Future Science - Accurate Diagnosis of Autism Phenotype using Brain Biopsies grown from Skin Cells

 

This is a post for the scientists among you, that like to separate science from pseudoscience.

You may not be aware that in recent years some revolutionary science has been developed allowing you to create a brain biopsy from a sample of skin.  I was rather taken aback when I first read about it, but it is true.  Researchers are now creating their own artificial biopsies, on which to experiment.

I first read about this in work being done at Stanford, looking at Timothy Syndrome.  I did not really focus on the importance of what they had done.  Without physical samples, it almost impossible to develop medical therapies and you cannot take brain biopsies from living people.  Rats and other lab animals are never going to be an adequate substitute.   

Here is a link to Johns Hopkins, where there used the same technology to make a brain sample with Huntington’s disease.

Turning Skin Cells Into Brain Cells

The true potential of this technology is amazing.  Today, it is being used to create samples, with which researchers can carry out experiments.  In future, it could be used as a diagnostic tool, just like when somebody goes to the gastroenterologist and has an endoscopy.  The doctor takes a sample from your intestines and sends it to the lab for analysis; hopefully a few days later you get a call saying the sample had no cancerous biomarkers, or other nasties.

In the case of autism, the skin sample would be turned into stem cells and then neurons from specific parts of the brain could be made.  These samples would then be tested to reveal specific dysfunctions.  This is exactly what was done at Stanford to make samples of Timothy Syndrome, a rare condition nearly always comorbid with autism.

Stanford Research

“We developed a way of taking skin cells from humans with Timothy syndrome and converting them into stem cells, then converting those stem cells into neurons,” says Ricardo Dolmetsch.

He then identified an experimental drug that could reverse the effects.  This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type-channel blocker.

Roscovitine, also known as Seliciclib or CYC202, is an experimental cancer drug produced by a Scottish/American company called Cyclacel. Don’t expect it to be a cheap drug; Goldman Sachs is one of the main shareholders.

Ricardo is from Cali in Colombia and so I had better shift him to my Dean’s List post-haste.

Conclusion

At some, not so distant point in time, people with a lot of money will be able to identify the precise phenotype of their child’s ASD and then they will know exactly what the optimal treatment is.

Once this technology becomes established, maybe in 50 years, I think Psychiatrists will become an endangered species.  We will need a lot of neurologists and for many people with mental health problems, there will at last be the chance of a very precise diagnosis and a matching drug therapy, created and tested on “human” brain samples made from stem cells.

Hopefully by then, the American Psychiatric Association’s DSM nonsense will be relegated to the trash can (or dustbin, for British readers).  Patients with brain disorders will be diagnosed and treated by neurologists and supported by psychotherapists, psychologists, behavioural analysts and other professionals.

 

 

 

 

Autism Phenotypes

Hardly a week goes by without somebody mentioning to me a wonder treatment or even “cure” for autism; the latest one being the GAPS diet.

I think all such reports are worthy of investigation, but many lead to nowhere.

Why is this?




  

·       Medical science has failed to adequately define autism, so we are not all talking about the same autism

·        Many people putting forward theories have not read even the most basic (and not contested) autism research.  Some are even, apparently, qualified “doctors”.
 

Autism Phenotypes

What is not disputed is that autism has many sub-types (phenotypes). Researchers tell us 10-20% of cases referred to as autism have a known genetic defect (Fragile X, SLOS, Timothy syndrome etc.).  80% do not have a known genetic marker/cause.

Autism can be subdivided into regressive (when a child loses speech and other learnt skills) and non-regressive (early onset).  Even this can be a subjective judgment, since it effectively relies on parents to determine it, after the event.

Then you have cases of autism which clearly have nothing to do with Kanner’s classic version.  In this blog I showed how even cerebral malaria in a child can lead to the onset of autism.  This clearly is a case of brain damage caused by malaria; but to the observer, months later, it would probably be classed as regressive autism or childhood disintegrative disorder.

 

Testing for Autism

Researchers and doctors keep repeating that there is no test for autism.  This is not strictly true, but it does explain why so many different conditions are all lumped together as “autism”.

In fact, if you read the research closely, you will see that there are many tests for autism; although they may not be perfect.

The only way to know for sure that it is genuine autism is to examine the brain itself.  The only way to do this 100% accurately is via post-mortem analysis of the brain.  Recently, non-invasive methods have been developed to confirm the same findings of brain malformation that occurred prior to birth.

So the kind of autism that relates to tissue held in brain banks is best understood.  But what kind of autism would that be?  Well, it refers mainly to children and young adults who died prematurely.  They died from things like seizures or drowning.  What does that tell us?  This tells us that these people were most likely severely affected by autism.  The mild, social difficulties, type of autism is, fortunately, hardly likely to make it to the brain tissue bank.

If the person interpreting the MRI of a child’s brain knows what to look for, they may very well be able to identify this type of autism.  The expert here is Eric Courchesne.

A similar approach can followed using Electroencephalography (EEG) to identify autism; but it would be smart to cross check this with Eric.

Regressive vs. Early-Onset

Then you have the difference between regressive and non-regressive autism.  Here again, from my Dean’s List of researchers, we look at Paul Ashwood’s research to see that kids with regressive autism have HIGHER levels of inflammatory markers in their blood.  These include cytokines like interleukin 6, which can be inexpensively measured in most laboratories.  This tells us that perhaps regressive autism is an entirely different condition from non-regressive/early onset autism.  As I would expect, increasing cytokine levels were associated with more impaired communication and aberrant behaviors. 

Elevated plasma cytokines in autism spectrum disorders provide evidenceof immune dysfunction and are associated with impaired behavioral outcome

 

Lab Testing

We have seen earlier in this blog that some very expensive lab tests exist for autism, but their usefulness and integrity is highly disputed.  There are, of course, many hundreds of other tests that are entirely validated by medical science.  Many of these tests are cheap and available all over the world.

Hormonal Screening

We know from the research that about 30% of people with autism have high blood serotonin. A standard lab test is required.

We know that many have high levels of insulin-like growth factor (IGF-1).  A standard lab test is required.

Thyroid hormone levels and in particular a blunted response of TSH to TRH (i.e. central hypothyroidism) can help define further phenotypes.

The TRH test is now not widely used, but TSH, FT3 and FT4 are cheap tests.

Growth Hormone (GH) is also implicated in autism, along with IGF-1; there is a lab test to measure pituitaryfunction to see how well GH is being produced.

By screening for hormonal dysfunction, it would be possible to identify phenotypes that would most likely benefit from therapies targeting those defects, like NNZ-25266.

Pancreatic Dysfunction

It is reported by Joan Fallon, of Curemark, that 50+% of kids diagnosed with “US autism” seem to have a pancreatic dysfunction.  This can be tested for by measuring fecal chymotrypsin level.  The test measures how well your pancreas is working, and is a standard test for people with cystic fibrosis.  Since the US diagnoses far more kids with autism than other countries, it seems highly plausible that “US autism” includes many more phenotypes than, say, “French autism”.

I was quoted about $8 for a chymotrypsin test.

Ion-Channel Diseases (Channelopathies)

Many diseases like Parkinson’s disease, Spinocerebellar Ataxia and Timothy Syndrome are caused by faulty calcium ion-channels.

The Bumetanide autism therapy, undergoing trials in Europe, is based on another channelopathy, this time a faulty chloride transporter NKCC1.

It is clear from reports I have received, that Bumetanide therapy is totally ineffective in some children with ASD, but in other children, like my son, it is effective.

So some types of autism have certain channelopathies and other types have different ones or, quite possibly, none at all.  

Conclusion

My conclusion today is pure conjecture.  I imagine that possibly as few as a quarter of cases of “US autism” are actually “real” autism, that is with all the brain damage/malformation that is identified in those post mortem brain studies and which forms the basis of 90% of autism research.

The other three quarters may be something entirely different, just like the case of the mosquito that bit the child, produced cerebral malaria and then later the full symptoms of autism.  Within the three quarters may be food allergies, digestive enzyme deficiencies, gut disorders, mastocytosis, blood brain barrier defects, undefined calcium ion-channel diseases etc.

This would account for those occasional amazing “recoveries” and the apparent success, in some cases, of diets like GAPS.  Sadly, diet is unlikely to 100% fix brain damage.  If you are lucky enough to totally “recover”, you cannot have had brain damage in the first place.  It is evident that in some phenotypes of autism, diet can reduce autistic behaviours.  This can only be proved in trials, if biomarkers are established for that specific phenotype.

Most likely the only biological thing all these “autisms” have in common is oxidative stress and neuroinflammation; but only a non-medical scientist, like me, can say such a thing.

 

 

Excess Calcium and Calcium Signalling in the Autistic Brain

Today's post is quite brief, but will get complicated if you watch the entire video that is included.

Some very smart Italian researchers have been looking into the various causes of autism to better understand the overall picture.

Two screen shots from the end of the presentation are below.  Professor Antonio Persico talks about his "epiphany" when he realized autism was not just about genetics and how he then redirected his research.

He came to a surprising conclusion.  Calcium.

He showed that in the autistic brain there is an excess of both physical calcium and calcium signalling (via ion channels)  We are not talking about rare types of autism, like Timothy Syndrome.

That expert on studies of autistic brains, Eric Courchesne, from the University of California in San Diego, seems to agree.  You can hear Persico mentioning Courchesne in the talk. 

Persico hereby joins Courchesne on my Dean's List of researchers.  This is the list of researchers that I think really know what they are doing.

The entire video should be a "must see" for any parent who really wants to know why their kid is different.  At around 50 minutes you get to the calcium part.



 

Here is the link to the video:-

Autism at the Crossroads between Genetics, Neurodevelopment and the Immune System

None of the good research papers published on this subject have free access.  You can google "Persico calcium autism".

Persico has plenty of other research ongoing, based on his large pool of Italian families affected by autism that provide him biological samples. He is looking for the trigger that starts autism.

Nowhere does he draw any therapeutic conclusions, which is a pity.














 

Hypokalemic Autistic Sensory Overload

Hypokalemic Autistic Sensory Overload (HypoASO) is a condition causing distress to autistic people.  Moderately loud sounds, like those around an indoor swimming pool, or shopping mall, can cause an autistic person great irritation, leading to covering their ears, a tantrum, or even self-injurious behaviour (SIB).  The same sensory overload can be caused by light, smell or touch.  

HypoASO is a condition that can be measured and treated.

HypoASO is related to two other conditions Hypokalemic Periodic Paralysis and Hypokalemic Sensory Overstimulation.

HypoASO is an ion-channel disorder triggered by intra/extra cellular concentrations of sodium and potassium.  Calcium may also play a role.  In simple terms, sodium is bad and potassium is good.

Therapy for HypoASO

The therapy for HypoASO is a diet rich in potassium but low in sodium; magnesium will also be beneficial, since it helps maintain the level of potassium.  People with HypoASO need to maintain a high level of potassium in their blood (> 5.0 mmol/L) in order to avoid triggering this ion channel disorder;  this is at the high upper level of the reference range for potassium.   Oral supplements of potassium with magnesium will also prove useful, but need to be spread out throughout the day, for best effect.  Time release tablets should be the most effective.  Very high levels of potassium are dangerous, so care is required.

Testing for HypoASO

Diet should not be changed on a whim.  A simple test can be carried out to check whether the individual is indeed affected by the disorder. 

1.       Find a sound which the person finds disturbing, like a baby crying.

2.       Download a recording of this sound.

3.       Set up a chair in a fixed location in a room with a strong sound system / Hi Fi

4.       Sit the subject in the chair and play the annoying sound at ever greater volume and see at what point the subject reacts strongly (e.g. covers ears)

5.       Repeat the experiment over  a few days to establish a steady base-line volume, at which the subject reacts, (for example volume setting 3, when the amplifier to goes 0-10)

6.       Give the subject an oral potassium supplement (say 250 mg) and wait 20 minutes

7.       Play the annoying sound and measure the volume at which ears are covered.

8.       If the volume is markedly higher than the base-line, you established earlier, then you have established HypoASO

9.       If the subject has an NT sibling, try it on them.  They will most likely show no difference with the potassium and do not have HypoASO



 

Seasonal Autistic Mastocytosis

 

 The degranulation process in a Mast cell. 1 = antigen; 2 = IgE; 3 = FcεRI; 4 = preformed mediators (histamine, proteases, chemokines, heparin); 5 = granules; 6 - Mast cell; 7 - newly formed mediators  leukotrienes, platelet-activating factor)

Source: Wikipedia 

Some of the most popular posts on my blog refer to my investigation into the role of histamine in autism.  The investigation was productive and lead to a highly effective treatment for the wild summertime flare-ups in autistic behaviour exhibited by Monty, aged 10 with ASD. 

Since I have found a therapy it is only reasonable to give the condition a name. 

Seasonal Autistic Mastocytosis (SAM)

Seasonal Autistic Mastocytosis (SAM), sometimes known as Airborne Autistic Mastocytosis, occurs when airborne allergens like pollen, cause mast cells in the eyes, nose, mouth and lungs to degranulate.  These mast cells contain many granules, themselves containing histamine, serotonin and heparin, a naturally occurring anticoagulant.  This mast cell activation also releases inflammatory cytokines, leukotrienes and platelet activating factor (PAF).  Some of these pro-inflammatory agents enter the brain and stoke up the ever-present neuro-inflammation, starting a downward spiral with further localized cytokine release.  In behavioural terms, the result is that all the earlier bad behaviours will return, but in a magnified form.  The observer may notice swings to aggression and self-injurious behaviour (SIB).  If the subject is verbal, he may complain of unpleasant itching on arms and legs, typical of mastocytosis.  The autistic subject, not understanding the reason for the itching, is likely to react with some form of tantrum, aggression or hitting that part of his body.

SAM should be considered even when only very minor symptoms of an allergy are visible, like red eyes or runny nose.  

The most effective therapy is to use mast cell stabilizers, but even a standard OTC H1 antihistamine will provide some relief within 20 minutes.  The dosage required to have an effect, may be much higher than the recommended dose for allergic rhinitis (hay fever), but should still be within safe limits.

 

An alternative therapy is simply to move to somewhere that is pollen free, even just for a weekend and observe the effect.

Depending on where you live, SAM may be possible for about 5 months of the year.

Mast cells are particularly present in the digestive tract, the lungs, skin, eyes, mouth and nose.  They undoubtedly also play a major role is asthma.

Mastocytic enterocolitis is a related condition.  This condition is acknowledged in the medical community.  I am not a gastroenterologist, but I think Messrs Wakefield and Krigsman may have really stumbled upon cases of Mastocytic enterocolitis, when they came up with their diagnosis of Autistic enterocolitis.  Incidentally, Krigsman recently published an interesting paper on genes and colitis in ASD.

Some of the frequently reported cases of GI problems in autism may also be caused by mast cell degranulation.  Some of the DAN doctors treat GI problems with mast cell stabilizers and allergists routinely prescribe them.

Note on Serotonin

Approximately 30% of people with autism have high blood serotonin; perhaps a contributing factor is serotonin released by the degranulation of mast cells.  We have already seen that there often appears to be central hypo-function of serotonin in autism (i.e. low brain serotonin).  The endocrine system functions using feedback loops, so high blood serotonin sends a signal to lower serotonin; thus you could get low brain serotonin but high blood serotonin.  Remember that serotonin does not cross the blood brain barrier.

Autism Clinical Trials, Arbaclofen (STX209), Curemark CM-AT and the Clever Chiropractor

 

In the world of clinical trials for drugs, judging success and failure can be highly subjective.  They try to make it as logical as possible and the method works pretty well for assessing things that you can measure objectively.

Primary and Secondary Endpoints

To quote Pfizer:

A trial endpoint of a clinical trial should fulfill three criteria: (1) be measurable and interpretable, (2) sensitive to the objective of the trial, and (3) clinically relevant. The endpoint can be either clinical or surrogate in nature.

If you are developing a drug to lower cholesterol or to increase survivability after a traumatic brain injury, it is pretty easy to define your endpoints.

When it comes to autism, one of the major hurdles is to define objective measurable endpoints.  As it stands today, none of the assessment tools are really fit for purpose, when Big Pharma is supposed to come along and invest hundreds of millions of dollars in some bright spark’s idea.

Arbaclofen, Seaside Pharma & Roche

The Swiss giant, Roche, recently had just such a problem.  They had partnered with a spinout company from MIT called Seaside Therapeutics.  One of the projects was to complete the trials of a fragile X targeted drug, called Arbaclofen (STX209).  During the 4+ years of trials Seaside had changed the primary endpoint.  Arbaclofen started out as drug to treat one aspect of behaviour, but by the time they got to phase 3  clinical trials this had been changed to lethargy and social withdrawal scores from the Autism Behavior Checklist (ABC).

Quite logically, Roche assessed the result of the stage 3 trial against its primary endpoint.  Based on the total cohort in the trial, Roche determined the drug to be a failure and pulled the plug on financing the drug further.

Open Label Extension Study of STX209 (Arbaclofen) in Autism Spectrum Disorders

The owner and developer of the drug, Seaside, even though they have recently raised $90 million, said they could no longer continue to fund the trial and all those kids in the trial would have to be weaned of Arbaclofen ASAP.

It turns out that among the families involved in the trial there were many reports of wonderful improvements on Arbaclofen.  They even formed a group to lobby for a continuation of the trial.  There website is interesting.

They wrote to big boss of Roche in Basel. 

And he wrote back to them.

It now appears that Seaside has had a rethink and will try again with a new trial with a new primary endpoint (mark 3).

Background on Arbaclofen

Some of the first studies of Arbaclofen were conducted in patients with Fragile X syndrome, a genetic condition caused by a change in a gene called FMR1, which normally is needed to make the brain grow properly. Fragile X is the most common form of inherited intellectual disability in boys and can be a cause of autism or related disorders. In those Phase 2 trials, Arbaclofen was shown to decrease social withdrawal and improve adaptive social function.

A  Phase 2a study conducted at 8 sites and involving 32 children showed significant positive behavioral outcomes, including improved scores on the Aberrant Behavior Checklist-Irritability Score (ABC-I) and on the ABC-Social Withdrawal Scale. The most common adverse events were agitation, irritability, fatigue, psychomotor hyperactivity, insomnia and diarrhea. Most resolved without dose changes, but one serious adverse effect did occur during down-titration of the medication.

In July 2011, Seaside Therapeutics, announced that 25 sites across the nation will be involved in a new clinical trial to involve approximately 150 ASD patients between the ages of 5 and 21.

STX209 is an orally-administered GABA-B agonist; the drug acts by stimulating the release of GABA, a neurotransmitter in the central nervous system. GABA inhibits the release of glutamate, an excitatory neurotransmitter, for which an overabundance negatively affects the ability of neurons to communicate with each other.

The GABA "A" receptor, is a chloride channel, while STX209 targets the GABA "B" receptor, which is a G-protein coupled receptor and regulates a different set of molecules from GABA "A".

The original basis for starting this blog was my success with bumetanide, which is affecting the GABA “A” receptor.  In the brain, bumetanide blocks the NKCC1 cation-chloride co-transporter and thus decreases internal chloride concentration in neurons.

Medicine as an art and a science

Mark Bear is a neuroscientist at MIT and he was the co-founder of Seaside Therapeutics.  He is clearly a very brainy guy.

There is a derivative of his Arbaclofen called Arbaclofen Placarbil.  I found it interesting that this substance was also being trialed as a therapy for Multiple Sclerosis and GERD.  GERD is the medical term for heartburn/indigestion.

Incidentally, Arbaclofen Placarbil failed both trials.

Now to the Clever Chiropractor and her Pancreatic Enzymes

People outside the US will find it very strange that in the US chiropractors and osteopaths have the same right to prescribe drugs as conventional medical doctors. 

Outside of the US, if you want to be a doctor you have to apply to medical school and in most countries the competition is very tough. There is no plan B if your exam grades slip.  In the US it is different, if your grades and resources are not taking you to the Harvard, you can opt to become an osteopathic physician or a chiropractic physician.

Rather than Harvard or MIT, Joan Fallon trained as a chiropractor at Palmer University.

Not surprisingly the scientific community is skeptical of her autism treatment, which is linked to pancreatic enzymes.  After all, how can a chiropractor know more than Ivy League neuroscientists?

Peter, on the other hand, thinks that Fallon is actually far more savvy than the very brainy people over at Seaside.  Her therapy may, or may not be effective, but her method of developing it is highly effective.

First she raised $6 million to start her company Curemark, then as trials progressed she very recently she raised another $18 million.

The reason I like what Fallon is doing is that she has figured out which sub-type of autism is helped by her therapy and she has identified a bio-marker for that subgroup.

Hallelujah, a street-smart autism researcher !

If you want to enroll in a clinical trial for Curemark’s CM-AT, first they will screen out the 50% that do not have the biomarker.  Fallon is making sure that her clinical trial results look as good as possible, by only including those subjects most likely to benefit.  This may sound like common sense, but in autism research this is a revolution.

CM-AT therapy and the biomarker

The reason the autism world are skeptical of Fallon, is that she is going on about Secretin and pancreatic enzymes.

Many years ago parents thought that Secretin was going to be the wonder cure for autism; it turned out not to be.  By reading her patents, it is clear that Fallon has some faith in the role of secretin, in addition to enzymes produced in the pancreas.

What impressed me was how she has screened the kids allowed into her clinical trials.

A Trial of CM-AT in Children With Autism 

Eligibility

·         Inclusion criteria:

o    Child is 3-8 years old

o    Child has a diagnosis of autistic disorder

o    Child must have a low fecal chymotrypsin level (we will measure)

·         Exclusion criteria:

o    Child must have no dietary restrictions (other than for a nut allergy)

o    Child may not have an allergy to pork products

o    Child may not have a history of severe head trauma or stroke

o    Child may not have had a seizure within the past year

o    Child may not be diagnosed with: HIV, cerebral palsy, endocrine disorder or pancreatic disease

o    Child may not be taking any enzyme product, amino acids, secretin product or stimulant medication currently 

“Low fecal chymotrypsin level” is a standard lab test available all around the world.  Over the years Fallon has found that it is a biomarker of the kids who benefit from her patented mix of enzymes sprinkled on their meals.

You actually can buy a very similar product called Creon, or Kreon, depending on which country you live in.  The reason why you cannot be in the trial if you have a pork allergy, is that they use the pancreas of dead pigs to make the enzymes.  This is bad news if you are Jewish, Muslim, a Seventh Day Adventist, or indeed the pig.

The active ingredient in Creon is Pancreatin. Pancreatin contains the pancreatic enzymes lipase, amylase and protease. These assist the digestion of fat, carbohydrates and proteins.

Update on trials of CM-AT

Here is a link to the always-helpful Simons Foundation, with the expected skeptical comments from experts:-

 https://sfari.org/news-and-opinion/news/2010/first-drug-for-autism-enters-final-stage-of-testing

Now Curemark have finished there phase 3 trial, and guess what? It met both primary and secondary endpoints and has been “fast-tracked” by the FDA.

Congratulations Joan !!!

One of the secrets of her success was to have the good sense to enroll herself in a course on clinical investigation run by Harvard/Massachusetts general hospital.

 

Curemark Begins NDA Submission for CM-AT Autism Treatment

CM-AT had previously been granted Fast Track status by the FDA, a designation given to drug candidates that treat serious or life-threatening conditions and demonstrate the potential to address unmet medical needs. The rolling submission process allows companies with a Fast Track designation to submit the NDA in sections to the FDA as they are completed.

“Initiating our ‘rolling NDA’ submission is a major step in the registration process for CM-AT,” said Dr. Joan Fallon, Curemark founder and CEO. “We have an extraordinary opportunity to help many children with autism improve the quality of their lives and we will continue to work closely with the FDA to make that happen.”

Curemark previously announced the successful completion of its Phase III multicenter clinical trial of CM-AT for autism. CM-AT met both primary and secondary endpoints in its double-blind, randomized, placebo-controlled study of children with autism at 3 to 8 years of age.



  

Conclusion

I would suggest those researchers who believe that diet can be an effective therapy in sub-types of ASD take good note of Joan Fallon's methods. You might indeed be right, but unless you can prove it, the skeptics will always hold sway.