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Sunday, 26 March 2017

Sensory Gating in Autism, Particularly Asperger's


Sensory gating is an issue in autism, schizophrenia and ADHD.   It is the neurological process of filtering out redundant or unnecessary stimuli in the brain; like the child who sits in his classroom and gets bothered by the noise of the clock on the wall.  He is unable to filter out and ignore this sound. He becomes preoccupied by the sound and cannot concentrate on his work.
There are also sometimes advantages to not filtering out environmental stimuli, because you would have more situational awareness and notice things that others miss.
An example of sensory gating is the fact that young children are not waken by smoke detectors that have high pitched siren, but are waken by a recorded human voice telling them there is a fire and to wake up.
There may be times when sensory overload in autism is not a case of too much volume from each of the senses, but rather too many inputs being processed by the brain, instead of some just being ignored.  It is more a case of information overload.
Note that this blog has already covered hypokalemic sensory overload in some depth, which is treatable.
Much is known about sensory gating because it has long been known to be a problem in schizophrenia.
An EEG (Electroencephalography) test measures your brain waves / neural oscillations. Many people with autism have EEGs, but mainly those in which epilepsy is a consideration.
In the world of the EEG, the P50 is an event occurring approximately 50 millisecond after the presentation of an auditory click.  The P50 response is used to measure sensory gating, or the reduced neurophysiological response to redundant stimuli.
Abnormal P50 suppression is a biomarker of schizophrenia, but is present in other disorders, including Asperger’s, post-traumatic stress disorder (PTSD) and traumatic brain injury (TBI).
In more severe autism abnormal P50 suppression was found not to be present in one study.  This might be because cognition and the senses are dimmed by the excitatory-inhibitory imbalance.
More broadly, sensory gating is seen as an issue in wider autism and ADHD.

Correcting P50 gating
It is known that α7 nicotinic acetylcholine receptor (α7 nAChR) agonists can correct the impaired P50 gating. It is also known that people with schizophrenia have less expression of this receptor in their brains than typical people.

One short term such agonist is the nicotine released from smoking.  This likely contributes to why people with schizophrenia can be heavy smokers.  The effect is thought to last for about 30 minutes.
Clinical trials using Tropisetron, a drug that is a α7 nAChR agonist and used off-label to treat fibromyalgia, have shown that it can correct defective P50 gating and improve cognitive function in schizophrenia.

An alternative α7 nAChR agonist that is widely available is varenicline, a drug approved to help people stop smoking.
So you might expect varenicline to improve P50 gating and improve cognition. You might also expect it to help people with fibromyalgia and indeed some other people with chronic inflammation, as shown by elevated inflammatory cytokines.

You may recall that the α7 nAChR is the key to stimulating the vagus nerve and this should be beneficial to many people with inflammatory conditions (from arthritis to fibromyalgia).


Abnormalities in CHRNA7, the alpha7-nicotinic receptor gene, have been reported in autism spectrum disorder. These genetic abnormalities potentially decrease the receptor’s expression and diminish its functional role. This double-blind, placebo-controlled crossover study in two adult patients investigated whether an investigational receptor-specific partial agonist drug would increase the inhibitory functions of the gene and thereby increase patients’ attention. An electrophysiological biomarker, P50 inhibition, verified the intended neurobiological effect of the agonist, and neuropsychological testing verified a primary cognitive effect. Both patients perceived increased attention in their self-ratings. Alpha7-nicotinic receptor agonists, currently the target of drug development in schizophrenia and Alzheimer Disease, may also have positive clinical effects in autism spectrum disorder.


A role for H3 and HI histamine receptors
It has also been suggested that histamine plays a role in sensory gating via the H1 and H3 receptors.

It had also been thought H3 receptors could be targeted to improve cognition in schizophrenia, but that research really did not go anywhere.

Histamine H1 receptor systems have been shown in animal studies to have important roles in the reversal of sensorimotor gating deficits, as measured by prepulse inhibition (PPI). H1-antagonist treatment attenuates the PPI impairments caused by either blockade of NMDA glutamate receptors or facilitation of dopamine transmission. The current experiment brought the investigation of H1 effects on sensorimotor gating to human studies. The effects of the histamine H1 antagonist meclizine on the startle response and PPI were investigated in healthy male subjects with high baseline startle responses and low PPI levels. Meclizine was administered to participants (n=24) using a within-subjects design with each participant receiving 0, 12.5, and 25 mg of meclizine in a counterbalanced order. Startle response, PPI, heart rate response, galvanic skin response, and changes in self-report ratings of alertness levels and affective states (arousal and valence) were assessed. When compared with the control (placebo) condition, the two doses of meclizine analyzed (12.5 and 25 mg) produced significant increases in PPI without affecting the magnitude of the startle response or other physiological variables. Meclizine also caused a significant increase in overall self-reported arousal levels, which was not correlated with the observed increase in PPI. These results are in agreement with previous reports in the animal literature and suggest that H1 antagonists may have beneficial effects in the treatment of subjects with compromised sensorimotor gating and enhanced motor responses to sensory stimuli.


The aim of this study was to investigate an established rat model of decreased PPI induced by administration of the NMDA antagonist, dizocilpine and the reversal of this PPI impairment by the histaminergic H1-antagonist, pyrilamine. H1-antagonism is a potential mechanism of the therapeutic effects of the atypical antipsychotic, clozapine, which improves PPI following dizocilpine administration in rats as well as in patients with schizophrenia. In the present study we show that chronic pyrilamine administration prevents the PPI impairment induced by chronic dizocilpine administration, an effect that is correlated with a reduction in ligand-binding potential of H1 receptors in the anterior cingulate and an increase in nicotinic receptor α7 subunit binding in the insular cortex. In light of the functional anatomical connectivity of the anterior cingulate and insular cortex, both of which interact extensively with the core PPI network, our findings support the inclusion of both cortical areas in an expanded network capable of regulating sensorimotor gating.

The brain histamine system has been implicated in regulation of sensorimotor gating deficits and in Gilles de la Tourette syndrome. Histamine also regulates alcohol reward and consumption via H3 receptor (H3R), possibly through an interaction with the brain dopaminergic system. Here, we identified the histaminergic mechanism of sensorimotor gating and the role of histamine H3R in the regulation of dopaminergic signaling. We found that H3R knockout mice displayed impaired prepulse inhibition (PPI), indicating deficiency in sensorimotor gating. Histamine H1 receptor knockout and histidine decarboxylase knockout mice had similar PPI as their controls. Dopaminergic drugs increased PPI of H3R knockout mice to the same level as in control mice, suggesting that changes in dopamine receptors might underlie deficient PPI response when H3R is lacking. Striatal dopamine D1 receptor mRNA level was lower, and D1 and D2 receptor-mediated activation of extracellular signal-regulated kinase 1/2 was absent in the striatum of H3R knockout mice, suggesting that H3R is essential for the dopamine receptor-mediated signaling. In conclusion, these findings demonstrate that H3R is an important regulator of sensorimotor gating, and the lack of H3R significantly modifies striatal dopaminergic signaling. These data support the usefulness of H3R ligands in neuropsychiatric disorders with preattentional deficits and disturbances in dopaminergic signaling.



Conclusion

Other than nicotine, varenicline would seem a good potential therapy for sensory gating.  There are α7-nicotinic acetylcholine receptor agonists in development.
There are many H1 histamine antagonists.  Histamine release in the brain triggers secondary release of excitatory neurotransmitters such as glutamate and acetylcholine via stimulation of H1 receptors. Centrally acting H1 antihistamines are sedating.

H3 antagonists have stimulant and nootropic effects. Betahistine is an approved drug in this class, there are many research drugs.

The aim of this study is to investigate the role of the neurotransmitter histamine in sensory and cognitive deficits as they often occur in schizophrenia patients (e.g. hearing voices, planning and memory problems). The ideal location to conduct the study and to obtain a unique learning experience is at the Institute of Psychiatry, London, United Kingdom, where staff comprises of leading experts in the field of schizophrenia and Magnetic Resonance Imaging of pharmacological effects. Current pharmacological treatment of psychotic symptoms including sensory and cognitive deficits remains partially unsuccessful due to side effects and treatment resistance. The neurotransmitter histamine seems to be a very promising target for new treatments. It has been found that histamine neurotransmission is altered in brains of schizophrenics, which may contribute to both the hallucinatory and cognitive symptoms. However, this specific role of histamine has not been investigated before. I will assess the effects of increased histaminergic activity, by administration of betahistine to healthy volunteers, on performance (sensory gating, executive functioning or planning and memory) and associated brain activity using fMRI. Altered performance and brain activity would support the importance of histamine in schizophrenia and would provide a research model and target for new treatments.



Thursday, 23 March 2017

Targeting Angiotensin in Schizophrenia and Some Autism





 A home run? Certainly worth further consideration.

Just when you thought we had run out of hormones to connect to autism and schizophrenia, today we have Angiotensin. 

Angiotensin is a hormone that causes vasoconstriction and a subsequent increase in blood pressure. It is part of the renin-angiotensin system, which is a major target for drugs (ACE inhibitors) that lower blood pressure. Angiotensin also stimulates the release of aldosterone, a hormone that promotes sodium retention which also drives blood pressure up.

Angiotensin I has no biological activity and exists solely as a precursor to angiotensin II.

Angiotensin I is converted to angiotensin II  by the enzyme angiotensin-converting enzyme (ACE).  ACE is a target for inactivation by ACE inhibitor drugs, which decrease the rate of Angiotensin II production.  

It turns out that Angiotensin has some other properties very relevant to schizophrenia, some autism and quite likely many other inflammatory conditions. 

Blocking angiotensin-converting enzyme (ACE) induces those potent regulatory T cells that are lacking in autism and modulates Th1 and Th17 mediated autoimmunity.  See my last post on Th1,Th2 and Th17. 

In addition, Angiotensin II affects the function of the NKCC1/2 chloride cotransporters that are dysfunctional in much autism and at least some schizophrenia.  

Drugs that reduce Angiotensin are very widely prescribed, so they are cheap and well understood. This means that yet another cheap generic has the potential to be repurposed to treat neurological disorders. 

As one paper puts it “modulation of the RAAS (renin-angiotensin-aldosterone system) with inexpensive, safe pharmaceuticals used by millions worldwide is an attractive therapeutic strategy for application to human autoimmune diseases.” 

No big profits then for big pharma. 


IL-17a 

We learnt all about the inflammatory cytokines IL-17 and IL-17a in a recent post. That post was about autism, but not surprisingly, elevated levels of IL-17a are a feature in big brother schizophrenia. Big brothers do tend to get more research attention.

In schizophrenia there is increased plasmatic Angiotensin Converting Enzyme (ACE) activity in patients compared to healthy controls, which is also associated to poor cognitive functioning. The ACE main product angiotensin II has known pro-inflammatory properties. 

So an ACE inhibitor looks an obvious choice for schizophrenia.  Very slowly research is indeed moving in that direction.

Angiotensin receptor blockers have even been proposed for bipolar disorder, autism’s other elder brother.
  

What about ACE and Autism? 

As we have got used to, kid bother autism has not had the same level of research attention as given to schizophrenia, but we do have this:- 



Autism is a disease of complex nature with a significant genetic component. The importance of renin-angiotensin system (RAS) elements in cognition and behavior besides the interaction of angiotensin II (Ang II), the main product of angiotensin-converting enzyme (ACE), with neurotransmitters in CNS, especially dopamine, proposes the involvement of RAS in autism. Since the genetic architecture of autism has remained elusive, here we postulated that genetic variations in RAS are associated with autism. 

Our data suggests the involvement of RAS genetic diversity in increasing the risk of autism.
   

Here is the supporting research:-  



The renin-angiotensin-aldosterone system (RAAS) is a major regulator of blood pressure. The octapeptide angiotensin II (AII) is proteolytically processed from the decapeptide AI by angiotensin-converting enzyme (ACE), and then acts via angiotensin type 1 and type 2 receptors (AT1R and AT2R). Inhibitors of ACE and antagonists of the AT1R are used in the treatment of hypertension, myocardial infarction, and stroke. We now show that the RAAS also plays a major role in autoimmunity, exemplified by multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Using proteomics, we observed that RAAS is up-regulated in brain lesions of MS. AT1R was induced in myelin-specific CD4+ T cells and monocytes during autoimmune neuroinflammation. Blocking AII production with ACE inhibitors or inhibiting AII signaling with AT1R blockers suppressed autoreactive TH1 and TH17 cells and promoted antigen-specific CD4+FoxP3+ regulatory T cells (Treg cells) with inhibition of the canonical NF-κB1 transcription factor complex and activation of the alternative NF-κB2 pathway. Treatment with ACE inhibitors induces abundant CD4+FoxP3+ T cells with sufficient potency to reverse paralytic EAE. Modulation of the RAAS with inexpensive, safe pharmaceuticals used by millions worldwide is an attractive therapeutic strategy for application to human autoimmune diseases.
  

In an effort to find a marker that predicts psychosis, postdoctoral researcher Lindsay Hayes, Ph.D., learned unexpectedly that mice and people with behavior disorders have abnormally low levels of a hormone system tied to blood pressure regulation and inflammation. In the cerebrospinal fluid of patients with first episode psychosis, she noticed abnormally low levels of the enzyme that makes the hormone angiotensin. To see if these results correlated to animals and could be studied in the lab, Hayes, who works in the laboratory of treated brain cells with angiotensin and inflammation activators in their mouse model for behavior disorders, then measured the output of proteins involved in inflammation. Compared to normal mice, the cells from the mouse with behavioral disorders released more inflammation protein when treated with low levels of angiotensin and less when treated with high levels. Next, she looked at gene expression levels of the angiotensin system components in the brain cells of the behavioral disorder mice. The gene expression levels for the receptor that detects angiotensin were abnormally low in a specific type of brain cell. Hayes says these specific cells in the behavior disorder mice seem to be less susceptible to angiotensin’s immunosuppressive properties, because they have less receptor to detect angiotensin than the same brain cells in normal mice. Hayes and Sawa plan to investigate whether targeting angiotensin could control inflammation and perhaps treat psychosis. 

Angiotensin converting enzyme activity is positively associated with IL-17a levels in patients with schizophrenia.

Abstract


Previous studies of our group showed increased plasmatic Angiotensin-I Converting Enzyme (ACE) activity in schizophrenia (SCZ) patients compared to healthy controls, which was also associated to poor cognitive functioning. The ACE main product angiotensin II (Ang-II) has pro-inflammatory properties. Activated immune-inflammatory responses in SCZ and their association with disease progression and cognitive impairments are also well-described. Therefore, we examined here the association of plasma ACE activity and inflammatory mediators in 33 SCZ patients and 92 healthy controls. Non-parametric correlations were used to investigate the association of the enzyme activity and the peripheral levels of immune inflammatory markers as interleukins, tumor necrosis factor (TNF-α), and interferon (IFN-γ). Although no significant correlations could be observed for ACE activity and measured cytokines levels in healthy controls, a significant positive correlation for ACE enzymatic activity and IL-17a levels was observed in SCZ patients. Correcting for gender did not change these results. Moreover, a significant association for ACE activity and IFN-γ levels was also observed. To our knowledge, this is the first study to show a significant association between higher ACE activity and the levels of cytokines, namely IL-17a and IFN-γ, in patients with SCZ. 

Cerebrospinal fluid angiotensin-converting enzyme (ACE) correlates with length of illness in schizophrenia. 

Abstract


The aim of the study was to evaluate a possible progression with time of cerebrospinal fluid (CSF) angiotensin-converting enzyme (ACE) levels in treated schizophrenia patients. CSF ACE was determined in duplicate by a sensitive inhibitor-binding assay (IBA) from morning CSF samples of 56 acute and chronic in-patients with schizophrenic psychoses diagnosed according to DSM-IV. CSF ACE correlated significantly with length of schizophrenic psychosis (r=0.39, p=0.003). There was also a positive significant correlation between CSF ACE and duration of current psychotic episode (r=0.39, p=0.003) as well as duration of current hospitalization (r=0.66, p<0 .001="" span=""> These significances were maintained even when patients who were not treated with antipsychotics at the time of sampling were excluded. The correlations also remained significant when controlling for current neuroleptic dose in chlorpromazine equivalents. Serum ACE did not correlate with any clinical variable. No significant correlations between serum or CSF ACE and age, diagnostic subgroup, gender, serum ACE, CSF to serum albumin ratios, or neuroleptic dose in chlorpromazine equivalents were detected. The elevation of CSF ACE seemed to be confined to a subgroup of chronic patients with few positive symptoms. Elevated CSF ACE may reflect an increased solubilization of ACE from cell membranes in the central nervous system or constitute an increased expression of the ACE gene in response to some stimuli. This may be a function of treatment or a result of the deteriorating schizophrenic process. 



The renin-angiotensin-aldosterone system (RAAS) is a major regulator of blood pressure. The octapeptide angiotensin II (AII) is proteolytically processed from the decapeptide AI by angiotensin-converting enzyme (ACE), and then acts via angiotensin type 1 and type 2 receptors (AT1R and AT2R). Inhibitors of ACE and antagonists of the AT1R are used in the treatment of hypertension, myocardial infarction, and stroke. We now show that the RAAS also plays a major role in autoimmunity, exemplified by multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Using proteomics, we observed that RAAS is up-regulated in brain lesions of MS. AT1R was induced in myelin-specific CD4+ T cells and monocytes during autoimmune neuroinflammation. Blocking AII production with ACE inhibitors or inhibiting AII signaling with AT1R blockers suppressed autoreactive TH1 and TH17 cells and promoted antigen-specific CD4+FoxP3+ regulatory T cells (Treg cells) with inhibition of the canonical NF-κB1 transcription factor complex and activation of the alternative NF-κB2 pathway. Treatment with ACE inhibitors induces abundant CD4+FoxP3+ T cells with sufficient potency to reverse paralytic EAE. Modulation of the RAAS with inexpensive, safe pharmaceuticals used by millions worldwide is an attractive therapeutic strategy for application to human autoimmune diseases.


African Americans have been shown to exhibit lower urinary potassium excretion when compared to Caucasians. Angiotensin II regulates both potassium handling by the kidney and the Na-K-2Cl (NKCC) cotransporter in vitro . However, little is known about the role of the reninangiotensin system (RAS) in human NKCC cotransport regulation in vivo. We hypothesized that regulation of RAS would induce concomitant alterations in NKCC activity in humans. The kidney and erythrocyte express NKCC-1 isoform. Therefore, we measured NKCC-1 activity in freshly isolated ex vivo red cells from 12 healthy blacks and 11 healthy whites in high (200 mmol/d) and low (10 mmol/d) salt balance, followed by a measure 24 h-post candesartan [16 mg] to block angiotensin II type I receptors on low salt diet. Baseline NKCC cotransport activity was significantly lower in Blacks when compared to Whites in balance on a typical high salt diet, and was reduced when the subjects were placed on a low salt diet in whites only. Administration of candesartan reversed the reduction seen with low salt diet in whites, where as in blacks there was no significant effect. These data suggest altered in vivo regulation of NKCC-1 via RAS in Blacks when compared to Whites, and provide a mechanism that may in part explain the altered potassium handling observed among otherwise healthy African Americans.


Conclusion

I think it is likely that some sub-types of autism would likely benefit from an ACE inhibitor. As a secondary benefit, it will also reduce any troubling high levels of leptin.

There are other ways to modulate Th1, Th2 and Th17, but if you have elevated Angiotensin Converting Enzyme (ACE), then an ACE inhibitor would appear the logical choice.

How about a clinical trial in adults with Asperger's?











Wednesday, 22 March 2017

Secretome, Microbiome/Hologenome, Proteome, Epigenome, Exome and Genome


Biologists clearly like "–omes".

A full understanding of all the –omes would lead to an understanding of pretty much all disease, including autism.  Science is still a long way from such an understanding.

More questions than answers

Even a partial understanding of the "-omes does help to see how things fit together and also it helps understand where the research is going and why. It also makes you realize how little we currently know, and that includes all those clever scientists. This is why we keep coming back to stumbled-upon and trial error as valid discovery methods.
You can also see why researchers and those who fund them, like Mr Simons, easily get lost in the detail. Behind the 5% of the detail they fully understand is another 95% that will take a hundred years to fully understand. 


Microbiome & Hologenome/Holobiont
The intestinal microbiome is currently very fashionable.

The intestinal microbiome describes the microorganisms that reside in your intestines/gut.  It includes bacteria, fungi, viruses and archaea/protists (single cell organisms). Microbiota have been found to be crucial for immunologic, hormonal and metabolic homeostasis of their host.
The microbiome and host emerged together during evolution as a synergistic unit from epigenetic and genetic characteristics, called a holobiont.  The hologenome concept of evolution, considers a human as a community, or a holobiont - the host plus all of its symbiotic microbes. The collective genomes of the holobiont form a hologenome.
This becomes very relevant in human disease because in modern life humans have become separated from part of their evolutional holobiont (symbiotic microbes).  As a result all kinds on immune disease have become more prevalent.
It became accepted that all dirt is bad and daily contact with other living non-human animals is bad.

Humans are colonized by many microorganisms and surprisingly your body contains more  types of non-human cells than human cells. Some estimates suggest it is many times more; food for thought.
Microbiota have been found to be crucial for immunologic, hormonal and metabolic homeostasis of their host.

Bacteria are transferred from mother to child through different pathways. As the newborn’s microbiome is established, bacteria quickly populate the gut, prompting a range of immune responses and programming the immune system with long-lasting effects.
Having pets at home during and after pregnancy has been shown to have beneficial health effects on the child. Your pet dog and his bugs are part of your holobiont. If you make drastic changes in your holobiont, do not be surprised if strange things happen; a case in point are the so-called Somali Autism Clusters. When Somali refugees moved to Sweden and the US, they produced children with a high incidence of autism and all the researchers wondered why.

Secretome
This was a new "–ome" to me, that we came across in a recent post.
The secretome is the totality of secreted organic molecules and inorganic elements by biological cells, tissues, organs, and organisms.
It turned out that human fat cells secrete over 600 different different proteins/hormones.  None of this was even known 25 years ago.
So your endocrinologist knows all about the usual endocrine glands: pineal gland, thymus gland, pituitary gland, thyroid/parathyroid gland, and adrenal glands; but what about the largest endocrine gland of all, your body fat (adipose tissue)?

Genome
I did write a post about genetics, more for completeness than in the expectation that many people would read it.

The genome includes a small part of so-called “coding DNA” that contains the instructions to make proteins, this is called the exome.  The remaining 98% is made up of non-coding DNA.  This 98% used to be called junk DNA, but it is not. It is suggested that 80% of human genomic DNA has biochemical activity, not just the 2% that is the coding DNA in the exome. 

Exome
The exome is the 2% of the genome that holds the codes to make the proteins that make up your body.

Epigenome
The epigenome are all the so-called epigenetic markers that you accumulate from the environment and that you inherited from your ancestors.  You could think about this as bruises and scars on your body.  Imagine if you inherited your Grandma’s appendix scar and Grandad’s scar from a skiing accident.  In epigenetics the fact that Grandad was a heavy smoker may have marked your genes that relate to oxidative stress.
Your personal epigenome is a record of all these markers and it would show which genes are likely miss-expressed.  It would be used to determine personalized medical therapies.
This would be very relevant to autism.  

Proteome
I rather like the proteome, because it would tell you what is happening right now, rather than what might happen (as in the genome, exome and epigenome).

Most people do not realize just how much in your body is changing all the time and is not fixed at all.
The proteome is the entire set of proteins expressed by the coding DNA (the exome part of the genome), at a certain time.
So it would tell you exactly what is happening in a specific part of your body at a specific time.
This would be invaluable in understanding autism, if it could be done in real time in different parts of the brain. This is not possible with today’s technology.









Monday, 20 March 2017

Progress towards the Approval of Bumetanide as a Drug to treat Core-Autism


This blog is seen by many as being  bumetanide-inspired, so it is only fair to highlight the recently published Bumetanide for autism Phase IIb results. This study probably will not get the attention it should get in the media, in part because it is French and not American. 




This was a trial based on 90 days of taking the drug, while some readers of this blog have been using bumetanide for years. 

Nonetheless, there are some interesting things in the study, most importantly is that you can see the guiding hand of the drug approving agency (the European Medicines Agency).  They have specified how to measure the results (the CARS rating scale), the level of severity of autism to test the drug on (severe) and the age group (2-18 years old).

In this blog we know that autism is very heterogeneous and that while bumetanide can be highly effective, there is a large group that do not respond.  This trial did not use any kind of biomarker and so it contains a broad mix of responders and non-responders.

The good news is that even though only about 30-50% of people with an autism diagnosis seem to be responders, this was more than enough to make the drug look effective in a trial of just 88 people.

The small sample size does throw up anomalies, like giving the impression that 0.5mg twice a day  is more effective than 1mg twice a day, on some measures. 

The most effective treatment, judged by the authors, was 2mg twice a day, but it was ruled out for the Phase III trial because of side effects.  

“Clearly this trial must be viewed as a source of data on the safety and dose-ranging usage of bumetanide and it provides further support to justify a large multisite European Phase III trial.

We report here the results of a multicenter phase II dose-ranging study conducted according to a pediatric investigation plan approved by the EMA to determine the optimum bumetanide dose in ASD children and adolescents aged 2–18 years. To achieve these aims, the study population was divided into four subgroups receiving three doses of bumetanide (0.5, 1.0, 2.0mg twice a day) or placebo. The distribution of age was the same in the four groups (2–4, 5–9, 10–13, 14–18 years old). The results of this trial confirm and extend our earlier observation that bumetanide improves the symptoms of ASD and that too in the full age range targeted. 

EMA imposed the age range from 2 to 18.

CARS has been selected by the EMA in their recent (2016) guidelines for evaluation of ASD core symptoms (Guidelines on the clinical development of medicinal products for the treatment of ASD, 25 December 2015).
The mean initial CARS scores were similar across all treatment groups in the six clinical centers and were above the cut-off for severe autism as required by EMA (>34). “

  
Placebo Effect

In the CARS scale, a score greater than 30 is the threshold for autism.  The higher the score, the more severe the autism. The regulator required that people in the trial had a CARS score greater than 34 to be eligible for the trial. The average CARS score for those in the trial was 41, so you would need an average improvement of 11 to potentially make them all “better” (this is a simplification).  This just shows the meaning of improving by 8 CARS points.  It is a big deal.

Here you see what happens when half a study group are not responders to bumetanide and you see a large group who either show minor improvement or get worse.  This is just the expected background noise in all autism trials.  There is even one person who improved greatly (>8 on the CARS scale) just by taking the placebo.




 Change in the completers of the total CARS score from screening to day 90 after bumetanide (blue bars; n=52) and placebo (orange bars; n=21). All changes were calculated from the initial values for each individual participant at screening. Note a significant amelioration of the CARS scale after the treatment period (>4) is almost entirely restricted to the bumetanide-treated patients (only placebo). CARS, Childhood Autism Rating Scale.


Thirty bumetanide- and five placebo-treated showed an attenuation of more than 4 of whom 23 treated and only one placebo showed an amelioration of more than six Childhood Autism Rating Scale (CARS) scores, and 13 of these and only one placebo showed an attenuation of more than 8 points. The differences between placebo- and bumetanide-treated patients having more than 4, 6 or 8 points attenuation are highly significant.




Side effects 

As we have seen in feedback on this blog the harmful side effects of bumetanide are dehydration and low levels of potassium. These side effects are entirely manageable, but that job is for the parents. 
I was interested in the chart below which measures the potassium levels during the trial of the four groups. I assume green (placebo), blue (0.5mg), red (1.0) mg, black (2.0mg)


  

 Points perhaps unbeknown to the authors

An important point we have seen on this blog is that intracellular levels of chloride vary under the influence of inflammation (which affects KCC2 expression).  This results in some people responding well to bumetanide at some times and apparently not at others.
We also noted that bumetanide does not cross the blood brain barrier very well and so in some people with high levels of intracellular chloride they may appear not to respond to bumetanide, not because they do not have elevated Cl-, but because it is just too high for bumetanide to show effect. 
Taken together the point is that if at any one time say 40% of people with autism are responders, there may be another x% who are potential responders to a similar but more potent therapy.
If you trial bumetanide in the summer and have a pollen allergy, it may appear not to work, as in the case of my son. Fortunately I did my trial in the winter.


New information


Dr Ben-Ari told Medscape Medical News that the phase 3 study, which is approved by the European authorities, will be performed in about 400 children in five EU countries. The patients will receive 1 year of treatment, and they will reflect the entire pediatric autism population.
"We hope to get the drug on the market in Europe for autism by the end of 2021," he said.
Source: http://www.medscape.com/viewarticle/877562#vp_2



As some wise older person told me many years ago, "things take time".  In the world of autism, things seem to take forever.





Friday, 17 March 2017

T helper cells in Autism - TH1 TH2 & TH17


Today’s post is about another complex and still emerging subject.  It should really be earlier in this blog.

There are lots of papers highlighted for those who like the details. The papers written by the autism researchers are generally much simpler to read than those by the mainstream researchers.  


First some biology:-

  

   


  
Differentiation of naïve T helper cells into particular subsets. T helper lymphocytes leaving the thymus (naïve or Th0) are not yet fully differentiated to perform their specific functions in peripheral lymphoid tissues. They are endowed of these properties in the process of their interactions with dendritic cells (DCs) that engulf, process, and present antigens to them.  DCs produce different cytokines.

If DCs produce IL-12, naïve T cells polarise into the Th1 subset

If DCS produce IL-4 into the Th2 subset

if DCs synthesise IL-6, naïve T helper cells will become the Th17 cells. 

Th2 helper cells are triggered by IL-4 and their effector cytokines are IL-4, IL-5, IL-9, IL-10 and IL-13

IL-10 suppresses Th1 cells differentiation and function of dendritic cells.  

Th2 over activation against autoantigen will cause Type1 IgE-mediated allergy and hypersensitivity. Allergic rhinitis, atopic dermatitis, and asthma belong to this category of autoimmunity. 

Effector Th cells secrete cytokines. 

Memory Th cells retain the antigen affinity of the originally activated T cell, and are used to act as later effector cells during a second immune response (e.g. if there is re-infection of the host at a later stage).


Regulatory T cells do not promote immune function, but act to decrease it instead. Despite their low numbers during an infection, these cells are believed to play an important role in the self-limitation of the immune system; they have been shown to prevent the development of various autoimmune diseases.  

***  

It has been pointed out by Paul Ashwood, and others, that people with autism fit into sub-groups based on their immune profile and could be treated as such.  In the jargon that becomes:-


“Children with ASD may be phenotypically characterized based upon their immune profile. Those showing either an innate proinflammatory response or increased T cell activation/skewing display a more impaired behavioral profile than children with noninflamed or non-T cell activated immune profiles. These data suggest that there may be several possible immune subphenotypes within the ASD population that correlate with more severe behavioral impairments.”



In my case I want more IL-10, less Th2, less Th17 (IL-17) and less IL-6.


The idea of Th1/Th2 balance that appears on parent internet forums no longer seems entirely valid, because in autism cytokines from both systems can be found elevated. It used to be thought that someone’s immune system could be skewed one way or the other.


Allergies have been thought of as generally Th2 driven and autoimmune disorders generally Th1 driven. Some people have both.
Under normal circumstances, the Th1 and Th2 systems balance one another by inhibiting each other's activity. Each type of helper T cell (Th) produces different kinds of cytokines, with the Th cell types defined by the cytokines they produce. These cytokines are termed interferons and interleukins. Within the Th1 system, the dominant cytokine is interferon gamma (IFN-gamma), which is responsible primarily for reactions against viruses and intra-cellular microbes, and is pro-inflammatory.
Th2 cells produce interleukins IL-4, IL-5, IL-9, (IL-10) and IL-13 among. These interleukins are important for stimulating production of antibodies and often have multiple functions. As part of the Th2 system, IL-4 and IL-13 are primarily anti-inflammatory (by inhibiting Th1 cells), but they also promote the growth and differentiation of other immune cells. IL-4 also has the very important role of producing the regulatory cytokine IL-10, which helps maintain the balance between the Th1- and Th2- produced cytokines.
Historically, the role of cytokines in the immune system dysregulation observed in studies of individuals with autism has not been conclusive, because different patterns of cytokine activation have been found.  It is necessary to great subgroups with similar profiles. 



Along came Th17 

The relative newcomer is Th17 which produce IL-17. Th17 is the target of much research into Crohn’s disease, MS and now even autism.  Inhibition of IL-17 is seen as having great merit for numerous diseases. There is also the IL-23 - IL-17 immune axis; since most cells that produce IL-17 cannot do so with IL-23 being present. In the research anti-IL-17 and anti-IL-23 treatments are remarkably effective for many immune-mediated inflammatory diseases. 

The autism research has shown that IL-17 can be inhibited in mouse models that show clear behavioral gains; but they use resveratrol doses of 20 and 40 mg/kg given by injection. We already know that resveratrol given orally has very low bioavailability. 

Th17 has been shown able to cause autism, via immune activation of the pregnant mother, but it has also been shown to be an ongoing issue, with elevated levels of IL-17 and IL-17a found in people with autism. 


Not to forget Tregs 

T regulatory cells (Tregs) are another component of the immune system that suppresses the immune responses of other cells. Impaired function, or just lack of Treg cells, is associated with various diseases including MS. 

Some autism studies show increased IL-6, increased IL-17 but a systemic deficit of Treg cells. 


In the middle seesaw we have plenty of Th1, Th2, Th17, known collectively as Teff, but few Tregs.  Things are not in equilibrium, but that is many people's autism.

The generation of both effector (Th1, Th2, Th17) and regulatory T cells (Tregs) is profoundly influenced by gut microbiota. 

You could see this as a lack of wide range of bacteria in the mother and baby resulting in a maladjusted immune system, or you could just see modifying the microbiota of an person with autism as a novel therapeutic strategy. 

Regular readers of this blog will be well aware that we have already looked at three different ways to use the gut to modify the immune system.


1.     Using the short chain fatty acid (SCFA) butyric acid you can increase Tregs and affect Th1. Th2 and Th17.  We saw this added to animal feed to improve immune health and a least one reader of this blog uses sodium butyrate. The mode of action is as an HDAC inhibitor. 


2.     The TSO helminth worms that are ingested every few weeks.  In order to avoid being rejected by the body these worms modify the host’s immune system. This seemed clever.  Potassium channels, Kv1.3 and KCa3.1, have been suggested to control T-cell activation, proliferation, and cytokine production. Recall the clever researchers in Australia determined the worm’s mode of action and are working to develop a pill. 



3.     Various probiotic bacteria and not the ones that produce SCFAs have been shown to affect Th1 Th2 and Th17 and increase Tregs. These are various different forms of Lactobacillus reuteri 


There is a lot of research on this subject, for those who are interested, even as an anti-obesity therapy and an anti-asthma therapy.  


  



A recent epidemiological study showed that eating ‘fast food’ items such as potato chips increased likelihood of obesity, whereas eating yogurt prevented age-associated weight gain in humans. It was demonstrated previously in animal models of obesity that the immune system plays a critical role in this process. Here we examined human subjects and mouse models consuming Westernized ‘fast food’ diet, and found CD4+ T helper (Th)17-biased immunity and changes in microbial communities and abdominal fat with obesity after eating the Western chow. In striking contrast, eating probiotic yogurt together with Western chow inhibited age-associated weight gain. We went on to test whether a bacteria found in yogurt may serve to lessen fat pathology by using purified Lactobacillus reuteri ATCC 6475 in drinking water. Surprisingly, we discovered that oral L. reuteri therapy alone was sufficient to change the pro-inflammatory immune cell profile and prevent abdominal fat pathology and age-associated weight gain in mice regardless of their baseline diet. These beneficial microbe effects were transferable into naïve recipient animals by purified CD4+ T cells alone. Specifically, bacterial effects depended upon active immune tolerance by induction of Foxp3+ regulatory T cells (Treg) and interleukin (Il)-10, without significantly changing the gut microbial ecology or reducing ad libitum caloric intake. Our finding that microbial targeting restored CD4+ T cell balance and yielded significantly leaner animals regardless of their dietary ‘fast food’ indiscretions suggests population-based approaches for weight management and enhancing public health in industrialized societies. 




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 H2 receptor. 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. 



 Conclusions: These results strongly support a role for nonantigen-specific CD4+CD25+Foxp3+ regulatory T cells in attenuating the allergic airway response following oral treatment with L. reuteri. (ATCC #23272). This potent immuno-regulatory action may have therapeutic potential in controlling the Th2 bias observed in atopic individuals. 


There is a rather complex paper that shows how the different short chained fatty acids (SCFAs) affect different element of the immune system. More work needs to done to see if only butyric acid has therapeutic merit.  



Microbial metabolites such as short chain fatty acids (SCFAs) are highly produced in the intestine and potentially regulate the immune system. We studied the function of SCFAs in regulation of T cell differentiation into effector and regulatory T cells. We report that SCFAs can directly promote T cell differentiation into T cells producing IL-17, IFN-γ, and/or IL-10 depending on cytokine milieu. This effect of SCFAs on T cells is independent of GPR41- or GPR43 but dependent on direct histone deacetylase (HDAC) inhibitor activity. Inhibition of HDACs in T cells by SCFAs increased the acetylation of p70 S6 kinase and phosphorylation rS6, regulating the mTOR pathway required for generation of Th17, Th1, and IL-10+ T cells. Acetate (C2) administration enhanced the induction of Th1 and Th17 cells during C. rodentium infection but decreased anti-CD3-induced inflammation in an IL-10-dependent manner. Our results indicate that SCFAs promote T cell differentiation into both effector and regulatory T cells to promote either immunity or immune tolerance depending on immunological milieu.


acetate (C2), propionate (C3), and butyrate (C4), are highly produced from dietary fibers and other undigested carbohydrates in the colon 

Effector T cells, such as Th1 and Th17 cells, fight pathogens and can cause tissue inflammation.12-15 Regulatory T cells, such as IL-10+ T cells and FoxP3+ T cells, counter-balance the activities of effector immune cells. Importantly, the generation of both effector and regulatory T cells is profoundly influenced by gut microbiota  

Once entered into T cells undergoing activation, SCFAs effectively suppress HDACs as demonstrated in this study. Acetylation of proteins including histones, transcription factors and various signaling molecules by HDACs can alter the functions of modified proteins 

A pathway, important for T cell differentiation and affected by HDAC inhibition demonstrated in this study, is the mTOR-S6K pathway. The mTOR pathway promotes the expression of key effector and regulatory cytokines such as IL-10, IFN-γ and IL-17.27, 39-41 In this regard, the sustained high mTOR-S6K activity in T cells cultured with SCFAs reveals a regulatory point for SCFAs in regulation of T cell differentiation. Consistently, metformin, an anti-diabetic drug that activates AMPK and negatively regulates the mTOR pathway, was effective in suppressing the SCFA effect on T cells. Along with the mTOR pathway, STAT3 activation was enhanced as well by SCFAs, which is involved in expression of the cytokines (IL-10, IFN-γ and IL-17) in T cells.


Our results indicate that the C2 function in regulation of T cells is modulated by cytokine milieu and immunological context. We observed that IL-10+ T cells were increased by SCFAs in the steady condition in vivo, whereas effector T cells were increased by C2 only during active immune responses. Moreover, IL-10 expression was promoted in all T cell polarization conditions tested in this study, whereas the expression of IL-17 and IFN-γ was promoted specifically in respective polarization conditions. IL-10 production by effector T cells is an important negative feedback mechanism to rein in the inflammatory activities of effector T cells.42, 43 This selective enhancement of effector versus IL-10+ T cells would be beneficial to the host in promoting immunity with the built-in negative feedback function of IL-10. An interesting observation made in this study in this regard was that induction of FoxP3+ T cells by SCFAs can occur in a low TCR activation condition. Taken together, SCFAs can induce both effector and regulatory T cells including IL-10+ T cells and FoxP3+ T cells in appropriate conditions. 

Our study provides an example how the host immune system harnesses commensal bacterial metabolites for promotion of specialized effector and regulatory T cells. The results identified SCFAs as key gut metabolites important for T cell differentiation into effector and regulatory cells in the body depending on SCFA levels and immunological context. The results have many practical ramifications in regulation of tissue inflammation and immunity.
   

What to do? 

It would make sense to group people with autism together by their immune profile and then develop practical therapies for each sub-group. When will this happen? Not soon, nobody seems to be in a hurry to translate their findings into therapies. 

There is no point treating imaginary dysfunctions.  


Numerous studies suggest that abnormal activation of the immune system plays a role in causing autism. Some behavioral problems in children have been traced back to viral infections in their mothers during pregnancy. Studies in experimental mice have shown that revving up the mother’s immune system during pregnancy results in offspring with altered gene expression in the brain and problems with behavioral development. More specifically, immune system changes and autoimmune disorders, such as inflammatory bowel disease, have been found in individuals with autism.
Dan Littman and his colleagues at New York University School of Medicine suspect that the link between immune function and autism lies in a newly discovered subset of immune cells called Th17 cells.
Th17 cells are so named because they produce the inflammation-inducing signaling molecule interleukin-17. Their normal role is thought to be in fighting bacterial and fungal infections, but if this defense mechanism goes awry, Th17 cells can cause inflammatory tissue damage that eventually leads to rheumatoid arthritis, multiple sclerosis, Crohn’s disease, psoriasis and other autoimmune and inflammatory diseases.

Viral infection during pregnancy has been correlated with increased frequency of autism spectrum disorder (ASD) in offspring. This observation has been modeled in rodents subjected to maternal immune activation (MIA). The immune cell populations critical in the MIA model have not been identified. Using both genetic mutants and blocking antibodies in mice, we show that retinoic acid receptor–related orphan nuclear receptor gamma t (RORγt)–dependent effector T lymphocytes [for example, T helper 17 (TH17) cells] and the effector cytokine interleukin-17a (IL-17a) are required in mothers for MIA-induced behavioral abnormalities in offspring. We find that MIA induces an abnormal cortical phenotype, which is also dependent on maternal IL-17a, in the fetal brain. Our data suggest that therapeutic targeting of TH17 cells in susceptible pregnant mothers may reduce the likelihood of bearing children with inflammation-induced ASD-like phenotypes 



Highlights 

·        We examined cytokine production and co-morbid conditions in children with autism.


·        Increased prevalence of asthma was observed in children with autism.
·        Children with autism produced increased levels of IL-17.


·        Increased production of IL-17 and IL-13 was associated with ASD cases with asthma.
·        Typically developing children with food allergies produced increased levels of IL-13.
Inflammation and asthma have both been reported in some children with autism spectrum disorder (ASD). To further assess this connection, peripheral immune cells isolated from young children with ASD and typically developing (TD) controls and the production of cytokines IL-17, -13, and -4 assessed following ex vivo mitogen stimulation. Notably, IL-17 production was significantly higher following stimulation in ASD children compared to controls. Moreover, IL-17 was increased in ASD children with co-morbid asthma compared to controls with the same condition. In conclusion, children with ASD exhibited a differential response to T cell stimulation with elevated IL-17 production compared to controls. 




Background:  

Autism spectrum disorder (ASD) is characterized by social communication deficits and restricted, repetitive patterns of behavior. Varied immunological findings have been reported in children with ASD. To address the question of heterogeneity in immune responses, we sought to examine the diversity of immune profiles within a representative cohort of boys with ASD.  

Methods:  

Peripheral blood mononuclear cells from male children with ASD (n = 50) and from typically developing age-matched male control subjects (n = 16) were stimulated with either lipopolysaccharide or phytohemagglutinin. Cytokine production was assessed after stimulation. The ASD study population was clustered into subgroups based on immune responses and assessed for behavioral outcomes.  

Results:  

Children with ASD who had a proinflammatory profile based on lipopolysaccharide stimulation were more developmentally impaired as assessed by the Mullen Scales of Early Learning. They also had greater impairments in social affect as measured by the Autism Diagnostic Observation Schedule. These children also displayed more frequent sleep disturbances and episodes of aggression. Similarly, children with ASD and a more activated T cell cytokine profile after phytohemagglutinin stimulation were more developmentally impaired as measured by the Mullen Scales of Early Learning.

 Conclusions:

Children with ASD may be phenotypically characterized based upon their immune profile. Those showing either an innate proinflammatory response or increased T cell activation/skewing display a more impaired behavioral profile than children with noninflamed or non-T cell activated immune profiles. These data suggest that there may be several possible immune subphenotypes within the ASD population that correlate with more severe behavioral impairments.





With support from Cure Autism Now, a study recently published in the Journal of Neuroimmunology has found that children with autism have a more active immune system. The research, led by Cynthia Molloy, MD, also identified a potential mechanism for this immune dysregulation. The authors suggest that a cytokine called interleukin-10 (IL-10) could be a key part of the mechanism that leads to alterations in the adaptive immune response in individuals with autism. This new finding about the role of IL-10 provides another piece of the puzzle in understanding the complex nature of immune dysfunction in autism.
As early as the 1970's, immunological factors were identified in autism. Over time, a growing body of evidence has indicated a role of immune dysfunction in individuals with autism, but the exact nature is not fully clear, and no causal function has been established. One potent area of research has been the study of cytokines, chemicals in the body that serve as signaling molecules and play a crucial role in mediating specific types of immune responses. Cytokines are essential components of both the innate immune system (immune defense mechanisms that are the first line of defense against any kind of invading substance, and present from birth) and the adaptive immune system (immune defense mechanisms that develop in response to specific invading substances, built up as immunities to infection from diseases we have been exposed to over our lifetimes.) These important messengers control the strength, length, and direction of immune responses, and are essential in regulating the repair of tissue after injury. The many individual cytokines play different roles; some act as stimulators of immune system activation, while others provide inhibitory functions. Together, the various cytokines work in an intricately coordinated system, the success of which is dependent on their well-timed production by the various cell types of the immune system.
Interested in the impact of immune regulation on the development of autism, in 2003 Dr. Molloy received a pilot project grant from CAN. Dr. Molloy is an Assistant Professor of Pediatrics at the Center for Epidemiology and Biostatistics at Cincinnati Children's Hospital Medical Center, and is also the mother of a 13 year-old daughter with autism. While she began her career in pediatric emergency medicine, the emphasis of her work changed in 1999, when Dr. Molloy started a research fellowship in developmental disabilities at Cincinnati Children's Hospital Medical Center. She joined the faculty in 2003, where her research currently focuses on immune phenotypes and the contribution of genes on chromosome 21 to autism. Dr. Molloy highlights the benefits of teamwork at Cincinnati Children's Hospital, where she works closely with Marsha Wills-Karp, Ph.D. "I have been fortunate to collaborate with an exceptional immunobiologist to work on understanding the extent to which the immune system contributes to the pathogenesis of autism."
In this study, Dr. Molloy and her colleagues were interested in the levels of certain cytokines that are produced by a specific type of immune cell in the adaptive immune system, called helper T cells (T cells are a type of white blood cell). Helper T cells contribute to the immune response by promoting the production of other types of T and immune cells. The research team studied two types of helper T cells that work as a system: Th1 and Th2. Under normal circumstances, the Th1 and Th2 systems balance one another by inhibiting each other's activity. Each type of helper T cell produces different kinds of cytokines, with the T cell types defined by the cytokines they produce. These cytokines are termed interferons and interleukins, and the research group concentrated on a certain subset. Within the Th1 system, the dominant cytokine is interferon gamma (IFN-gamma), which is responsible primarily for reactions against viruses and intra-cellular microbes, and is pro-inflammatory. Among others, Th2 cells produce interleukins IL-4, IL-5, and IL-13. These interleukins are important for stimulating production of antibodies (immune proteins that identify specific foreign substances for destruction) and often have multiple functions. As part of the Th2 system, IL-4 and IL-13 are primarily anti-inflammatory (by inhibiting Th1 cells), but they also promote the growth and differentiation of other immune cells. IL-4 also has the very important role of producing the regulatory cytokine IL-10, which helps maintain the balance between the Th1- and Th2- produced cytokines.
Historically, the role of cytokines in the immune system dysregulation observed in studies of individuals with autism has not been conclusive, because different patterns of cytokine activation have been found. Some studies of the adaptive immune system in autistic individuals have shown that the cytokines of the Th1 cells are elevated, while other studies have found elevations in the cytokines of the Th2 system. Interestingly, a study of patient registries in Europe found that many individuals suffered from both allergies (generally Th2 driven) and autoimmune disorders (generally Th1 driven). Typically, autoimmune diseases and allergies are not seen together in an individual, because both Th systems are not usually overactive at the same time. One goal of Dr. Molloy's study was to determine if direct measures of the cytokine levels themselves (as opposed to measures of the allergic/autoimmune disorders produced by imbalances in these systems) would show the same simultaneous hyper-activation in individuals with autism.
To examine the adaptive immune system, Dr. Molloy's team measured cytokine production of children's immune cells in a cell culture, both at a baseline level and after stimulation by an allergen and a toxin. The team compared individual cytokine levels in blood samples from twenty children with autism and twenty unaffected controls matched on the basis of age, race, gender and date of study visit; this careful one-to-one matching was important for controlling some of the variability that has made previous studies of immune function in autism hard to interpret.
At baseline, the researchers found that immune cells of children with autism produced higher levels of both the Th1 and Th2 cytokines, including IFN-gamma and IL-4, -5, -13, than the cells cultured from the control group. In contrast, in the experiment using stimulation by an allergen or toxin, there was no difference between cases and controls, indicating that the cells in both groups were equally capable of producing the cytokines and generating an immune response.
These findings demonstrate that, in children with autism, both the Th1 and Th2 cytokines are more highly activated in the immune system's resting state, indicating potential underlying hypersensitivity to exposures in the general environment. Dr. Molloy's study shows that immune dysregulation is found in the adaptive immune system, as has been previously shown for the innate immune system, confirming that children with autism exhibit hyper-sensitivity in both innate and adaptive systems. Dr. Molloy's research has found increases in both pro- and anti- inflammatory cytokines in the Th1 and Th2 system which is indicative of dysregulation in the two systems. Instead of focusing on the exact role of the anti- or pro- inflammatory cytokines, the study highlights the importance of balanced regulation between these two systems in the adaptive immune system.
In an intriguing twist, although baseline levels of almost all the cytokines measured were higher in children with autism than in control individuals, Dr. Molloy found an exception in the relatively lower levels of the critical regulatory cytokine, IL-10, in individuals with autism. If both Th1 and Th2 cells are just generally overactive in individuals with autism, elevated IL-10 production would have been predicted as well. Dr. Molloy explains that "it is unusual to see both the Th1 and Th2 arms of the adaptive immune response so active at the same time; it is even more unusual to see this increased activation without a proportional increase in the regulatory cytokine IL-10, which is involved in Th1 and Th2 system regulation." Although previous research has shown that IL-10 regulates the Th1 and Th2 systems, the exact mechanisms contributing to the balance within the two systems is currently not known. Dr. Molloy proposes that "many of the paradoxical findings that have been reported about immune responses in autism could possibly be explained by the general dysfunction of IL-10." The finding that IL-10 levels were not elevated in individuals with autism, even when the levels of both Th1 and Th2 cytokines were elevated, suggests that the immune response dysfunction seen in autism may be a problem with regulating the cytokine system. Dr. Molloy hypothesizes that "children with autism may not be able to down-regulate their Th1 and Th2 systems" either because of a dysfunction in the production of IL-10 or because of a dysfunction with the activity of IL-10 itself.
Dr. Molloy's research contributes a crucial piece of information to the ability to determine how these cytokines function within the complex interactions of an adaptive immune system response. Further study of IL-10 is needed to determine how it contributes to the balance between the Th1 and Th2 systems.     

Role of Regulatory T Cells in Pathogenesis and Biological Therapy of Multiple Sclerosis













Figure 1: Differentiation of naïve T helper cells into particular subsets. T helper lymphocytes leaving the thymus (naïve or TH0) are not yet fully differentiated to perform their specific functions in peripheral lymphoid tissues. They are endowed of these properties in the process of their interactions with dendritic cells (DCs) that engulf, process, and present antigens to them. Moreover, DCs in dependence of the processed antigens produce different cytokines. If DCs produce IL-12, naïve T cells polarise into the TH1 subset, if IL-4 into the TH2 subset and eventually, if DCs synthesise IL-6, naïve T helper cells will become the TH17 cells.









Autism appears to be the middle seesaw


Figure 2: Causes of impaired Treg cells function in autoimmunity development. Failures of regulatory T (Treg) cell-mediated regulation can include: inadequate numbers of Treg cells owing to their inadequate development in the thymus, for example, due to a shortage of principal cytokines (IL-2, TGF-β) or costimulatory signals (CD28), and so forth. Further, the number of Treg cells can be in a physiological range; however, there are some defects in Treg-cell function that are intrinsic to Treg cells, for example, they do not synthesise sufficient quantity of immunosuppressive cytokines (IL-10, IL-35, and TGF-β), or there is a breakdown of their interaction with effector T cells. Ultimately, pathogenic effector T cells (Teff) are resistant to suppression by Treg cells owing to factors that are intrinsic to the effector cells or factors that are present in the inflammatory milieu that supports effector T cells resistance.  

Regulatory T cells play a vital role in the regulation of immune processes. Based on the induction of autoimmune processes caused by the FOXP3 gene mutation, it was supposed that defective Treg cells might also contribute to the development of immunopathological processes in “more common” autoimmune disorders. This supposition has been confirmed.


Dysregulation of Th1, Th2, Th17, and T regulatory cell-related transcription factor signaling in children with autism.


Abstract


Autism is a neurodevelopmental disorder characterized by stereotypic repetitive behaviors, impaired social interactions, and communication deficits. Numerous immune system abnormalities have been described in individuals with autism including abnormalities in the ratio of Th1/Th2/Th17 cells; however, the expression of the transcription factors responsible for the regulation and differentiation of Th1/Th2/Th17/Treg cells has not previously been evaluated. Peripheral blood mononuclear cells (PBMCs) from children with autism (AU) or typically developing (TD) control children were stimulated with phorbol-12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A. The expressions of Foxp3, RORγt, STAT-3, T-bet, and GATA-3 mRNAs and proteins were then assessed. Our study shows that children with AU displayed altered immune profiles and function, characterized by a systemic deficit of Foxp3+ T regulatory (Treg) cells and increased RORγt+, T-bet+, GATA-3+, and production by CD4+ T cells as compared to TD. This was confirmed by real-time PCR (RT-PCR) and western blot analyses. Our results suggest that autism impacts transcription factor signaling, which results in an immunological imbalance. Therefore, the restoration of transcription factor signaling may have a great therapeutic potential in the treatment of autistic disorders. 





Autism spectrum disorder (ASD) is a neurodevelopmental disorder. It is characterized by impaired social communication, abnormal social interactions, and repetitive behaviors and/or restricted interests. BTBR T + tf/J (BTBR) inbred mice are commonly used as a model for ASD. Resveratrol is used widely as a beneficial therapeutic in the treatment of an extensive array of pathologies, including neurodegenerative diseases. In the present study, the effect of resveratrol administration (20 and 40 mg/kg) was evaluated in both BTBR and C57BL/6 (B6) mice. Behavioral (self-grooming), Foxp3, T-bet, GATA-3, RORγt, and IL-17A in CD4+ T cells were assessed. Our study showed that BTBR control mice exhibited a distinct immune profile from that of the B6 control mice. BTBR mice were characterized by lower levels of Foxp3+ and higher levels of RORγt+, T-bet+, and GATA-3+ production in CD4+ T cells when compared with B6 control. Resveratrol (20 and 40 mg/kg) treatment to B6 and BTBR mice showed substantial induction of Foxp3+ and reduction of T-bet+, GATA-3+, and IL-17A+ expression in CD4+ cells when compared with the respective control groups. Moreover, resveratrol treatment resulted in upregulated expression of Foxp3 mRNA and decreased expression levels of T-bet, GATA-3, RORγt, and IL-17A in the spleen and brain tissues. Western blot analysis confirmed that resveratrol treatment decreased the protein expression of T-bet, GATA-3, RORγ, and IL-17 and that it increased Foxp3 in B6 and BTBR mice. Our results suggest that autism is associated with dysregulation of transcription factor signaling that can be corrected by resveratrol treatment. 

Recent studies have demonstrated that Th17, Th1, Th2, and Treg cells have a dominant central role in the progress and development of neurological disorders through a composite system of contacts among cells and their cytokines.

Previous investigation demonstrated that patients with autism had a significantly lower number of Treg cells than did healthy children 

Because Tregs play an important role in preventing immune activation and inhibiting self-reactivity, a deficiency in their numbers could underlie a link between autism and the immune system 

RORγt has been identified as a Th17-specific transcription factor [17]. Because RORγt is a critical regulator of the IL-17A pathway, its role in contributing to ASD-like behaviors in mouse offspring has been investigated [18]. Several recent studies have reported an increased production of IL-17A in children with ASD [19, 20]. Th17 cells are intricately associated with the development of a variety of and inflammatory autoimmune diseases. Initiation and propagation of Th17 cells are linked to the suppression of Treg cells  

Resveratrol Regulates Immunological Imbalance through Decreasing IL-17A Cytokine 

Treatment of B6 mice with resveratrol also caused a marked decrease in IL-17A mRNA expression levels (Fig. 6b). Correspondingly, IL-17 protein expression levels were significantly higher in BTBR control mice when compared with that of B6 control mice. Resveratrol treatment of BTBR mice also significantly reduced IL-17 protein expression when compared with that of BTBR control mice (Fig. 6c). These results indicated that resveratrol could reverse the appearance of inflammatory cytokines and signal transducers related with differentiation and production of Th17 cells.
  

Elucidating the mechanisms and pathways associated with n eurodevelopmental disorders such as autism is essential.


This will provide an understanding of the etiology of these disorders and also help to discover early diagnostic markers and prophylactic therapies. Resveratrol prevents social deficits in an animal model of autism [26] and improves hippocampal atrophy in chronic fatigue syndrome by enhancing neurogenesis [39]. Resveratrol is widely recognized as an anti-oxidant and as an anti-inflammatory, anticancer, cardioprotective, and neuroprotective compound [40, 41]. It has been shown to inhibit increases in levels of proinflammatory factors [42]. Resveratrol has also been found to provide a neuroprotective effect on dopaminergic neurons [43]. The mechanism of action of resveratrol against neuroinflammation appears to involve targeting activated microglia.

This results in a decrease in levels of pro-inflammatory factors through the modulation of key signal transduction pathways [43]. In addition, it has been reported that resveratrol inhibits the activation of NF-κB, decreases levels of IL-6 and TNF-α cytokines [42], and prevents suppression of Treg cells [9]. In the current study, we explored the effects of resveratrol on Th1, Th2, Th17, and Treg cell-related transcription factors.


Our results demonstrated that resveratrol was effective in reducing a prominent repetitive behavior in the BTBR mouse model of autism. Doses of 20 and 40 mg/kg i.p. reduced repetitive self-grooming. The efficacy of resveratrol in reducing repetitive behavior is a novel finding and adds to the potential therapeutic indications of resveratrol for the treatment of autism. BTBR is an inbred strain of mice which displays social deficits, reduced ultrasonic vocalizations in social settings, and high levels of repetitive self-grooming [44]. Learning and memory defects have been reported for BTBR mice when they are assessed in fear conditioning, water maze reversal, discrimination flexibility, and probabilistic reversal learning tests [45, 46]. Stereotypy and behavior rigidity are widely known as core and defining features of ASD [47].


In the present study, we explored the effect of resveratrol on Foxp3 expression in BTBR mice. We found a significant upregulation of Foxp3 expression on CD4+ T cells following resveratrol administration to BTBR mice. The expression of Foxp3 plays an important role in regulating the development and function of Treg. Our results suggest that immune dysfunction, specifically in Treg cells, is associated with the modulation of behaviors and core features of autism. Treg cells have been identified as important mediators of peripheral immune tolerance. A functional defect caused by Foxp3 dysregulation has been demonstrated to lead to several autoimmune diseases [48, 49]. Autoimmune neuroinflammation is considered to result from a disrupted immune balance between effector T cells such as Th1/Th2/Th17 and suppressive T cells such as Treg [50]. Several attempts have been made to elevate the numbers of Treg cells to suppress ongoing autoimmunity in experimental autoimmune disorders [51].

In the present study, we observed that the high T-bet expression in CD4+ T cells of control BTBR mice could be reversed by resveratrol treatment. This may suggest that resveratrol can downregulate expression of T-bet in autistic individuals. Several studies suggest that expression of T-bet plays an important role in disease initiation and progression of experimental autoimmune disorders [52]. T-bet enhances IL-17 production by central nervous system (CNS)-infiltrating T cells and this may be linked to neuroinflammation [53].


Our study also demonstrated that the high GATA-3 expression levels in CD4+ T cells and spleen of BTBR mice could be reversed by treatment with resveratrol. This suggests that resveratrol may correct neurodevelopment dysregulation in autism through regulation of Foxp3 expression. GATA-3 is involved in the development of serotonergic neurons in the caudal raphe nuclei [15] and regulates several processes in the body including cell differentiation and immune response [54]. The GATA-3 transcript is detected in the pretectal region, mid-brain, and most of the raphe nuclei [55]. Intriguingly, disturbances in these processes are considered involved in the etiology of ASD in human or autism-like behaviors in animals [56]. Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system [57]. A recent study reported higher GATA-3 levels in lymphoblastic cell lines derived from the lymphocytes of autistic children as when compared to that of their non-autistic siblings [58], suggesting the importance of GATA-3 in this neurodevelopmental disorder. Valproate- and thalidomide-use may also be linked to autism through induction of GATA-3 expression [16].


Another key transcription factor associated with the Th17 lineage is RORγt [59]. Suppression of RORγt ameliorates CNS autoimmunity [33]. Alzheimers disease patients have increased expression levels of RORγt in the brain, cortex, and hippocampus [60]. Th17 cell signature cytokines have a confirmed role in ASD. For example, IL-17A administration promotes abnormal cortical development and ASD-like behavioral phenotypes [18]. Elevated levels of IL-17A have been detected in autistic children [61]. In line with these observations, our data showed that resveratrol treatment inhibits RORγt and IL-17A expression in CD4+ T cells and spleen in BTBR mice, suggesting their importance in regulation of autistic behavior. Recent data also suggest that therapeutic targeting of Th17 cell, or its transcription factor, in susceptible pregnant mothers may reduce the likelihood of children being born with SD-like phenotypes [18]. 


Conclusions 

Our results indicate that resveratrol treatment can improve social behaviors in a BTBR mouse model of autism through suppression of Th17, Th2, and Th1 cell-related transcription factors and induction of Treg cell-related transcription factor. Our data also suggest that resveratrol may be a promising candidate for the treatment of ASD and other immune mediated neurological disorders. 


A heavyweight mainstream study:-  



IL-23-IL-17 immune axis: Discovery, Mechanistic Understanding, and Clinical Testing 

With the discovery of Th17 cells, the past decade has witnessed a major revision of the T helper subset paradigm and significant progress has been made deciphering the molecular mechanisms for T cell lineage commitment and function. In this review, we focus on the recent advances on the transcriptional control of Th17 cell plasticity and stability as well as the effector functions of Th17 cells—highlighting IL-17 signaling mechanisms in mesenchymal and barrier epithelial tissues. We also discuss the emerging clinical data showing anti-IL-17 and anti-IL-23 treatments are remarkably effective for many immune-mediated inflammatory diseases.


 “Type 17” subsets of cells ubiquitously express RORγt and IL-23R. Their development is Thymic dependent with the exception of Group 3 ILCs. Adaptive CD4+ IL-17-producing cells require IL-6 signaling during initial TCR-mediated activation. All other subsets do not require IL-6 activation and are capable of responding to IL-1 and IL-23 signaling upon emigrating from the thymus. These “innate” immune cells are poised to produce IL-17 upon sensing inflammatory cytokines as well as stress and injury signals. While the adaptive Th17 cells reside primarily in secondary lymphoid organs, the “innate” Type 17 cells are situated in a broad range of peripheral tissues, where they directly survey the interface between the host and the environment. 



Company
Agent
Target
Indications
Stage
Clin Trial ID
Eli Lilly
Ixekizumab
(Ly2439821)
IL-17A
Psoriasis
Rheumatoid arthritis
Phase 3
Ph 2
complete
Novartis
Secukinmab
(AIN457)
IL-17A
Psoriasis
Rheumatoid arthritis
Ankylosing
spondylitis
Psoriatic arthritis
Asthma
Multiple sclerosis
Type 1 Diabetes
Crohn’s disease
Phase 3
Ph 3
Ph 3
Phase 3
Ph 2
Ph 2
Ph 2
Ph
2terminated
Amgen/
MedImmun
e
Brodalumab
(AMG 827)
IL-17
Receptor A
Psoriasis
Psoriatic arthritis
Asthma
Crohn’s disease
Phase 3
Ph 3
Ph 2
Ph
2suspended
Abbott
AbbVie
ABT-122
IL-17A/
TNFa
Rheumatoid arthritis
Phase 1
Johnson &
Johnson
Janssen
Biotech
Stelara
(Ustekinumab)
(CNTO 1275)
p40 subunit
of IL-12 and
IL-23
Psoriasis
Crohn’s disease
Ankylosing
spondylitis
Rheumatoid arthritis
Psoriatic arthritis
Multiple sclerosis
GvHD
Atopic dermatitis
Approved 2009
Phase 3
Phase 2
Phase 2
Phase 2
Phase 2
Phase 2
Phase 2
Abbott
Briakinumab
ABT-874
p40 subunit
of IL-12 and
IL-23
Crohn’s disease
Psoriasis
Multiple Sclerosis
Ph
2terminated
Phase 3
Phase 2
Merck
Tildrakizumab
(MK 3222)
(SCH 900222)
IL-23p19
Psoriasis
Phase 3
Johnson &
Johnson
Janssen
Biotech
Guselkumab
CNTO 1959
IL-23p19
Psoriasis
Rheumatoid arthritis
Phase 2
Phase 2
Amgen/
MedImmun
e
AMG 139
IL-23p19
Psoriasis
Crohn’s disease
Phase 1
Phase 1
Eli Lilly
LY3074828
IL-23p19
Psoriasis
Phase 1
Boehringer
Ingelheim
BI 655066
IL-23p19
Ankylosing
spondylitis
Crohn’s disease
Psoriasis (single
rising dose)
Phase 2
Phase 2
Phase 2

Table 2 -human diseases being treated with anti-p40, anti-p19, anti-IL-17, and anti-IL-17RA 

Conclusions and perspectives

Since the discovery of the IL-23-Th17 immune pathway a decade ago, immunologists and clinicians have worked diligently to bring this novel therapeutic strategy to the clinic, which is now showing encouraging results for psoriasis, Crohn’s disease, rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. However, this treatment strategy is complex. It was initially assumed that IL-23 controls the production of pathogenic IL-17 and that these cytokines are ‘duplicate’ targets. Recent clinical results suggest that is not the case at all. We are now beginning to appreciate that anti-IL-23p19 versus anti-IL-17 treatments each has its own beneficial effects as well as unique challenges in different disease settings. For example, anti-IL-17 showed good therapeutic efficacy for the treatment of psoriasis—even surpassing anti-TNF therapy, but failed in Crohn’s disease. The search for better clinical efficacy biomarkers is critically needed to improve patient stratification and disease indication selection. In addition, better understanding of Th17 biology and cellular mechanisms would allow discovery of additional targets for inflammatory diseases. 


Blog post conclusion

There are so many known ways to modify the immune system; you would think that this aspect of many people’s autism really should be widely treated.

Very slowly in the literature we are moving towards defining inflammatory subtypes, which is a first step.

Modifying the immune system can have a profound effect on some types of autism.

We had the case of Stewart Johnson, who pioneered the TSO helminth therapy for his son with severe autism.  He teamed up with his son’s doctor Dr. Eric Hollander, Director of the Seaver York Autism Center at Mount Sinai Medical Center in New York, to try and make this a wider used therapy.  Ultimately the clinical trial was terminated and a company that was trying to commercialize the therapy gave up.

He documented his story here:

          http://autismtso.com/about/the_story/

We have our reader Alli from Switzerland, whose investigated the science and found that the Swedish variants of Lactobacillus reuteri should help; and they did.  In addition she uses 500mg sodium butyrate which will be converted into butyric acid.  Via its HDAC inhibiting properties it will further tune the immune system.  Sodium butyrate and butyrate-producing bacteria are widely used to improve immune health in animals.

What is clear is that there is no “cure-all” for autism, but that is hardly surprising.  There is no cure-all for cancer, which is equally heterogeneous.

The solution looks obvious to me and it is not hundreds of millions of dollars of research, it is to gather together all the existing knowledge and examine it fully.  This is how the world outside medicine generally operates.