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[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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[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.

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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.

 

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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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Junk Sciences Sciences Uncategorized Vaccines

[Sciences/Junk Sciences/Vaccines] HPV vaccine safety – a tale of two studies

“It was the best of times, it was the worst of times” Charles Dickens – A Tale Of Two Cities

If you are following a bit the current news about immunization and vaccines, you likely heard about two studies in regards of the effect of HPV vaccines on population safety, in particular in terms of risk of developing complications or chronic conditions.
Interestingly two studies (to be honest one study and one comment) weighing the pros and cons of HPV public immunization were published within weeks.
One of them was claiming that HPV vaccines increased the risk of cervical cancer in the Swedish population, the other the incidence of autoimmune diseases in the Canadian population, in particular in the population of Ontario province.
One of them was published in a society journal with an reasonably impact factor (IF~8 and Scimago score of 1.7), the other in a journal with inexistent impact factor (Scimago score of 0.2) showing behavior similar to “predatory journals”.
One of them was published “in a peer-reviewed general medical journal that publishes original clinical research, commentaries, analyses, and reviews of clinical topics, health news, clinical practice updates and thought-provoking editorials.”; the other “is a multi-disciplinary academic journal providing a platform for publication of original material and discussion on all aspects of healthcare ethics and the humanities, relevant to and/or from the perspective of India and other developing countries.
One of them was a full-length study with several authors, the other one a rapid communication labelled as “comment” and purposely falsified the author’s name and affiliation (hiding behind an outlook email address), claiming the fear of retaliation by the opposing group.
One of them was a controlled population study, investigating two groups (vaccinated versus unvaccinated) with a sample size of 100’000+ each; the other one tossed epidemiological data together without further stratification and cherry-picked the information.
One of them was concluding the safety of HPV vaccine and absence of increased risk of autoimmunity, the other questioned the safety of HPV vaccine straight from the abstract as “I discuss the possibility that HPV vaccination could play a role in the increase in the incidence of cervical cancer by causing instead of preventing cervical cancer disease in women previously exposed to HPV. A time relationship exists between the start of vaccination and the increase in the incidence of cervical cancer. The HPV vaccines were approved in 2006 and 2007, respectively and most young girls started to be vaccinated during 2012–2013.
After a media firestorm and the strange support of the editorial board towards the author (and still consider the data as legitimate), it got finally retracted. The corresponding author has also been informed he will have four other retractions upcoming.
Guess which one was the flawed study published with a falsified author information and in a journal with a scope outside the content of the article published?
You can these publications here and here.

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Blood-Brain Barrier Junk Sciences Junk Sciences Neurosciences Sciences Uncategorized

[BBB/Autism/Junk Sciences] Autism, bleach and the blood-brain barrier: how the CD/MMS cult is promoting child abuse on bogus scientific claims.

I have been blogging about quack medicine, charlatanisms and debunking claims about the blood-brain barrier for few years now. But nothing reach the level of indignation and anger than the treatment reserved for children diagnosed as “on the spectrum” (for autism spectrum disorder or ASD), especially those treated with the “CD/MMS protocol” aka “the bleach protocol”, as recently discussed in various blogs and in news outlet here and there.

Introduction:

“Autism spectrum disorders” that is an umbrella medical definition that is defining children presenting deficiencies in social skills, a particular focus on patterns or objects including certain rituals or organization (e.g. sorting toys by their colors, lining cars in a perfect order, bed linen to be perfectly folded), hyper-sensibility to environmental cues (sounds, light, colors….) and in some cases neurodevelopment or communication delays. Not all autistic children are equals, with very different types of syndromes or conditions (e.g. Asperger’s Syndrome, Rett’s Syndrome……).

Although the etiology of ASD is deeply anchored into genetics as a major risk factor (followed by neuroinflamamation during gestation due to infectious diseases), the diagnostic still remains flexible and have been standardized only recently through the “Diagnostic and Statistical Manual for Mental Disorders”, currently in the fifth edition. Such standardization is as recent that a notable number of adults often get diagnosed “on the spectrum” late in their life, often during their adulthood.

Until now, there is no therapies to address such condition and mostly involves medication for treating other conditions associated with the disease (epilepsy is often diagnosed in children on the spectrum) or behavioral therapy (also known as applied behavioral analysis).

Because the diagnosis of autism is perceived and feared amongst parents and the lack of therapies are obvious, such environment creates a fertile ground for charlatans and snake oil seller preying on fear to make profit, selling parents a “cure-it all” potion or interventions, using these children as “guinea-pigs” by pushing protocols or treatment that are best have poorly fared in the scientific literature (most of the time published in low-impact factor journals) if not completely bogus.

A few example of such doubtful or quack remedies are dietary restrictions (gluten-free/casein-free diets), injection of biologics (GcMAF), if not dangerous interventions such as the use of hyperbaric oxygen treatment or use of chelation therapy. But amongst them reside one of the worst treatment: the CD/MMS protocol, or as we should call it the “bleach protocol”.

The CD/MMS protocol: a fancy name for a bleach protocol targeting autistic children

CD stands for chlorine dioxide (O=Cl=O) a bleaching agent used mostly for industrial purposes. CD shares similarities with the household bleach (O=Cl-) and both as referred as chlorinated bleaching agents.

Until recently, Kerri Rivera has been actively promoting the CD/MMS protocol as a “cure for autism” through her book co-authored with other charlatans named “Healing The Symptoms Known As Autism”. in this book, they promote the use of CD via ingestion of droplets or via enema administration. Such aggressive chemical is enough to damage the mucosal layer lining the luminal wall of the gastrointestinal (GI) tract and its detachment. Such detached mucosal layer is often labelled as “parasites” which indeed any respected parasitologist will quickly debunk such fallacious claims.  Kerri Rivera promotes the use of this protocol to cure “autistic children” and up until recently was promoting such treatment in the Autism One conference. She discussed in details about this protocol on the Chapter 8 of her book and makes disturbing claims about the blood-brain barrier.

Fallacious things Kerri Rivera said about the blood-brain barrier in her book:

The first fallacious claim from Kerri Rivera appears on Chapter 3, pages 48 and 49. In this chapter, she promotes the gluten-free/casein-free/soy-free diet as a treatment for autism with the excuse of the “leaky gut syndrome” as the following: “This results in poor digestion, which facilitates the entry of these harmful proteins [gluten and casein] directly into the bloodstream, where they can cross the blood-brain barrier.“. I never heard about gluten and casein crossing the BBB, especially considering that these are large peptides and therefore have to use transporters and receptors. Of course, her claims is not backed by a reference to a study.
Then she refers to this ” Improperly digested gluten and casein fragments can both enter the bloodstream and cross the blood-brain barrier. Because of their opioid properties, these peptides can react with opiate receptors in the brain to cause effects similar to those of an opiate drug such as heroin or morphine.7 These opiates are called gluteomorphin (or gliadorphin) and casomorphin, and can react with some parts of the brain, for example, the temporal lobes, which are actively involved in the process of the integration of language and hearing. Interestingly, these are two of the areas most affected by autism.

She cites this page for her claims. Interestingly, if you look at the page this claim is based on making a parallel between celiac disease and a speculation and hypothesis as cited: “Now in terms of autism, the situation is somewhat different because children with autism generally do not have celiac disease and do not have the DQ2 genotype problem. Whereas the problem of celiac disease is well proven in scientific studies, the problem with gluten sensitivity in autism is less well studied. The autism hypothesis involves, like celiac disease, the toxic effects of small peptides, generally in the range of five to seven amino acids in length (termed casomorphin and gliadorphin, as noted below). It is believed that these peptides from gluten, as well as certain peptides from cow milk protein (casein), can somehow cross the intestinal microvillus barrier and reach the blood stream.”

In a previous edition, Rivera went further and cited two papers to back her claims (now this claims has been watered down and put in the FAQ section of this chapter):  a study from Reichelt1 and colleagues and a  review from Shattock and colleagues2. Firstly, the citation of Shattock review is outdated and only provide an exhaustive overview of published studies supporting or dismissing the theory of opioid-excess. It has no scientific value as it does not provide a direct evidence of such claim. More troublesome is the following study led by Hunter and colleagues published by Hunter and colleagues in 2003 investigating the presence of opioids mimetics in patients urine and published in Developmental Medicine & Child Neurology3, a journal with an acceptable impact factor (IF=3.29). Using liquid chromatography coupled with mass-spectrometry (a common analytical technique used for measuring metabolites in biological fluids), the authors have investigated the presence of opioids in a cohort of 10 children with ASD and used siblings as controls. Interestingly, the authors failed to notice notable differences (as defined by presence of unique peaks) in the urine chromatogram of ASD children compared to controls. The authors further investigated the presence of opoid peptides previously cited by Shattock, in particular beta-casomorphin (a peptide byproduct obtained from casein degradation) and alpha-gliadin (a peptide byproduct obtained from gluten degradation). The authors failed to identify the presence of both peptides, based on retention time compared to standard or based on the m/z index.  This publication irated enough Shattock to be followed  by a comment to Editor and a scientific joust between Shattock and Hunter4, however an  editorial published by John F Mantovani resumes well the context in which the initial statement of Shattock was published5. At this time, ASD etiology was completely unknown and remained highly speculative. The publication (and subsequent retraction) of the so-called “Wakefied study” 6 linking MMR vaccines to ASD cases, but also documenting the presence of inflammatory bowel disorder in ASD patients, such condition is known to triggered by gluten and casein in patients suffering from celiac diseases. As Mantovani mentioned, the adoption of the theory of gluten and casein was correlating with the same approach than the vaccine without any scientific rationale. The study from Hunter indeed showed the lack of evidence about the claim made by Shattock. The amount of studies linking autism and exorphin remains very low. A query on Pubmed (the database of the National Library of Medicine) using the keywords “autism” and “exorphin” results in only 7 publications with 3 publications from Reichelt, KL and two publications from Brudnak, MA.
This brings the concern of data reproducibility. In order to have a scientific claim that have strong significancy you need two factors: a significant number of publications that investigated such statement and the publications of findings from different research groups. Having the monopoly of such investigation solely on a single research laboratory raises the issue of data reproducibility and reliability.
In this case, the study of Reichelt is very interesting, as its publication quality appears dubious at different levels. The journal of Microbial Ecology and Health Disease has recently adopted the “open-access” policy. Prior its publication as open-access, the journal has an 2013 unofficial impact factor of 0.933. The “open access” and the low IF raise red flags: such journal may be a potential “predatory journal” (a term coined by Retractionwatch.org, a website tracking scientific articles retraction). In this model, the cost of open-access is levied by the payment of hefty publication fees ($3000-5000) usually higher than subscription-based journals. Because of such financial gain, the peer-review process may be altered and even may be completely omitted, removing the quality control accomplished by peer-review. This lack of peer-review process is particularly blatant by the absence of clearly structured “methods” sections, odd wordings for a scientific (“ELISA typed as Elisa, thaw over night, eight-hundred microliters”), the source of samples (Association Planet Autism (Italy), samples from Slovenia, Serbia and Australia) and the overall format of the paper figures with some appearing as a screenshoot of a Powerpoint presentation or from printed copies. It raises some skepticism about why the author (based in Norway) failed to collect samples from Norwegian ASD patients.

In the previous edition, Rivera linked these studies to a “leaky bowel syndrome”. A major flaw in this claim is the absence of citing the original publication for Hsaio and colleagues7 that have demonstrated the presence of a “leaky gut syndrome” in mice showing an ASD phenotype. Instead Rivera cites the Gluten Free Society webpage as a source of information (http://www.glutenfreesociety.org/gluten-free-society-blog/dr-fasano-on-leaky-gut-syndrome-and-gluten-sensitivity/).

Dr. Alessio Fasano is certainly a respected researcher in celiac diseases but as noted with pseudoscience and activists groups lacking the scientific knowledge, cherry-picking and extraordinary extrapolation. In particularly in this case by the Gluten Free Society, those as their Facebook webpage mentions, identify themselves as alternative and holistic health society. This is again a red flag on the mission and purpose of this society that have little or no scientific evidence to support their claims except deviating, cherry-picking and reformulating genuine studies to push for their agenda.

Under normal conditions, the intestinal and the blood-brain barrier (BBB) (Figure 1) provides a tight cell monolayer creating a gut-blood and a blood-brain barriers respectively. Under normal conditions, such barrier is achieved by the presence of tight junctions complexes stopping the diffusion of electrolytes and water between the two compartments. Only digestion byproducts such as amino-acids or glucose are transported through dedicated nutrient transporters or solute carriers, whereas bigger entities such as peptides, proteins and pathogens have virtually no diffusion). Only lipids (fatty acids, cholesterol…) and drugs (designed as lipid-soluble chemicals) can passively diffuse across the barrier by mixing themselves with the phospholipid bilayers making the cell membranes.

Picture1

In the study from Hsiao, the authors demonstrated indeed the presence of a “leaky gut” as measured by an increase in FITC-dextran permeability with an estimated size of 4kDa (that’s about the size of a peptide of 36 amino acids). Even is such peptides can cross a “leaky gut”, they still have to cross the BBB. Some scientific studies have demonstrated the biological activity of opioids analogs obtained from digestion byproducts, including gluten and casein. Yet, a review from Lister and colleagues 8 denoted that a majority of these studies were based on intracerebroventricular (ICV) injections (or intracranial). This drug delivery approach allows to bypass the BBB but also is a very invasive approach that is used in clinical settings only for emergency and severe cases.

If such peptides were to cross the BBB and exert the biological activity discussed by Rivera, they have to have a dedicated peptide transporter that can deliver such peptides from the blood to the brain side. The number of peptides capable to cross the BBB has been recently reviewed by Banks 9, a well-established BBB scientist in the transport and delivery of peptides and inflammatory cytokines across the BBB. There is no mention about any of the opioids mentioned by Reichelt or Shattock publications. Furthermore, the increase in gut permeability appears unlikely or indirectly related to gluten or casein-sensitivity, as the authors demonstrated a change in the gut microbioma, in particular changes in Bacteorides fragilis as well as changes in metabolites discovered in serum plasma. However this study has to be taken with a lot of precaution due to the differences related to interspecies variation, the behavioral representation of mice to model ASD and more importantly, similar studies in human patients investigating samples from stools and plasma levels to observe if similar trends or biomarkers are noted in humans.

The next chapter that talks about the BBB is the Chapter 5, in which she discussed about the use of CD/MMS protocol, claiming to hunt their imaginary parasite inside the brain as mentioned by the following: ” In early 2011, we added enemas to the protocol to kill the pathogens causing dysbiosis in the large intestine (we didn’t know about parasites yet). We wanted to get the chlorine dioxide into the blood stream so it could kill the biofilm that exists in the blood. In this way, the blood can carry the CD past the blood-brain barrier to kill pathogens in the brain
When we are detoxing, it is absolutely critical to keep the colon moving and avoid the reabsorption of toxins through the intestinal walls. Enemas allow us to do just this. Some toxins can exit the intestine through the intestinal wall (more so if leaky-gut syndrome is present), and cross the blood-brain barrier, therefore affecting cognition and behavior. When we cleanse the colon, we get those out before they can cross into the brain, and we detoxify the lymphatic system, liver, and gallbladder.
The following argumentation of Rivera is very interesting as she is referring to Dr. Andreas Kalcker and the parasites at the base of her bleaching-based therapy. Let’s first identify Dr. Kalcker. According to his official biography (http://www.andreaskalcker.com/en/biography.html), he studied economics in Barcelona and has earned a Ph.D. in biophysics and alternative health without mentioning his alma matter. This is very puzzling, as any genuine Ph.D. holder will mention the institution that granted his/her degree. Furthermore, the deliverance of a Ph.D. in biological and biomedical sciences (and I believe in any scientific domains) requires the publication of at least one publication in a peer-reviewed journal. Notably, the search of Dr. Kalcker publication in either Pubmed (NLM) or in Sciencedirect (Elsevier) database leads to inconclusive results. At this stage, his Ph.D. degree claim is highly doubtful and raises concern about the credentials of Andreas Kalcker to hold such title.
The main question that can arise is on which expertise Dr. Kalcker discusses about autism, parasites, blood-brain barrier and nutrition? The author of this critique has 11 years of scientific research experience in the blood-brain barrier, 15 peer-reviewed publications.
The gut-brain axis is still a fairly new concept in the BBB field. Up to now, there is only one study that have demonstrated the beneficial effects of gut microbioma on the BBB development during gestation 10 and requires more studies to further confirm this single report. Furthermore, ASD diagnosis and mechanisms of disease have highly progressed since the original retracted publication of Wakefield and colleagues. It is now a consensus that ASD is triggered by two major factors: a genetic and an environmental factor11-14.
The current consensus is the predominance of the genetic factor that set the risk of ASD development and different factors in particular exposure to environmental toxins may trigger the onset of the condition. This second aspect is very interesting, as the penetration of such toxins across the BBB is poorly understood and believed that the presence of efflux drug transporters and phase II metabolism enzymes would void the penetration of such compounds across the BBB and target neurons. Such statement is supported by the ability of the BBB to act as a very strong barrier towards xenobiotic (drugs and toxins), we estimate than less than 5% of current drugs are capable to cross the BBB. The presence of such BBB is a main challenge for drug delivery 15, 16. However, scientific literature yet has to demonstrate how such environmental polluants mar the BBB and how they may affect brain development during gestation that leads to the ASD onset.
Therefore, we can reasonably ask the following question:

  1. On which scientific basis Kelly Rivera supports the claim of parasitic infection? There is no published scientific literature supporting her claim.
  2. Furthermore, under which expertise and scientific literature Dr. Kalcker built his theory on the improper digestion?

According to the Merriam-Webster Dictionary (http://www.merriam-webster.com/dictionary/theory), the definition of theory is “the analysis of a set of facts in their relation to one another”. Neither Kerri Rivera nor Dr. Kalcker have the credential to set a theory because there is no scientific facts to support their theory.

Therefore their tentative to explain their rationale is deeply flawed and should be considered as wrong until a significant number of studies with the adequate scientific quality and neither Rivera or Dr. Kalcker have demonstrated the credentials to exercise a diagnosis or establish a treatment regimen and are legally unlicensed to practice medicine (diagnosis) or pharmacy (treatment) and may face severe legal issues to do so.

The most compelling fact of Rivera and Dr. Kalcker are their active participation in the sell of MMS and CD as a treatment from autism. Such behavior is a clear sign of conflict of interest, a modern form of snake oil sell and a deliberate act of poisoning. Such misuse of public trust and poisoning has lead to the arrest of Dr. Kalcker in Spain in 2014 as reported by the bancdmms website (http://www.bancdmms.com/#!about1/c157n) as well as pro-MMS groups.

In conclusion, until now there is not direct evidence of a gut-brain axis interaction triggering ASD is until now a fallacious statement. There is no clear evidence of such statement, only a series of meticulous cherry picking studies from predatory journals and retracted articles. The direct evidence of gluten and casein peptides in ASD patients is weak and doubtful and would requires a substantial re-evaluation of such claims until other independents research groups demonstrates similar outcomes under controlled conditions.

Furthermore, the etiology of ASD as presented by Rivera and Dr. Kalcker is pure fallacy as none of them have the expertise, credentials and the scientific evidence to make such claims but also have deliberately ignored a sustained and solid publication records concerning the diffusion of peptides across the BBB and the etiology of ASD as a neurological disorder with a high genetic background (supplemented by an environmental factors).

Because the etiology of ASD at this time remains elusive, the treatment of ASD by medication remains until now undocumented, even using pre-clinical models. Only an early diagnosis and intervention by behavioral therapy have been proven successful to improve behavioral and social outcome in ASD patients.

Using common tactics of pseudoscience to distract a non-scientific literate audience, Rivera shows her ability to build an argument on fallacious statements with a an obvious conflict of interest (the endpoint is to sell her MMS/CD cure), as well as a documented harmful outcome of such treatment.

References:

  1. Reichelt KL, Tveiten D, Knivsberg AM, Bronstad G. Peptides’ role in autism with emphasis on exorphins. Microb Ecol Health Dis 2012; 23.
  2. Shattock P, Whiteley P. Biochemical aspects in autism spectrum disorders: updating the opioid-excess theory and presenting new opportunities for biomedical intervention. Expert Opin Ther Targets 2002; 6(2): 175-83.
  3. Hunter LC, O’Hare A, Herron WJ, Fisher LA, Jones GE. Opioid peptides and dipeptidyl peptidase in autism. Dev Med Child Neurol 2003; 45(2): 121-8.
  4. Shattock P, Hooper M, Waring R. Opioid peptides and dipeptidyl peptidase in autism. Developmental Medicine & Child Neurology 2004; 46(05).
  5. Mantovani JF. Not knowing. Developmental Medicine & Child Neurology 2003; 45(02).
  6. Wakefield AJ, Murch SH, Anthony A, Linnell J, Casson DM, Malik M et al. Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children. Lancet 1998; 351(9103): 637-41.
  7. Hsiao EY, McBride SW, Hsien S, Sharon G, Hyde ER, McCue T et al. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 2013; 155(7): 1451-63.
  8. Lister J, Fletcher PJ, Nobrega JN, Remington G. Behavioral effects of food-derived opioid-like peptides in rodents: Implications for schizophrenia? Pharmacol Biochem Behav 2015.
  9. Banks WA. Peptides and the blood-brain barrier. Peptides 2015.
  10. Braniste V, Al-Asmakh M, Kowal C, Anuar F, Abbaspour A, Toth M et al. The gut microbiota influences blood-brain barrier permeability in mice. Sci Transl Med 2014; 6(263): 263ra158.
  11. Fakhoury M. Autistic spectrum disorders: A review of clinical features, theories and diagnosis. Int J Dev Neurosci 2015.
  12. Correia C, Oliveira G, Vicente AM. Protein interaction networks reveal novel autism risk genes within GWAS statistical noise. PloS one 2014; 9(11): e112399.
  13. Pinto D, Delaby E, Merico D, Barbosa M, Merikangas A, Klei L et al. Convergence of genes and cellular pathways dysregulated in autism spectrum disorders. American journal of human genetics 2014; 94(5): 677-94.
  14. Rossignol DA, Genuis SJ, Frye RE. Environmental toxicants and autism spectrum disorders: a systematic review. Transl Psychiatry 2014; 4: e360.
  15. Cucullo L, Aumayr B, Rapp E, Janigro D. Drug delivery and in vitro models of the blood-brain barrier. Curr Opin Drug Discov Devel 2005; 8(1): 89-99.
  16. Abbott NJ. Blood-brain barrier structure and function and the challenges for CNS drug delivery. Journal of inherited metabolic disease 2013; 36(3): 437-49.

 

Categories
Junk Sciences Neurosciences Sciences Uncategorized

[Neurosciences/Aluminum] Does the latest paper from Exley show a link between ASD and aluminum?

Someone brought my attention today about the most recent Exley paper out in the press titled “Aluminium in brain tissue in autism” (the title could have been better but well….) and published in the journal “Journal of Trace Elements in Medicine and Biology“.
Let me put this straight, this is not a paper that has evidence of scientific fraud or data manipulation. There is no duplicated images, no suspicious blots. The problem I have with this paper is its deep experimental flaws and data analysis that nonetheless should not have passed through the peer-review filter.

  1. Before we dive into the paper, lets put the paper into context
    Lets just put the paper in the context. It was received on October 26th (Thursday). Came back in its revised form on November 21st on Tuesday and accepted for publication on November 23rd (Thanksgivings for the US, but since the editor-in-chief (EIC) is in Europe no Thanksgiving here). Let that sink it a bit: in a bit more than three weeks, it got send to review, came back from review and got revised in 26 days. In my standard of reviewing for journals and publishing my papers, thats some faster-than-light peer-reviews. I usually wait 4-5 weeks by the time I submit mine and get the editor reply to my submission with the infamous reviewers comments. Does a fast-reviewed manuscript means a bad manuscript? Not necessarily, but it can mean that maybe the peer-reviewed was not optimal, rushed or even worse just botched. Based on the quality of the data presented, I am leaning towards a botched review. Thats quite disappointing because the journal holds a decent impact factor (~3 for 5-year impact factor) and you expect an okay review.
    Then comes another problem. Exley published this paper (as well as few others) in the journal…..in which he holds a seat in the editorial board. Nobody can exclude the possible conflict of interest. Consider that: if you were an EIC, would you provide the same rigor and objective decision on a paper submitted by a colleague sitting in your editorial board than a paper submitted by Doe and colleagues?
    Not forbidden, but if you can avoid it, avoid it. Transparency is key and publishing in diversified journals (unless it is society-official journals) is an indicator of an healthy research.
    Finally, the last thing to keep in mind before I deconstruct the paper is the funding source. According to the acknowledgment section “The research is supported by a grant from the Children’s Medical Safety Research Institute (CMSRI), a not-for-profit research foundation based in Washington DC, USA.”  Behind the fancy name is just another anti-vaccine foundation that will play “the vaccine safety” card to peddle their pseudosciences. So we can claim that Exley is a shill for CMSRI, since he received monetary support for his research. Does that mean the research is completely bogus? No, but it means it will require further scrutiny, especially when the claim of the study goes against the consensus in the field (aluminum in vaccines is safe).
    Same goes if a study funded by Big Tobacco claimed the absence of correlation between lung cancer and smoking or if Big Sugar claimed the absence of correlation between type 2 diabetes mellitus and consumption of sweetened beverages.
  2. So what is wrong with this paper?
    For those who wants to read the paper with me, you can download it here (I assume it is open-access, so you should not have an issue with the paywall). Exley has a publication record on aluminum, especially when it comes to its possible ecotoxicity and the impact of aluminum on certain biological processes.
    The introduction is damn short, half a page of a double-spaced document but set the tone, this study will investigate the relationship between autism and aluminum in the brain.
    Samples are obtained from the Oxford Brain Bank, but felt short to indicate the source of the tissue (like a catalog number) and how this source of materials was complying with the institutional review boards (IRB). Basically, for any research involving human subjects or human tissues, you have to comply with the IRB that such specimens are used for a certain and defined use and foremost been anonymized.
    We have 5 patients that were diagnosed as on the autism spectrum and immediately we can pinpoint an important issue: there are no controls and that’s one of the big and unforgiving flaw of this paper.
    The authors then used two techniques to localize and quantify the Al in different cortical regions (and sometimes hippocampal regions). They have used three technical replicates (random sampling from the same cortical lobe) for measuring the Al content using an atomic absorption spectrometry and used lumogallion (aka4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzene-1-sulphonic acid, a fluorescent dye initially described to localize Al in plant roots). This dye have an excitation/emission spectra close from FITC/Alexa Fluor 488. It has been also used for live cell imaging , in particular to study how macrophages process Al present in vaccines adjuvants (http://www.sciencedirect.com/science/article/pii/S0022175915001222).
    Considering the equipment mentioned in the method, the microscope used provides the right excitation bandwidth filter and provide a long pass emission filter for anything over 510nm.Then things get weird, in the result sections, the authors mention the following:”We examined serial brain sections from 10 individuals (3 females and 7 males) who died with a diagnosis of ASD and recorded the presence of aluminium in these tissues (Table S1).“Where is the number coming from? Why don’t we have the same numbers in the Materials and methods?The other problem is the over interpretation of the data. To be brief, the lumogallion will show some punctuated pictures. The authors show some brightfield pictures overlapping to show the tissue structure but does not really help the reader. A DAPI stain (to stain cell nuclei) as counterstain would have been much more informative, it would helped to distinguish background noise from possible Al inclusion. Again, keep in mind we have no controls. The other issues with immunostaining is the high risk to cherry pick the data. You will be naturally inclined to show the presence of a positive risk but this cannot be used for quantitation. Thus, the use of the second method is welcomed as a complementary technique.
    For those not familiar with fluorescence, there is an important notion to keep in mind when analyzing the data: ensuring you keep the same exposure time, the same brightness or contrast and foremost have a negative control to set your exposure time. You can see a sketch explaining here on one of my fluorescence staining (based on my data, I concluded the expression was weak if not negative).

    Slide2

    The background subtraction is also a bit weird. I acknowledge the assessment of autofluorescence is a good control, but you expect to see a low staining. But foremost, you cannot overlap two distinct slices, as proximal as it can be. For instance, in Figure 1, you see some lumogallion staining and below the fluorescence  from the “control” using the adjacent slice. The lumogallion also seems to have a very high background.
    Picture1
    It seems lipid-rich environment increase dramatically the fluorescence of lumogallion (if you look at the spectra, the dissolution of the dye in Triton-X100 solution (b, a detergent) dramatically increase the excitation and emission spectra compared to water (a)).
    What I found troubling is this sentence in the results section: “We examined serial brain sections from 10 individuals (3 females and 7 males) who died with a diagnosis of ASD and recorded the presence of aluminium in these tissues (Table S1). Excitation of the complex of aluminium and lumogallion emits characteristic orange fluorescence that appears increasingly bright yellow at higher fluorescence intensities. Aluminium, identified as lumogallion-reactive deposits, was recorded in at least one tissue in all 10 individuals. Autofluorescence of immediately adjacent serial sections confirmed“.
    If you are a bit a fluorescence microscopy savvy, you know that the “emission color” we see in the objective is never caught by the CCD camera. These camera have in the most majority a B&W output for the simple reason that they have a much higher sensitivity than color cameras. You can always re-create colors in the micrograph pictures using various “lookup tables” (LUTs) that will give a pseudo color based on the level of grays. This is very useful when you samples different excitation/emission channels (for instance, samples stained with DAPI and two antibodies, one conjugated with Alexa Fluor 488 and the other with Alexa Fluor 546 or further down).
    The problem inherent with fluorescence is you can make thing fluoresce or end up with a false-positive signal if you increase the light beam (usually never happens because it is set) or if you increase the exposure time of your camera (this is the most common issue). As you increase exposure, you increase the risk to capture non-specific signal like autofluorescence signals.
    The other problem here is how to explain this sudden shift from orange to yellow?  This seems more like a subjective observation than something caught on camera.  That can be due to different things. You can have some bleed-through of the dye that is normally emitting in a certain wavelength but if it is strong enough can appears in neighboring emission channels. This thing rarely happens with a good fluorescence microscope that have defined filter cubes that allows the diffusion of certain emission wavelengths (for instance, my microscope have a DAPI, Alexa Fluor 488 and Alexa Fluor 555 cubes that only let the respective emission wavelengths  with 20nm-margin error to cross through the objective and reach the camera and binocular).
    Usually, we have to deal with bleed-through when you use flow cytometry and usually is solved using fluorescent dyes latex beads and by following a protocol called “compensation” (this has the result of removing any noise and keeping only the signals).
    We cannot also exclude that such fluorescence is just an autofluorescence from lipofuscine inclusion bodies. Lipofuscin is a lipid-based compound naturally produced by our cells. It has an important concentration in the central nervous system, however it is normally cleared out by cells. Failure in the clearance of lipofuscin is associated with different diseases called “lipofucsinosis” such as Batten’s disease. Even the author admit the possible presence of lipofuscin inclusions “Intracellular aluminium was identified in likely neurones and glia-like cells and often in the vicinity of or colocalised with lipofuscin (Fig. 5).” Lipofuscin is also capable of autofluorescence, although it is more in the wavelengths matching DAPI. Lipofuscin has an excitation/emission peaks at 360 and 435nm respectively but has been reported to also show fluorescence at 510nm when excited at 488nm (https://www.sciencedirect.com/topics/neuroscience/lipofuscin).
    Compared to the lumogallion excitation/emission spectra (507/567), we cannot exclude the presence of a phenomenon called “FRET” (Fosterman Resonance Energy Transfer) in which the excitation of lipofuscin (as the microscope excitation bandwidth is 470-495nm) provide enough energy to the photons emitted by the lipofucsin to excite nearby lumogallion dyes. Because the microscope setting used in this paper has no restricted bandwidth (it let pass any photons harboring a wavelength of 510nm and more), it may explain this orange-to-yellow transition noted by the author. The presence of a DAPI nuclear stain would greatly helped to identify this region as grey matter (rich in cells) or white matter (rich in lipid-rich myelin sheets). Thus, we can legitimately questions the nature of these as it these punctae labelled as “Al inclusion” are simply lipid inclusion or some artificial noise due to the tissue processing. This is where controls come as critical, it can help you sort the signal from the noise.

     

    The second big issue with this paper is the over-interpretation of what the experimenter see. The experimenter wants to see Al inclusion in monocytes? So be it: “Aluminium-loaded mononuclear white blood cells, probably lymphocytes, were identified in the meninges and possibly in the process of entering brain tissue from the lymphatic system“. Or maybe these are astrocytes, or neurons, or microglial cells, or blood vessels….or whatever the author wants to believe in: “Aluminium could be clearly seen inside cells as either discrete punctate deposits or as bright yellow fluorescence. Aluminium was located in inflammatory cells associated with the vasculature (Fig. 2). In one case what looks like an aluminium-loaded lymphocyte or monocyte was noted within a blood vessel lumen surrounded by red blood cells while another probable lymphocyte showing intense yellow fluorescence was noted in the adventitia (Fig. 2b). Glial cells including microglia-like cells that showed positive aluminium fluorescence were often observed in brain tissue in the vicinity of aluminium-stained extracellular deposits (Figs. 3&4). Discrete deposits of aluminium approximately 1m in diameter were clearly visible in both round and amoeboid glial cell bodies (e.g. Fig. 3b). Intracellular aluminium was identified in likely neurones and glia-like cells and often in the vicinity of or colocalised with lipofuscin (Fig. 5). Aluminium-selective fluorescence microscopy was successful in identifying aluminium in extracellular and intracellular locations in neurones and non-neuronal cells and across all brain tissues studied (Figs.1-5). The method only identifies aluminium as evidenced by large areas of brain tissue without any characteristic aluminium-positive fluorescence (Fig. S1).
    This is the second big mistake of this paper. If the author wants to make the claim he proposed here, then he has the obligation to show a counterstain using selective markers for neurons (e.g. MAP2, bIII-tubulin, NeuN….), astrocytes (e.g. GFAP), microglial cells (CD11b), leukocytes (CD3), macrophages (CD45), blood vessels (e.g. PECAM-1, claudin-5). This could have been easily performed (using a secondary antibody conjugated with Alexa Fluor 555 or better Alexa Fluor 647)  and would have give support to this claim.
    If the author can identify cells by the naked eye, he is either equipped with  Superman X-ray eyes or he is just imagining things.

    The discussion quickly gets into an anti-vaxxer diatribe and throws the minimal amount of scientific data under the bus.
    For example, the author throws this sentence as is: “We recorded some of the highest values for brain aluminium content ever measured in healthy or diseased tissues in these male ASD donors including values of 17.10, 18.57 and 22.11 g/g dry wt. (Table 1).” Firstly, where does it get this data? You cannot sum technical replicates, you have to average them (even with considering the huge variability between technical replicates). Secondly, how can the author make a claim like this without providing values from controls (well there are no controls) or from the literature. It is like “we have recorded the highest amount of leukocytes in ASD patients blood samples with values of 11.3, 12.0 and 11.5 x10e3 cells/mm3.” I cannot make an interpretation or conclusion without knowing the reference from the normal population (normal range 4.5-11x 10e3 cells/mm3) or from control groups. The average Al level was 2.38-4.79 microg/g tissues in male ASD and 1.15 in the female ASD patient. Such levels were very similar to those reported in samples from patients suffering from familial form of Alzheimer’s disease.
    Slide3

    The data is interesting but we are lacking additional female samples to make a claim as he did: “All 4 male donors had significantly higher concentrations of brain aluminium than the single female donor.” He lacks the proper conditions to run the statistics (you need same number of patients in male and female to make such claims) and even the important inter-individual variability makes it unlikely that he could achieve the statistical significance. This is a statement that would put a graduate student in shame for overconfidence in the data.
    Then goes the tirade “What discriminates these data from other analyses of brain aluminium in other diseases is the age of the ASD donors. Why, for example would a 15 year old boy have such a high content of aluminium in their brain tissues? There are no comparative data in the scientific literature, the closest being similarly high data for a 42 year old male with familial Alzheimer’s disease (fAD) [19].” (another Exley paper published…..in the same journal). We are dealing with the same issues (lack of controls, huge variability in the technical replicates…..).
    Now if you plot the average patient Al levels agains the age, regardless of the condition, you end up with an homogenous cloud. Now, two things have to be noted here: seems there is no impact of Al levels based on the disease (only age seems to matter between ASD and AD) and there is no correlation between increase in brain Al and age, at least in the very small sample size.
    Data 2

    No pun intended, but the data scatter looks vaguely like the United States map. Again, it shows the need of data from asymptomatic patients to estimate the burden of Al in the brain.
    Since we have not access to Al content in the brain, we have to see some values in the literature. A study by Andrasi and colleagues (https://content.iospress.com/articles/journal-of-alzheimers-disease/jad00432) provide some Al levels in control samples. According to their study, the average Al content in control samples were between 1.4 to 2.5µg/g dry tissue. We are indeed not far from the value reported by this study, especially when you consider the important standard deviation in these samples.

    Maybe it is also to consider the other study by Exler on Al level in brain samples from patients associated with familial form of Alzheimers disease (fAD) and familial dementia. In that study, all reported with Alzheimers (some with early onset, some with late onset based on age), the Al values reported were ranging from 0.34microg/g tissue (male) to 6.55microg/g (female, presenting a mutation in the PSEN1 gene, a known gene in FAD). So are we just measuring noise and try to extrapolate data from noise? Thats some bold statement that should have been smashed already by a decent reviewer in the field of neurosciences.
    But seeing these two papers went through in a apparent free ride is not looking good for the journal integrity.

  3. Conclusive Remarks
    To make a claim is one thing, to back it up with robust data is another thing. I think Exley jumped the shark a while ago and started to aluminum as the big bad wolf in every little things. But a wolf can be tamed, kept out from showing danger to the community and somehow co-exist. But for Exley, like Shaw, like Gherardi, aluminum is the devil incarnate. God forbid it has been used for 70 years and showed barely more than simple coincidence in its association with some disease, aluminum is their dead horse that worth being beaten again and again. If your funding sponsor will give you money for showing a link between aluminum and autism, lets give them what they want. Ethically it is insane, but when you need to keep your lab and your faculty position afloat, sometimes making the pact with the devil and throwing the scientific integrity and the philosophism that is given to you  following your thesis defense can be tempting. Sometimes, it feels that anti-vaccines researchers are like Faust and succumbed to the offer made by Mephistopheles offer. But this come with a price and a hefty price to pay: the loss of your integrity as a scientist.
    So my question is what is coming next to patients on the spectrum: does this study will be used to support the anti-vaccine agenda (another reason to yell “Aluminum is a chemikillz” in parenting groups?) and breakdown the herd immunity? Bogus remedies by bleach enemas and drops (the infamous CD/MMS)? or give a support to chelation therapy? gluten-free/casein-free diet? Or like Exley once claimed have these people drink ad nauseam silicon-rich water like Fiji water or Volvic water with the magic claims that the silicon with drain your brain from the Al contained inside it?
    This kind of deeply-flawed studies, lacking proper controls and driven by an ideology over the facts are dangerous because they prey on the meek and enrich modern snake oil sellers.

 

Categories
Junk Sciences Junk Sciences Sciences Uncategorized

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

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

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

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

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

Background for experimental design flaws: PK and species issues

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

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

Selection of genes to measure:

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

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

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

22016544_10102969159317918_1868648690_n

The unaltered figure 4.

22052798_10102969159312928_192591888_n

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

Semi-quantitative RT-PCR:

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

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

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

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

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

1-s2.0-S0162013417300417-gr1_lrg

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

Slide1

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

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

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

image

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

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

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

And below the close up of Fig.5A

Slide3

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

 

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

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

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

 

Conclusions:
Based on the methods that were used in this paper, Shaw et al. went too far in declaring that aluminum adjuvants cause autism. But there are six other key points that limit what conclusions can be drawn from this paper:
1) They selected genes based on old literature and ignored newer publications.
2) The method for PCR quantification is imprecise and cannot be used as an absolute quantification of expression of the selected genes.
3) They used inappropriate statistical tests that are more prone to giving significant results which is possibly why they were selected.
4) Their dosing regime for the mice makes assumptions on the development of mice that are not correct.
5) They gave the mice far more aluminum sooner than the vaccine schedule exposes children to.
6) There are irregularities in both the semi-quantitative RT-PCR and Western blot data that strongly suggests that these images were fabricated. This is probably the most damning thing about the paper. If the data were manipulated and images fabricated, then the paper needs to be retracted and UBC needs to do an investigation into research misconduct by the Shaw lab.

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

Categories
Blood-Brain Barrier Junk Sciences Sciences Uncategorized

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

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

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

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

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

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

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

 

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

 

 

Categories
Junk Sciences Junk Sciences Sciences Uncategorized

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

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

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

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

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

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

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

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

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

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

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

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

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

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