Boosting “Tregs” in Autism, IBD, MS and even Obesity with Short-Chain Fatty Acids (SCFAs)

 T Rex - for what turned out to be rather a monster post

If the title of this post already makes sense, you probably do not need to read it.

It is about regulatory T cells (Tregs), which are an interesting way to treat what I have termed the over-activated immune system in autism.  The same ideas can be extended to other conditions related to mast cells, and also potentially Multiple Sclerosis (MS), Irritable bowel Disease (IBD) and even obesity.

Take Home Summary

For those more interested in what can be done, rather than why, here is the conclusion from this post:-

There are at least four possible ways to increase the number of regulatory T cells (Tregs), which should reduce pro-inflammatory cytokines (particularly IL-6) and increase anti-inflammatory cytokines (like IL-10).  

It should also reduce obesity, protect against diabetes and protect against organ damage in those already diabetic.

The simplest method is to increase production of small-chain fatty acids, which are the main metabolic products of bacteria fermentation that occurs naturally in the intestines.  You either eat more fibre or eat the specific bacteria, that causes the fermentation.

1.     Increase specific gut microbiota, namely B. fragilis and Clostridia

2.     Increase natural production of small-chain fatty acids (SCFAs) by eating more fibre.  Here using soluble maize fibre.

3.     Add supplemental SCFAs to your diet.  You just eat a source rich in some of the following:- Formic acid, Acetic acid, Propionic acid, Butyric acid (eat butter), Isobutyric acid, Valeric acid, Isovaleric acid

4.     Have a bone marrow transplant (not recommended)

  

For regular readers you may recall that B. fragilis appeared in an earlier post:-

Antibiotics andAutism(s) – Pass the Bacteroides Fragilis?

'Friendly' bacteria treat autism-like symptoms in mice

Why this post?  - Bumetanide has stopped working

I recently received a comment from a lady who has tried Bumetanide in her child with autism.  After the expected two week delay, she noticed lots of positive behavioral changes, but sadly latter on the Bumetanide “stopped working”.

In the past I received comments about “NAC has stopped working”.

Since I also experienced the same effect of “everything stops working” in the summer, I know how these people feel.

In reality, as I eventually discovered, it is not that Bumetanide/NAC has stopped working, but rather something else has started working.  I wrote once about autism being a Dynamic Encephalopathy, which to be fair was Martha Herbert’s idea and not mine.  This is one reason that a new type of doctor will be needed if autism is ever to be treated.  It is a moving target.

http://epiphanyasd.blogspot.com/2013/12/autism-dynamic-encephalopathy-indeed.html

In some types of autism it seems that the immune system can switch to an over-activated state and when in this state all my clever autism drugs appear to stop working.

In some people the problem is driven by so-called mast cells.  Mast cells play a key role in the inflammatory process. When activated they release granules and various hormonal mediators.  Histamine and the pro-inflammatory cytokine IL-6 are produced and this wreaks havoc in the brain, undoing all the good done by Bumetanide, NAC etc.

  

Regulatory T cells (Tregs)

In earlier posts I think I have exhaustively covered mast cells and to how to stabilize them.  However, I decided to look further back up the chain in the immune system at what may modulate the mast cells. Regulatory T cells caught my attention.

The regulatory T cells (Tregs), formerly known as suppressor T cells, are a subpopulation of T cells which modulate the immune system, maintain tolerance to self-antigens, and abrogate autoimmune disease. These cells generally suppress or downregulate induction and proliferation of effector T cells.

T regulatory cells are a component of the immune system that suppress immune responses of other cells. This is an important "self-check" built into the immune system to prevent excessive reactions.

The immune system must be able to discriminate between self and non-self. When self/non-self discrimination fails, the immune system destroys cells and tissues of the body and as a result causes autoimmune diseases. Regulatory T cells actively suppress activation of the immune system and prevent pathological self-reactivity, i.e. autoimmune disease

The immunosuppressive cytokines TGF-beta and Interleukin 10 (IL-10) have also been implicated in regulatory T cell function.

Recent evidence suggests that mast cells may be important mediators of Treg-dependent peripheral tolerance.

Regulatory T cells come in many forms with the most well-understood being those that express CD4, CD25, and Foxp3 (CD4+CD25+ regulatory T cells).

Foxp3⁺ Treg cells are known to produce IL-10 in the colon (Round and Mazmanian, 2010).

Regulatory T cells enhance mast cell production of IL-6 via surface-bound TGF-β.

Abstract

Mast cell degranulation is a hallmark of allergic reactions, but mast cells can also produce many cytokines that modulate immunity. Recently, CD25(+) regulatory T cells (Tregs) have been shown to inhibit mast cell degranulation and anaphylaxis, but their influence on cytokine production remained unknown. In this study, we show that, rather than inhibit, Tregs actually enhance mast cell production of IL-6. We demonstrate that, whereas inhibition of degranulation was OX40/OX40 ligand dependent, enhancement of IL-6 was due to TGF-β. Interestingly, our data demonstrate that the Treg-derived TGF-β was surface-bound, because the interaction was contact dependent, and no TGF-β was detectable in the supernatant. Soluble TGF-β1 alone was sufficient to enhance mast cell IL-6 production, and these supernatants were sufficient to promote Th17 skewing, but those from Treg-mast cell cultures were not, supporting this being surface-bound TGF-β from the Tregs. Interestingly, the augmentation of IL-6 production occurred basally or in response to innate stimuli (LPS or peptidoglycan), adaptive stimuli (IgE cross-linking by specific Ag), and cytokine activation (IL-33). We demonstrate that TGF-β led to enhanced transcription and de novo synthesis of IL-6 upon activation without affecting IL-6 storage or mRNA stability. In vivo, the adoptive transfer of Tregs inhibited mast cell-dependent anaphylaxis in a model of food allergy but promoted intestinal IL-6 and IL-17 production. Consequently, our findings establish that Tregs can exert divergent influences upon mast cells, inhibiting degranulation via OX40/OX40 ligand interactions while promoting IL-6 via TGF-β.

Treg cells are reduced in people with Autism

The following study showed that 73% of subjects with autism had reduced levels of Tregs and in particular those with allergies of a familial history of autoimmune disease.

Those in the 73% with allergies are the ones who fit my over activated immune system category.

Frequency of CD4+CD25 high regulatory T cells in the peripheral blood of Egyptian children with autism

Abstract

Autoimmunity may have a role in autism, although the origins of autoimmunity in autism are unknown. CD4( +)CD25(high) regulatory T cells play an important role in the establishment of immunological self-tolerance, thereby preventing autoimmunity. The authors are the first to study the frequency of CD4(+)CD25( high) regulatory T cells in the blood of 30 autistic and 30 age- and sex-matched healthy children. Patients with autism had significantly lower frequency of CD4(+)CD25(high) regulatory T cells than healthy children (P < .001). These cells were deficient in 73.3% of children with autism. Autistic patients with allergic manifestations (40%) and those with a family history of autoimmunity (53.3%) had a significantly lower frequency of CD4(+)CD25(high) regulatory T cells than those without (P < .01 and P < .001, respectively). In conclusion, CD4(+)CD25( high) regulatory T cells are deficient in many children with autism. Deficiency of these cells may contribute to autoimmunity in a subgroup of children with autism. Consequently, CD4(+)CD25(high) regulatory T cells could be new potential therapeutic targets in these patients.

This study was about autism, but for some therapeutic insights we need to go over to Wendy Garrett’s lab at Harvard.

Her group are not researching autism, they are researching inflammation, particularly in the colon. 

But inflammation can occur anywhere.

Their recent work and some relating to it is covered in the following excellent article is from the Multiple Sclerosis Discovery Forum.  It is very readable.

  

Gut Bacteria Boost Anti-Inflammatory Immune Cells inMice

Common compounds made by gut microbes that break down dietary fiber appear to boost the number and function of regulatory T cells (Tregs) in the colons of mice, a new study found. The findings expand the known ways that intestinal bacteria can influence Tregs, which can dial down an immune response and may be malfunctioning in autoimmune and inflammatory disorders, including multiple sclerosis (MS) and inflammatory bowel disease (IBD).

The microbial metabolites, known as short-chain fatty acids (SCFAs), restored the depleted Tregs of germ-free mice, the researchers reported. In mice with normal intestinal bacteria, supplemental SCFAs expanded the existing Treg population and activity. In a mouse model of colitis, SCFAs in drinking water reduced intestinal inflammation by enhancing Treg function.

"It's a terrific paper," said Sarkis Mazmanian, Ph.D., a microbiologist at the California Institute of Technology in Pasadena, in an interview with MSDF. Mazmanian first reported that PSA on the surface of B. fragilis converts CD4⁺ T cells into Foxp3⁺ Treg cells that produce IL-10 in the colon (Round and Mazmanian, 2010). "We have been working with a specific organism that makes a molecule unique to B. fragilis that induces Tregs and suppresses inflammation, and Wendy has discovered a more general metabolite produced by multiple bacterial groups that does something similar."

The study builds on discoveries (Nagano et al., 2012) showing that Tregs are dependent upon gut microbiota, specifically B. fragilis and Clostridia, Garrett told MSDF in an email. "We all may not have B. fragilis," she wrote. "In addition, human and mice both have many different strains of Clostridia. However, all healthy humans have regulatory T cells. Since SCFA are such abundant microbial metabolites, we hypothesized that SCFA may regulate Tregs in the colon."

"SCFA exert so many different effects on Tregs by altering molecules that affect the structure of DNA, making some areas of the DNA more open and available for transcription," Garrett wrote in an email. "In this way, SCFA can affect several different Treg functions."

For Garrett and others, the findings advance the therapeutic potential of dietary-based interventions using the SCFA mix and perhaps other molecules that boost signaling through GPR43 to improve Treg function in patients with inflammatory bowel disease and other autoimmune diseases. The concept was also advanced in another new study from Kenya Honda, M.D., Ph.D., of the RIKEN Center for Integrative Medical Sciences in Yokohama, Japan, in a recent Nature paper. A mixture of 17 strains of human-derived Clostridia designed to expand and differentiate Tregs relieved symptoms of colitis and allergic diarrhea in mouse models (Atarashi et al., 2013).

The full paper is here:-

The microbial metabolites, short chain fatty acids, regulate colonic Tregcell homeostasis

So much for the colon, what about the effect of increasing Treg in autism?

We already know that in the MIA (maternal immune activation) mouse model of autism, treating mice pups with B. fragilis reduces their autistic behaviours.

'Friendly' bacteria treat autism-like symptoms in mice 

That is a pretty good start, since we know that B. fragilis causes more SCFAs to be produced in the intestines.

The most effective way to reset an immune system would be a bone marrow transplant.  The following article from SFARI looks about what happens in mice. 

Bone marrow transplant curbs autism symptoms in mice

  

An altered immune system can cause autism-like behaviors, suggests a study published 31 July in the Proceedings of the National Academy of Sciences¹. The researchers found that a bone marrow transplant, which restores the animals’ immune system, alleviates some of their symptoms, including anxiety and repetitive behavior.

Such transplants are too dangerous for treating people with autism, but the findings suggest other treatments targeting immune cells, the researchers say.

When confronted with foreign cells — for example, when infected with a virus — the body typically activates immune cells called T cells to release signaling molecules called cytokines. A different set of T cells, called regulatory T cells, then keep that immune response in check by suppressing the activated T cells.

In the study, researchers injected pregnant mice with a mock flu virus that sets off their immune response. The offspring carry overly responsive T cells and have too few regulatory T cells throughout their lifetime, the study found. These two things together point to an immune system that's overly reactive.

The findings are provocative because several lines of evidence, including studies of postmortem brain tissue, cerebrospinal fluid and blood, suggest that some individuals with autism have an overactive immune system that leads to high levels of inflammation. One study has also reported that some children with the disorder have a dearth of regulatory T cells in their blood²

Studies on the effect of Small Chain Fatty Acids (SCFAs) on Humans

The good news is that numerous studies show that Wendy Garrett’s findings seem to apply far beyond the colon.

The reason is that SCFAs are able to cross the Intestinal Epithelium (i.e. cross from the gut to the bloodstream)

Potential Role of Regulatory TCells in Reversing Obesity-Linked Insulin Resistance and Diabetic Nephropathy

CONCLUSIONS Data suggest a potential therapeutic value of Tregs to improve insulin resistance and end organ damage in type 2 diabetes by limiting the proinflammatory milieu.

Short-Chain Fatty Acids protectagainst High-Fat Diet-Induced Obesity via a PPARγ-dependent switch from lipogenesis to fat oxidation

Abstract

Short-chain fatty acids (SCFAs) are the main products of dietary fiber fermentation and are believed to drive the fiber-related prevention of the metabolic syndrome. Here we show that dietary SCFAs induce a peroxisome proliferator-activated receptor (PPAR) γ-dependent switch from lipid synthesis to utilization. Dietary SCFA supplementation prevented and reversed high-fat diet-induced metabolic abnormalities in mice by decreasing PPARγ expression and activity. This increased the expression of mitochondrial uncoupling protein 2 and raised the AMP/ATP ratio, thereby stimulating oxidative metabolism in liver and adipose tissue via AMP-activated protein kinase. The SCFA-induced reduction in body weight and stimulation of insulin sensitivity were absent in mice with adipose-specific disruption of PPARγ. Similarly, SCFA-induced reduction of hepatic steatosis was absent in mice lacking hepatic PPARγ. These results demonstrate that adipose and hepatic PPARγ are critical mediators of the beneficial effects of SCFA on the metabolic syndrome, with clearly distinct and complementary roles. Our findings indicate that SCFAs may be used therapeutically as cheap and selective PPARγ modulators.

  

Recall that from earlier posts, I am already on the look out for selective PPARγ modulators (like Tangeretin)

The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism

Increased intake of dietary carbohydrate that is fermented in the colon by the microbiota has been reported to decrease body weight, although the mechanism remains unclear. Here we use in vivo¹¹C-acetate and PET-CT scanning to show that colonic acetate crosses the blood–brain barrier and is taken up by the brain. Intraperitoneal acetate results in appetite suppression and hypothalamic neuronal activation patterning. We also show that acetate administration is associated with activation of acetyl-CoA carboxylase and changes in the expression profiles of regulatory neuropeptides that favour appetite suppression.

Short-Chain Fatty Acids May Stimulate Fat Utilization

Tregs and Allergies

Fortunately some researchers have indeed looked at Tregs and allergies, but they did not seem to know about SCFAs.

T regulatory cells: an overview and intervention techniques to modulate allergy outcome.

Abstract

Dysregulated immune response results in inflammatory symptoms in the respiratory mucosa leading to asthma and allergy in susceptible individuals. The T helper type 2 (Th2) subsets are primarily involved in this disease process. Nevertheless, there is growing evidence in support of T cells with regulatory potential that operates in non-allergic individuals. These regulatory T cells occur naturally are called natural T regulatory cells (nTregs) and express the transcription factor Foxp3. They are selected in the thymus and move to the periphery. The CD4 Th cells in the periphery can be induced to become regulatory T cells and hence called induced or adaptive T regulatory cells. These cells can make IL-10 or TGF-b or both, by which they attain most of their suppressive activity. This review gives an overview of the regulatory T cells, their role in allergic diseases and explores possible interventionist approaches to manipulate Tregs for achieving therapeutic goals.

Regulation of Inflammation by Short Chain Fatty Acids

Here is a very good paper from Brazil, for those who need more convincing.

Regulation of Inflammation by Short Chain Fatty Acids

Short chain fatty acids (SCFAs), which are the major metabolic products of anaerobic bacteria fermentation, have been suggested to be the link between microbiota and host tissues. The concentration of these fatty acids in the GI tract and blood may predispose to or prevents pathological conditions such as IBD, cancer and diabetes. Modifications in the concentrations or the ability of host

tissues to use SCFAs have been described in these conditions.

SB =  Sodium butyrate

Mode of action of SCFAs

If anyone is interested in how SCFAs work their tricks, this is what they say in Brazil:

The main mechanism described for these effects is the attenuation of HDAC activity. Among the SCFAs, butyrate is the most potent, whereas acetate is the least potent inhibitor of HDAC.

This enzyme, together with the histone acetyltransferases (HAT), controls the degree of protein acetylation. By inhibiting the HDAC activity, SCFAs increase the acetylation of histone and non histone proteins including NFκB, MyoD, p53 and N-FAT [57] and, consequently, modulate gene

expression.

The production of prostaglandin E2 (PGE2) is also modified by SCFAs. These fatty acids stimulated the in vitro production of this eicosanoid by human monocytes [58]. In accordance with this result, induction of PGE2 production was observed three hours after intraplantar injection of SCFAs and LPS in rat paws [34]. PGE2 has been considered an anti-inflammatory prostanoid due to its ability to attenuate the production of IL-1β and TNF-α by macrophages and Th1 differentiation. However, there is now evidence in favor of a pro-inflammatory action of this molecule [59]. PGE2, through activation of its receptor EP4, facilitates Th1 differentiation and Th17 expansion, two subsets of T helper involved in immune inflammation [59,60]. Considering these findings, SCFAs may also affect T cell differentiation.

In addition to the classical eicosanoids, such as PGE2, other lipid mediators are also generated from polyunsaturated fatty acids including lipoxins, resolvins, protectins and maresins [61]. Despite their relevance to the resolution of the inflammatory process [61], at the moment, no study has been conducted in order to investigate the effect of SCFAs on the production of these lipid mediators.

Anti-inflammatory actions of SCFAs have been also observed in neutrophils. Acetate, propionate and butyrate at 30 mM reduce TNF-α production by LPS-stimulated human neutrophils [62].

Propionate and butyrate inhibit the expression of pro-inflammatory mediators (TNF-α, CINC-2αβ and NO) in rat neutrophils, an effect that seems to involve attenuation of NF-κB activation [21].

Microglial cells are resident immune cells of the central nervous system (CNS). Activation of these cells leads to production of several inflammatory mediators (e.g., cytokines and NO) that participate in the defense reaction of the CNS against insults including microorganisms and damaged cells [63].

Chronic or excessive activation of these cells has detrimental effects on the CNS and seems to be involved in the initiation and progression of neurodegenerative diseases including Alzheimer and Parkinson’s disease. In spite of some controversy about the effect of SCFAs on microglial production of inflammatory mediators [52,53], most of the studies indicate that these fatty acids attenuate microglial activation, an effect that seems to involve HDAC inhibition [53,54]. These observations and the data obtained in vivo [64] support the proposition that SCFAs and other inhibitors of HDAC may be useful in preventing inflammation in the CNS. Indeed, Kim et al. [64] have shown that butyrate, valproic acid and trichostatin A (all inhibitors of HDAC activity) present antineuroinflammatory and neuroprotective effects in the ischemic brain of rats.

Effectors Mechanisms of Phagocytes

Once in the inflammatory site, neutrophils and macrophages internalize, kill and digest bacteria and fungi through mechanisms including production of reactive oxygen species (ROS) and release of granule enzymes. SCFAs affect the production of ROS and the phagocytic capacity of phagocytes.

This effect is important in the course of anaerobic bacteria infection. Both inhibition [65,66,68] and stimulation [4,68] of neutrophil phagocytosis by SCFAs have been described. In macrophages, butyrate reduce the phagocytic activity, an effect that probably arises from its inhibitory action on cell differentiation and maturation [69].

The effects of SCFAs on ROS production by neutrophils remain controversial. Some groups have found that SCFAs induce ROS production [4,70,71], whereas others have shown inhibition [65,67,72–74].

The discrepancy in the results obtained may be explained by differences in the protocols used such as the concentrations of SCFAs, measurement of ROS by using different methodologies (e.g., lucigenin-amplified chemiluminescence or reduction of cytochrome c), stimuli (e.g., PMA or fMLP), solution pH, source and state of neutrophil activation (e.g., neutrophils isolated from human blood or elicited rat neutrophils).

3.3. Lymphocyte Activation and Response

Lymphocytes are involved in the adaptive immune response. These cells display membrane receptors that recognize a broad range of non-self antigens and allow them to generate specific responses to  liminate invading pathogens and infected or tumoral cells. SCFAs modify lymphocytes function as follows:

T-cell proliferation: butyrate inhibits lymphocyte proliferation in response to several stimuli including concanavalin-A and immobilized anti-CD3 monoclonal antibody [41,75].

Production of cytokines: incubation of lymphocytes with butyrate reduces the production of interleukin-2; this cytokine stimulates growth, differentiation and survival of antigen-selected

T-lymphocytes, and interferon-γ (IFN-γ) after stimulation with concanavalin-A or anti-CD3 and anti-CD8 [76,77]. This latter cytokine is particularly important in response to viral infection, tumor cells and in auto-immune conditions. On the other hand, butyrate presents an opposite effect on the production of IL-10 by lymphocytes [75].

Production of regulatory T (Treg) cells: this subpopulation of T cells actively suppresses immune function and is considered an attractive target for the treatment of immunological and inflammatory pathologies. HDAC inhibitors enhance the production and suppressive function of regulatory T cells [77]. Considering that SCFAs, as previously described, also suppress the activity of HDAC, we hypothesize that these fatty acids may also exert their effects on inflammation and immune responses through regulation of this subset of T cells.

Conclusion

Within reasonable limits, short chain fatty acids (SCFAs) are good for you.

Particularly if you have an inflammatory condition or need to lose some weight.

You already produce them and some people would benefit from some more.

P.S. for the Diehards

Proprionic Acid (PPA) in Rats

There is also research indicating that injecting large amounts of one particular SCFA, Propionic acid into the brains of rats does them no good at all.  In fact the opposite of all the good things notes by the Brazilians and others.

Having read an awful lot of autism research, I have to point out that sometimes a little of what does you harm, can actually do you some good.  For example the Valproate mouse model of autism is based on feeding Valproic Acid to the female mouse to make her pup be born with autistic features.  Yet the same drug Valproic Acid, in lower doses, is an effective treatment for autism with seizures in humans.

In pregnant humans the risk of Valproate is slightly different.  According to Harvard:-

Valproate. It’s best to avoid taking valproate (Depakote) during pregnancy, especially during the first trimester, as this drug increases the risk of neural tube defects such as spina bifida. Risk increases with dose. In absolute terms, researchers estimate that one to six babies out of every 100 exposed to valproate in the first trimester of fetal development are born with some type of neural tube defect.

  

Neurobiological effects of intraventricular propionic acid in rats: Possible role of short chain fatty acids on the pathogenesis and characteristics of autism spectrum disorders

Abstract

Clinical observations suggest that certain gut and dietary factors may transiently worsen symptoms in autism spectrum disorders (ASD), epilepsy and some inheritable metabolic disorders. Propionic acid (PPA) is a short chain fatty acid and an important intermediate of cellular metabolism. PPA is also a by-product of a subpopulation of human gut enterobacteria and is a common food preservative. We examined the behavioural, electrophysiological, neuropathological, and biochemical effects of treatment with PPA and related compounds in adult rats.

Intraventricular infusions of PPA produced reversible repetitive dystonic behaviours, hyperactivity, turning behaviour, retropulsion, caudate spiking,

and the progressive development of limbic kindled seizures, suggesting that this compound has central effects. Biochemical analyses of brain homogenates from PPAtreated rats showed an increase in oxidative stress markers (e.g., lipid peroxidation and protein carbonylation) and glutathione S-transferase activity coupled with a decrease in glutathione and glutathione peroxidase activity. Neurohistological examinations of hippocampus and adjacent white matter (external capsule) of PPA treated rats revealed increased reactive astrogliosis (GFAP immunoreactivity) and activated microglia (CD68 immunoreactivity) suggestive of a neuroinflammatory process. This was coupled with a lack of cytotoxicity (cell counts, cleaved caspase 3_ immunoreactivity), and an increase in phosphorylated CREB immunoreactivity. We propose that some types of autism may be partial forms of genetically inherited or acquired disorders involving altered PPA metabolism. Thus, intraventricular administration of PPA in rats may provide a means to model some aspects of human ASD in rats.

Relative abundance of short chain and polyunsaturated fatty acids in propionic acid-induced autistic features in rat pups as potential markers in autism

The short chain fatty acids (SCFAs) acetate (C2), propionate (C3) and butyrate (C4) are the main metabolic products of anaerobic bacterial fermentation in the intestine. In addition to their important role as fuel for intestinal epithelial cells, SCFAs modulate different processes in the gastrointestinal (GI) tract such as electrolyte and water absorption. These fatty acids have been recognized as potential mediators of the effects of the gut microbiota on intestinal immune function and gut-brain axis interaction [4]. Recently it was reported that the three types of SCFAs (acetate, propionate, and butyrate) reduce the production of proinflammatory factors, including TNF-α, IL-1β, IL-6, and NO. Additionally, SCFAs enhance the production of the anti-inflammatory cytokine IL-10 in low concentrations (1–1,200 μmol/L) [5].

In spite of the protective effects of SCFAs, propionic acid (PPA) neurotoxicity was recently demonstrated via intraventricular direct infusion into rat brains [6], passage from the gut to the brain in the case of acute PPA orally administered to rat pups [7] or Chronic administration on postnatal days 5–28 [8] and, most recently, subcutaneous injection once a day (500 mg/kg) in pregnant rats on gestation days G12–16 [9].

I am very much minded to go with Wendy, the Brazilians and the Egyptians (who found Trep low in autism). 

I think the Saudis, with their PPA-neurointoxicated rats, are barking up the wrong tree.

In fact, the Saudis say that PPA is low in humans with autism.

Low SCFAs, like PPA, help produce low Trep, which helps produces high IL-6 and low IL-10, just as I expect to find in autism.