Showing posts with label TNF. Show all posts
Showing posts with label TNF. Show all posts

Tuesday 6 September 2016

Histamine Reaction to Bio Gaia Gastrus

Alli from Switzerland discovered the autism benefits of Bio Gaia Gastrus.

This probiotic contains two different bacteria:-

·        Lactobacillus reuteri 17938 (Lactobacillus reuteri Protectis)

 ·        Lactobacillus reuteri ATCC PTA 6475

These two bacteria have different effects.

The first bacteria is very well researched and recently was shown to increase oxytocin in autism mouse studies.  It is available on its own and this is the product most people I know are using.

The second bacteria is included in Bio Gaia Gastrus specifically for its additional anti-inflammatory effects.

Recent comments on this blog have shown that some people have a negative “histamine-y" reaction to Bio Gaia Gastrus.  This is entirely logical since the mode of action of the second bacteria is to generate histamine to activate H2 receptors in the gut.

This might sound rather odd since histamine is thought of as inflammatory, but the researchers working for Bio Gaia have shown that histamine can produce the opposite effect, suppressing TNF via Modulation of PKA and ERK Signaling.

Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H2receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases.

This may mean that people who respond well to H2 histamine antagonists (Zantac, Tagamet etc) are unlikely to benefit from Lactobacillus. reuteri ATCC PTA 6475.

It might also mean that people who respond negatively to Bio Gaia Gastrus might get benefit from H2 histamine antagonists.

It might be worthwhile people trialing the single bacteria Bio Gaia product (Protectis), if they have a negative reaction to Gastrus.

Thursday 20 March 2014

Dr Chez’s Trial of Lenalidomide, a TNF- α and IL-6 Inhibitor in Autism


An interesting trial of a TNF- α and IL-6 inhibitor in autism has been brought to my attention.  It was by Michael Chez, the neurologist from Sacramento, who has made several appearances on this blog.
By coincidence, a copy of his book arrived this week.  The book is called “Autism and its Medical Management”, Chez is one of the few mainstream doctors who does try and treat autism.  The book is rational, readable and in no way radical, so you could show it to your family doctor without upsetting him/her.  Chez does particularly focus on distinguishing regressive from non-regressive autism, as do I. His view is that it is regressive autism, even if it was regression from slightly abnormal.  The important part is that some learned skills, like language, were lost sometime after 12 months of age.  He believes that regressive autism has a different basis to non-regressive autism; he has his own ideas about this, but he admits there is no concrete proof.

The book is a few years old and Chez has published much work in the intervening few years.    
The paper I was referred to is:-

Lenalidomide, an analogue of thalidomide, has the potential to invoke significant changes in TNF-α and other immunomodulatory cytokines.  If thalidomide sounds familiar, it is the drug from the 1950s, that turned out to be very unsafe for use in pregnant women and around the world 10,000 babies were born with malformation of the limbs.

Lenalidomide has been used to successfully treat both inflammatory disorders and cancers in the past 10 years. There are multiple mechanisms of action.  It is extremely expensive, according to NICE:-

“Lenalidomide 25 mg capsules cost £4368 per 21 capsules (excluding VAT; ‘British national formulary’ [BNF] edition 55). Dosage is continued or modified based upon clinical and laboratory findings. For example, if lenalidomide is continued for ten 28-day cycles without dose reduction, the cost would be £43,680.”
Dr Chez does not mention the cost of Lenalidomide, but he uses a tiny dose of 2.5 mg; This would cost £20, or $30, a day.  This might also explain the small number (7) of participants in the trial.
“2.2. Drug and Dosing. Lenalidomide 2.5mgs was given daily
for 12 weeks. This low dose was selected to minimize the risk
of adverse effects. In addition, because this was a pilot study,
the goal was to test the lowest dose that could potentially lead to improvements.”
The drug did reduce TNF-α levels and there were some behavioral improvements, but nothing dramatic.  Perhaps a higher dosage would have had a greater effect?
There were only seven participants and the data on the seven is not complete; also the dose of Lenalidomide was very low.  I think it is really only fair to conclude that the trial is interesting but that a much cheaper drug would need to be found and tested on a much larger number of participants.

“Despite the limitations, to our knowledge, this open-label study represents the first attempt to treat autism by specifically targeting elevated innate inflammatory cytokine levels. Safety monitoring and pharmacokinetic data were successfully completed during this pilot study and exploratory observations of clinical and cytokine changes suggest a trend towards improvement. Correlating treatment outcomes with cytokine level changes may be a target in future autism spectrum treatment, especially in those with known maternal or postnatal immunological risk factors. Larger blinded and placebo-controlled studies assessing cytokine measurement and cytokine-targeted treatment in autism patients with TNF-α or other inflammatory cytokine elevation are warranted.”

To his credit, unlike the researchers in Athens who trialed Neuroprotek in a recent post, Chez went about his pilot study in a scientific manner and collected both the biological and the behavioral data.  In other words, he measured the before and after levels of the inflammatory cytokines and the before and after behavioral rating scales.  Well done Dr Chez.

Monday 17 March 2014

Let’s be Serious about the Data - Flavonoids, Cytokines & Autism

You may be wondering why, with so many research papers written about autism, so little progress has been made.  It is a very complex task, but nobody is coordinating it.

How do you find a Boeing 777 missing somewhere in Asia?  Another daunting challenge, but with the right people and resources it can be done.  With the wrong people, it will prove to be impossible.
Ashwood et al have documented the level of various inflammatory markers in autism.  Very helpfully, they created three groups: typical children, children with non-regressive autism, and children with regressive autism.

Table 2, on the third page, tells us what we need to know.  Certain cytokine levels are markedly elevated in regressive autism, including IL-6 and TNF-alpha.  Furthermore, the difference between the two types of autism is dramatic; rather implying the existence of two distinct conditions.

So now, I move on to what could have been an amazingly helpful study, had they spent 1% more time on it and collected some blood samples and split the kids into regressive and non-regressive groups.

Last year in Athens, a study was done using Theoharides’ mix of luteolin and quercetin flavonoids to look at the effect of mast cell stabilization on behaviour in autism.  From recent posts, you will recall that these flavonoids reduce the level of inflammatory cytokines, histamine and nerve growth factor, by stabilizing so called mast cells.  In effect, the study was looking at the impact of inhibiting certain cytokines on behaviour in autism.

This sounds great and just what I wanted to find.  Get 40 kids with ASD measure their level of these cytokines/histamine and assess their behaviour.  Give them the cytokine inhibitor/mast cell stabilizer for six months, measure the levels in their blood and assess the behaviour again.
Sadly, they did not bother to take the before and after blood samples and send them downstairs to the hospital’s laboratory.
So we have a paper that took years of planning that tells us that the flavonoids do seem to help; but we do not know exactly why and we cannot correlate improvement in behaviour with change in cytokine levels.
What a pity.  


Wednesday 12 March 2014

Single Dose of IL-6 Antibodies or TNF-ᾳ Inhibitor as Potential Disease-Changing Autism Therapies

We have noted in earlier posts that autism is a dynamic encephalopathy and this may help explain why a therapy that works in a child aged 10, may be of little help to another child aged 3.  Not only are there many sub-types of autism, but each sub-type is evolving, as the child matures.

None of the autism drug therapies I have implemented have permanent disease changing effects, they all seem to work, but the effect is lost once you stop taking them.  Today’s post is about drugs that you take just once.  For a parent trying to find a drug that works in the sub-type affecting their child, this has a big advantage.  No need to keep trying for months to see if the drug has any effect.
Perhaps the most important time to intervene with drug therapy is as soon as possible after the diagnosis; but with what?
In an earlier post on trying to get a non-verbal child to talk, I suggested the use of corticosteroids to arrest on-going neuroinflammation.  Drugs like prednisone are potent, but they these have nasty side-effects if used long term. In that post, Dr Michael Chez, an eminent neurologist from Sacramento, was upbeat on their potential as immunomodulators.  We will refer back to him in this post as well.
In this post I will give more background about the role of a cytokine called Interleukin 6, or just IL-6, in autism.  You will see how science can both create a mouse with autism using IL-6 and reverse it again using IL-6 antibodies.
We will also look at another cytokine called   TNF-ᾳ and see how a single dose of a TNF-ᾳ inhibitor can improve chronic neurological dysfunction following a stroke, TBI and indeed autism.  It is effective even a decade after the original traumatic event.
Both the IL-6 and TNF-ᾳ drugs are developed for arthritis and these drugs cost tens of thousands of dollars a year, but in the case of neurological conditions they may have a disease-changing effect when used just once. Remarkably, both drugs are already approved for long term use in very young children with Juvenile Idiopathic Arthritis.

Why am I interested in Cytokine inhibition?
My very first attempt to reduce neuroinflammation in Monty, aged 10 with ASD, was a very surprising, but resounding success.  That followed my research into cytokine storms and statins.  I know it works, because when I stop the statin, the very same behavioural improvement is lost in a day or so.
Are there randomized trials of atorvastatin in autism? Sadly, not; but it is a safe intervention that works in my mouse model.
Are there further potential benefits from such therapy? Quite possibly, but higher doses of statins have side effects.
We saw in recent posts that PEA, quercetin and luteolin also inhibit pro-inflammatory cytokines.  Is there a potential disease-changing therapy?  We will only find one, if we look.

The Cytokine IL-6 and Autism
Thanks to Dr Wei, we have some excellent research linking specifically the cytokine IL-6 to autism.  He suggests that elevated levels of IL-6 may cause much of the damage in autism and he went as far as to prove it in a mouse model.

A single injection of IL-6 into a pregnant mouse, produced a mouse pup with social deficits.  When the mother received a dose of IL-6 antibodies the resulting mouse pup has normal behaviour.  Humans are not mice, but we do already know from Ashwood and others that people with ASD have elevated levels of IL-6 and in particular those people with regressive autism.  
Autism is a severe neurodevelopmental disorder characterized by impairments in social interaction, deficits in verbal and non-verbal communication, and repetitive behavior and restricted interests. Emerging evidence suggests that aberrant neuroimmune responses may contribute to phenotypic deficits and could be appropriate targets for pharmacologic intervention. Interleukin (IL)-6, one of the most important neuroimmune factors, has been shown to be involved in physiological brain development and in several neurological disorders. For instance, findings from postmortem and animal studies suggest that brain IL-6 is an important mediator of autism-like behaviors. In this review, a possible pathological mechanism behind autism is proposed, which suggests that IL-6 elevation in the brain, caused by the activated glia and/or maternal immune activation, could be an important inflammatory cytokine response involved in the mediation of autism-like behaviors through impairments of neuroanatomical structures and neuronal plasticity. Further studies to investigate whether IL-6 could be used for therapeutic interventions in autism would be of great significance

Background: Although the cellular mechanisms responsible for the pathogenesis of autism are not understood, a growing number of studies have suggested that localized inflammation of the central nervous system (CNS) may contribute to the development of autism. Recent evidence shows that IL-6 has a crucial role in the development and plasticity of CNS. 

Methods: Immunohistochemistry studies were employed to detect the IL-6 expression in the cerebellum of study subjects. In vitro adenoviral gene delivery approach was used to over-express IL-6 in cultured cerebellar granule cells. Cell adhesion and migration assays, DiI labeling, TO-PRO-3 staining and immunofluorescence were used to examine cell adhesion and migration, dendritic spine morphology, cell apoptosis and synaptic protein expression respectively.

Results: In this study, we found that IL-6 was significantly increased in the cerebellum of autistic subjects. We investigated how IL-6 affects neural cell development and function by transfecting cultured mouse cerebellar granule cells with an IL-6 viral expression vector. We demonstrated that IL-6 over-expression in granule cells caused impairments in granule cell adhesion and migration but had little effect on the formation of dendritic spines or granule cell apoptosis. However, IL-6 over-expression stimulated the formation of granule cell excitatory synapses, without affecting inhibitory synapses.

Conclusions: Our results provide further evidence that aberrant IL-6 may be associated with autism. In addition, our results suggest that the elevated IL-6 in the autistic brain could alter neural cell adhesion, migration and also cause an imbalance of  excitatory and inhibitory circuits. Thus, increased IL-6 expression may be partially responsible for the pathogenesis of autism.  

Brain IL-6 elevation causes neuronal circuitry imbalances and mediates autism-like behaviors.
Abnormal immune responses have been reported to be associated with autism. A number of studies showed that cytokines were increased in the blood, brain, and cerebrospinal fluid of autistic subjects. Elevated IL-6 in autistic brain has been a consistent finding. However, the mechanisms by which IL-6 may be involved in the pathogenesis of autism are not well understood. Here we show that mice with elevated IL-6 in the brain display many autistic features, including impaired cognitive abilities, deficits in learning, abnormal anxiety traits and habituations, as well as decreased social interactions. IL-6 elevation caused alterations in excitatory and inhibitory synaptic formations and disrupted the balance of excitatory/inhibitory synaptic transmissions. IL-6 elevation also resulted in an abnormal change in the shape, length and distributing pattern of dendritic spines. These findings suggest that IL-6 elevation in the brain could mediate autistic-like behaviors, possibly through the imbalances of neural circuitry and impairments of synaptic plasticity. 


Schizophrenia and autism are thought to result from the interaction between a susceptibility genotype and environmental risk factors. The offspring of women who experience infection while pregnant have an increased risk for these disorders. Maternal immune activation (MIA) in pregnant rodents produces offspring with abnormalities in behavior, histology, and gene expression that are reminiscent of schizophrenia and autism, making MIA a useful model of the disorders. However, the mechanism by which MIA causes long-term behavioral deficits in the offspring is unknown. Here we show that the cytokine interleukin-6 (IL-6) is critical for mediating the behavioral and transcriptional changes in the offspring. A single maternal injection of IL-6 on day 12.5 of mouse pregnancy causes prepulse inhibition (PPI) and latent inhibition (LI) deficits in the adult offspring. Moreover, coadministration of an anti-IL-6 antibody in the poly(I:C) model of MIA prevents the PPI, LI, and exploratory and social deficits caused by poly(I:C) and normalizes the associated changes in gene expression in the brains of adult offspring. Finally, MIA in IL-6 knock-out mice does not result in several of the behavioral changes seen in the offspring of wild-type mice after MIA. The identification of IL-6 as a key intermediary should aid in the molecular dissection of the pathways whereby MIA alters fetal brain development, which can shed new light on the pathophysiological mechanisms that predispose to schizophrenia and autism.

Effects of exogenous cytokines

Our pilot studies indicated that maternal administration of IL-6, but not IL-1α, tumor necrosis factor α (TNFα), or IFNγ, causes PPI deficits in the adult offspring. PPI is the inhibition of a startle response when the startling stimulus is immediately preceded by a smaller, nonstartling stimulus of the same modality and is a measure of sensory-motor gating, attention, and distractibility. PPI deficits are observed in several mental disorders, including schizophrenia and autism. Furthermore, PPI deficits in the offspring elicited by maternal influenza infection respond to antipsychotic and psychomimetic drugs, and the PPI deficit resulting from poly(I:C) MIA is present in adult but not juvenile rats, mimicking the adult onset of schizophrenia. The changes seen in this very relevant behavior prompted further study of the effects of maternal IL-6 administration

Thus, a single injection of IL-6 on E12.5 causes deficits in two relevant behaviors (LI and PPI) in the adult offspring.
Abnormal behavior in MIA offspring is prevented by maternal treatment with anti-IL-6 antibody
f, In the social interaction test, control mice show a strong preference for the social chamber [defined as (percentage of time in social chamber) – (percentage of time in opposite chamber)], whereas the offspring of poly(I:C)-treated mice show no such preference. Again, the deficit is corrected by maternal administration of IL-6 antibody

Tocilizumab / Actemra
Wei has made a pretty solid case that IL-6 is implicated in autism and that IL-6 inhibition could be a very interesting therapy.  While we have a range of interventions that can do just that, the ultimate therapy would be IL-6 antibodies.
This therapy does actually exist as a recent option in treating arthritis. Tocilizumab, brand name Actemra, is an immunosuppressive drug made of humanized monoclonal antibodies  against the interleukin-6 receptor (IL-6R)  In 2013 Actemra was approved by the FDA for children as young as 2 years old, as an ongoing treatment for arthritis.

This drug is frighteningly expensive and in arthritis you need to keep taking it regularly.
Now let us look at another related very expensive drug. Etanercept (trade name Enbrel).  Enbrel is another immunosuppressive drug for arthritis , but this time it is not inhibiting IL-6 but rather tumor necrosis factor (TNF).
This drug also treats a condition called psoriasis.  There is a case of a 53 year old Italian lady only partially verbal and by the sound of it, autistic, living with her mother.  She had her psoriasis treated with Enbrel and suddenly she became social and her speech improved.  Now an example of one is definitely interesting, but it does not prove anything.
But, remember Dr Chez from Sacramento?  Tucked away in his excellent paper of immunomodulation in autism.

"A single case of repetitive regression, with bouts of inflammatory colitis in an 8-year-old with regressive autism after age 3, has shown elevated serum TN alpha levels and rapid colitis, as well as behavioral and language improvements after injections of etanercept (unpublished data, personal communication Y. Davies and M. Chez 2008)."

At the time, I did not pay much attention since who can afford an ongoing therapy costing tens of thousands of dollars a year?
But, there is more.
In the US, a controversial doctor has been treating various chronic neurological dysfunctions with single dose etanercept.  He was criticized both for his marketing and the lack of published research to back up his claims.  To his credit, he is now publishing his work and has patented his therapy.

Here is a press article.

Here is an abstract of the study:-

Selective TNF inhibition for chronic stroke and traumatic brain injury: an observational study involving 629 consecutive patients treated with perispinal etanercept.



Brain injury from stroke and traumatic brain injury (TBI) may result in a persistent neuroinflammatory response in the injury penumbra. This response may include microglial activation and excess levels of tumour necrosis factor (TNF). Previous experimental data suggest that etanercept, a selective TNF inhibitor, has the ability to ameliorate microglial activation and modulate the adverse synaptic effects of excess TNF. Perispinal administration may enhance etanercept delivery across the blood-CSF barrier.


The objective of this study was to systematically examine the clinical response following perispinal administration of etanercept in a cohort of patients with chronic neurological dysfunction after stroke and TBI.


After approval by an independent external institutional review board (IRB), a chart review of all patients with chronic neurological dysfunction following stroke or TBI who were treated open-label with perispinal etanercept (PSE) from November 1, 2010 to July 14, 2012 at a group medical practice was performed.


The treated cohort included 629 consecutive patients. Charts of 617 patients following stroke and 12 patients following TBI were reviewed. The mean age of the stroke patients was 65.8 years ± 13.15 (range 13-97). The mean interval between treatment with PSE and stroke was 42.0 ± 57.84 months (range 0.5-419); for TBI the mean interval was 115.2 ± 160.22 months (range 4-537). Statistically significant improvements in motor impairment, spasticity, sensory impairment, cognition, psychological/behavioural function, aphasia and pain were noted in the stroke group, with a wide variety of additional clinical improvements noted in individuals, such as reductions in pseudobulbar affect and urinary incontinence. Improvements in multiple domains were typical. Significant improvement was noted irrespective of the length of time before treatment was initiated; there was evidence of a strong treatment effect even in the subgroup of patients treated more than 10 years after stroke and TBI. In the TBI cohort, motor impairment and spasticity were statistically significantly reduced.


Irrespective of the methodological limitations, the present results provide clinical evidence that stroke and TBI may lead to a persistent and ongoing neuroinflammatory response in the brain that is amenable to therapeutic intervention by selective inhibition of TNF, even years after the acute injury.


Excess TNF contributes to chronic neurological, neuropsychiatric and clinical impairment after stroke and TBI. Perispinal administration of etanercept produces clinical improvement in patients with chronic neurological dysfunction following stroke and TBI. The therapeutic window extends beyond a decade after stroke and TBI. Randomized clinical trials will be necessary to further quantify and characterize the clinical response.

Now I am fully aware that author, Dr Tobinick,  has got into trouble with the Medical Board of California for his marketing approach.  Here is a link for those interested.  This does not mean his off-label use of etanercept is without merit.
Etanercept (trade name Enbrel) is a biopharmaceutical that treats autoimmune diseases by interfering with tumor necrosis factor (TNF; a soluble inflammatory cytokine) by acting as a TNF inhibitor. It has U.S. F.D.A. approval to treat rheumatoid, juvenile rheumatoid and psoriatic arthritis, plaque psoriasis and ankylosing spondylitis. TNF-alpha is the "master regulator" of the inflammatory (immune) response in many organ systems. Autoimmune diseases are caused by an overactive immune response. Etanercept has the potential to treat these diseases by inhibiting TNF-alpha.
Other comorbidities

You might view arthritis and psoriasis as as being related rather than being comorbid with autism.  Are there other comorbid conditions where anti-cytokine therapy is used?

One example is Irritable Bowel Disease (IBD), where several anti-TNF-alpha drugs have been shown to be effective and are widely prescribed.  IBD includes ulcerative colitis (UC) and the more severe Crohn’s disease.  UC does appear to be comorbid with autism and indeed UC itself does seem to be associated with mild autistic behaviours.  You will find adults with UC debating whether or not they have Asperger’s.

Here is a short video on anti-TNF therapy in IBD.

The complete set of video on IBD can be found here:-

For those scientists among you here is a full paper on this subject:- 
Pro-Inflammatory Cytokines in the Pathogenesis of IBD


I am surprised that nobody has sought to do even a very small trial of Etanercept/Enbrel or Tocilizumab/Actemra in autism. These potent immunomodulatory drugs can have side effects with long term use, but the case reports suggest that a single dose can be disease changing in neurological conditions, like autism.
In all likelihood only a single dose would be needed, so you really would not need the usual years of delay to complete a trial.  There is a lot of interest in GH and IGF-1 therapy in autism, which both require ongoing injections. To trial Etanercept and Tocilizumab would be so easy, in comparison.
Because the mechanism of action is fully understood, and IL-6 and TNF-ᾳ are easy to measure, it would later be possible to identify the people most likely to benefit from the cytokine lowering therapy.  Quite possibly it would be people with regressive autism who would benefit most, since they have the highest level of inflammatory markers, as highlighted by Ashwood.
If indeed the therapy worked, it is not going to be cheap; but at least it would be a one-off cost of $1,000 to $2,000, rather than a monthly cost as in severe arthritis.
I think our new friend Dr Wei would favour Tocilizumab/Actemra. If you live in California, Dr Tobinick would be the one to ask about Etanercept/Enbrel, but it won’t be cheap.

If medicine was a true science, we would have longitudinal autism studies that showed the level of inflammatory cytokines over time.  Then we would be able to say, for example, when regression occurs there is acute neuroinflammation with a spike in IL-6,TNF-ᾳ and other cytokines. 
Perhaps this inflammation does some long term damage that might be halted with immediate immunomodulatory therapy.  If the data did show this, we could look for correlations between later behavioral improvement and falling level in inflammatory cytokines. 
In children with regressive autism and who do not improve much, do the inflammatory cytokines stay at high levels?  Are the behavioral problems caused by the current level of inflammatory cytokines, or is the problem caused by the long term damage the cytokines already caused?  With data, all these questions could be answered.  Without data it is just conjecture.
All you need to do this research are regular blood samples.  The tests themselves are cheap.  Then you could compare cheap immunomodulatory therapy using steroids versus the expensive arthritis injections used one-off.

Friday 20 December 2013

Amyloids, APP, ADAM17 and Autism

Tonsil biopsy in variant CJD, source: Wikipedia

Amyloid may sound like someone’s name, but in fact it is something rather sinister and is related to many brain disorders.  It appears that, at least in severe cases, they may be implicated in autism, or least the precursor is.
Proteins that are normally soluble undergo a process called amyloidosis, which makes them insoluble and allows deposits to accumulate in various organs, including the brain.  There are many known examples, including Alzheimer’s  and Mad Cow Disease (Creutzfeldt–Jakob disease).  A number of years ago there was a huge public health scare in the UK, when humans were affected by Mad Cow Disease, after eating the brains of cows in processed food.
Symptoms vary widely, depending upon where in the body amyloid deposits accumulate. Amyloidosis may be inherited or acquired.
The precursor to amyloid is naturally called  Amyloid Precursor Protein (APP).
APP exists in all of us and is not necessarily bad.  Its function is not fully understood (see later in this post). 

Alzheimer’s                           Autism

Affects female > Mmale                                   Affects male > female 

Brain atrophy                                                     Macrocephaly
                                                                              (enlarged brain in child)

Amyloid plaques  

Degenerative                                                      Decline followed by stable 

High αβ, low sAPPα                                           High sAPPα, low αβ

Amyloid Precursor Protein (APP)

The gene related to Amyloid Precursor Protein (APP), was only identified in 1987 and the biology surrounding it is only very partially understood.  Much of the experimental work is related to Alzheimer’s, but some of these researchers are also looking at implications for autism.
For the bold, here is a very recent paper on APP:-

A power-point style presentation is here:-

The research proved the hypothesis:-

APP metabolites follow nonamyloidgenic pathway (i.e., high sAPP, sAPPα, low Aβ 40) in brain tissue of children with autism, compared to age matched controls

Here is the data:-



For those of you who want to read a full paper by the same authors from Indianapolis, here it is:-

Biologists do make their work sound very complicated; generally it is the terminology that may make it look unintelligible on first reading.  Just read it again and look up the confusing terms.  They also seem to have up to 5 different names for the same molecule.

Compared to other areas of science like Fluid Mechanics, which I had to study, and Wikipedia rather understated describes as “Fluid mechanics can be mathematically complex”,  biology is just a lot of knowledge; none is really intellectually challenging, at least not until the amyloids start growing.
Just use the amazingly up to date resources of Wikipedia.

  =  beta amyloid   = amyloid β-peptide     The most common isoforms are Aβ40 and Aβ42
βAPP = β-amyloid precursor protein = amyloid-β precursor protein  = AβPP

 sAPPα = soluble APPα = soluble amyloid precursor protein α

β-secretase = Beta-secretase 1  = BACE1 = beta-site APP cleaving enzyme 1 = beta-site amyloid precursor protein cleaving enzyme 1

 γ-secretase = Gamma secretase

Gamma secretase can cleave APP in any of multiple sites to generate a peptide from 39 to 42 amino acids long.

Generation of the 42  Aβ (amyloid β-peptides) that aggregate in the brain of Alzheimer's patients requires two sequential cleavages of APP.  Extracellular cleavage of APP by β-secretase (BACE) creates a soluble extracellular fragment and a cell membrane-bound fragment referred to as C99. Cleavage of C99 within its transmembrane domain by γ-secretase releases the intracellular domain of APP and produces Aβ (amyloid-β).
However a single residue mutation in APP reduces the ability of β-secretase to cleave it to produce amyloid-beta and reduces the risk of Alzheimers and other cognitive declines.
Inhibitors of amyloid deposition include the enzymes responsible for the production of extracellular amyloid such as β-secretase and γ-secretase inhibitors.  Currently the γ-secretase inhibitors are in clinical trials as a treatment for Alzheimer's disease.

Amyloid Precursor Protein
Amyloid precursor protein (APP) is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Its primary function is not known, though it has been implicated as a regulator of synapse formation, neural plasticity and iron export. APP is best known as the precursor molecule whose proteolysis generates beta amyloid (Aβ), a 37 to 49 amino acid peptide whose amyloid fibrillar form is the primary component of amyloid plaques found in the brains of Alzheimer's disease

Biological function
Although the native biological role of APP is of obvious interest to Alzheimer's research, thorough understanding has remained elusive.

Synaptic formation and repair
The most-substantiated role for APP is in synaptic formation and repair; its expression is upregulated during neuronal differentiation and after neural injury. Roles in cell signalling, long-term potentiation, and cell adhesion have been proposed and supported by as-yet limited research. In particular, similarities in post-translational processing have invited comparisons to the signaling role of the surface receptor protein Notch.
APP knockout mice are viable and have relatively minor phenotypic effects including impaired long-term potentiation and memory loss without general neuron loss. On the other hand, transgenic mice with upregulated APP expression have also been reported to show impaired long-term potentiation.
The logical inference is that because Aβ accumulates excessively in Alzheimer's disease its precursor, APP, would be elevated as well. However, neuronal cell bodies contain less APP as a function of their proximity to amyloid plaques. The data indicate that this deficit in APP results from a decline in production rather than an increase in catalysis. Loss of a neuron's APP may affect physiological deficits that contribute to dementia.

Iron export
A different perspective on Alzheimer's is revealed by a mouse study that has found that APP possesses ferroxidase activity similar to ceruloplasmin, facilitating iron export through interaction with ferroportin; it seems that this activity is blocked by zinc trapped by accumulated Aβ in Alzheimer's. It has been shown that a single nucleotide polymorphism in the 5'UTR of APP mRNA can disrupt its translation.
The hypothesis that APP has ferroxidase activity in its E2 domain and facilitates export of Fe(II) is possibly incorrect since the proposed ferroxidase site of APP located in the E2 domain does not have ferroxidase activity.
Hormonal regulation
The amyloid-β precursor protein (AβPP) and all associated secretases are expressed early in development and plays a key role in the endocrinology of reproduction – with the differential processing of AβPP by secretases regulating human embryonic stem cell (hESC) proliferation as well as their differentiation into neural precursor cells (NPC). The pregnancy hormone human chorionic gonadotropin (hCG) increases AβPP expression and hESC proliferation while progesterone directs AβPP processing towards the non-amyloidogenic pathway, which promotes hESC differentiation into NPC.
AβPP and its cleavage products do not promote the proliferation and differentiation of post-mitotic neurons; rather, the overexpression of either wild-type or mutant AβPP in post-mitotic neurons induces apoptotic death following their re-entry into the cell cycle. It is postulated that the loss of sex steroids (including progesterone) but the elevation in luteinizing hormone, the adult equivalent of hCG, post-menopause and during andropause drives amyloid-β production and re-entry of post-mitotic neurons into the cell cycle.

Recently, amyloid precursor protein (APP) origin was demonstrated with arthritogenic animals. The source noted is breakdown of immune complexes, where the amyloid aggregates are left degraded and bind together to form coil like structures that are not reabsorbed. Also, it induces secondary inflammation, which may cause local damage.

ADAM17 is understood to be involved in the processing of tumor necrosis factor alpha (TNF-α) at the surface of the cell. This process, which is also known as 'shedding', involves the cleavage and release of a soluble ectodomain from membrane-bound pro-proteins (such as pro-TNF-α), and is of known physiological importance. ADAM17 was the first 'sheddase' to be identified, and is also understood to play a role in the release of a diverse variety of membrane-anchored cytokines, cell adhesion molecules, receptors, ligands and enzymes.

Even though it does sound complicated, there are some conclusions.

Amyloid Precursor Protein (APP) can either be processed towards so-called amyloidogenic pathways in the brain that lead to Alzheimer’s, or it can follow so-called non-amyloidogenic pathways, as appears to be the case in autism.  The direction taken seems to depend on α, β and γ–secretases, which are themselves regulated by neurotransmitters and other signalling molecules.
But why are there elevated levels of APP in autism?

As is often the case in autism research, some are thinking biomarker and some are thinking about therapeutic interventions.  I am with the latter.
By the way, now we have dealt with Amy, what about Adam? (the final chart above)
Functional ADAM17 has been documented to be expressed in the human colon, with increased activity in the colonic mucosa of patients with ulcerative colitis, a main form of inflammatory bowel disease.  But remember, that paper by Wakefield was retracted and so there should not be evidence linking autism with colitis.  Tell Adam to keep quiet.

ADAM17 = ADAM metallopeptidase domain 17  =  TACE  = (tumor necrosis factor-α-converting enzyme) = TNF α-converting enzyme 

TNF are a group of cytokines that cause cell death.