 1.07 +/- 1.06 [Note: Zeolite A], 0.67 +/- 0.27, 0.75 +/- 0.31, 0.44 +/- 0.17 mg/L [Note: Aluminum Hydroxide] for Zeolite A, sodium aluminosilicate, magnesium trisilicate, and aluminum hydroxide respectively. Although mean silicon AUC and Cmax values were elevated when compared to baseline after administration of the silicon containing compounds, only the AUC from Zeolite A reached statistical significance (p = 0.041). The mean plasma silicon Tmax values (+/- S.D.) were 7.9 +/- 6.4, 5.8 +/- 4.6, 6.9 +/- 6.3 and 8.5 +/- 3.4 hrs for Zeolite A, sodium aluminosilicate, magnesium trisilicate and aluminum Hydroxide respectively.”. Since we have a Cmax and AUC value for Zeolite A and aluminum hydroxide very similar, we can assume that both compounds may likely show similar bioavailability. Considering the bioavailability of Al is very low (0.3%), it is very likely that zeolite and CLI may not show a higher value that this one. Thus, out of 100g ingested of zeolite, maybe less than 0.3g will likely reach the bloodstream. In conclusion the amount of zeolite capable to cross the GI is very small and considering the volume of a TRS “Detox” (28mL), the amount of zeolite capable to cross the GI tract after swallowing a whole bottle of it is likely to be ZERO.

3. What about the rest of the claims?
As far we have seen:
1) CLI absorption at the GI tract is likely close to ZERO, even if you sip a whole bottle at once (see 2).
2) CLI cannot cross the BBB because of the physicochemical constrains (see 1). The only paper listed in Pubmed is a letter written to a journal with no scientific evidence or experimental data backing up the claim (https://www.ncbi.nlm.nih.gov/pubmed/23224491).
3) The claim of detox is utterly BS: there are two organs that do it for you. The liver and the kidneys. Thats it.
4) Heavy metal detox mostly occurs via renal (kidney) filtration. Even if zeolites can trap ions like Na+ or K+, I still have to find a paper that shows me it can trap heavy metals (Cd2+, Pb2+, Hg2+…..). CLI has been shown to only trap three ions (Ca2+, Na+ and K+) with the poorest ability.
5) Claiming that autism be cured is not fallacious but criminal. Until now, there is no cure for autism. There is no evidence that chelating ions cure autism (chelation therapies have even been proven to be dangerous and responsible for the death of at least one boy). There is also no published mechanism of action demonstrating how a treatment can reverse a condition mostly identified as genetic.

 

Categories
Junk Sciences Junk Sciences Sciences Uncategorized

[Sciences/Junk Sciences] Reconsideration of the immunotherapeutic pediatric safe dose levels of aluminum (Lyons-Weiler and Ricketson, J Trace Elem Med Biol 2018)

This is a post I wanted to write about a long time ago but for some reasons, I have been putting on the back burner for many different reasons.
You know what can be the most irritating to read? Papers from anti-science in general. You see, if the data was sound, the experimental setup was robust then I would consider their arguments are valid and sound. The problem with the anti-science papers I have been reading so far (anti-vaccines, anti-GMOs) are most of the time written by scientists that are lacking the expertise and credentials (in terms of publication record) to discuss on a topic, are based on speculation (the experimental data to support their hypothesis is at best paper-thin), the experimental data are most of the time missing the rigor and paradigm needed to make an objective outcome and often cherry-pick the literature.
Under normal conditions, such papers would not even pass a normal peer-review filter and would have been rejected outright. Yet, such papers found their way in very low impact factor journals or in predatory journals (that will publish any garbage study, as long as there is a valid payment method).

1. Who are the authors?
This is the case of this manuscript written by James Lyons-Weiler and Robert Ricketson. In this study, they claim that the current immunization schedule is dangerous, blaming on the extraordinary amount of aluminum and using questionable and speculative pharmacokinetics to support their claims (of course, there is no experimental data to support their claims, only speculation). A tenet in scientific publication is to assess how credible the authors are in the field, this can be judged by the authors affiliation and publication records. James Lyons-Weiler has  (according to his LinkedIn profile) a PhD in Ecology, Evolution and Conservation Biology and currently affiliated to the “Instittute for Pure and Applied Knowledge”. This is not a scientific institute as the Salk Institute, but rather an frontstore for some quackery posing as a “scientific institute”. The second author, Robert Ricketson, is no better. Indeed, he is even worse. Apparently Dr. Ricketson has a history of medical malpractice as a spine surgeon, and has been implicated in a medical malpractice lawsuit in 2001 for inserting a screwdriver in a patient spine. At the publication date, Ricketson affiliation is another “scientific institute” named “Hale O’mana’o Research” in Edmond, OK. A quick verification on his LinkedIn profile suggest that these two Ricketson are the same Ricketson. To summarize, we have two authors with ZERO expertise in pharmacokinetics (including one doctor that got fined over $5 millions for medical malpractice), working in institutes with questionable scientific credientials but established anti-vaccine stance, under the disguise of “vaccine safety” (here and here), published in a journal in which a notorious anti-vaccine scientist is sitting in the editorial board. Is it surprising? For me, it is not. Just a classical MO for anti-vaccine scientist.

2. What the paper is about?
You can find the paper here, since it is behind paywall I cannot legally share the information, so I would request the reader to corroborate my claims by getting the full-text. In this study, the authors consider the safety studies done in animals are not correct and underestimate the toxicity of aluminum because they are based on animal body weight. And thats where the trouble start. The authors solely consider the amount of aluminum injected into animals and patients SOLELY based on the body weight.
They ignore the administration route, they ignore the existing literature and even questions the outcomes and recommendation of the World Health Organization as mentioned as “We found two important errors in the provenance and derivation of provisional aluminum intake levels from World Health Organization (WHO; Supplementary Material) which, unfortunately, led to overestimation of safe exposure levels.” That’s a bold statement by the beginning, coming from two non-experts in pharmacokinetics and toxicokinetics.
So how do they ended up using such claim? By using a derived version of the Clarke’s equation:

Child dose (mg) = Adult Dose (mg) * (child bodyweight (lbs)/adult bodyweight (lbs))

The Clarke’s equation is a common equation used for therapeutic dosing, as we commonly refer to administer doses as x mg/kg. Knowing the patient weight, you can easily calculate the dose administered.  This formula is great…….if you already know the target concentration (or the average plasma concentration) you aim to target. This is usually supported by empirical data and further confirmed by lab tests (you can dose the drug in the patient plasma and assess if such amount falls within the therapeutical window). But this equation tells you nothing about the pharmacokinetics of the drug, or about the bioavailability of the drug, or differences in the administration routes.
It only tells you one thing “How many miligrams of X should I administer to obtain a plasma concentration of X falling into therapeutical range?” That’s it. You assume a dosing regimen (mg/kg), you know your patient weight (in kgs) and thus you can obtain the dose needed (loading dose or maintenance dose).  However, the authors manipulated the equation to be able to transpose the minimal risk level from adults to children as the following:

CED (mg/kg)= HED(adult) mg/kg x [BW(child) (kg)/BW(adult) (kg)]

The rest of the paper is SOLELY based on speculation, no experimental data to support the claim (we rather have a post hoc ergo fallacy unfolding). If the authors wanted to make their claims valid, they would provide experimental data (in forms of blood sampling) for 2 months babies before immunization (baseline control) and 6-24 hours after immunization to show that Al levels in plasma are significantly altered by the immunization. But they never show that data.
Indeed, what they show is a blatant misuse of the data and recommendation of the FDA and a serious miscalculation that a 12th grader would not even do.
They compared the dietary MRL as “JECFA provisional tolerable daily intake from dietary and additive exposures of 140 μg/kg/day and current provisional tolerable daily intake of 290 μg/kg/day per day both before and after the safety factor of 10 is applied (Fig. 3).
We end up in the classical cases of “apples versus oranges” and trying to make the claim they are the same. Which they are not. Yes, both are extravascular routes and follow similar fate. But in the same time, we have to compare the physics-chemical aspects and the bioavailability of Al in both routes. One is administered by oral route, the other by intramuscular route. In both cases, the bioavailability falls within the same range, with the oral showing about 0.3% and the IM from 0.6% (based on Flarend et al., Vaccine 1997) and 0.9% (Yokel and McNamara estimate, Pharm Tax 2001).

What the authors show us is basically a graph that assume the WHOLE Al injected in 100% bioavaialable at once, exceeding the MRL adjusted to pediatrics) as seen in Figure 4:
Lyons_Weiler_2018_Fig4

There is one thing to consider: The ATSDR. The ATSDR considers the MRL of 1mg/kd/day of ingested aluminum (that is about 100x lower than the NOAEL and adjusted to the bioavailability, as described here: https://www.atsdr.cdc.gov/toxprofiles/tp22-c8.pdf). If we assume a bioavailability of 0.3%, then we expect that out of 1mg/kg/day ingested, we can estimate that about 3microg (or 0.003mg)/kg/day would contribute in the total burden in the Al plasma level. This graph would be correct if 100% of the aluminum injected ended up in the systemic circulation at ONCE and spiked Al levels significantly high. But thats not the case, and the authors blatantly ignored this critical information, coming from previous studies. In order to compare these two items, you have to compare and estimate how much of each of these routes will contribute in the total Al plasma/blood levels.
You cannot just plot the total amount injected (adjusted per kg) and assume it is representing the same variable than estimated plasma levels from the MRL. Now, let consider that the Al injected is available at the rate of 1% a day, the graph will look more like that.

Lyons_Weiler_2018_Fig4_Adjusted

You see, we have a complete different scenario. If we consider that the aluminum is slowly released into the body at a rate of 1% per day we are now being way under the MRL and within safe levels. Again, we consider the MRL of 1mg (1000microg)/kg/day. If we consider a 0.3% bioavailability and difference in 5th and 95th percentiles weight (grey bars), we conclude that the daily burden of Al via dietary route should not exceed a value ranging from 13.20-18.72microg/day. Our values matches the MRL from Lyons-Weiler. In other words, our assumption is correct. If we consider an average weight of 5.35kg (50th percentile) at 2 months and 1mg as a cumulative dose of the immunization occurring the same day (conservative estimate), the amount delivered that day would be 0.187mg or (187microgram). Considering a bioavailability of 1% per day via IM, we have about 1.87microgram of burden from the vaccine added each day to a maximum MRL of 16.05microgram/day for the 50th percentile (weight 50th percentile=5.35kgs). Thats about 11.7% aluminum to be removed from the daily MRL, but within negligible range to have a statistical significance (you need at least 30% to consider it as statically meaningful).  This of course has to be confirmed by studies assessing plasma levels of Al after injections but there are already a literature out there with such data avaialable and reported here and here. Both studies concluded no changes in total Al plasma levels in regard of the vaccination status, including 6-24 hours after immunization.

3. Concluding remarks

Anti-science know how to bangs for their bucks, by sensationalizing claims knowing that the lay person will not or be capable to verify their claim. Most of the times, such claims come from persons that are legitimate scientists in their field, but completely speak out of their expertise domain. This is a common trope we see when people cite Linus Pauling, Otto Warburg or Luc Montagnier. Each of them have done remarkable discoveries in their field, got their Nobel Prizes but once they speak outside their expertise have proven to be wrong or have seen their claims manipulated by quack-peddlers. Lets take Linus Pauling that has been incremental in modern chemistry by describing the chemical bonds, but later claimed cancer(s) can be cured with Vitamin C. Coincidentaly, he died of prostate cancer in 1994.
Same applies in this paper. We have two authors with ZERO knowledge of pharmacokinetics, yet they have given themselves the role to demonize aluminum at all cost, bending and occulting facts to fit their narrative and their conclusion. This paper is the evidence that they are not serious about “vaccine safety”. They are staunch anti-vaccines, and they will use their status of scientists to vilify it at all costs, even if it means reaching outside their expertise, make extraordinary claims without extraordinary evidences (they did not have evidence for this study) and get published in a journal that will favor their claims and obviously lacked the rigor in the review.
Negating the neurotoxic effects of aluminum is not a correct statement either. Aluminum is neurotoxic, but as anything in toxicology it is all about the dose. And one parameter that is critical to assess the safety of aluminum is its plasma level. This safety level is driven by how much aluminum access the systemic circulation (from IV parenteral nutrition bags or from extravascular routes such as vaccines or dietary exposure). What matter at the end is the Al plasma levels and the FDA set a limit on that daily exposure (5 micrograms/kg/day via IV route). This is a problem encountered by patients suffering from non-functional kidneys (95% of aluminum is cleared via renal route) and from patient continuously fed via IV route (total parenteral nutrition).
Both Lyons-Weiler and Ricektson failed to applied basic concepts of pharmacokinetics, ignored the differences between vascular and extravascular routes and willfully used a calculation method that is not appropriated for this purpose. I would even what is worse is that none of their claims is supported by hard data, making their claims even more questionable.
Unfortunately, such “junk paper” felt through the cracks of peer-review and has been used repeatedly used by anti-vaxxers as supporting evidence. As Andrew Wakefield has his second paper removed after 16 years, how long it will take to remove that paper?
I dont know but the damage is done, and until “vaccine safety” scientists come with robust and foul-proof studies published in highly respected journals, they will be considered by me and others as junk scientists, keeping on feeding the literature with their garbage studies that should have been wiped out by a rigorous peer-review process.

 

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Blood-Brain Barrier Neurosciences Sciences Uncategorized

[Neurosciences/BBB] Brain Endothelial Erythrophagocytosis and Hemoglobin Transmigration Across Brain Endothelium: Implications for Pathogenesis of Cerebral Microbleeds (Chang et al., Frontiers Cell Neurosci 2018)

I usually don’t post BBB papers on my blog because most of the time they address concepts or answers questions that are not relevant for the public in general, but I thought this one was an interesting paper to share. This is an original article published by Rudy Chang and colleagues in Frontiers in Cellular Neuroscience las month. It is open-access, that allows everyone to access to it. Another interesting feature is the disclosure of the reviewers that help improve the peer-review process and transparency.
Why I found this paper interesting? Its because it propose a novel mechanism of cerebral microbleeds, without affecting the tight junction complexes. In the field, when we consider brain bleeds, we consider a loss of the barrier function and a massive brain leakage. Such phenomenon occurs when you have an hemorrhagic stroke due to an aneurysm, or due to a arterioveinous malformation resulting in an unstable blood vessel. The presence of blood in the brain parenchyma is harmful for two reasons:
1) You are injecting a volume inside a closed space (cranium) that will lead to an increased mechanical pressure (intracerebral pressure) and ultimately brain damage by tissue crunching.
2) Red blood cells (RBCs) may be damaged (hemolysis) and release their content into the extracellular space. Heme is toxic at high concentration (through mechanisms that yet to be identified) and can further damage neurons via generations of free radicals and other damage-inducing signaling pathways.
In this article, they demonstrate that you may achieve a similar outcome than brain bleeds without having a leaky BBB. In particular, this study demonstrate the ability of damaged RBCs to cross the BBB via transcytosis (via an engulfment inside the BBB and the exit to the other side). For this study, they used bEND.3 cells (an immortalized mouse brain endothelial cells) and compared mouse RBC that were considered healthy or induced damage via tert-butylhydroperoxide (t-BHP). t-BHP induces oxidative stress (via the release of radical oxygen species such as hydroxyl radicals), in this case leading to the exposure of a particular phospholipid named “phosphatidylserine” (PS) from the inside to the outside of the cell surface membrane.
In this study, they demonstrated that oxidative stress mattered in RBC cell adhesion to b.End3 cells. Treatment of b.End3 cells with t-BHP or LPS (a bacterial membrane lipid, commonly used to induce an inflammation state at the BBB) failed to yield similar results. in addition to demonstrating the ability of RBC to adhere on b.End3 cell surface (the first step needed for cellular transcytosis), they also demonstrated the ability to have these cells to engulf and get trapped into these cells. This process appeared slow and it took about 18-24h to see a significant number of RBCs inside the cells. Finally, they show that such RBCs were capable to migrate through the b.End3 monolayers and popped out in the other side. Similar outcome was observed in vivo, but to a certain extent.
It is a very interesting study, because it can maybe explain some aspect of diseases associated with RBCs such as cerebral malaria (we can imagine that Plasmodium may use RBCs as a Trojan horse to cross the BBB). However, I also have some criticism of the study. First, it uses the b.End3 that has fairly poor barrier function (TEER<100Ohms.cm2), much less than the tightness expected in vivo (>2000Ohms.cm2). The second issue is inherent to working with non-human cells. Do we have the same outcome when it comes to the human BBB and human RBCs? Maybe this phenomenon is exclusive to rodents and may have limited impact in humans. Finally, and as seen with the in vivo data, RBCs maybe able to cross the BBB but they maybe likely get retained by 1) the basement membrane supporting brain endothelial cells and 2) rest in a limbo state in the perivascular space (a virtual space between the basement membrane and an external protein mesh called “glia limitans” wrapped around cerebral blood vessels). We have some hints as RBCs appear juxtaposed near the vasculature, as stuck by a mesh surrounding the vessels. I don’t think that RBCs can cleave such mesh because I assume they don’t have the molecular machetes (matrix metalloproteinases) to cut their way through. Yet, I think it is a very interesting paper, that work to be investigated with a human BBB model, using a more robust model.