[BBB/Alzheimer] Study: Blood-Brain Barrier Leakage in Patients with Early Alzheimer Disease

An interesting article came today by the research group of Walter H. Backes, PhD published in the journal Radiology further strengthen the connection between Alzheimer’s disease pathogenesis and blood-brain barrier (BBB).

In this study, the authors investigated changes in paracellular leakage using gadolinium as a contrast agent and post-processing algorithms. They observed an increased vascular leakage to gadolinum in patients diagnosed with an early stage of AD compared to matched control patients.

This is a fairly observational study but again strengthen the link between BBB dysfunction and AD. Yet the mechanism leading to such dysfunction remains unclear and if such leakage is a cause of AD or rather a consequence of it.

 

Source: Radiology: Blood-Brain Barrier Leakage in Patients with Early Alzheimer Disease

[Metal] Lacuna Coil – Delirium (85%)

Today Lacuna Coil released their 9th album entitled “Delirium” today. Earlier this year, Cristina and Andrea announced that this album will be much heavier than their more recent release.
At their 18th year anniversary of their freshman album “In Reverie” and an important change in the line-up, how does the band stands with this 9th album?
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Well, you gonna have to read this post to get my thoughts (after keeping it in loop in my office and in my car the whole day). The short answer? It is heavy and loud, much heavier than their previous releases.
The long answer? It is a good album, much more metal than their previous release but still I wish they went further back to their roots that made their first albums “In A Reverie”, “Unleashed Memories” or “Comalies” such a great experience. Because the album have a heavy thematic on asylum and mental insanity, I have decided to cluster the album into different categories of mental illnesses.
The album starts with “House of Shame”, certainly one of the best songs of the whole album. It feels like suffering from a maniaco-depressive episode, being shaken by Andrea brutal tones nicely reflecting  the guilt and shame that someone undergo when hit by depression episode. Andrea symbolizes the darkness and hostile feelings you get during a depressive. Such episode is nicely alternating with the clean and angelic voice of Cristina and spartan musical arrangement giving this nice feeling of euphoria. With Cristina, we have wings of euphoria, heading to the lights of heaven. This stark contrast between Andrea and Cristina nicely play on the bipolar aspect.
The asylum visit continues with “Broken Things”, “Delirium”, “Take Me Home”, “You Love Me ‘Cause I Hate You”, “Live To Tell” that are representative of the Korsakoff syndrome: the ability of someone to remember the past and very old events but cannot recall recent events. It is like relistening to the early years of Lacuna Coil and kind to occulting their more recent release. Oh man, listening to “Downfall” and “Claustrophobia”  made me feel so good to listen to the Lacuna Coil I fell in love during my college years. That Lacuna Coil that was playing on gothic metal thematics of melancholy and went gone into memory lane as they watered down their artistic direction to be more accessible as I  went further down the rabbit hole into metal Wonderland.
Some tracks are made to feel being into a schizophrenia panic like “Ghost in The Mist”, “My Demons”, “Ultima Ratio” with voices and hallucinatory sounds assaulting your mental self-consciousness. It puts your auditory cortex into a rollercoaster ride that you cannot predict the next turn or loops you will experience. Being shattered between contradictory thoughts and cognitive processes.
The album overall is good, it is damn heavy and yet fairly accessible. My own personal bias is that such album sounds perfectly suited for being aired on the US radios, it has this sound of hard rock/metal that you grow accustomed to listen on the US FM stations.
However, this also mean you have several songs that sounds the same with little variations, giving the feeling of deja vu, especially a certain number of songs repeating the magic formula of “House of Shame” that saturate your hippocampal region and caudate nucleus fast enough.
Nevertheless, this album gets the title of “Album of the Month” for really brining memories from my youth, when I was discovering the voice of Anneke (The Gathering) and got introduced to Cristina via a bootleg CD of “Unleashed Memories”.

[Neurosciences/BBB] Thiomersal and the blood-brain barrier: where does the science stand?

I decided to write down this article as one follower of this page asked me several questions about the blood-brain barrier (BBB), the scientific rationale of a “leaky” BBB and whether such claims were applicable for delayed vaccines schedule, with a special emphasis on ethylmercury and ethylmercury precursor “thiomersal” or “Thimerosal”.

Following a series of discussions through social media messaging, I thought it would be good to compile the science behind it and share the information.

 

1.What is the difference between inorganic mercury and organic mercury and why we used it for vaccines?

The first part of this post will focus on setting the science on mercury chemistry, understand the different forms and why we used one form of it inside vaccines.

Mercury (Hg) is considered as a heavy metal due to its high molecular weight (~200g/mol). It is also a particular type of metal called transition metal and it is known for its particular feature of being a liquid metal at room temperature.

Mercury exists as different ionized forms, such as Hg+ (as a bimercury form +Hg-Hg+) and Hg2+, the latter being the most commonly found, in particular as the inorganic mercury form HgCl2.

In addition to its inorganic form, mercury is also found as an organic form, either chemically bound to a methyl (CH3-) group (methylmercury or CH3-HgCl) or to a ethyl (CH3-CH2-) group (ethylmercury or CH3-CH2-HgCl).

Because of its bactericide activity, mercury has been used for a long time in medicine, with use of inorganic form of mercury (cinnabar) in Traditional Chinese Medicine. It was also used as a treatment of syphilis (from the 15th century until the discovery of penicillin) and as a dental amalgam for both its bactericidal properties and its compliance and longevity to the buccal environment compared to resin-based fillings.

Various organic formulation of mercury has disinfection of superficial wound (merbromin, also known as Mercurochrome) and as a preservative for vaccines (thiomersal or thimerosal, both names refers to the same compounds). The structure of thiomersal is the following:

512px-thiomersal-svg

Inside the body, thiomersal is metabolized into ethylmercury:

640px-ethylmercury-2d-svg

as well as elemental mercury (as HgCl2 form)

Elemental mercury (Hg), as any other heavy metals (lead, cadmium….) is highly toxic as it has a long biopersistence (it stays a long time inside the body) and by its chemical properties.

Inorganic mercury (as its HgCl2) is considered as the most toxic heavy metal, and classified as highly to extremely toxic (LD50 = 1mg/kg in rats via oral route, source: http://www.rsc.org/images/health-concerns-heavy-metals-and-metalloids_tcm18-210187.pdf). Methylmercury LD50=24-30mg/kg) is higher than inorganic mercury (LD50=23-30 mg/kg with some age variability in rats, with the highest tolerance noted in young rats  (Lin, Malaiyandi et al. 1975), whereas ethylmercury has an LD50 of 40mg/kg (http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=N11940&brand=SUPELCO). Thimerosal has the highest LD50 of all of them, with an LD50 of 70mg/kg (https://www.nwmissouri.edu/naturalsciences/sds/t/Thimerosal.pdf).

Inside the body, thimerosal breaks down into various species but classically follows these metabolic steps as recently documented by Carneiro and colleagues (Carneiro, Oliveira Souza et al. 2014). In this study, the authors have injected a single intramuscular dose (20micrograms) of thimerosal and followed the accumulation inside different organs as well as the fate of it. They found that thimerosal rapidly accumulated into the kidney (30 minutes after injection) as ethylmercury and inorganic mercury forms. 70% of thimerosal metabolites were found in the kidney and reported the lowest concentrations in the brain. They also reported some pharmacokinetic parameters including half-lives (the time you need to remove 50% of a chemical out of your body) of 10.7 days and 45.8 days for the brain and kidney tissues respectively.

Thimerosal has been in vaccines since the 1930’s following the tragic death of 12 children during an diphtheria immunization campaign in 1928. Such deaths were due to tainted batch of vaccines that were preservative-free and following an expert panel, such lot was contaminated by a live strain of staphylococcus. (http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/VaccineSafety/UCM096228). Thiomersal concentrations in vaccines have been variable, with reported values oscillating between 0.001% to 0.01% (0.01g thimerosal/100mL solution). At such concentration, a vaccine shot (0.5mL) contains about 50 micrograms of thimerosal (~400g/mol) which represent about 25 micrograms of mercury element (~200g/mol). A common practice in modern medicine is to phase out and replace older generations with newer generations of medicines that have shown a better efficacy and/or lesser side effects.

Thiomersal has been used for almost one hundred year in vaccines with no reported fatalities or morbidities with the exception of the retracted Lancet article from Andrew Wakefield that was claiming a causal link between autism and MMR vaccines (the original publication can be viewed here: http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(97)11096-0/abstract).

There are several flaws in this studies that are too lengthy to explain in this post and that have been already refuted many times by different studies. However, for a lay person one important criteria in a scientific study involving population study is the sample size (or n). It tells you how many individuals have been enrolled or followed in this study. In the Wakefield study, this number was 12. This is a very small number, especially if you are using it to make bold statements. There is a strong bias with small numbers because you may have a very homogenous group based on their age, gender, ethnicity and geographical localization. These different parameters matter when you deal with statistics and try to identify if a phenomenon or feature in a population is real or it if it just some background noise.

To give you an idea, the latest meta-analysis that investigated the relationship between autism and MMR vaccines by Taylor and colleagues (Taylor, Swerdfeger et al. 2014) in which by comparing studies done in over 1’000’000 children (in other means 100’000 times the sample size of the study by Wakefield) failed to find any association between thimerosal and autism, as well as any association between mercury compounds and increased incidence of autism, as well as no association between the administration route and increased risk of autism.

Why this matters? Because size matters. What can maybe true in a small population may not be true when you are expanding the population size and diversify the population studied. This is a common feature that happens in clinical trials. Clinical trials are the crucial steps for assessing the efficacy of a novel medicine. It is divided into three phases. In Phase I, we assess the toxicity and side effect of a drug candidate as well as its fate inside the human body, usually we are talking about a small number of healthy volunteers (n=12-20). This is usually following pre-clinical studies done in animals that already provided us with some information. If there is any severe side effects noted, the drug is immediately phased out.

After assessing the safety, clinicians proceed to Phase II trials in which a small number of patients are recruited and split into two groups: placebo (sugar pill) and treatment (drug candidate). Note that most of the case neither the patient nor the physician know which group they have been assigned. This is what we refer to the double-blind study. Only when the experimentation timeline is completed that the identity is revealed. Upon conclusive Phase II, a drug candidates move into Phase III in which we have now hundreds of patients distributed worldwide that receive the drug candidate and compare to placebo. This is where often a promising drug candidate that have shown some encouraging results in Phase II fails to show the same effects when the population size is increased.

This is why sample size matters, it allows to increase the statistical power to sort real and predictive effect from just pure luck or from a lucky strike.

2. The “leaky” blood-brain barrier in the newborn: fact or fiction?

A common thematic that comes from certain anti-vaxxers or those promoting a delayed vaccination schedule is that the newborn and infant blood-brain barrier is not fully mature and leaky, thus may present an increased risk to absorb and retain mercury or any other chemicals included inside vaccines formulation.

So, in this section we will dive deeper into that question and see what the current science says about it. For this section, I will rely on existing reviews on that topic that have been published in the last five years (Liebner, Czupalla et al. 2011, Ek, Dziegielewska et al. 2012, Lippmann, Al-Ahmad et al. 2013, Obermeier, Daneman et al. 2013).
Before we dig into the concept of developmental blood-brain barrier, it is important to define what is a blood-brain barrier (BBB).

The blood-brain barrier (BBB) is a component of the neurovascular unit, mostly found in brain microvessels.

As illustrated in the picture below, the BBB is formed by a multicellular unit formed by specialized brain microvascular endothelial cells (BMECs) lining the brain blood vessels. These BMECs makes tightly firmed cell-cell junctions, as noted by the presence of tight junctions (TJ) complexes that strictly restrict the diffusion of water, ions and small solutes between the blood tissue and the brain compartment.

For molecules to enter the brain, they have to be either very lipophilic (soluble in fat and oils) or have a set of carriers that can transport in and out the two compartments.

Even being a lipophilic compound is not sufficient to guarantee a transport as there is an array of drug efflux pumps that highly limit the diffusion across the BBB. We estimate about 95% of drugs cannot cross the BBB.

The BBB is a feature we refer as induced as pioneering studies using in vivo (animal studies) and in vitro  (Janzer and Raff 1987, Tao-Cheng, Nagy et al. 1987) have shown that astrocytes can induce such barrier properties in both endothelial cells isolated from brain microvessels and from non-brain microvessels. For a long time, we thought that astrocytes were the only key players in the induction of the barrier function. In addition, other studies that investigated anatomical and functional features noted changes between brain microvessels from embryonic stage compared to post-natal stage animals (Senjo, Ishibashi et al. 1986, Butt, Jones et al. 1990, Keep, Ennis et al. 1995, Kniesel, Risau et al. 1996) suggested that the fetal BBB was probably immature and that it gets the maturity only during late in gestation, when astrocytes differentiate from their precursor cells, undergo maturation and wrap brain microvessels with their astrocytic end-feet process.

However, more recent studies and techniques using genetically engineered animals (knock-out mice) found that indeed such BBB may be already present as pericytes, another cell types lining the vasculature has shown to play a role as important as astrocytes. Unlike astrocytes, these pericytes are already present during development much earlier than astrocytes (Braun, Xu et al. 2007, Virgintino, Girolamo et al. 2007, Daneman, Zhou et al. 2010, Al Ahmad, Taboada et al. 2011).

By the 3rd trimester of gestation, the developing fetus has already a functional BBB and capable to work as well as the adult BBB, in particular such concept has been extensively discussed by Pr. Norman Saunders (University of Melbourne, VIC, Australia)  that have been discussing that the misconception of a leaky BBB in developing brain was the consequence of inappropriate or flawed techniques that wrongly brought such conclusions (Saunders, Ek et al. 2011, Ek, Dziegielewska et al. 2012, Saunders, Liddelow et al. 2012, Saunders, Dreifuss et al. 2014, Saunders, Dziegielewska et al. 2016, Saunders, Habgood et al. 2016).

3. Mercury and the blood-brain barrier: what do we know?

As we have been discussed now, mercury exists into different forms and we have a functional BBB in both the newborn and the adult. So our next step is to understand and use the existing literature to assess the ability of mercury to cross the BBB.

In this post, we are discussing about thiomersal and its two major metabolites: ethylmercury and inorganic mercury. Another form of organic mercury that is problematic and see often and wrongly associated to ethylmercury is methylmercury.
Unlike ethylmercury (CH3-CH2-HgCl), methylmercury (CH3-HgCl) is a form of organic mercury that has serious toxicity issues.

Methylmercury is formed by bacterial metabolism from the biotransformation of inorganic mercury present in the environment into methylmercury. In particular such transformation is occurring preferentially in aquatic systems (Ullrich, Tanton et al. 2001). Unlike ethylmercury, methylmercury is a potent environmental pollutant from industrial processes that have shown ability to bioaccumulate in fish through the food chain and by the relative important half-life of methylmercury (~72 days in aquatic environment).

The uptake of methylmercury occurs through the L-system amino-acid transporter-1 (LAT-1, SLC7A5) both at the human intestinal wall and at the BBB (Wade and Brady 1981, Mori, Yamamoto et al. 2012).

An important caveat that needs to be noted is the relative paucity of the literature in regards of the BBB uptake of the different forms of mercury. Such caveat maybe explained by the relative poor in vitro models of the BBB lacking the tightness needed for studying the diffusion of metals across the BBB.

Two studies worth citing and further discussion: the study from Toimela and collagues (Toimela, Maenpaa et al. 2004) using a co-culture models based on immortalized cell lines and the study from Lohren and colleagues  co-authored by Pr. Hans-Joachim Galla (University of Muenster, Muenster, Germany) (Lohren, Bornhorst et al. 2015) that investigated the effect of mercury on the blood-CSF-barrier (CSF: cerebrospinal fluid). The blood-CSF-barrier (or BCSFB) is the second barrier of the central nervous system located in the choroid plexus.

In the study of Toimela and colleagues using RBE4 monolayers (an immortalized rat brain endothelial cell line), the authors found an impaired BBB function (as marked by increase in fluorescein permeability) for concentrations of HgCl2 and CH3HgCl of 100 micromole/L and 0.1micromole/L following 24 hours treatment. Cell toxicity were noted for HgCl2 and CH3HgCl on SH-5YSY (an immortalized human neuroblastoma cell line) concentrations of 10 micromole/L and 1 micromole/L respectively (24 hours incubation). Toxicity of HgCl2 and CH3HgCl were noted for values of 10 micromoles/L for both respectively.

In contrast, the study by Lohren and colleagues investigated the effects of various concentrations of elemental mercury (HgCl2), methylmercury (CH3HgCl) and ethylmercury on primary choroid plexus epithelial cells. Following an incubation of 72 hours in presence of different concentrations, the authors found a decrease in cell viability for HgCl2, CH3HgCl and CH3CH2HgCl for concentrations of 40, 6 and 2 micromoles/L.
To put into context, we have to remember that the average vaccine content in thimerosal is 50 micrograms for an average volume of 0.5mL.

If we consider that the intramuscular injection yields a 100% bioavailability (all the thimerosal injected ends up in the systemic circulation) and consider such injection in a newborn (~200mL blood), the final circulating maximal circulation following a vaccine shot would be 50/200=0.25microgram/mL or 0.25 mg/L or (0.25/400) = 0.00625 mmol/L, that is equal to 0.625micromol/L. In the worst case scenario situation (one-shot injection in a newborn with a consideration that thiomersal remains non-metabolized), the peak plasma concentration is still three times less than those shown to have in vitro. Now you have to remember, thiomersal is rapidly degraded into ethylmercury and elemental mercury.

Now another interesting piece of data from the study is the relative absence of diffusion of elemental mercury across the BCSFB and the presence of an active efflux for both methylmercury and thiomersal. In other words, the BCSFB may act as a drainage system for both thiomersal and methylmercury, diminishing their probability to accumulate inside the brain tissue.

Finally the last study that is worth discussing in another study from Lohren and colleagues (Lohren, Blagojevic et al. 2015) using  immortalized human neuronal cell line LUHMES (http://www.atcc.org/products/all/CRL-2927.aspx) and astrocytes isolated from a glioblastoma multiforme (primary brain tumor patient CCF-STTG1 (http://www.atcc.org/products/all/CRL-1718.aspx). This is one major point of criticism is how reliable can be tumor derived cell lines to assess cell toxicity and viability (unless you are screening for novel anticancer drugs).

Again, this study supports that organic mercury have a higher cell toxicity than the elemental mercury, with about 10-fold increased toxicity in thiomersal compared to elemental mercury. Toxicity values were in the range of 1 micromole/L for neurons and 8 micromole/L for astrocytes. You have to keep in mind such toxicity was achieved for a prolonged (24 hours) and direct insult. As we have discussed, the amount of thiomersal or methylmercury reaching these neurons and astrocytes is surely much less as we know by now that both the BBB and BSCFB hinder the penetration of both molecules.

Thus the main source of concern comes from methylmercury rather than thiomersal or ethylmercury. Again, both studies failed to measure the toxicity of ethylmercury, that is the major thiomersal metabolite.

If expecting mothers and new parents would have to worry about, it is certainly methylmercury toxicity. Therefore, the primary source of methylmercury uptake that is of concern is uptake from fish and its effect on health. In particular, methylmercury is a legitimate cause of concern for gestating women and for infants.

The Food and Drug Administration (FDA) in collaboration with the Environmental Protection Agency (EPA) has written down an exhaustive report on the current situation of the impact of methylmercury on the brain development in the US and in other countries, such report can be downloaded here (http://www.fda.gov/downloads/Food/FoodborneIllnessContaminants/Metals/UCM396785.pdf).
A take home message from the report are multiples. The average methylmercury concentration in US fish consumption is about 0.072 ppm (particle per millions), that’s the equivalent of saying 0.072mg/L or 72micrograms/L. The CDC upon EPA recommendation set methylmercury levels for drinking water to 2 parts per billions (ppb) or 2micrograms/L and from fish and seafood to 1ppm or 1mg/L (source: http://www.atsdr.cdc.gov/toxfaqs/tf.asp?id=113&tid=24). Unless someone eat astronomical amount of fish and seafoodon daily basis, the levels of methylmercury should not be a concern for expecting mothers and for infants.

4. Conclusion and summary

In this post, I have been covering the possible neurotoxicity of thiomersal on brain development. Several take home messages to take from this post:

1) Thiomersal introduction about 100 year ago was crucial to ensure vaccine safety and sterility, but as science progress thiomersal became an obsolete preservative and as early as 1997, the FDA was developing a framework to phase it out from the market. As today, only certain forms of dTaP vaccine has thiomersal. If thiomersal

2) The association between thiomersal and neurodevelopmental disorders as suggested by Andrew Wakefield has been since refuted by a number of meta-analysis. Of course, anti-vaxxers will not change their mind in front of evidence and either called a conspiracy or decided to move their goalpost to other vaccines components (such as the aluminum used in adjuvants, that will be discussed in another post, or formaldehyde).

3) Contrary to some logical fallacy, the concept of “leaky” blood-brain barrier in infants is not supported anymore. This concept was considered in the light of science about 30 years ago but since modern techniques refuted this concept. Therefore, using such outdated concept to refuse or delay a vaccination schedule is not only fallacious but clearly underlie a serious issue of continuous medical education (CME) from healthcare professionals proponent of refusing vaccinations or promoting a delayed schedule (CME as part of an important continuous education to ensure healthcare professionals are up-to-date with scientific knowledge, such CME are earned as credits by attending national and international conferences and symposia, with a minium number of credits to be fulfilled within a certain period).

4) The nature of the mercury discussed is very important: elemental mercury is not methylmercury. Methylmercury is not ethylmercury. Thiomersal has shown some toxicity in vitro but it remains important to put into context. However, pharmacokinetic data suggest that thiomersal is rapidly degraded into ethylmercury and inorganic mercury, the latter being the least neurotoxic), as well as the presence of defense mechanism at the BBB and BCSFB.

5) If there is one form of mercury that is non-occupational that expecting moms should worry about is the methylmercury found in fish and seafood. However, the beneficial gains of such food category rich in poly-unsaturated fatty acids (PUFAs, such as omega-3 and omega-6) is essential for ensuring the proper fetal brain development. A equilibrated diet with the advice of the reputable dieticians and physician will ensure the optimal use of fish and seafood without having to worry about detrimental effects due to methylmercury.

 5. References

Al Ahmad, A., C. B. Taboada, M. Gassmann and O. O. Ogunshola (2011). “Astrocytes and pericytes differentially modulate blood-brain barrier characteristics during development and hypoxic insult.” J Cereb Blood Flow Metab 31(2): 693-705.

Braun, A., H. Xu, F. Hu, P. Kocherlakota, D. Siegel, P. Chander, Z. Ungvari, A. Csiszar, M. Nedergaard and P. Ballabh (2007). “Paucity of pericytes in germinal matrix vasculature of premature infants.” The Journal of neuroscience : the official journal of the Society for Neuroscience 27: 12012-12024.

Butt, A. M., H. C. Jones and N. J. Abbott (1990). “Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study.” J Physiol 429: 47-62.

Carneiro, M. F., J. M. Oliveira Souza, D. Grotto, B. L. Batista, V. C. de Oliveira Souza and F. Barbosa, Jr. (2014). “A systematic study of the disposition and metabolism of mercury species in mice after exposure to low levels of thimerosal (ethylmercury).” Environ Res 134: 218-227.

Daneman, R., L. Zhou, A. A. Kebede and B. A. Barres (2010). “Pericytes are required for blood-brain barrier integrity during embryogenesis.” Nature 468(7323): 562-566.

Ek, C. J., K. M. Dziegielewska, M. D. Habgood and N. R. Saunders (2012). “Barriers in the developing brain and Neurotoxicology.” Neurotoxicology 33(3): 586-604.

Janzer, R. C. and M. C. Raff (1987). “Astrocytes induce blood-brain barrier properties in endothelial cells.” Nature 325: 253-257.

Keep, R. F., S. R. Ennis, M. E. Beer and A. L. Betz (1995). “Developmental changes in blood-brain barrier potassium permeability in the rat: relation to brain growth.” J Physiol 488 ( Pt 2: 439-448.

Kniesel, U., W. Risau and H. Wolburg (1996). “Development of blood-brain barrier tight junctions in the rat cortex.” Brain Res Dev Brain Res 96: 229-240.

Liebner, S., C. J. Czupalla and H. Wolburg (2011). “Current concepts of blood-brain barrier development.” Int J Dev Biol 55(4-5): 467-476.

Lin, F. M., M. Malaiyandi and C. R. Sierra (1975). “Toxicity of methylmercury: effects on different ages of rats.” Bull Environ Contam Toxicol 14(2): 140-148.

Lippmann, E. S., A. Al-Ahmad, S. P. Palecek and E. V. Shusta (2013). “Modeling the blood-brain barrier using stem cell sources.” Fluids Barriers CNS 10(1): 2.

Lohren, H., L. Blagojevic, R. Fitkau, F. Ebert, S. Schildknecht, M. Leist and T. Schwerdtle (2015). “Toxicity of organic and inorganic mercury species in differentiated human neurons and human astrocytes.” J Trace Elem Med Biol 32: 200-208.

Lohren, H., J. Bornhorst, H. J. Galla and T. Schwerdtle (2015). “The blood-cerebrospinal fluid barrier–first evidence for an active transport of organic mercury compounds out of the brain.” Metallomics 7(10): 1420-1430.

Mori, N., M. Yamamoto, E. Tsukada, T. Yokooji, N. Matsumura, M. Sasaki and T. Murakami (2012). “Comparison of in vivo with in vitro pharmacokinetics of mercury between methylmercury chloride and methylmercury cysteine using rats and Caco2 cells.” Arch Environ Contam Toxicol 63(4): 628-636.

Obermeier, B., R. Daneman and R. M. Ransohoff (2013). “Development, maintenance and disruption of the blood-brain barrier.” Nat Med 19(12): 1584-1596.

Saunders, N. R., J. J. Dreifuss, K. M. Dziegielewska, P. A. Johansson, M. D. Habgood, K. Mollgard and H. C. Bauer (2014). “The rights and wrongs of blood-brain barrier permeability studies: a walk through 100 years of history.” Front Neurosci 8: 404.

Saunders, N. R., K. M. Dziegielewska, K. Unsicker and C. Joakim Ek (2016). “Delayed astrocytic contact with cerebral blood vessels in FGF-2 deficient mice does not compromise permeability properties at the developing blood-brain barrier.” Dev Neurobiol.

Saunders, N. R., C. J. Ek, M. D. Habgood, P. Johansson, S. Liddelow and K. M. Dziegielewska (2011). “Assessing blood-cerebrospinal fluid barrier permeability in the rat embryo.” Methods Mol Biol 686: 247-265.

Saunders, N. R., M. D. Habgood, K. Mollgard and K. M. Dziegielewska (2016). “The biological significance of brain barrier mechanisms: help or hindrance in drug delivery to the central nervous system?” F1000Res 5.

Saunders, N. R., S. A. Liddelow and K. M. Dziegielewska (2012). “Barrier mechanisms in the developing brain.” Front Pharmacol 3: 46.

Senjo, M., T. Ishibashi, T. Terashima and Y. Inoue (1986). “Correlation between astrogliogenesis and blood-brain barrier formation: immunocytochemical demonstration by using astroglia-specific enzyme glutathione S-transferase.” Neurosci Lett 66: 39-42.

Tao-Cheng, J. H., Z. Nagy and M. W. Brightman (1987). “Tight junctions of brain endothelium in vitro are enhanced by astroglia.” J Neurosci 7: 3293-3299.

Taylor, L. E., A. L. Swerdfeger and G. D. Eslick (2014). “Vaccines are not associated with autism: an evidence-based meta-analysis of case-control and cohort studies.” Vaccine 32(29): 3623-3629.

Toimela, T., H. Maenpaa, M. Mannerstrom and H. Tahti (2004). “Development of an in vitro blood-brain barrier model-cytotoxicity of mercury and aluminum.” Toxicol Appl Pharmacol 195(1): 73-82.

Ullrich, S. M., T. W. Tanton and S. A. Abdrashitova (2001). “Mercury in the Aquatic Environment: A Review of Factors Affecting Methylation.” Critical Reviews in Environmental Science and Technology 31(3): 241-293.

Virgintino, D., F. Girolamo, M. Errede, C. Capobianco, D. Robertson, W. B. Stallcup, R. Perris and L. Roncali (2007). “An intimate interplay between precocious, migrating pericytes and endothelial cells governs human fetal brain angiogenesis.” Angiogenesis 10: 35-45.

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[Neurosciences/Junk Sciences] MTHFR, folate, folic acid, spina bifida, autism…..Oh my!

Lately a lot of media attention has been revolving about folic acid (or folate which is strictly the same I will explain to you later) and I have seen a lot of worried and less worried mommy bloggers panicking about folate or obviously making comments about being diagnosed with MTHFR mutation, that folic acid is not folate, that the former is the “all-natural” and the latter is not absorbed, that folate-supplemented food is “evil”.
Add to it the recent oral communication presented at the International Society of Autism Research (ISNAR) Annual meeting in Baltimore that suggested an association between excessive folate consumption and increased risk for autism (as reported by ScienceDaily here) that was blown out of context in mainstream media (because of course mainstream media has a fond for blowing up single studies and junk science as fact).
You have the perfect storm for a major sanitary disaster to happen and the right condition for quacks to prey on panicking expecting mothers.
I feel it is important that we desacralize and demystify the “folate/MTHFR” scare using science and facts to better understand folate and folic acid, the physiological function of folic acid, why folic acid is essential and have been added to wheat and corn flour and why you should not stop taking folic acid without recommendation of your physician and pharmacist.

1. What is folic acid?
Folic acid  is also known as folate or vitamin B9. Some people will argue that it is not the same molecule. The fact is folate=folic acid, even Sigma-Aldrich (one of the main chemical manufacturer for scientific research) agree on me with this term (Sigma-Aldrich technical data sheet can be found here). Both share the same chemical identification number known as CAS (CAS-59-30-3).
The only difference is subtle and has to do whether you are referring to the non-ionized form (folic acid) or the salt (or ionized form, folate) form of folate. To understand it, you have to have a basic in organic chemistry but if you remember your high school science class, you should be able to understand the chemical structure below.2000px-folat-svg
As you can see we have two carboxylic acid groups (COOH<->COO- + H+) and one basic primary amine (NH2 + H+ <->NH3+) that is conjugated with two neighboring secondary amines.
If you remember your chemistry class, carboxylic acids are what we call weak acids, same for amines (weak bases). Each weak acid and base has a particular  feature called the pKa value. This pKa value is important as  giving the properties of a pH buffer solution. When pH=pKa, we have a 50%/50% ratio between ionized and non-ionized form and this gives the buffer properties of a solution. Most biological solutions have a defined composition that results in a particular pH. Most body fluids (with some exception such as stomach), have a pH defined by various chemicals. In blood and tissular fluid, this pH is set at 7.4. You should go slightly below (pH=7.3) or over (pH=7.5) and you will enter into an acidosis or alkalosis situation that can kill you if your body does not remediate. This is why the idea of alkaline diet or trying to alkalinize your body with lemon juice (an acid made mostly of citric acid) is completely useless and based on junk science (I have been discussing this aspect of junk physiology in an earlier post).
The pKa of folic acid/folate is about 3.37. At intestinal pH (~5.5), you are roughly 2 pH units over the pKa of folic acid, that means 99% of folic acid will be under form of folate and will harbor its two carboxyl groups as COO-.

2. What is folate biological function?

Folate is actively transported from the intestinal lumen and converted into different metabolic intermediates but usually will end up as 5-methyltetrahydrofolate (5-MeTHF).

gr2b1

Like any vitamin, Vitamin B12 plays an important function as a co-factor. You don’t need much of it, but you need it to have some enzymes to work. Without it, enzymes cannot work and you may develop severe vitamin malnutrition. For instance, a lack of vitamin A (beta-carotenoid) is a important cause of Vitamin A deficiency resulting in children blindness in South East Asia (this is the raison d’être for the genetically-modified rice or “golden rice” that can provide a steady Vitamin A supply in the region). This is also the case for vitamin C (ascorbate), an important co-factor for prolyl-hydroxylase, an enzyme synthesizing collagen that form your connective tissue. Without it, you end up with scurvy.

In the case of 5-methylfolate (named mTHF in the graph below), it is a pivotal cofactor in several biochemical reactions as depicted below:
nrc3557-f3

mTHF carry a very important chemical called methyl (CH3-) group. This methyl is given to the homocysteine that becomes methionine, an important amino acid. This methionine can in turn serve as a co-substrate for methyltransferase to transfer such methyl into an another molecule. Why methylation matters? Because methylation is a key element of epigenetics (epi=over). Epigenetics is a branch of genetics that studies how living organisms can modify DNA without altering its sequences. Mutations affect the DNA sequences, epigenetic do not affect the sequences. It is the same than putting your smartphone into a lock position. If you don’t enter the password, it will remains locked.
It is a way for the living organisms to switch off certain DNA regions that they do not need anymore for their function. This explains why you have nerve cells, blood cells, muscle cells, bone cells despite having the same DNA sequence in all of them. This explains why some bees will become queen and the rest will remains workers, foragers or soldiers. This explain why your hair become grey over time.

Remember the methyl group that was for donation? Here we go, it goes into one nucleotides forming our DNA, cytosine. Once added, cytosine becomes 5-methylcytosine.

zakhari01

3. What about folate and spina bifida?

Now you may have heard about folate deficiency and spina bifida. Folate plays an important role during embryonic development, as it will serve as a co-factor for various enzymes responsible for the neural tube closure.
The neural tube is an important embryonic structure as it will give rise to the central nervous system. In particular, there is a step that is in crucial need for folate, it is the closing of the neural tube (step D) on both the rostral (head) and the caudal (tail) region, to close the brain and spinal cord. Folate deficiency impairs such closure and results in two dramatic and lethal congenital malformations: spina bifida and anencephaly.

 

tmp1411

Because such condition can be prevented by ensuring a minimum daily folate uptake, the Food and Drug Administration adopted the enrichment of wheat flour with folate in 1995.
As you can see, upon introduction of folate-enriched flour, we can see a net decrease in the number of cases of spina bifida:
usa_spina_bifida_rates

However, this public health intervention was missing an important population at risk for folate deficiency, the Latin-American community, as corn flour rather than wheat flour is their staple food. The recent FDA approval to enrich masa corn flour should help to reduce such levels.

4. MTHFR mutation: Fact or fad?

In recent years, we have seen a sudden urge in number of persons claiming to have an MTHFR mutation and claim all their issues related to such mutation including fatigue, dizziness and other ailments. The problem encountered is the recent development of genetic testing is a wild wild West in which modern snake oil sellers claim to have accurate genetic testing . Science-based Medicine has done an extensive job to debunk this fallacy and novel type of quackery currently flourishing.
To be concise, I will say that MTHFR polymorphism is not an important concern for most individuals. However, it can be a serious risk for mother to be and pregnant women as their MTHFR enzyme maybe working at slower pace than normal MTHFR and therefore impair proper methionine metabolism. Because patients become aware of such mutation only after having an history of miscarriage or abortion due to spina bifida or anencephaly diagnosis during ultrasound maternal screening and constitute only a small population (~10% of the US population), the supplementation with folic acid is an easier and safer intervention.
If you want to know more about MTHFR mutation, please visit the adequate page at the National Institute of Health (NIH).

5. Folate supplementation and autism risk: what you should know

Asia was reported in the news, a recent study presented in a scientific conference associated an excessive folate intake with an increased risk of autism. Let it be clear, this is an oral presentation presented in front of other scientists at the meeting. This has not been processed through peer-review yet. So we have a lot of unknown and in particular we have to assess the quality of the data.
I want to be clear about this: if you are pregnant and on folate supplementation, do not change your treatment without having your physician (real physician, not a DC or a ND. A real physician in a real hospital) or pharmacist recommendation. By stopping your folate supplementation, you are putting your pregnancy at risk to develop a spina bifida or anencephaly that so far exceed the risk of autism.
This is also a grim reminder on a lot of supplements prescribed by quack doctors and naturopaths may have not the quality control to ensure you receive the correct amount of folate supplementation and you may have a risk of overdosing on it. Do not consider such supplement harmless and ask your physician and pharmacists to direct you and counsel you for the right folate dosing and source.

 

 

[Junk Sciences] Is reviving brain dead patients with stem cells a scientific breakthrough or just another Dr. Frankenstein Monster fantasy?

I am sure that everyone heard about that clickbait headlines that run over on social media, with viral posts and videos proposed by a company named “Bioquark” (US) in association with another company named “Revita Life Sciences” (India). Among some websites that have reported that information, I decided to share the article posted by Futurism earlier this week:
http://futurism.com/brain-dead-brought-back-life-thanks-new-biotech-experiments/

Of course, my first reaction was surprise and shock. Surprised because no one came as far to make a claim on resurrecting dead people.  Oh well, not surprised if you look at fictions they only time I heard people resurrecting dead were with various methods including electrical energy (Frankenstein’s Monster), trioxin 245 (Return of the living dead), cybernetic implants (Robocop) or some classified scientific methods codenamed “Lazarus project” (Mass Effect 2).
Because with extraordinary claims comes extraordinary proof and evidence. Usually this does not end up very well and usually ends up flat like an underbaked souffle. Just see the epilogue of Theranos and remind yourself to never sell the bears hide before killing it.

What is being “brain dead”?

Brain death is defined according to the Merck Manual by “a loss of function of the entire cerebrum and brain stem, resulting in coma, no spontaneous respiration, and loss of all brain stem reflexes. Spinal reflexes, including deep tendon, plantar flexion, and withdrawal reflexes, may remain. Recovery does not occur.”  (source: http://www.merckmanuals.com/professional/neurologic-disorders/coma-and-impaired-consciousness/brain-death).
This definition is very important as it is inclusive of both absence of cortical function as well as vegetative functions emanating from the brain stem. Brain stem is an important nervous structure that controls all the vegetative functions including control of the respiration, bowel movements, blood pressure, pH and smooth muscles activity. When a patient in such condition, this is a point of no-return. There is virtually no chances patient recover. The patient seems alive because the patient is on respiratory aid and still has the heart beating. The heart will keep pumping as long it is properly perfused with oxygen and nutrient.
One test to assess the brain status of a patient in comatose stage is to assess its ability to breath by its own. This is what we refer the apnea test. The human body is designed that it will by survival reflex induces breathing even when we force apnea. In these patients, the loss of brain stem viability will fail to induce this respiratory reflex.
In the case of this news, we will have to look at the application packet submitted to the NIH clinicaltrials.gov portal that can be consulted here.

According to the application, the companies have in their inclusion criteria:
Individuals declared Brain dead from a traumatic brain injury having diffuse axonal injury on MRI Not willing for organ donation
Written informed consent from the legally acceptable representative of the patient

This is an important criteria for ethical standpoint, because these volunteers fall into the uniform anatomical gift act (http://www.uniformlaws.org/shared/docs/anatomical_gift/uaga_final_aug09.pdf).

Because they can considered as anatomical gift, there is little or no of the ethical issues commonly raised with living and healthy patients, that require absolutely an institutional review board (IRB).
In the press release, the company  or companies as it involves Bioquark and Revita Life Sciences claim they receive approvals from the Institutional Review Boards and by the National Institute of Health to perform their experiments on volunteers that donated their bodies to science and that refused to sign as potential organ donors.
It is also important to note this trial will be performed outside the United States, especially held into the Anupam Hospital, Rudrapur City, Uttrakarand State in India (based on the email address of the collaborator listed on the trial).
This is an important issue to raise, as we have these trials performed in India. India has currently a fairly laziest policy in terms of ethics in biomedical research as raised recently by an article in the World Health Organization (WHO) referenced here: http://www.who.int/bulletin/volumes/86/8/08-010808/en/.

Although running experiment on clinical dead persons is by itself not an ethical violation as long as the body is utilized and treated following medical and ethical consideration, we cannot however fully guarantee that these donors and their relatives have provided their consent into a fully informed and without any financial incentives.

What is the science behind the trial?

The second issue that I have with this study is the science behind it. In their application, the authors listed the outcomes as the following:

Primary Outcome Measures:

  • Reversal of brain death as noted in clinical examination or EEG [ Time Frame: 15 days ] [ Designated as safety issue: No ]

Secondary Outcome Measures:

  • Cerebrospinal fluid (CSF) analysis of color consistency, cell counts, and microbial evaluation [ Time Frame: 15 days ] [ Designated as safety issue: Yes ]
    To signify any signs of aseptic or bacterial meningitis
  • MRI analysis to analyze any changes in meninges [ Time Frame: 15 days ] [ Designated as safety issue: Yes ]
    To signify any signs of aseptic or bacterial meningitis
  • Pulse [ Time Frame: 15 days ] [ Designated as safety issue: No ]
  • O2 saturation [ Time Frame: 15 days ] [ Designated as safety issue: No ]
  • Blood Pressure [ Time Frame: 15 days ] [ Designated as safety issue: No ]
  • Respiration changes [ Time Frame: 15 days ] [ Designated as safety issue: No ]

The idea of resurrection the clinical dead and the reality of the experiment is way different and definitely less eye-catching and sensationalized. The paradigm consist of implementing stem cells inside the brain and assess the presence of a re-establishemnt of an electrical activity as measured by an EEG.
Several studies has documented protocols to differentiate human pluripotent stem cells (including embryonic and induced pluripotent stem cells) into neurons capable of physiological activity, in other terms of capable to general an action potential or in other words electrical activity.
The first issue I have in this case, where, when and what of stem cells will be used are never mentioned, either on the press release or on Bioquark Inc. presentation slide.
The second issue I have is about the credentials of the investigators, Dr. Himanshu Bansal. By submitting a query on Pubmed with the keyword “Bansal H Stem“, we have only four results listed with two directly relevant studies published in the Journal of Stem Cells. Such journal is published by Nova Science Publishers, a predatory publisher as reported by Scholarly Open Access. Add to it there is no Thomson-Reuters ISI impact factor listed raise the question is this journal is legitimate or will publish anything as long as you give them a valid credit card number. Remember the adage “garbage in, garbage out

Things are even more murky when you visit Dr. Himanshu Bansal webpage and the tab about his stem cell results. For information, this is a screenshot of the page:
BansalStemCell

This is fairly impressive yet highly questionable when someone boast such claims without linking to the literature, considering that until now there is NO FDA approval for stem cell treatment.

If it looks like a quack and sounds like a quack

Now things are getting suddenly sour and fishy. The more we dive in and the more things are looking. Now one criteria I consider important for assessing a reliability of a startup biotech is its portfolio and if not the quality of the data presented. This is not looking good either.
Unfortunately, Bioquark has little to show and have rather laconic statements, no published studies, no data demonstrating the activity of their drug candidates nothing. For my standpoint, I am always skeptical when I see a biotech company making a living on cosmetic and beauty products, as seen on their page:
BioquarkCosmetics.png

This is raising already some issues about the company credential, I am not even talking about Revita Life Sciences. Both company have such a horrible web design but also looks a lot like websites I commonly label as “quackery”, especially the second website. Having on the frontage treatment like “Naturopathy, Bioactive Molecules, Laser Stimulation, Ayurveda….”. If it sounds like a quack and looks like a quack…..

Now, if you pay attention to the Slideshare, the quality of the data is simply horrible and just look like a botched data. One thing that particularly caught my eye was this slide:
BQA_Melanoma

The gold standard in cancer research using mouse models is the xenograft model in which you inject sub-cutaneously human cancer cell line and follow the growth of the tumor like in this study.
f1-large1

In their study, rationale of injection of tumor in the paw is never mentioned, not even mentioning the questionable quality of the pictures, with the identification number changing over time in terms of quality. Indeed the paw seems to display an edema as shown in an another study. Edema is a swelling of connective tissue due to various reasons. In that case, I question whether the authors injected some vital dye (Trypan blue or Evan’s blue) into the paw with a volume enough to induce an edema and allow it to resorb over time, as seen in a study found online.
If I was an investor at this point, I would run away from this project.

Conclusion
To conclude on this breaking news that has its weight of clickbait, I sincerely raise questions and skepticism on that study that boast to revigorate patients that are clinically dead. There are a lots of smoke screen, an outcome that is very limited with very or little significance in terms of science. Here are my major concerns about that experiment:
1. 
There are no data from preclinical studies done in animal models to support the information proposed for the clinical studies. This is contradicting the common scientific process in which we transition from the bench to the bedsides.
2. The outcome measured as proposed in the NIH application package is much less extraordinary that the claim posted on mainstream media. That is fine but really raises the relevance of this study. Stem cells may be able to survive and differentiate in the brain parenchyma to an extent they can generate electrical activity measurable by EEG. However, there is virtually no chances these cells will provide an axon rewiring capable to result in functional activity. So no zombies.
3. There is a lot of smoke screen in terms of the experimental paradigms. We don’t know which stem cells will be used, where such stem cells will be implanted and how they will address the outcomes beyond the EEG. It sounds impressive in terms of science but once we think on how this advance our current knowledge, we are dropping short. We are ending up with a similar outcome than Dr. Frankenstein monster: it sounds very impressive and defying but the relevance of the monster was virtually none.
4. Finally, the public data and the lack of credentials of both companies and the investigators in terms of experimental data, peer-reviewed studies quality and records and the tendency of both companies to rely on healthcare products if not on alternative medicine as a source of revenue is sincerely raising questions on the ability of these companies to deliver. In particular, some claims of therapeutical success of Dr. Bansal on treating neurological diseases using stem cells are highly questionable as there is until now no evidence or approval by the FDA for any stem cell treatment beyond bone marrow stem cell transplants for patients suffering from leukemia.