The role of the microbiome in aggression. Gut microbe imbalances that predict autism and ADHD. Biogaia trial for Autism.

 

By December 2020 7.3% of the Swedish cohort born in 1997-9 had been diagnosed with a Neurodevelopmental Disorder (ND). This can be predicted by samples previously collected.

Today’s post is all about the microbiome and covers three different areas covered recently in the research. Eight years after I wrote a post about our informal trial of Biogaia probiotics for autism, we now have a published paper.

Aggression and self injurious behavior (SIB) affects at least half of those diagnosed with level 3 autism at some point in their lives. SIB can become the overriding concern for care givers.

Our first paper looks at the role of the microbiome in aggression.

Gut-brain axis appears to play a critical role in aggression

A series of experiments on mice has found that they become more aggressive when their gut microbiome is depleted. Additionally, transplanting gut microbiota from human infants exposed to antibiotics led to heightened aggression in mice compared to those receiving microbiome transplants from non-exposed infants. The research was published in Brain, Behavior, and Immunity.

In the past decade, scientists have discovered a complex communication pathway linking gut microbiota—the trillions of microorganisms living in the human gut—with the brain. This pathway is called the microbiota-gut-brain axis. It regulates various physiological functions, including digestion and immunity, but also affects mood and behavior. The gut microbiota produces neurotransmitters and other metabolites that can influence brain function through neural, immune, and endocrine pathways.

Recent studies have demonstrated that symptoms of various disorders, once considered primarily psychological or neurological, can be transferred to rodents by transplanting gut microbiota from humans with these disorders. For example, researchers have shown that transplanting gut microorganisms from people with Alzheimer’s disease into mice (whose gut microbiota had been depleted to enhance transplant effectiveness) resulted in cognitive impairments in the mice. Similarly, symptoms of anxiety have been induced in mice by transplanting gut microbiota from humans with social anxiety.

For the humanized mice, the researchers obtained fecal samples from infants who had been exposed to antibiotics shortly after birth, as well as from unexposed infants. These samples were transplanted into five-week-old germ-free mice. The researchers then waited for four weeks before testing the mice for aggression.

To measure aggression, the researchers employed the resident-intruder test, a well-established behavioral assay in which a male mouse (the “resident”) is introduced to another unfamiliar male mouse (the “intruder”) in its home cage. Aggression was quantified based on the latency to the first attack (how quickly the resident mouse attacked the intruder) and the total number of attacks during a 10-minute period.

The results showed that mice raised without gut bacteria (germ-free) and those treated with antibiotics exhibited higher levels of aggression compared to the control group. These mice attacked more frequently and were quicker to initiate aggressive behavior in the resident-intruder test.

The researchers found that humanized mice receiving fecal microbiota from antibiotic-exposed infants were significantly more aggressive than those receiving transplants from non-exposed infants. Even though the infants’ microbiomes had a month to recover after antibiotic exposure, the aggressive behavior was still evident in the recipient mice.

Biochemical analyses revealed that aggressive mice (both germ-free and antibiotic-treated) had distinct metabolite profiles compared to control mice. Specifically, levels of tryptophan—a precursor to serotonin, a neurotransmitter associated with mood and behavior—were elevated in these mice. Additionally, the levels of certain metabolites associated with microbial activity, such as indole-3-lactic acid, were reduced in the aggressive mice, suggesting that the absence of a healthy microbiome might alter key biochemical pathways involved in aggression.

Here is the link to the original paper:

A gut reaction? The role of the microbiome in aggression

Recent research has unveiled conflicting evidence regarding the link between aggression and the gut microbiome. Here, we compared behavior profiles of control, germ-free (GF), and antibiotic-treated mice, as well as re-colonized GF mice to understand the impact of the gut microbiome on aggression using the resident-intruder paradigm. Our findings revealed a link between gut microbiome depletion and higher aggression, accompanied by notable changes in urine metabolite profiles and brain gene expression. This study extends beyond classical murine models to humanized mice to reveal the clinical relevance of early-life antibiotic use on aggression. Fecal microbiome transplant from infants exposed to antibiotics in early life (and sampled one month later) into mice led to increased aggression compared to mice receiving transplants from unexposed infants. This study sheds light on the role of the gut microbiome in modulating aggression and highlights its potential avenues of action, offering insights for development of therapeutic strategies for aggression-related disorders

Note the ABX means antibiotics

We include a study of humanized mice using unique fecal samples of 1-month-old infants, collected nearly a month after early-life ABX administration. In previous work (Uzan-Yulzari et al. 2021, Nat Comm), we have demonstrated that ABX in this critical period of life can have lasting effects of childhood growth. Here, we extend these findings using samples from the same cohort. Using fecal samples collected weeks after ABX administration also reduces the direct chemical effects of ABX on the host, highlighting the causative role of the dysbiotic host microbiome and associated metabolome in driving aggressive behavior. We demonstrate that infant microbiota, perturbed within the first 48 h of life, has a lasting signature through 1 month of age that, when transplanted into GF mice, results in increased aggression (3–5 weeks after transplant) when compared to effects of stools of infants not exposed to any early-life antibiotics. The findings are revolutionary as they show how ABX-altered microbiota during a critical development window can lead to persisting behavioral deficits.

 

Gut microbe imbalances could predict a child’s risk for autism, ADHD and speech disorders years before symptoms appear.

Study Identifies Gut Microbe Imbalances That Predict Autism And ADHD

We are researchers who study the role the microbiome plays in a variety of conditions, such as mental illness, autoimmunity, obesity, preterm birth and others. In our recently published research on Swedish children, we found that microbes and the metabolites they produce in the guts of infants – both found in poop and cord blood – could help screen for a child’s risk of neurodevelopmental conditions such as autism. And these differences can be detected as early as birth or within the first year of life. These markers were evident, on average, over a decade before the children were diagnosed. 

The imbalance in microbial composition – what microbiologists call dysbiosis – we observed suggests that incomplete recovery from repeated antibiotic use may greatly affect children during this vulnerable period. Similarly, we saw that repeated ear infections were linked to a twofold increased likelihood of developing autism.

Children who both repeatedly used antibiotics and had microbial imbalances were significantly more likely to develop autism. More specifically, children with an absence of Coprococcus comes, a bacterium linked to mental health and quality of life, and increased prevalence of Citrobacter, a bacterium known for antimicrobial resistance, along with repeated antibiotic use were two to four times more likely to develop a neurodevelopmental disorder.

Antibiotics are necessary for treating certain bacterial infections in children, and we emphasize that our findings do not suggest avoiding their use altogether. Parents should use antibiotics if they are prescribed and deemed necessary by their pediatrician. Rather, our study suggests that repeated antibiotic use during early childhood may signal underlying immune dysfunction or disrupted brain development, which can be influenced by the gut microbiome. In any case, it is important to consider whether children could benefit from treatments to restore their gut microbes after taking antibiotics, an area we are actively studying.

Another microbial imbalance in children who later were diagnosed with neurodevelopmental disorders was a decrease in Akkermansia muciniphila, a bacterium that reinforces the lining of the gut and is linked to neurotransmitters important to neurological health.

Even after we accounted for factors that could influence gut microbe composition, such as how the baby was delivered and breastfeeding, the relationship between imbalanced bacteria and future diagnosis persisted. And these imbalances preceded diagnosis of autism, ADHD or intellectual disability by 13 to 14 years on average, refuting the assumption that gut microbe imbalances arise from diet.

We found that lipids and bile acids were depleted in the cord blood of newborns with future autism. These compounds provide nutrients for beneficial bacteria, help maintain immune balance and influence neurotransmitter systems and signaling pathways in the brain.

The full paper is here: 

Infant microbes and metabolites point to childhood neurodevelopmental disorders 

Highlights

• Infant microbes and metabolites differentiate controls and future NDs

• Early-life otitis lowers Coprococcus and increases Citrobacter in future NDs

• Preterm birth, infection, stress, parental smoking, and HLA DR4-DQ8 increase ND risk

• Linolenic acid is lower and PFDA toxins higher in the cord serum of future ASD

Summary

This study has followed a birth cohort for over 20 years to find factors associated with neurodevelopmental disorder (ND) diagnosis. Detailed, early-life longitudinal questionnaires captured infection and antibiotic events, stress, prenatal factors, family history, and more. Biomarkers including cord serum metabolome and lipidome, human leukocyte antigen (HLA) genotype, infant microbiota, and stool metabolome were assessed. Among the 16,440 Swedish children followed across time, 1,197 developed an ND. Significant associations emerged for future ND diagnosis in general and for specific ND subtypes, spanning intellectual disability, speech disorder, attention-deficit/hyperactivity disorder, and autism. This investigation revealed microbiome connections to future diagnosis as well as early emerging mood and gastrointestinal problems. The findings suggest links to immune-dysregulation and metabolism, compounded by stress, early-life infection, and antibiotics. The convergence of infant biomarkers and risk factors in this prospective, longitudinal study on a large-scale population establishes a foundation for early-life prediction and intervention in neurodevelopment.

ABIS = All Babies in Southeast Sweden cohort

NDs = Neurodevelopmental disorders

Young children later diagnosed with ASD or exhibiting significant autistic traits tend to experience more ear and upper respiratory symptoms. In ABIS, infants who had otitis in their first year were found to be more prone to acquiring NDs if they lacked detectable levels of Coprococcus or harbored Citrobacter. The absence of Coprococcus, despite comparable levels in controls irrespective of otitis, raises questions about microbial community recovery. This potential failure of the microbiome to recover following such events may serve as a mechanism connecting otitis media to ND risk. Moreover, antibiotic-resistant Citrobacter was more prevalent in these infants. The presence of strains related  to Salmonella and Citrobacter, labeled in this investigation as SREB, was significantly higher in infants who later developed comorbid ASD/ADHD (21%), compared to controls (3%). This disruption may have consequences on neurodevelopment during a critical period. Salmonella and Citrobacter have shown the ability to upregulate the Wingless (Wnt) signaling. The Wnt pathway is vital for immune dysregulation and brain development, and its disruption has been implicated in ASD pathogenesis. 

Two fatty acid differences were notable in the stool of future ASD versus controls: omega-7 monounsaturated palmitoleic acid, (9Z)-hexadec-9-enoic acid (below the level of detection in 87.0% of future ASD but present in 43.5% of controls), and palmitic acid (elevated in future ASD). Palmitoleic acid has been associated with a decreased risk of islet and primary insulin autoimmunity. Conversely, palmitic acid, a saturated fatty acid, has been linked to neuronal homeostasis interference. Its effects are partially protected by oleic acid, which although approaching significance, was lower in the cord serum of future ASD.

Few metabolites were higher in stool of infants with future ASD, but there are a few notable examples: α-d-glucose, pyruvate, and 3-isopropylmalate. Coprococcus inversely correlated with 3-isopropylmalate, suggesting gut-brain connections and a possible imbalance in branched-chain amino acid (BCAA) pathways given the role of 3-isopropylmalate dehydrogenase in leucine and isoleucine biosynthesis. An increase in dehydroascorbate suggests potential disruptions in vitamin C metabolism, crucial for neurotransmitter synthesis and antioxidant defense, while elevated pyruvate suggests disturbance of neurotransmitter synthesis or energy production early in life. Pimelic acid elevation, found in disorders of fatty acid oxidation, suggests disruption of mitochondrial pathways for fatty acid oxidation.

Akkermansia and Coprococcus, absent or reduced in infants with future NDs, positively correlated with signals in stool representing neurotransmitter precursors and essential vitamins in stool. Specifically, Akkermansia correlated with tyrosine and tryptophan (i.e., catecholamine and serotonin precursors, respectively) and Coprococcus with riboflavin. Disruption of BCAA metabolism in ASD has been documented, involving coding variants in large amino acid transporters (LATs) and reduced utilization of trypotphan and large aromatic amino acids along with increased glutamate and decreases in tyrosine, isoleucine, phenylalanine, and tryptophan in children with ASD. Oxidative stress, a diminished capacity for efficient energy transport, and deficiencies in vitamins (like vitamin B2) essential for neurotransmitter synthesis and nerve cell maintenance have been implicated. Riboflavin as an antioxidant reduces oxidative stress and inflammation, demonstrating neuroprotective benefits in neurological disorders, possibly through maintenance of vitamin B6, which is necessary for glutamate conversion to glutamine and 5-hydroxytryptophan to serotonin.

Together, these findings support a hypothesis of early-life origins of NDs, mediated by gut microbiota. This provides a foundation for research and for developing early interventions for NDs.

 

Today’s final paper was highlighted recently in a comment on a post I wrote eight years ago, when we were trialing Biogaia probiotics. This original interest was prompted by a reader sharing her successful experiences of treating her son with severe autism. Perhaps she left the recent comment?

The two bacteria involved are both types of L. reuteri.

L. reuteri 6475 is sold as Biogaia Osfortis

L. reuteri 17938 is sold Biogaia Protectis

The combination of L. reuteri 17938 and L. reuteri 6475 is sold as Biogaia Gastrus.

My old post from 2016:-

Epiphany: Biogaia Trial for Inflammatory Autism Subtypes

The recently published trial:

Precision microbial intervention improves social behavior but not autism severity: A pilot double-blind randomized placebo-controlled trial -

Highlights

• L. reuteri (6475 + 17938) improves social functioning in children with autism

• L. reuteri does not improve overall autism severity or repetitive behaviors

• L. reuteri does not significantly alter microbiome composition or immune profile

•  Only the 6475 strain reverses the social deficits in a mouse model for autism

we performed a double-blind, randomized, placebo-controlled, parallel-design pilot trial in children with ASD. Importantly, we found that L. reuteri, compared with placebo, significantly improved social functioning, both in terms of reducing social deficits, as measured by the social responsiveness scale (SRS^31,32), and increasing adaptive social functioning, as measured by the social adaptive composite score of the Adaptive Behavior Assessment System, Second Edition (ABAS-2³³). L. reuteri did not improve overall autism severity, restricted and repetitive behaviors, and co-occurring psychiatric and behavioral problems, nor did it significantly modulate the microbiome or immune response. Thus, this safe microbial manipulation has the potential for improving social deficits associated with ASD in children.

I had to amend my old post with a warning long ago.

UPDATE: A significant minority of parents report negative reaction to Bio Gaia, this seems to relate to histamine; but more than 50% report very positive effects without any side effects; so best to try a very small dose initially to see if it is not well tolerated. 

Histamine Reaction to BioGaia gastrus

Conclusion

The gut microbiota does indeed play a key role in how your brain functions, but the gut-brain axis works in both directions. What goes on in your brain can affect your gut and not just the other way around. It is called bidirectional signaling.

Antibiotics taken during pregnancy, or during early childhood, will have unintended consequences. Often there is no choice, like for those readers whose baby experienced sepsis at birth (bacterial blood stream infection); you have to give antibiotics to avoid death.

In today’s second paper we see that the researchers are thinking about therapeutical implications. Perhaps the newborn’s gut flora should be repopulated during the weeks after the antibiotic treatment?

I receive many questions about how to treat self injurious behavior that does not respond to anything the doctor has prescribed. Rifaximin, an antibiotic used to treat irritable bowel syndrome with diarrhea, is one therapy that does help some types of SIB (and SIBO, small intestinal bacterial overgrowth, of course). This probably would not surprise the authors of today’s first paper.

Biogaia Gastrus (L. reuteri 6475 + 17938) from today’s third paper worked wonders for the SIB of one reader’s child.

Not surprisingly fecal microbiota transplantation (FMT) can improve SIB in some people.

The Swedish data shows interesting insights such as that lipids and bile acids were depleted in the cord blood of newborns with future autism. The researchers think they can predict the diagnosis of autism or ADHD. The question is and then what? Even when there is a diagnosis of autism, not much changes for most children.

How to increase Oxytocin (OT) effects in the autistic brain? OT nasal spray, L. reuteri DSM 17938, Magnesium, Estradiol, Nicotinamide riboside …

 Struggle to make friends? Consider Oxytocin

Today’s post was going to be about FMT super-donors, but instead we have a post about new insights into using oxytocin to treat autism.  From personal experience I can say that you really can target oxytocin receptors to affect mood/behavior; I have no personal experience of FMT (fecal microbiota transplants), but thousands of people use it for many conditions.  The FMT post will be next.

Oxytocin and vasopressin are two hormones, made in the hypothalamus, that are established targets for autism treatment. They are released into the bloodstream where they carry out their best-known functions, but they are also released from the hypothalamus directly into the brain where these hormones have entirely different functions.

Both oxytocin and vasopressin can be given as nasal sprays to enter the central nervous system (CNS) rather than just the blood stream.  This means you get the brain effects of the hormone, also known as the “central effects”.

As was discussed previously in this blog and is highlighted more recently in the article below, you can use certain bacteria in the gut to signal to the hypothalamus to produce more oxytocin.  This is really clever and it works in humans, not just research animals.  It also has the advantage of producing a more continuous effect than is found using the intranasal method to deliver oxytocin. 

When you sever the vagus nerve, the bacteria in the gut continues to produce the required chemicals, but the signal to the brain has been lost. The hypothalamus no longer produces increased oxytocin and so the behavioral/mood effect is lost. This has been proven in the research.

Gut microbes may treat social difficulties in autism mice

In science speak, “the results suggest that a peptide or metabolite produced by bacteria may modulate host oxytocin secretion for potential public or personalized health goals”.  It also appears that oxytocin improves wound healing. So perhaps old people with leg ulcers, which never seem to get better, might benefit from a daily dose of L. reuteri DSM 17938, it also might make them feel better due to those central effects.

Oxytocin in the brain acts via oxytocin receptors

As we learned years ago in this blog, you can increase the effect (turn up the volume) of receptors using a PAM (positive allosteric modulator).  Interestingly, magnesium is a PAM of the oxytocin receptor (OTR).  Many people with autism are supplementing magnesium, perhaps those using intranasal oxytocin should join them. 

A very recent paper has investigated in detail how oxytocin receptors function.

Crystal structure of the human oxytocin receptor

The peptide hormone oxytocin modulates socioemotional behavior and sexual reproduction via the centrally expressed oxytocin receptor (OTR) across several species. Here, we report the crystal structure of human OTR in complex with retosiban, a nonpeptidic antagonist developed as an oral drug for the prevention of preterm labor. Our structure reveals insights into the detailed interactions between the G protein–coupled receptor (GPCR) and an OTR-selective antagonist. The observation of an extrahelical cholesterol molecule, binding in an unexpected location between helices IV and V, provides a structural rationale for its allosteric effect and critical influence on OTR function. Furthermore, our structure in combination with experimental data allows the identification of a conserved neurohypophyseal receptor-specific coordination site for Mg2+ that acts as potent, positive allosteric modulator for agonist binding. Together, these results further our molecular understanding of the oxytocin/vasopressin receptor family and will facilitate structure-guided development of new therapeutics. 

Magnesium and mood disorders: systematic review and meta-analysis

Another consequence of ERβ under-expression in autism

Also interesting to those following autism research, is the role of ERβ (estrogen receptor beta).  It is well known that in the brains of those with autism, there is a lack of ERβ.  A lack of ERβ is likely to lead to lower oxytocin in the brain and CSF (spinal fluid).  In many types of autism, we know that the level of oxytocin in CSF is reduced.

If you activate ERβ you both increase expression of oxytocin receptor (OTR) and also increase the level of oxytocin measured in the CSF.  You can activate ERβ with estrogens, like estradiol or even phytoestrogens like soy.  The ideal therapy to use would be DHED.

https://epiphanyasd.blogspot.com/2018/02/dhed-delivering-estradiol-only-to-brain.html

The cheap diuretic spironolactone may very well indirectly increase the level of oxytocin in CSF.

Oxytocin and Estrogen Receptor β in the Brain: An Overview

Oxytocin (OT) is a neuropeptide synthesized primarily by neurons of the paraventricular and supraoptic nuclei of the hypothalamus. These neurons have axons that project into the posterior pituitary and release OT into the bloodstream to promote labor and lactation; however, OT neurons also project to other brain areas where it plays a role in numerous brain functions. OT binds to the widely expressed OT receptor (OTR), and, in doing so, it regulates homeostatic processes, social recognition, and fear conditioning. In addition to these functions, OT decreases neuroendocrine stress signaling and anxiety-related and depression-like behaviors. Steroid hormones differentially modulate stress responses and alter OTR expression. In particular, estrogen receptor β activation has been found to both reduce anxiety-related behaviors and increase OT peptide transcription, suggesting a role for OT in this estrogen receptor β-mediated anxiolytic effect. Further research is needed to identify modulators of OT signaling and the pathways utilized and to elucidate molecular mechanisms controlling OT expression to allow better therapeutic manipulations of this system in patient populations.

NAD and Nicotinamide Riboside to boost Oxytocin

Today we see that recent research from Japan shows that in those people with autism who have reduced NAD, they may well be able to improve behavior/mood by increasing the level of their oxytocin using Nicotinamide Riboside (NR).

Nicotinamide riboside (NR) is a special form of vitamin B3, sold as an expensive supplement.  The FDA say it is safe for use in humans.

Nicotinamide riboside supplementation corrects deficits in oxytocin, sociability and anxiety of CD157 mutants in a mouse model of autism spectrum disorder

Oxytocin (OT) is a critical molecule for social recognition and memory that mediates social and emotional behaviours. In addition, OT acts as an anxiolytic factor and is released during stress. Based on the activity of CD38 as an enzyme that produces the calcium-mobilizing second messenger cyclic ADP-ribose (cADPR), CD157, a sister protein of CD38, has been considered a candidate mediator for the production and release of OT and its social engagement and anti-anxiety functions. However, the limited expression of CD157 in the adult mouse brain undermined confidence that CD157 is an authentic and/or actionable molecular participant in OT-dependent social behaviour. Here, we show that CD157 knockout mice have low levels of circulating OT in cerebrospinal fluid, which can be corrected by the oral administration of nicotinamide riboside, a recently discovered vitamin precursor of nicotinamide adenine dinucleotide (NAD). NAD is the substrate for the CD157- and CD38-dependent production of cADPR. Nicotinamide riboside corrects social deficits and fearful and anxiety-like behaviours in CD157 knockout males. These results suggest that elevating NAD levels with nicotinamide riboside may allow animals with cADPR- and OT-forming deficits to overcome these deficits and function more normally.

NR elevates brain NAD⁺ and cerebrospinal OT

Social preference deficit and anxiety of CD157KO males are best corrected at a relatively low dose of NR

The results demonstrated that the daily oral administration of NR rescued the social behavioural impairments observed in male CD157KO mice. NR had essentially no effects on social behaviour in wild-type male mice. The beneficial effects of NR appear to depend on restoration of CSF OT levels because the NR-induced OT elevation was only detected in CD157KO mice, which have a CSF OT deficit.

In the course of identifying a nutritional intervention for CD157KO mice, we reproduced the anxiety-like and social-avoidance-like deficits reported previously. Reproducibly lower levels of CSF OT in male CD157KO mice make these mice an attractive model of autism, anxiety disorder, or social avoidance in neurodegenerative diseases. Significantly, this model responds to both OT and NR as a treatment.

The challenge of polygenic diseases of incomplete penetrance is that they are difficult to understand mechanistically. Multiple genetic and environmental (biochemical) factors may converge to dysregulate pathways that are altered in common conditions such as ASD. We note that one potentially hopeful point when studying polygenetic diseases is that brain systems are redundant, and thus, it may be possible to increase normal functions that are only partially encoded by genetically damaged circuitry.

NAD⁺ is consumed by CD38 in formation of cyclic ADP-ribose. It then participates in OT release in the hypothalamus. In our study, ADP-ribosyl cyclase activity was maintained at a similar range as that in wild-type animals (data not shown). A recent study suggested that NR supplementation did not change CD38 expression. However, in vitro studies have shown that NAD⁺ applied to the mouse hypothalamus leads to OT release. It is reasonable to assume that an elevation in NAD⁺ levels by NR in the hypothalamus is responsible for repair of the OT release.

Future work will probe CD38 dependence and the cell-type dependence of the beneficial effects of NR on CD157KO behaviour, the potential benefits of NR in other ASD models, and the potential of NR to become a safe nutritional intervention, in addition to OT, for at least some types of ASD in human populations.

NAD+ is reduced in older people

There is a lot of research into combating the effects of aging.  It is agreed that the older you get, the less NAD+ you have and so research has looked at numerous ways to raise it.

The CD157KO mice model of autism does feature reduced NAD+, but nobody knows how common reduced NAD+ is in autism.

If you have low levels of NAD+ there will be negative consequences.

I think you can consider NAD+ depletion in a similar way to oxidative stress, both are inevitable and damaging features of aging.

Most healthy younger people are likely wasting their time and money worrying about oxidative stress and NAD+.  These are the people with “detox” diets and juices.

However, most old people and some young people with autism really stand to benefit from correcting oxidative stress and any reduced NAD+.

  

Therapeutic potential of NAD-boosting molecules: the in vivo evidence

Hallmarks of NAD homeostasis

NAD⁺ is not merely a redox co-factor, it is also a key signaling molecule that controls cell function and survival in response to environmental changes such as nutrient intake and cellular damage. Fluctuations in NAD impact mitochondrial function and metabolism, redox reactions, circadian rhythm, immune response and inflammation, DNA repair, cell division, protein-protein signaling, chromatin and epigenetics.

There are many ways to boost NAD+.

Classes of NAD+ boosting molecules and known effects in humans

NAD+ Precursors              

Niacin/ nicotinic acid (NA), Nicotinamide riboside (NR) Nicotinamide (NAM) etc.

CD38 Inhibitors                 

Flavonoids (Quercetin, Luteolin, Apigenin, fisetin, rutin and naringin)             

Luteolinidin.  Kuromanin/ Chrysanthemin, an anthocyanin (food pigment)    

PARP Inhibitors    

BGB-290, Olaparib, Rucaparib, Veliparib, CEP-9722, E7016, Talazoparib, Iniparib, Niraparib, PJ34, DPQ, 3-aminobenzamide

                       

SARM Inhibitors

XAV939                    

NAMPT Activators

P7C3 

Conclusion

Some readers of this blog do give intranasal oxytocin as a therapy.  There have been numerous studies on children with autism, some discussed in earlier posts.  Oxytocin needs to be kept chilled, not to lose its potency.

Eleven previous posts in this blog refer to Oxytocin.

https://epiphanyasd.blogspot.com/search/label/Oxytocin

As to whether stimulating oxytocin receptors is going to be worthwhile in your case of autism, you will just have to try it and see.

I found that the Biogaia Protectis probiotic (L. reuteri DSM 17938) had very clear effects, which were very much hallmark effects of oxytocin.  This is easy and inexpensive to try.

Some readers of this blog do use Nicotinamide Riboside (NR), which we saw today can increase oxytocin by increasing NAD+.

There are very many reasons why you do not want to be lacking in NAD+, other than oxytocin, but if you already have plenty NAD+ you will unlikely see a benefit from yet more.

Magnesium is a very common autism supplement; it is often given with vitamin B6; both can be used to treat stress.

Superiority of magnesium and vitamin B6 over magnesium alone on severe stress in healthy adults with low magnesemia: A randomized, single-blind clinical trial

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:-

  

   

Source: https://www.hindawi.com/journals/mi/2013/963748/fig1/

  

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. 

Kv1.3 channel-blocking immunomodulatory peptides from parasitic worms: implications for autoimmune diseases

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.  

Resetting microbiota by Lactobacillus reuteri inhibits T reg deficiency–induced autoimmunity via adenosine A_2A receptors

  

Microbial Reprogramming Inhibits Western Diet-Associated Obesity

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. 

Histamine Derived from Probiotic Lactobacillus reuteri Suppresses 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 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. 

Lactobacillus reuteri–induced Regulatory T cells Protect against an Allergic Airway Response in Mice

 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.  

Short chain fatty acids induce both effectorand regulatory T cells by suppression of histone deacetylases and regulation ofthe mTOR-S6K pathway

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.  

Regulation of inflammatory Th17 cells in autism spectrum disorder

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.

The maternal interleukin-17apathway in mice promotes autism-like phenotypes in offspring 

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 (T_H17) 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 T_H17 cells in susceptible pregnant mothers may reduce the likelihood of bearing children with inflammation-induced ASD-like phenotypes 

Increased production of IL-17 in children with autism spectrum disorders and co-morbid asthma 

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. 

Immune Endophenotypes in Children WithAutism Spectrum Disorder

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.

Study Finds Children with Autism Have More Active Adaptive Immune System

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

https://www.hindawi.com/journals/mi/2013/963748/fig1/

Figure 1: Differentiation of naïve T helper cells into particular subsets. T helper lymphocytes leaving the thymus (naïve or T_H0) 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 T_H1 subset, if IL-4 into the T_H2 subset and eventually, if DCs synthesise IL-6, naïve T helper cells will become the T_H17 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. 

Resveratrol Ameliorates Dysregulation of Th1,Th2, Th17, and T Regulatory Cell-Related Transcription Factor Signaling in aBTBR T + tf/J Mouse Model of Autism

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]. Alzheimer’s 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	{"type":"clinical-trial","attrs":{"text":"NCT01597245","term_id":"NCT01597245"}}NCT01597245 {"type":"clinical-trial","attrs":{"text":"NCT00966875","term_id":"NCT00966875"}}NCT00966875
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	{"type":"clinical-trial","attrs":{"text":"NCT01544595","term_id":"NCT01544595"}}NCT01544595 {"type":"clinical-trial","attrs":{"text":"NCT01770379","term_id":"NCT01770379"}}NCT01770379 {"type":"clinical-trial","attrs":{"text":"NCT01358175","term_id":"NCT01358175"}}NCT01358175 {"type":"clinical-trial","attrs":{"text":"NCT01892436","term_id":"NCT01892436"}}NCT01892436 {"type":"clinical-trial","attrs":{"text":"NCT01478360","term_id":"NCT01478360"}}NCT01478360 {"type":"clinical-trial","attrs":{"text":"NCT01874340","term_id":"NCT01874340"}}NCT01874340 {"type":"clinical-trial","attrs":{"text":"NCT02044848","term_id":"NCT02044848"}}NCT02044848 {"type":"clinical-trial","attrs":{"text":"NCT01009281","term_id":"NCT01009281"}}NCT01009281
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	{"type":"clinical-trial","attrs":{"text":"NCT01708590","term_id":"NCT01708590"}}NCT01708590 {"type":"clinical-trial","attrs":{"text":"NCT02024646","term_id":"NCT02024646"}}NCT02024646 {"type":"clinical-trial","attrs":{"text":"NCT01902290","term_id":"NCT01902290"}}NCT01902290 {"type":"clinical-trial","attrs":{"text":"NCT01150890","term_id":"NCT01150890"}}NCT01150890
Abbott AbbVie	ABT-122	IL-17A/ TNFa	Rheumatoid arthritis	Phase 1	{"type":"clinical-trial","attrs":{"text":"NCT01853033","term_id":"NCT01853033"}}NCT01853033
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	approved {"type":"clinical-trial","attrs":{"text":"NCT01369329","term_id":"NCT01369329"}}NCT01369329 {"type":"clinical-trial","attrs":{"text":"NCT01330901","term_id":"NCT01330901"}}NCT01330901 {"type":"clinical-trial","attrs":{"text":"NCT01645280","term_id":"NCT01645280"}}NCT01645280 {"type":"clinical-trial","attrs":{"text":"NCT01009086","term_id":"NCT01009086"}}NCT01009086 {"type":"clinical-trial","attrs":{"text":"NCT00207727","term_id":"NCT00207727"}}NCT00207727 {"type":"clinical-trial","attrs":{"text":"NCT01713400","term_id":"NCT01713400"}}NCT01713400 {"type":"clinical-trial","attrs":{"text":"NCT01945086","term_id":"NCT01945086"}}NCT01945086
Abbott	Briakinumab ABT-874	p40 subunit of IL-12 and IL-23	Crohn’s disease Psoriasis Multiple Sclerosis	Ph 2terminated Phase 3 Phase 2	{"type":"clinical-trial","attrs":{"text":"NCT00562887","term_id":"NCT00562887"}}NCT00562887 {"type":"clinical-trial","attrs":{"text":"NCT00626002","term_id":"NCT00626002"}}NCT00626002 {"type":"clinical-trial","attrs":{"text":"NCT00086671","term_id":"NCT00086671"}}NCT00086671
Merck	Tildrakizumab (MK 3222) (SCH 900222)	IL-23p19	Psoriasis	Phase 3	{"type":"clinical-trial","attrs":{"text":"NCT01729754","term_id":"NCT01729754"}}NCT01729754
Johnson & Johnson Janssen Biotech	Guselkumab CNTO 1959	IL-23p19	Psoriasis Rheumatoid arthritis	Phase 2 Phase 2	{"type":"clinical-trial","attrs":{"text":"NCT01483599","term_id":"NCT01483599"}}NCT01483599 {"type":"clinical-trial","attrs":{"text":"NCT01645280","term_id":"NCT01645280"}}NCT01645280
Amgen/ MedImmun e	AMG 139	IL-23p19	Psoriasis Crohn’s disease	Phase 1 Phase 1	{"type":"clinical-trial","attrs":{"text":"NCT01094093","term_id":"NCT01094093"}}NCT01094093 {"type":"clinical-trial","attrs":{"text":"NCT01258205","term_id":"NCT01258205"}}NCT01258205
Eli Lilly	LY3074828	IL-23p19	Psoriasis	Phase 1	{"type":"clinical-trial","attrs":{"text":"NCT01947933","term_id":"NCT01947933"}}NCT01947933
Boehringer Ingelheim	BI 655066	IL-23p19	Ankylosing spondylitis Crohn’s disease Psoriasis (single rising dose)	Phase 2 Phase 2 Phase 2	{"type":"clinical-trial","attrs":{"text":"NCT02047110","term_id":"NCT02047110"}}NCT02047110 {"type":"clinical-trial","attrs":{"text":"NCT02031276","term_id":"NCT02031276"}}NCT02031276 {"type":"clinical-trial","attrs":{"text":"NCT02054481","term_id":"NCT02054481"}}NCT02054481

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.