[Sciences/BBB/Drug Delivery] Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude (Brenner et al., Nat Comm 2018)

I know, I know I have been fairly quiet. I have to tell that between attending a scientific meeting, teaching a summer course, taking care of two grants proposals and finally handling three manuscripts. However, sometimes I also like to share some papers published in the field that are interesting or bringing novel ideas or concepts. This one has been suggested by one of the author and this is his quick summary:
We bind nanoparticles to the surface of red blood cells. These nanoparticles are going to be released from the red cells once they pass the first capillary bed. Therefore we can concentrate the nanoparticles in the target tissue. In addition, these nanoparticles potentially can carry one or multiple selected drug(s). So if we inject intravenously the particles are going to be release in the lungs but if we inject through the brain arteries the nanoparticles will be release into the brain vasculature.
It is a very nice of work here published in Nature Communication (I consider this is one of the top OA journal to get published in) and you can download the full-text here.
You can also appreciate this paper likely went into at least one round of review, with a time period of about 7 months between it got accepted for review and accepted for publications.

In this paper, the authors have been working on trying to develop alternative drug delivery carrier, in that case use red blood cells as “piggyback” cells to enhance drug delivery. They tried different formulations on isolated RBCs and identified some suitable for carrying antibodies or proteins.  They called these piggybacking RBCs as RBC-Hitchiking (RH) (I wonder if it is some Easter egg towards the “Hitchiking Guide to Galaxy”).

Upon identification of the right nanoparticle materials, the authors investigated the distribution and the delivery of the conjugates in different organs, as liver and lungs. What is interesting is the amount of injected dose recovered is much higher in the RH than the free-circulating one, in particular in lungs, whether they are bare-naked or bound with a protein. These nano-carriers can be delivered to endothelial cells.

But the interesting snack-bite from this paper is the intra-arterial injection in the carotid artery, in which there was a significant increase in nano carriers delivery in the brain.  Nano-carriers alone show a %ID lesser than 1% (that is about what you expect from delivering antibodies from an IV route towards the brain)  to over 10%.   The delivery was also maintaining the ipsilateral injection site, which is good considering you are likely to treat one brain hemisphere.

Now, time for me to be picky and kind of wish we had information on the PK profile, especially if this approach increased the stability of the nanocarriers. It would be also interesting to see how this technique fare in a experimental disease model (for instance a xenograft brain tumor and see if you can deliver targeted chemotherapy in mice).

Nevertheless it is a good paper that take us out from the classical nanoparticles formulation and try here an innovative and novel approach in drug delivery.

 

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[Vaccines/Junk Sciences] “Murine hypothalamic destruction with vascular cell apoptosis subsequent to combined administration of human papilloma virus vaccine and pertussis toxin” (Aratani et al., Sci Rep 2016 – RETRACTED) Lesson from a paper that went completely fritz on the scientific method

You maybe heard about the recent retraction notice of a study published in Scientific Reports that raised concern about the safety of Gardasil(R) (HPV vaccine) and got retracted this week. Another anti-vaccine paper that bit the dust. I would have say, I am not surprised at all. Anti-vaccine studies have this very annoying habit of either being a proven fraud (remember the latest Shaw paper?), botching the experimental protocol with omission of proper controls (thats the Exley paper I have reviewed) or conveniently sweeping under the rug some data that are not fitting the narrative (that goes for one recent paper published by Gherardi).
But this one is interesting at several levels, because there is a bit of blood-brain barrier in  it, and also adds to the list of papers retraction in Scientific Reports and recent threats by scientists in the editorial board to resign from their positions due to ambiguous and unjustified decision on a flawed paper (Disclaimer: I have a study authored in this journal and I have peer-reviewed for them a couple of times).

To be honest, I only heard about this paper during the last weekend and took some times to read the paper. I will be honest, I don’t see any scientific fraud in the sense of data manipulation. What concerns me is how such a botched study could even pass through peer-review process? Considering I self-impose a quality of standard in my manuscripts and still get challenged by peer-reviewers, seeing such junk studies getting a free pass is a bit vexing. I agree that open-access may not have the same stringency in terms of peer-review filter but considering Scientific Reports as part of Nature Publishing Group, you expect a rigor found for any Nature-related journals applied in this journal too.

But lets go through the paper, it is retracted but you can still access it here.

What is the wrong with this paper?

1. The experimental design in terms of groups

The first problem arises from Figure 1.

Aratani_Fig1

We have six groups: vehicle (PBS), pertussis toxin (PTX), Gardasil(R) (MSD, HPV vaccine 4-strains) (G), G+PTX, EAE and EAE+PTX. Let’s breakdown first these oddities.  Why the author has included a PTX group, even more adding PTX with Gardasil? There is not much explanation in the text explaining the rationale (also the writing style is odd, very odd. I completely understand that the first author is not a Native English speaker. As an ESL myself, I completely understand that). Also, I am not aware about a higher risk on contracting pertussis upon vaccines.
The second aspect is the use of the EAE mouse model. EAE stands for experimental autoimmune encephalitis. It is the “gold standard” for a mouse model of multiple sclerosis (MS). The idea behind is to inject a brain protein (myelin basic protein or MBP), which will trigger an immune reaction (as the brain is a immune-privileged organ) and result in symptoms similar to MS. I would understand to compare the incidence of Gardasil(R) on MS patients by comparing EAE mice versus non-EAE mice but that is never the case (they even administer PTX to a sub-group).
So here we already start with a wrong experimental design: it just make no sense. A more rationale approach would have been the following:
Vehicle (PBS), Gardasil (G), EAE, EAE+G. That would have saved two groups and precious lives sacrificed in another useless study.

2. The experimental design in terms of statistics and power of analysis

Another important issue is the blatant dismissal of consideration of biostatistics and the power of analysis in the experimental design. For those that are not familiar with scientific research, you have to ensure you have a statistical meaning to your data to ensure the effect observed is real and not due to simple coincidence. This statement is especially true when working with vertebrates animals. Any animal experiment has to be approved by the institutional IACUC that ensure you have a clear idea of what are the purpose of your experiments, how you will ensure a humane treatment to animals and also have an optimal number of animals to achieve a statistical significance.
An important aspect for in vivo (animal) studies is to achieve at least a sample size of 8 or more animals per group (n=8). From Figure 1a, we have already a violation of this as only the G and the G+PTX have enough animals (n=14 and 21 respectively). All other groups are below the n=8 threshold. In addition, you have very different number of animals per groups (control has n=6, EAE has n=5) making the statistical power weak and also restricting the use of common statistical methods such as the use of ANOVA (ANOVA recommends that all groups have an equal number of samples).

3. The experimental procedure and treatment

This is where the firestorm came in: the experimental data. So lets bring this to the table: “Groups of 11 week-old female C57BL/6 mice were intramuscularly administrated 100 μ l of Gardasil or phosphate-buffered saline (PBS) for a total of five times. Ptx was intraperitoneally administrated 2 and 24 hours after immunization. The Gardasil vaccine or Ptx were administrated at 2- weeks or 4-week intervals“.
A key element in a paper is the methods section and this one utterly failed. Based on this information I have absolutely no idea why they injected five times (Gardasil immunization is maximum 3 times), why they injected the PTX right after the immunization (2 and 24 hours, suggesting a double-induction) and when they injected the Gardasil and PTX (how do they separate the 2-weeks versus the 4-weeks? When did they started?).
Also the use of 11 week-old female is not reflective of a human case scenario. If we approximate 1 human year to about 3.6 mice-days, you would expect to use young mice (males and females, to have a gender-balanced study) that are about 6-weeks old (~43.2 days). That would be about 12 years, the age of puberty.
Here we are basically injecting HPV to adult females, which is known to not provide an additional benefit as such population may have already been exposed to HPVs.
The next problem is the injection dose. It is 100microL, thats the equivalent of 0.1mL. A single dose of HPV is 0.5mL. Lets ignore the scale law and assume a mouse is an equivalent to a human. A 50th percentile weight at age 12 is about 40kgs. Lets assume a 6-weeks old mice to be about 20g.
If we assume 0.1mL injection to a 20g mouse, then the human dose-equivalent would be about 200 dose-equivalent injected at once! This is a serious issue because there is absolutely no chance that such things to happen in a lifetime (at grand maximum you may have 3-4 HPV injections, spaced in time). Also, if we assume the age scale, we are expecting to have mice receive their two doses within 48 to 96 hours, not 2 to 4-weeks.
I am not even entering the rationale to inject PTX right after immunization, which is utterly no-sense and just scramble defining the effect of HPV versus the effect of PTX. Another disastrous example of how this paper was flawed from the beginning.

4. Failure to report weight and clinical score

If we want to follow an EAE protocol, it is important to show the evolution of animal weight over a period of times (up to 15-21 days) as well as a clinical score. The clinical score is a well-described protocol in which features found in EAE mice are score from mild (tail flaccid) to severe (inability to move hindlimb or complete immobility).
These two graphs are almost present in any EAE paper outside in the literature.
I assume this is what Figure 1b and 1c wanted to show but very poorly. Indeed Figure 1c does not really show up anything. We dont know when these data have been taken, we have no idea about the onset time of symptoms and furthermore we have no indication of a statistical differences. This is already a waste of data.

5. The constant cherry-picking of the data and incomplete picture

The methods used are honestly laughable: some hematoxylin-eosin staining (a common histological stain that does not tell much unless you have massive brain damage or the growth brain tumor),  Kluver-Barrera staining (for myelin staining), TUNEL stringing (for apoptosis), a behavioral test relegated in Supplementary Figure S1 (in which the author thinks that a P-value of 0.1 has a statistical meaning).

Aratani_Fig2

Aratani_FigS1
Where is the Evans blue extravasation staining to show a disrupted BBB? Where are the GFAP staining to show astrocytes activation? Where are the CD11b and F4/80 staining to show microglial cells activation and macrophages infiltration? Nowhere to be seen. We have to comptent ourselves with some miserable histological staining in Figure 2 and 3. Also no-one of the data about EAE is never shown past Figure 1. Figure 4 is even more laughable as the author only shows the staining of the G+PTX, giving the middle finger to the reader to how such staining looks like in vehicle, or G.

Aratani_Fig3

How can the author be confident that it was the Gardasil treatment, not the PTX treatment (despite being mentioned as a BBB disrupting toxin) being the sole contributor of all this?

6. Conclusions

Another anti-vaccine study, another case of botched science resulting in a junk paper, the sacrifice of animals over a useless experiment. That should not have been passing through the peer-review filter at all because of its deficiencies, yet was able to go through. If I was the reviewer behind it, I would have been ashamed to have this paper not outright rejected for major flaws in the study. Should we assume that the author recommended some complacent reviewers  to this paper? Or should we question the integrity of the editorial board in accepting papers for publication that fail to address some scientific integrity? Again, anti-vaccine studies shows that they cannot challenge vaccine safety and can only make fool of themselves by producing junk studies like this time.

The first and senior authors of this paper produced a paper that is so bad, they should feel ashamed to even had published at first.

 

[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.

 

[Stroke/Junk Sciences] Does a needle can save you from a stroke injury? No! No! No!

Some of you have seen this video going around, claiming you can save someone suffering from stroke injury using a needle. The idea behind this video, according to HealthyChoices365, is that a Chinese “professor” claimed this will save the person’s life following a stroke.
This is kind of the thing that, as a basic scientist in the field, boils me for the last few days. First, it is plain quackery. The needle prick has nothing to do with the stroke event: it is distal from the site to have an effect. Second, using this technique on a patient has a direct impact on the patient’s stroke outcome and recovery. Let me explain why this is bullshit and should be called for what it is: A gazillion pile of bullshit that has much more weight that all the coal West Virginia has and had since the geological formation of that region (no pun intended, W. Va has one of the highest number of stroke per capita in the US, since it is Xmas season the lump of coal is simply appropriate).
In brief, stroke is the 5th cause of death in the US (3rd amongst women) and a leading cause of disability. We have two types of stroke: ischemic (85%) and hemorrhagic (15%) with the later accounting for 40% of stroke-related deaths. We estimate that about one US citizen will experience a stroke event every 5 minutes.

1. Stroke 101: Back to basics
In the ischemic stroke, we have a clot (usually formed at the carotid artery bifurcation) that is formed due to the presence of atherosclerotic plaques. These plaques can become unstable and crumble over time. These crumbles are made of clot that navigate through the carotid artery that irrigate the brain. Such clot will act as a plug or a cap, once it reaches a vessel with a diameter smaller than the clot, it will occlude it and block the blood flow.
This create what we call an ischemic situation. In such ischemic situation, the brain is deprived of both oxygen (20% of all oxygen is wired to the brain) and nutrient (in particular, glucose. The brain accounts for about 25% of the total glucose level utilization in the whole body). Neurons are the most sensible brain cells to stroke injury. They cannot adapt to hypoxia (lack of oxygen). Few minutes of hypoxia is enough to cause severe and irreversible brain damage. We estimate about 1 million neurons die every minutes that a stroke is left untreated.
Furthermore, neurons are post-mitotic cells. They cannot divide anymore. When a neuron is gone, it is gone, as well as its neuronal circuitry. You see, each minute matters because what is lost is lost.
Stroke signs can be resumed by the “FAST” acronym: Face droop, Arm weakness, Speech issues, Time to call 911. By the time you are showing signs, it has been already a couple of hours your brain has been starving off glucose and oxygen. It is important that once you have the signs to call 911 and asked the paramedics to direct you to the closest stroke center.
The most important thing to happen in stroke diagnosis is to determine which type of stroke the patient is undergoing: ischemic or hemorrhagic? These two are very different and confusing one with another can have a deadly effect. You don’t want to give a clot-buster to someone with hemorrhagic stroke because it will make the bleeding worse. You don’t want to give a clotting agent to a patient with ischemic stroke because you will increase the risk to develop a second stroke.
The current procedure is the use of endovascular intervention: the neurosurgeon insert a catheter in the femoral artery and using an angiography method to see blood vessels “live on screen” reach the site of stroke injury to either remove the clot or to put a stent in place to stop the bleeding process. From discussing with a physician, this takes about 10-15 minutes once the patient is in the OR.

2. Why this video is BS and should be called BS:
Now, lets see why I call this video BS.
First, the idea of finger prick to treat stroke is BS. We are trying to act on the stroke from a remote site. The thing is, the clotting process occurs in a very local fashion. So trying to act on a stroke with pricking a finger with a needle is mostly useless.
Second, as I said, it is important to know which type of stroke we are treating. You cannot identify which type of stroke is involved just by the clinical signs. You need imaging (CT scan or MRI) to be able to distinguish ischemic stroke from hemorrhagic stroke.
Third, this useless procedure is a formidable waste of time on the patient. As we said, each minute lost is a precious minute lost that will condition the outcome and the recovery. How long should we waste before calling 911 because we noted no improvement: 15 minutes? 30 minutes? 60 minutes? By the time the patient realized this intervention is bogus, his/her chance to survive and recover from the stroke injury are almost close to zero.

To conclude, let me finish this post with a call: PLEASE! PLEASE! PLEASE! Whenever you or a loved one is showing the FAST signs, CALL 9-1-1!!!! Know your nearest hospital with a certified Stroke Center and have the paramedics bring you there. THERE IS NO THERAPY FOR STROKE! OUR BEST BETS ARE PREVENTION (80% of stroke events can be prevented) AND INTERVENTION (by keeping the “door-to-bed” to a minimum).

[Sciences/BBB] Why the vitamin K shot in newborn matters

I have seen the topic of vitamin K (VitK) shot coming over and over in various discussion groups, with some parents weighing the need of the VitK in newborns. One of the main argument in favor for the injection of VitK in newborn is its ability to reduce the risk of cerebral bleeding (cerebral hemorrhage).
I thought a post on this topic would provide a great help in understanding the physiological role of VitK, the consequence of brain hemorrhage and conclude on the importance of the VitK shot.

1.What is the vitamin K?

Vitamin K is a fat-soluble vitamin that is mostly obtained by our gut microbiota and accessory from our food intake (in particular leafy greens and liver).

220px-Phylloquinone_structure.svg

During gestation, the fetus obtains it from the mother, as such vitamin passes through the placenta barrier. Vitamin K plays an important role through its biochemical cycle called “the Vitamin K cycle”. Vitamin K can convert glutamyl residues present in proteins into gamma-carboxylglutamyl residues as depicted in the picture below:

F1.large

Such modified glutamyl residues are present in particular set of proteins called “coagulation factors”. These coagulation factors are important pieces of what we refer as the “coagulation cascade”.
400px-coagulation_full-svg

I know this graph is complicated but what we care here is the final part of the cascade. The presence of intrinsic damage or trauma, we have the activation of several coagulation factors. Amongst those that are VitK-dependent, we have factor VII (seven), IX (nine) and X (ten). Prothrombin, upon activation by factor X  is converted into thrombin, which in turn cleaves the soluble fibrinogen into the insoluble fibrin. Fibrin acts as a mesh and forms a fibrin clot that will patch the bleeding area. This is an important physiological response when you rupture a blood vessel. The coagulation cascade will create a clot that will stop the bleeding process, saving you from a risk of loosing too much blood and entering an hypovolemic shock. One organ is particularly sensible to brain bleed, this organ is the brain.

2. Brain hemorrhage: small numbers, big damage

In this section, I will mostly discuss about brain bleeds in regards of hemorrhagic stroke but you can apply the same pathophysiology to brain bleeds induced by brain trauma. Brain bleeds are the second type of stroke. They account for about 15% of total stroke events, but account for 40% of stroke-related deaths.

subarachnoid-800x416

We have different types of brain bleeds. In stroke, we usually have a type of brain bleed called “intracerebral hemorrhage” (ICH) that happens deep inside the brain. There are other types of hemorrhage called “sub-arachnoid hemorrhage”. In that case, the brain bleeds occurs in the sub-arachnoid space, a space between the brain and the skull. This type of bleed results into an ischemic stroke (due to a lack of blood perfusion in blood vessels beyond the bleed site) and a brain swelling (resulting in the crushing of the brain tissue due to increase intracranial pressure).

During the injury heme (from damaged astrocytes, neurons and red blood cells) is released in the extracellular space. Heme is a very strong pro-oxidant molecule resulting in the formation of radical oxygen species (ROS) such as anion superoxide (O2*-) and hydrogen peroxide (H2O2), which in turn further induce oxidative stress and cellular damage.

The major type of cells that suffers of such damage at the greatest extent are neurons. Neurons are highly sensible to such injury and unlike other cell types neurons do not divide anymore (post-mitotic cells). A dead neuron is a dead neuron. There are some studies suggesting a possible regeneration of neurons in certain brain regions in rodents (mice, rats), yet the presence of an evidence pointing out at similar mechanism in humans are yet to be demonstrated. Furthermore, there is still no evidence that stem cells (including cord blood stem cells from umbilical cord) can provide a repair of such brain region following injury.

As of today, a dead neuron is a dead neuron. The ability of a damaged brain region to recover is very limited.

3. Why Vitamin K shots?

As we just have explained here, we know that VitK is essential in coagulation and we also understood the impact of brain bleed on the brain. Thus, reducing such brain bleed can be done in the short-term by the induction of the coagulation cascade.
As we mentioned, babies get their VitK from the placenta, but by the time they are born, they are already coming with a low VitK. We also mentioned that the VitK is primarily produced by the gut microbiota. It will take weeks if not months for babies to get a gut microbiota that is functional enough to produce the VitK (I speculate that such microbiota is not present until the age of 12 months when baby eat a diet similar to adults). We can speculate that food (breast milk or baby formula) should provide a source of VitK but providing a steady and standardized intake from dietary is near impossible to achieve.
Furthermore, there is no lab tests or techniques that can predict the onset of a brain bleed. Furthermore, brain bleed has a very high mortality rate and very high morbidity rate including cerebral palsy and other brain damage.
Therefore, ensuring a source of VitK right at birth is the best approach to ensure the baby has enough VitK to have a functional coagulation cascade. In case of a brain bleed, we can expect to have a rapid response of the body to ensure a emergency clotting process ongoing until the doctors can intervene and stop such bleeding to happen and clean any possible brain bleed.
This is why it is important to opt-in for a VitK shot. Once a brain tissue is damaged, there is no evidence yet that there is regeneration of such area. Neurons do not divide anymore by birth and there is no evidence yet of stem cells (including stem cells from cord blood) able to repair such damage.

 

[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.

 

[Sciences/BBB] Acute Necrotizing Encephalitis (of childhood), a blood-brain barrier perspective.

This is a blog post following a request by a page follower on my Facebook account to provide an “layman” perspective on acute necrotizing encephalitis (ANE), also referred as acute necrotizing encephalitis of childhood (ANEC). This is a very short and surely incomplete summary but it should be a great starter to give the current perspective of this condition through the lens of the blood-brain barrier.

It is a condition that was firstly discovered by Mizuguchi and colleagues in 1995 firstly described in infants and toddlers (http://jnnp.bmj.com/content/jnnp/58/5/555.full.pdf). It was firstly described in patients from Asian origin (Japan). It was initially described to occur during the winter period, in particular with region that had experienced an influenza A outbreak. The main clinical feature of the disease marked by the presence in the magnetic resonance imaging (MRI) of increased water content inside the brain, mostly associated with edema (brain swelling). This increased water content can only be explained by the opening of the blood-brain barrier.

Water diffusion between the blood and the brain is tightly regulated by the blood-brain barrier (BBB). The BBB provides two kinds of barrier: a physical barrier (by the presence of tight junctions) and a chemical barrier (by the presence of solute carriers and drug efflux pumps). The case of water as a molecule (H2O) is very interesting. Water is a very small molecule (the molecular weight is 18g/mol or also 18 Daltons) but also a very polarized molecule. Hydrogens and the oxygen atoms forming H2O are not completely neutral, hydrogen carries a tiny positive-charge and oxygen carries two tiny-negative charges (we refer in chemistry as electronegative charges). Think about having a tiny magnet. In the opposite, cell membranes are made of phospholipids. As their name say, they are lipids by definition or what we commonly call them as “fatty acids”. Lipids have a distinct composition, they are mostly formed by carbons and hydrogens. Carbon is not much a magnet atom, it neither likes to carry positive charges nor negative charges. This is why lipids are commonly referred as apolar molecules. Now, polar and apolar molecules behave like water and oil mixed together: they simply do not mix and will sequestrate themselves, usually forming a oil droplet surrounded by water. Water entrance inside the brain is believed to occur mostly via paracellular route, as depicted in the picture below (source: http://www.nature.com/nrn/journal/v7/n1/full/nrn1824.html?foxtrotcallback=true).

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Tight junctions are very tights, letting water fall through the cracks only in a tiny amount. Imagine having a very good rooftop that only let water fall through one drop every hour. The problem with the opening of the BBB following various factor is the massive entrance of water. Think about having a hole in your rooftop and facing a tropical storm shower outside: you are facing now a massive and unregulated entrance of water inside the brain, leading to a brain swelling.
In peripheral tissue, edema (swelling) formation can naturally expand, resulting in a swollen tissue. The problem with the brain is its anatomical structure: it is encased inside a rigid shell (skull) that has no exit route for the penetrating water. This results in an increased pressure inside the brain (we usually referring as increase in intracranial pressure or ICP). This increased pressure induce a mechanical stress, crushing brain cells via mechanical stress and ultimately neuronal cell death. Such swelling appears to occur in specific brain regions, with a primary lesion site in the gray matter (neurons), with persistent deposition of hemosiderin and white matter (axon fibers) cysts during and after the recovery phase. Until now, we don’t exactly know what cause such disease, but appears as following a viral infection including flu (influenza A and B, swine flu (H1N1), parainfluenza virus), varicella, measles, rubella and various herpesviruses (HHV-6, HHV-7) (https://www.hindawi.com/journals/mi/2015/792578/#B1), although the presence of such viral agents (detection by polymerase chain reaction) in spinal tap as well as post-mortem signs of brain inflammation remains anecdotal.

Interestingly, it seems that patients suffering from ANE undergo a very severe immune response commonly referred as “cytokine storm”, as several studies noted an increase in inflammatory markers (in particular interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha (TNF-alpha) making this phenomenon the most prevalent hypothesis.

Immune cells communicate to each other via a common language called “cytokines”. Cytokines are like a “RED ALERT” system, they signal some breach in security or incoming danger.
Brain microvascular endothelial cells (BMECs) lining the blood side of the BBB can also understand the “cytokine” language and understand such signal as “RED ALERT – OPEN THE BBB SIGNAL” as depicted in the picture below (source: https://www.researchgate.net/profile/Nicolas_Weiss/):

Now where are these cytokines coming from and how they are triggered? It is a very good question. This is where the viral infection comes in. I will not details much about the immune response to viruses, but you can ask @TheMadVirologist for any questions related to viruses. For this I will use a figure that resume the immune response to viruses (source: https://www.researchgate.net/profile/Francoise_Stoll-Keller/).

figure-1-function-of-dendritic-cells-in-the-immune-response-to-virusesfollowing-the

Upon infection, infected cells will display viral particles on the cell surface and will also secrete a protein called “interferon-gamma”. This is a sort of cellular “SOS Danger” to the immune system. Natural killer cells, dendritic cells and macrophages may start the early response, also known as “innate immunity” to contain the viral infection. In addition, free circulating viruses can be spotted by B cells through their array of surface antibodies and trigger what we refer to as “acquired immunity”. Viral infection will trigger an immune response and we can think that maybe an overactive immune system may exaggerate the danger resulting into the “cytokine storm”. Another hypothesis is that such cytokine storm maybe triggered by natural killer (NK) cells.

This hypothesis is further supported by the presence of a higher count of natural killer leukocytes in ANE patients during the recovery phase. Natural killer (NK) cells are immune cells normally targeting cancer cells and cells infected by viruses.  This “cytokine storm” maybe the causative agent of the blood-brain barrier disruption (BBB) by different mechanisms (source: http://stroke.ahajournals.org/content/strokeaha/42/11/3323/).

but appears to occurs via an matrix-metalloproteinase (MMPs) dependent pathway. Under the stimulation of such cytokines, brain endothelial cells and astrocytes may increase the production and releases of MMPs locally. These MMPs act as little scissors that can chop the extracellular matrix supporting brain endothelial cells and astrocytes end-feet processes. In addition, these MMPs can also chop tight junction proteins that are involved in tight junction (TJ) complexes. These TJs are very important as they provide the barrier limiting the diffusion of water and solutes between the blood and the brain.

In addition to the cytokine storm hypothesis, it seems that other factors maybe involved in the pathophysiology of the disease. Until now, Ran binding protein 2 (RANBP2) (http://www.cell.com/ajhg/fulltext/S0002-9297(08)00630-7). RANBP2 is a protein involved in the nuclear pore complex, yet the relevance of this mutation at the blood-brain barrier remains unknown. In neurons, it is associated with cellular structures different from the cell nucleus, in particular it is associated with mitochondria (power house of cells) and microtubules.

Another protein of interest associated is EphB2, a receptor for ephrins (https://www.ncbi.nlm.nih.gov/pubmed/?term=ephb2+blood-brain+barrier). Ephrins play an important role in brain wiring during development (axon guidance) but also play a role in the formation of the vascular tree.

The function of EphB2 and ephrins at the blood-brain barrier remains unclear. However, a recent study identified the expression of EphB2 at the cell surface of endothelial cells including primary human non-BBB (HUVECs) and BBB (HBMECs) endothelial cells. Furthermore, a case report from a patient suffering from systemic lupus erythromatous (SLE), an autoimmune disorder, presenting the case of ANE showed the presence of antibodies in the serum capable to bind selectively to EphB2.

Yet, at this point we don’t know if this antibody binding is enough to trigger the BBB disruption or it requires the recruitment of immune cells to trigger such disruption.