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Tuesday 14 March 2023

Differentially expressed immune-related genes (dIRGs) in Changsha and Rapamycin/mTOR


 


I did write about an interesting paper last year concerning calcium channels and intellectual disability; it was from a city in China called Changsha.

Epiphany: Calcium channelopathies and intellectual disability

Changsha is on the old train line and the new high speed line from Beijing to Hong Kong. So like many other people, I must have passed by this city of 10 million on the old line, as a backpacking student many years ago.

After three years of closure, China announced that it is reopening to foreign visitors. China is well worth a visit and their high speed trains make travel much easier than it used to be.

Before moving on to today’s paper, I will mention the case study below from one of China’s top hospitals, the PLA hospital in Beijing.  They used the well known mTOR inhibitor Rapamycin to successfully treat an 8 year old boy with idiopathic (of unknown cause) autism.  This drug has been used in models of autism. The mTOR inhibitor Everolimus is approved as adjunctive therapy for a single gene autism called TSC to treat seizures. Click on the link below to read the one page case report.

Rapamycin/Sirolimus Improves the Behavior of an 8-Year-Old Boy With Nonsyndromic Autism Spectrum Disorder

Some readers have mentioned this case study and at least one has made a trial.  In that case the drug was well tolerated but did not moderate autism symptoms.

Mammalian target of rapamycin (mTOR) regulates cell proliferation, autophagy, and apoptosis by participating in multiple signaling pathways in the body. Studies have shown that the mTOR signaling pathway is also associated with cancer, arthritis, insulin resistance, osteoporosis, and other diseases including some autism.

Today we return to Changsha for another interesting paper about the altered immune system in autism and other neurological conditions.  It is an interesting study because it is based on samples from 2,500 brains of controls and patients with six major brain disorders - schizophrenia, bipolar disorder, autism spectrum disorder, major depressive disorder, Alzheimer’s disease, and Parkinson’s disease.

One of the reasons so little progress has been made in treating any neurological condition is the inability to take physical samples to experiment with.  All the 2,500 brain samples are taken from brain banks, not live people.

When it comes to autism that means the sample likely reflects severe autism (DSM3 autism).  No self-identified autism in today’s samples, their brains are unlikely to be donated to medical science. 


Immunity-linked genes expressed differently in brains of autistic people 

Genes involved in immune system function have atypical expression patterns in the brains of people with some neurological and psychiatric conditions, including autism, according to a new study of thousands of postmortem brain samples.

Of the 1,275 immune genes studied, 765 — 60 percent — showed elevated or reduced expression in the brains of adults with one of six conditions: autism, schizophrenia, bipolar disorder, depression, Alzheimer’s disease or Parkinson’s disease. The expression patterns varied by condition, suggesting that there are distinct “signatures” for each one, says lead researcher Chunyu Liu, professor of psychiatry and behavioral sciences at Upstate Medical University in Syracuse, New York.

The expression of immune genes could potentially serve as a marker for inflammation, Liu says. Such immune activation — particularly while in utero — has been associated with autism, though the mechanisms are far from clear.

“My impression is the immune system is not really a very minor player in brain disorders,” Liu says. “It is a major player.”

It’s impossible to discern from this study whether immune activation played a role in contributing to any condition or whether the condition itself led to altered immune activation, says Christopher Coe, professor emeritus of biopsychology at the University of Wisconsin-Madison, who was not involved in the work.

“A study of the postmortem brain is informative,” Coe says. “But not definitive.”

Liu and his team analyzed the expression levels of 1,275 immune genes in 2,467 postmortem brain samples, including 103 from autistic people and 1,178 from controls. The data came from two transcriptomics databases — ArrayExpress and the Gene Expression Omnibus — and other previously published studies.

Brains from autistic people had, on average, 275 genes with expression levels that differed from those of controls; brains from people with Alzheimer’s disease had 638 differentially expressed genes, followed by those with schizophrenia (220), Parkinson’s (97), bipolar disorder (58) and depression (27).

Autistic men’s expression levels varied more than those of autistic women, whereas the brains of women with depression showed more variation than those of men with depression. The other four conditions showed no sex differences.

The autism-related expression pattern more closely resembled those of the neurological conditions — Alzheimer’s and Parkinson’s — than the other psychiatric ones. Neurological conditions, by definition, must have a known physical signature in the brain, such as Parkinson’s characteristic loss of dopaminergic neurons. Researchers have not found such a signature for autism.

“This [similarity] just provides some kind of additional direction we should look into,” Liu says. “Maybe one day we will understand the pathology better.”

The findings were published in Molecular Psychiatry in November.

Two genes, CRH and TAC1, are the most commonly altered among the conditions: CRH is downregulated in all of the conditions but Parkinson’s, and TAC1 is downregulated in all but depression. Both genes affect the activation of microglia, the brain’s immune cells.

Atypical microglial activation may be “derailing normal neurogenesis and synaptogenesis,” Coe says, disrupting neuronal activity similarly across the conditions.

Genes involved in astrocyte and synapse function are similarly expressed in people with autism, schizophrenia or bipolar disorder, a 2018 study of postmortem brain tissue found. But microglial genes are overexpressed in autism alone, that study found.

People with more intensely upregulated immune genes could have a “neuroinflammatory condition,” says Michael Benros, professor and head of research on biological and precision psychiatry at the University of Copenhagen in Denmark, who was not involved in the work.

“It could be interesting to try to identify these potential subgroups and of course provide them more specific treatment,” Benros says.

Most of the expression changes observed in the brain tissue samples did not appear in datasets of gene expression patterns in blood samples from people with the same conditions, the study shows. This “somewhat surprising” finding indicates the importance of studying brain tissue, says Cynthia Schumann, professor of psychiatry and behavioral sciences at the University of California Davis MIND Institute, who was not involved in the study.

“If you want to know about the brain, you have to look at the brain itself,” Schumann says.

 

I am always reminding people not to think that blood samples are going to tell them how to treat autism.  The above commentary also highlights this fact.  If you want to know what is going on in the brain, you have to look there or in spinal fluid.  Looking just at blood samples may send an investigation in completely the wrong direction. Spinal fluid flows around the brain and spinal cord to help cushion them from injury and provide nutrients. Testing spinal fluid requires an invasive procedure, parents do not like it and so it is very rarely carried out until adulthood.  Time has then been lost.

 

Here is the link to the full paper and some highlights I noted.

 

Neuroimmune transcriptome changes in patient brains of psychiatric and neurological disorders 

Neuroinflammation has been implicated in multiple brain disorders but the extent and the magnitude of change in immune-related genes (IRGs) across distinct brain disorders has not been directly compared. In this study, 1275 IRGs were curated and their expression changes investigated in 2467 postmortem brains of controls and patients with six major brain disorders, including schizophrenia (SCZ), bipolar disorder (BD), autism spectrum disorder (ASD), major depressive disorder (MDD), Alzheimer’s disease (AD), and Parkinson’s disease (PD). There were 865 IRGs present across all microarray and RNA-seq datasets. More than 60% of the IRGs had significantly altered expression in at least one of the six disorders. The differentially expressed immune-related genes (dIRGs) shared across disorders were mainly related to innate immunity. Moreover, sex, tissue, and putative cell type were systematically evaluated for immune alterations in different neuropsychiatric disorders. Co-expression networks revealed that transcripts of the neuroimmune systems interacted with neuronal-systems, both of which contribute to the pathology of brain disorders. However, only a few genes with expression changes were also identified as containing risk variants in genome-wide association studies. The transcriptome alterations at gene and network levels may clarify the immune-related pathophysiology and help to better define neuropsychiatric and neurological disorders. 

 

Multiple lines of evidence support the notion that the immune system is involved in major “brain disorders,” including psychiatric disorders such as schizophrenia (SCZ), bipolar disorder (BD), and major depressive disorder (MDD), brain development disorders such as autism spectrum disorder (ASD), and neurodegenerative diseases such as Alzheimer's disease (AD), and Parkinson's disease (PD). Patients with these brain diseases share deficits in cognition, blunted mood, restricted sociability and abnormal behavior to various degrees. Transcriptome studies have identified expression alterations of immune-related genes (IRGs) in 49 postmortem brains of AD, PD, ASD, SCZ and BD separately. Cross disorder transcriptomic studies further highlighted changes in IRGs. At the protein level, several peripheral cytokines showed reproducible disease-specific changes in a meta-analysis. Since brain dysfunction is considered the major cause of these disorders, studying immune gene expression changes in patient brains may reveal mechanistic connections between immune system genes and brain dysfunction. Most previous studies were limited to the analysis of  individual disorders. There is no comprehensive comparison of the pattern and extent of inflammation-related changes in terms of immune constructs (subnetworks), neuro-immune interaction, genetic contribution, and relationship between diseases.  Neuroinflammation, an immune response taking place within the central nervous system,  can be activated by psychological stress, aging, infection, trauma, ischemia, and toxins. It is regulated by sex, tissue type and genetics, many of which are known disease risk factors for both psychiatric and neurological diseases. The primary function of neuroinflammation is to maintain brain homeostasis through protection and repair. Abnormal neuroinflammation activation could lead to dysregulation of mood, social behaviors, and cognitive abilities. Offspring who were fetuses when their mothers’ immune system was activated (MIA) showed dopaminergic hyperfunction, cognitive impairment, and behavioral abnormalities as adults. Alternatively, acute and chronic neuroinflammation in adulthood can also alter cognition and behavior. In animal models, both adult and developmental maternal immune activation in the periphery can lead to increases in pro-inflammatory cytokines in the brain , similar to what is found in humans with major mental illness.  Previous studies identified immune gene dysregulations in brains of patients with several major brain disorders. For example, Gandal et al. found that up-regulated genes and isoforms in SCZ, BD, and ASD were enriched in pathways such as inflammatory response and response to cytokines. One brain co-expression module up-regulated specifically in MDD was enriched for genes of cytokine-cytokine interactions, and hormone activity pathways. The association of neurological diseases such as AD and PD with IRGs has also been reported. These studies examined the changes of immune system as a whole without going into details of specific subnetworks, the disease signature, or genetic versus environmental contribution. We hypothesize that expression changes of specific subsets of IRGs constitute part of the transcriptome signatures that distinguishes diseases. Since tissue specificity, sex and genetics all could influence such transcriptome signatures, we analyzed their effects. Furthermore, we expect that neurological diseases and psychiatric disorders bear transcriptomic changes that may help to address how similar immunological mechanisms lead to distinct brain disorders. The current boundary between neurological diseases and psychiatric disorders is primarily the presence of known pathology. Neurological diseases have more robust histological changes while psychiatric disorders have more subtle subcellular changes. Nonetheless, pathology evidence is always a subject to be revised with new research.  To investigate immune-related signatures of transcriptome dysregulation in brains of six neurological and psychiatric disorders, we studied a selected list of 1,275 genes known to be associated with neuroinflammation and interrogated their expression across disorders. We collected and analyzed existing transcriptome data of 2,467 postmortem brain samples from donors with AD, ASD, BD, MDD, PD, SCZ and healthy controls (CTL). We identified the differentially expressed IRGs shared across disorders or specific to each disorder, and their related coexpression modules (Fig. S1). These genes and their networks and pathways provided important insight into how immunity may contribute to the risk of these neurological and psychiatric disorders, with a potential to refine disease classification.

 

The two most shared dIRGs are Corticotropin-releasing hormone (CRH) and Tachykinin Precursor 1 (TAC1), which were differentially expressed in five of the six diseases (Fig. 2D). They both involve innate immunity according to the databases we used and literature. CRH was downregulated in five of the six disorders; the exception was PD. CRH can regulate innate immune activation with neurotensin (NT), stimulating mast cells, endothelia, and microglia. TAC1 was down-regulated in five of the six disorders, the exception being MDD.  TAC1 encodes four products of substance P, which can alter the immune functions of activated microglia and astrocytes. Independent RNA-seq data confirmed both CRH and TAC1 findings. These transcripts are also neuromodulators and have action on neurons so they have roles in addition to immune functions. 

This indicated that even though immune dysfunction is widespread in the six disorders, signature patterns of the subset innate immune genes are sufficient to differentiate neurological from psychiatric disorders. 

Disease-specific IRMs in AD, ASD, and PD imply distinct biological processes.

We also searched for disease-specific IRMs for each disorder. We used rWGCNA to construct brain co-expression networks in the brains of each disorder and of controls, then compared them against each other to identify disease-specific IRMs (Fig.5A). Based on preservation results of one disease versus controls and against all other diseases (Fig. 5B, z-summary < 10), as well as immune gene enrichment results (Table S9; enrichment q.value < 0.05), we identified six disease-specific IRMs, including one for AD, three for ASD, and two for PD. We did not detect disease-specific IRMs for SCZ, BD, or MDD, which are considered psychiatric disorders. The disease-specific IRMs were enriched for various functions (Fig. 5C, Table S9). The AD specific IRM was enriched for neuron part (GO:0097458, q.value= 4.57E-4) and presynapse (GO:0098793, q.value = 4.57E-4). The PD-specific IRM was enriched for positive regulation of  angiogenesis (GO:0045766, q.value = 9.65E-06) and secretory granule (GO:0030141, q.value= 220 6.31E-06). The ASD-specific IRMs were enriched for developmental biological processes such as negative regulation of cell proliferation and growth factor receptor binding. 

Our reader Eszter will be pleased to see that the research links the differentially expressed genes more with Alzheimer’s than with Bipolar or Schizophrenia.  She has noted the overlap in effective therapies between Alzheimer’s and autism. 

We came up with four major findings of the neuroimmune system in brains of different neuropsychiatric disorders: 1) the innate immune system carries more alterations than the adaptive immune systems in the six disorders; 2) the altered immune systems interact with other biological pathways and networks contributing to the risk of disorders; 3) common SNPs have a limited contribution to immune-related disease risks, suggesting the environmental contribution may be substantial; and 4) the expression profiles of dIRGs, particularly that of innate immune genes, group neurodevelopment disorder ASD with neurological diseases (AD and PD) instead of with psychiatric disorders (BD, MDD, and SCZ) Dysregulation of the innate immune system is a common denominator for all six brain disorders. We found that more than half of the shared dIRGs and dIRG-enriched pathways were related to the innate immune system. The two most shared dIRGs, TAC1 and CRH, have known effects on innate immune activation(66, 67). Both genes were downregulated in patient brains. Additionally, TLR1/2 mediates microglial activity, which could contribute to neuronal death through the release of inflammatory mediators. Furthermore, innate immunity is critical in maintaining homeostasis in the brain. For example, the innate immune system has been reported to function in the CNS's resilience and in synaptic pruning throughout brain growth. When homeostasis is disrupted, the abnormal innate immunity may impact a wide range of brain functions.

 

Microglia are affected specifically in autism and Alzheimer’s.

Microglia are highlighted in the immune changes in brains of AD and ASD in this study. Microglia is the major cell type participating in the brain’s immune system. Our analyses showed that the IRM12 coexpression module was enriched for microglia genes and associated with inflammatory transcriptional change in AD and ASD but not the other four diseases. Does this suggest that microglial dysfunction contributes more to AD and ASD than to the other disorders? The PsychENCODE study showed the microglial module upregulated in ASD and downregulated in SCZ and BD(16), but the fold changes in SCZ and BD were much smaller than that in ASD (Fig 7.B in original paper(16)). Larger sample size may be needed to detect microglia contribution to other disorders such as SCZ and BD. 

Sex contributes to the disease-related immune changes too. Our results revealed sex-bias dysregulation of IRGs in brains of ASD and MDD but not in other disorders. These two  disorders are known to have sex differences in prevalence. Previous studies also have suggested that sex differences in stress-related neuroinflammation might account for the overall sex bias in stress-linked psychiatric disorders, including female bias in MDD and male bias in ASD. We did not observe sex-biased IRGs in other diseases with known sex-biased prevalence, such as SCZ and AD suggesting that sex differences in SCZ and AD may not involve IRG changes. 

Our results showed how immune system dysregulation may influence gene expression of the networked other non-immune genes and contribute to the pathology of these diseases specifically. Six disease-specific IRMs were detected in AD, ASD, and PD, showing that several functions of the immune-related networks also involved in corresponding disorders such as presynaptic related AD-IRM and Growth factor receptors-related ASD-IRMs. Presynaptic proteins are essential for synaptic function and are related to cognitive impairments in AD(85). Growth factor receptors and N-acetylcysteine are involved in the etiology of ASD. Secretogranin may be a pivotal component of the neuroendocrine pathway and play an essential role in neuronal communication and neurotransmitter release in PD (88). Furthermore, the immune system has been found to regulate presynaptic proteins(89), EGFR(90), and secretogranin(88). Our results indicate that alterations of the immune network can be disease-specific, affecting specific coexpression networks and driving distinct risk of each disorder. 

To our surprise, neurodevelopment disorder ASD was grouped with neurological diseases (AD  and PD) instead of with psychiatric disorders (BD, MDD, and SCZ) according to the changes of IRGs, particularly innate immune genes. Hierarchical clustering analysis based on the effect size of IRGs placed the presumed psychiatric disorder ASD with other neurological diseases. Previous studies have reported that ASD patients exhibited more neurological and immunological problems(99-102) compared to healthy people and to other brain disorders. As more etiologies are uncovered, the traditional classification of these diseases is increasingly challenged(93). Furthermore, we found that dIRGs change more in neurological diseases (AD, PD, and ASD) than in the psychiatric disorders (BD, SCZ, and MDD). It suggested that neuroimmunity dysregulation is more severe in neurological diseases than in psychiatric disorders, led by AD. Neuroimmunity may help to redefine disease classification in the future.

 


Conclusion 

It is good to see there is excellent research coming from China. Our reader Stephen has noted some interesting research underway in Russia. Look both East and West.

Intranasal Inhalations of M2 Macrophage Soluble Factors in Children With Developmental Speech Disorders

In today’s paper the focus was just on immune related genes.  That in itself is a big step forward, since in this blog we are well aware of the key role of the immune system in autism.

In this study all of autism was grouped together, when we know there will be many subgroups with totally different profiles.  In terms of treatment, you would need to know which subgroup you are part of.

But it does tell you that part of your autism therapy is going to have to account for an altered immune status. 

I would have to say that it does follow Western research in getting a bit lost in the detail.  We know that they found 275 of the immune genes mis-expressed in autism.

How about presenting a simple list of the 275 with whether the genes were over or under expressed ?

There are vast spreadsheets in the supplemental data, but nothing as down to earth and common sense as that.

Instead the researchers were preoccupied with overlaps between different conditions and churning out statistics.

It is notable from the first paper I mentioned today that one of the very top Chinese hospitals is actually trying to apply personalized medicine using Rapamycin for autism and publishing a case history. Bravo !!

A logical next step after trying to modify mTOR would be to try epigenetic modification therapy using HDAC inhibition.

One issue here is the age at which therapy begins, not surprisingly some therapies need to commence at birth (or ideally before) and do not give much effect later in life.

Romidepsin is one HDAC inhibitor used in the research.

In the studies below Chinese researchers in the US are making progress. 

In 2018:

Autism's social deficits are reversed by an anti-cancer drug

Using an epigenetic mechanism, romidepsin restored gene expression and alleviated social deficits in animal models of autism.

"In the autism model, HDAC2 is abnormally high, which makes the chromatin in the nucleus very tight, preventing genetic material from accessing the transcriptional machinery it needs to be expressed," said Yan. "Once HDAC2 is upregulated, it diminishes genes that should not be suppressed, and leads to behavioral changes, such as the autism-like social deficits."

But the anti-cancer drug romidepsin, a highly potent HDAC inhibitor, turned down the effects of HDAC2, allowing genes involved in neuronal signaling to be expressed normally.

The rescue effect on gene expression was widespread. When Yan and her co-authors conducted genome-wide screening at the Genomics and Bioinformatics Core at UB's New York State Center of Excellence in Bioinformatics and Life Sciences, they found that romidepsin restored the majority of the more than 200 genes that were suppressed in the autism animal model they used.

In 2021:

Synergistic inhibition of histone modifiers produces therapeutic effects in adult Shank3-deficient mice

 We found that combined administration of the class I histone deacetylase inhibitor Romidepsin and the histone demethylase LSD1 inhibitor GSK-LSD1 persistently ameliorated the autism-like social preference deficits, while each individual drug alone was largely ineffective.

 

We now need some leading researchers/clinicians in China to actually translate this approach to humans and see if it works.  Hopefully the PLA hospital in Beijing are keeping an eye out on what Zhen Yan is up to at the University of Buffalo, NY.  With luck they will not wait 20 years to try it!





93 comments:

  1. Thanks for the new post Peter. I would to add Bumetanide on the list of drugs that alter immune system.

    https://pubmed.ncbi.nlm.nih.gov/30102895/

    -Stephen

    ReplyDelete
  2. many drug and supplement effect to microglia. what you recommend?

    ReplyDelete
    Replies
    1. Microglia may function like an "immunostat" reflecting immune status elsewhere in the body. So you can either target microglia directly, which may not be effective, or target inflammation more broadly. A wide range of immune dysfunctions may be present in autism, so you just have to try different therapies until you find ones that are beneficial. What works for one person maybe ineffective in the next person. So I my recommendation is to make trials until you are successful.

      Delete
    2. Currently doing trials with Rapamycin. It works however the side effects are hard to ignore. My son is prone to viral infections and ever time I start a new trial a cold comes along and knocks him down.

      -Stephen

      Delete
  3. Maybe the objective is to modulate macrophages. Drugs that helped Covid-19 would work.

    Hydrochloride- stops m1 & m2

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079743/

    Ivermectin- polarized m1 to m2

    This is not a covid drug but itraconazole (Sporanox). Just type autism and sporanox into google.

    https://journals.lww.com/ijdv/fulltext/2019/12000/itraconazole_promotes_macrophage_m1_polarizationGoogle.

    And basically all of Peter's polypill.

    -Stephen

    ReplyDelete
    Replies
    1. Stephen, what microglia modulator you use for your child? What differences do you notice?

      Delete
    2. Janu this is the drug I want.

      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9397913/

      -Stephen

      Delete
    3. https://www.nature.com/articles/s41591-020-1007-0

      Delete
    4. Stephen, looks like you have a plan. Please keep us posted on your trials and findings.

      What are your thoughts on Pioglitazone and Fenofibrate which are studied in the context of Autism? https://link.springer.com/article/10.1007/s12035-022-02819-1
      https://www.sciencedirect.com/science/article/pii/S0361923018308736?via%3Dihub

      Delete
  4. Ivm- m1 to m2 polarization

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8350903/?report=reader

    Verapamil- Decreases macrophages

    https://www.sciencedirect.com/science/article/pii/S0753332222001718

    ReplyDelete
  5. Drug list of m1 to m2 converters

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915167/

    ReplyDelete
  6. Azithromycin-

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684391/

    ReplyDelete
    Replies
    1. Long term antibiotic therapy may be an effective treatment for children co-morbid with Lyme disease and autism spectrum disorder

      https://pubmed.ncbi.nlm.nih.gov/22361005/

      Crosses BBB

      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3957884/

      Decrease IL-6 and increases M2

      https://pubmed.ncbi.nlm.nih.gov/18230686/

      Delete
    2. There was a series of posts in this blog about the immunomodulatory effects of specific types of antibiotics.

      https://www.epiphanyasd.com/search/label/antibiotic

      Many people report improvement in autism when taking a specific antibiotic.

      Delete
  7. Cbd oil- m1 to m2

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767553/

    ReplyDelete

  8. Antabuse- potent macrophage inhibitor

    https://www.nature.com/articles/s41467-020-14338-5

    Thank you Dragos.

    ReplyDelete
  9. Hi Peter, how are you?
    Great to see that at least chinese cientists are advancing the research, sadly it is too slow.
    Seen your book on Amazon, congrats, will buy it. Do you know if anytime it will have a portuguese version? If i had time i would translate it myself with Google translate help, maybe in the future.
    By the way, my son Bento is trying bumetanide for almost tree months now with help of a parent of autistic boy in Mexico (he send to me, but i will have to find another way to keep it because he probably will no longer provide more, he said that farmacy starts to demand a prescription and i dont want to abuse his kindness).
    About neuroimunoregulation will you recommend study in any particular medication? It is so so difficult to even find a doctor specialized in that matters, so frustating, the time passes and our sons is losing opportunitys to get better.
    Thanks again for your effort in provide information to the community! Hugs from Brazil, if anytime you want to visit here, south region, will be glad to have you.

    ReplyDelete
    Replies
    1. you can search https://mexmeds4you.com.mx/product/product?product_id=4258
      this guy from mexico and sell bumetanid

      Delete
    2. Herson, different immunomodulatory therapies work for different people.

      Some people like low dose naltrexone, for example. Not a bad one to try.

      What works for my son include: Atorvastatin, Ponstan, Verapamil, Pioglitazone.

      I think looking at comorbidities and what makes autism better or worse in your specific case can help guide you.

      No current plans for a Portuguese book. It is really a niche subject.

      Delete
    3. Peter, I purchased your book and started with Bumetanide for my 6 yr old son so far its been a week but no noticeable changes. However, I was thinking to add Rapamycin next month. Are there any contraindications if we do both together. Thank you so much

      Delete
    4. You would expect it to take 2 weeks or more before bumetanide would show a benefit.

      Rapamycin is a potent drug. Some people have reported a benefit. There are no interactions listed with bumetanide.

      Delete
    5. If you do start rapamycin use a 0.5mg/daily dose. It will lessen the side effects. My biggest problem during my trials was colds and the constant runny nose.

      -Stephen

      Delete
  10. Do you know of any mTOR inhibitors besides Rapamycin? It is impossible to get in Denmark. Even if you found a pro active doctor, the pharmacies can only deliver this medication to the hospitals....

    ReplyDelete
    Replies
    1. The potent mTOR inhibitors are all drugs.

      There are foods like curcumin and supplements like green tea polyphenol that have mTOR inhibiting properties. I think they will lack potency unless you consume large amounts. Curcumin is very good for you if you eat a lot of it like in India. Adding it to cooked fatty food with piperine/ pepper increases is bioavailability.

      Delete
    2. i can't buy rapamycin. what elses i should try, statin?

      Delete
    3. Rapamycin is a potent drug. There likely are not any non drug equivalents.

      Look up mTOR inhibitors and you will see some supplements like EGCG and curcumin. Both are very good for you, but they are not real alternatives to Rapamycin.

      Delete
    4. I think intermittent fasting reduces MTOR but I don't know how much compared to rapamycin. E.g. Only eating in an 8-hour window each day, could be between 08:00-16:00, or 12:00-20:00 for example.

      Aspie2

      Delete
    5. Peter, can i buy online rapamycin, do you know any online store ?

      Delete
    6. Itraconazole is also an mtor inhibitor.

      https://pubmed.ncbi.nlm.nih.gov/28179296/

      -Stephen

      Delete
  11. So Peter I sent some emails out this morning in regard to this macrophage hypothesis. Dr. Naviaux nicely responded.

    "Macrophage polarization happens because the mitochondria inside the macrophages are polarized as either M1 (inflammatory) or M2 (anti-inflammatory). This is part of the CDR hypothesis at the foundation of treatment. "

    https://naviauxlab.ucsd.edu/science-item/healing-and-recovery/

    Dr. James Adams also responded and suggested looking into secretory IgA.

    -Stephen

    ReplyDelete
    Replies
    1. Anti-inflammatory drugs: Chronic inflammation can have negative effects on gut health, and medications that reduce inflammation, such as nonsteroidal anti-inflammatory drugs (NSAIDs), may help promote sIgA production.

      Delete
    2. so what drugs dr Naviaux recommend ?

      Delete
    3. He didn't give any suggestions but I bet the answer would be suramin.

      Delete
    4. suramin not exist at my country, any else?

      Delete
  12. Some examples of drugs that target macrophage polarization include:

    Thiazolidinediones: These drugs are used to treat type 2 diabetes and can also induce M2 polarization of macrophages by activating peroxisome proliferator-activated receptor-gamma (PPAR-γ).

    Interferon-gamma (IFN-γ) inhibitors: IFN-γ is a pro-inflammatory cytokine that induces M1 polarization of macrophages. Drugs that inhibit IFN-γ, such as monoclonal antibodies, can shift macrophage polarization towards the M2 phenotype.

    Statins: Statins are cholesterol-lowering drugs that have been shown to induce M2 polarization of macrophages by inhibiting the mevalonate pathway and activating PPAR-γ.

    Glucocorticoids: Glucocorticoids are anti-inflammatory drugs that can induce M2 polarization of macrophages by inhibiting the production of pro-inflammatory cytokines.

    Toll-like receptor (TLR) inhibitors: TLRs are important signaling receptors on macrophages that recognize microbial products and activate the immune response. Inhibitors of TLR signaling, such as small molecules and monoclonal antibodies, can modulate macrophage polarization towards the M2 phenotype.

    ChatGpt answer

    ReplyDelete
  13. I just try to find the lead author of this paper:
    https://www.atgu.mgh.harvard.edu/people/yu-chen/

    Would you like to contact her for the question you have Peter?

    ReplyDelete
    Replies
    1. Thanks, I think I have too much info as it is!

      I am happy with my current therapy, I think it is probably as good as it can get. All the current issues are being addressed. Hopefully no new issues will arise, but surprises do happen.

      Delete
  14. It is impossible to remove environment from this or any other genetic study: are these genes ‘bad’ or are they just ‘bad to have’ in a polluted environment? are people with these genes just canaries in the mine, signalling that the environment we live in js not fit to live anymore for us, or are they truly causing trouble by themselves?

    ReplyDelete
    Replies
    1. These are genes we all have.

      They are not mutated but they are over/under expressed.

      Delete
  15. Stephen, in our country we regularly test secretory iga in kids handled by a specific doctor and its off all the time.

    ReplyDelete
    Replies
    1. Interesting, blood or fecal secretory iga?

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    2. secretory iga is made by mucous surfaces so it is tested via saliva and stool. both are often low in asd kids. supposedly its one more proof of mitochondria being behind this problem.

      Delete
  16. Hi Peter,

    I can’t find the exact study, but here’s a link to an overview of a study at Yale that suggests that human Microglia cells are themselves actually a type of immune cell. So I wonder if there’s some type of synchronicity with the rest of the immune systems.

    https://news.yale.edu/2022/08/25/what-makes-human-brain-different-yale-study-reveals-clues

    ReplyDelete
    Replies
    1. Microglia are the brain"s immune cells plus they have other duties like synaptic pruning.

      There is cross talk with the immune system outside the brain.

      Delete
  17. Nitazoxanide-

    The M1/M2 ratio of monocytes/macrophages decreased the M1 and increased the M2 subpopulation by NTZ.

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8699442/

    -Stephen

    ReplyDelete
  18. Thanks as usual Peter
    Have you looked at butyrate as a HDAC inh compered to Romidepsin

    https://www.nature.com/articles/s41380-022-01566-y#Sec10

    "Specifically, SB had a prominent effect on reducing inflammatory cytokines in the BTBR brain; however, the treatment with Romidepsin showed a similar but more limited trend (Fig. 3c)"

    ReplyDelete
    Replies
    1. Yes, this came up as a topic a few years ago.

      Some people found Sodium Butyrate to be beneficial. It was dose dependent in at least one case - a low dose had a benefit, but a larger dose had no benefit.

      Some people with poor gut health will lack butyric acid and may be producing more propionic acid. There is a reversible mouse model of autism produced by injecting the mouse with propionic acid. Giving NAC switches the mouse back to "normal"/baseline.

      You can increase fiber in diet to increase butyric acid produced by fermentation in the gut. You can also buy a butyrate supplement or buy some butter and leave it out of the fridge.

      Delete
    2. butter should have something between 3-4% butyric acid if the come from grass-fed. I prefer to use Tributyrin over butyric acid as entrocytes will just eat up the butyrate leaving nothing to go to blood for that also I use mega doses actually just to make sure some will hit the blood and hopefully the brain. Regarding the gut my professor used to say it is the holey grail of the gut everything will cock down to gut permeability and nothing as good as it to tighten the junctions

      Delete
  19. i think microglia M1 is problem of all autism kid, and the first drug we should try is anti inflamma drug. Peter, Please synthesize the drugs you find out

    ReplyDelete
  20. One cheap and safe suplement that can help regulate the expression of genes is simply vitamin D. In Rett mouse models it was found so:

    > Supplementing with Vitamin D rescued the altered activity of genes associated with Rett syndrome and improved behaviors in a mouse model, a study showed.

    > The findings indicate that supplementation could provide a simple, cost-effective therapeutic option to help Rett patients, the scientists said.

    https://rettsyndromenews.com/news/vitamin-d-supplementation-improves-behaviors-rett-mouse-model/#:~:text=Supplementing%20with%20Vitamin%20D%20rescued,Rett%20patients%2C%20the%20scientists%20said.

    https://reader.elsevier.com/reader/sd/pii/S0969996122000274?token=FD0406D1F4D9A6E2340D0D42480967A9DE4CDF3AD98EC7409943DF4CD989C932C02C35535BFE1782082CA2FDEA14521F&originRegion=eu-west-1&originCreation=20230317054102

    ReplyDelete
    Replies
    1. Many people lack vitamin D in winter. In countries where people cover up, like Egypt, children with autism were found to lack vitamin D year-round.

      It is very easy and cheap to add vitamin D from a dropper every day. I do it myself.

      Delete
    2. There is some debate about doctors prescriving vitamin D2 to simply reach what's considered as normal level of vitamin "D" instead of what's considered to be optimal for a human being.

      Normal in the modern world would be below 30. Optimal in the jungle or the savane would would be below 100 depending on race and genetics.

      A level below 30 might favour a state of cronic low grade inflamation with slightly elevated C-reactive protein levels. Once you start suplementing with vitamin "D" you could see C-reactive protein levels diminish.

      A similar case goes with calprotectin which can effectively be treated by removing lectins, gluten, lactose... and suplementing L-threonine.

      I think it really makes sense to test vitamin D, calprotectin and PCR and then suplement accordingly.

      Delete
  21. Peter, use statin every day will make problem with liver, like my kid . can i change drug, what else?

    ReplyDelete
    Replies
    1. All drugs and supplements may cause side effects in some people.

      There is a long list of anti-inflammatory therapies that people use. It is specific to the person, some people choose something like low dose naltrexone, some people curcumin. Some people are injecting humira, it is a long list.

      You have to find what is safe and effective in your specific case.

      Delete
  22. guy, metformin is cheaper maybe can inhibit mtor too. how much the dose of metformin per kg is safe and work, did you used metformin or rapamycin to your kid ?

    ReplyDelete
    Replies
    1. I have not used either.

      Metformin has been shown to increase IQ in kids with Fragile X. It also has been shown to lower cancer risk.

      I know that people do trial metformin. It is cheap and widely available.

      Look up "Fragile X metformin" and see what dose is used.

      Delete
  23. yesterday my kid used betamethasone, steroid drug, i see he talk something alot. i dont know why, but i think its mean bumetanide maybe work with my kid. he have a problem with E/I

    ReplyDelete
  24. Hi Peter/ all

    can someone guide me what can be given to my 4 yr ASD child. our main concerns are his dark under eye and he is extremely picky fussy eater. he has eczema from birth. which seems in control now but he has dry pale skin,. we give daily citrizine. he is weak and gets frequent colds which lasts 3 to 4 weeks. I think his allergy and picky eating is connected. he is verbal and plays well with other kids at nursery. generally happy no tantrums.

    ReplyDelete
    Replies
    1. There is no pill for picky eaters that I am aware of. Behavioral strategies from a young age can work well to broaden diet, if you wait till teenage years it will be too late. Involve him in buying and preparing food and reward trials of new foods.

      Many people with autism benefit from mast cell stabilizers, like Ketotifen, cromolyn sodium, rupatadine etc.

      Poor diet will create further problems and is best addressed sooner rather than later.

      Delete
  25. https://jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-022-02456-4

    Fun Bumetanide facts

    -Stephen

    ReplyDelete
    Replies
    1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325955/

      Other fun Nkcc1 inhibitors

      https://www.cell.com/trends/pharmacological-sciences/fulltext/S0165-6147(21)00176-0

      Delete
    2. guy, can not use piperin every day, especially kid. OTC is week, just buy drugs

      Delete
  26. Peter,

    Thoughts on tight junctions.

    https://www.molbiolcell.org/doi/full/10.1091/mbc.E22-03-0073

    -Stephen

    ReplyDelete
  27. https://www.mdpi.com/2218-1989/12/2/104

    ReplyDelete
  28. Interestingly, CBD has been shown to prevent tight junctions alterations in inflammatory conditions, allowing a better maintenance of intestinal epithelial barrier.Apr 8, 2021

    https://www.frontiersin.org/articles/10.3389/fphar.2021.641210/full#:~:text=Interestingly%2C%20CBD%20has%20been%20shown,maintenance%20of%20intestinal%20epithelial%20barrier.

    -Stephen

    ReplyDelete
  29. Statin pretreatment could overcome the effect of doxorubicin in reducing endothelial tight junction formation and prevent increased permeability.

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655747/

    ReplyDelete
  30. Chinese medicine drug to increase BBB tight junctions.

    https://pubmed.ncbi.nlm.nih.gov/30995438/

    -Stephen

    ReplyDelete
  31. Insightful article. Heat shock proteins and BBB.

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1065276/

    -Stephen

    ReplyDelete
    Replies
    1. Fever effect.

      https://pubmed.ncbi.nlm.nih.gov/18055656/

      Delete
  32. Hi Peter and other readers,
    Curious if there are parents/caregivers here to those with dual diagnosis of asd and bipolar? I’ve started a Facebook group in hopes to share treatment research, add-on therapies etc. Is anyone interested?
    ~Tanya

    ReplyDelete
    Replies
    1. Hi Tanya, I am sure there will be older children/adults in North America with that dual diagnosis. I think in other countries people would not get a second diagnosis.

      I think you will find people via the NCSA.

      You could also ask for a mention from Treating Autism (UK) in their Twitter feed.

      https://twitter.com/TweetingAutism

      Also, TACA might be a good source in the US.

      Delete
    2. Thank you Peter for the ideas. NCSA has made a post and I’ve reached out to the UK group via Facebook msg.

      Delete
  33. Hi Peter,
    Thank you for sharing your insights on autism treatment. You are doing a great service for those affected by Autism.

    I’m 30 and was diagnosed with Autism as an adult a few years ago. I have recently been taking the following supplements: B12, D, Omega-3, and Sulforaphane, supplements. (Previously, just Dexedrine for ADD)

    I’ve noticed the following subtle social and communication improvements since taking these supplements:
    1) I’m able to recognize emotions clearer in faces (i.e. reading people)
    2.) It feels more natural to look people in the eye
    3.) Decrease in social anxiety
    4.) Decrease in general anxiety
    5.) Improved emotional regulation

    I wanted to try Bumetanide for the cognitive awareness benefit you’ve seen in Monthy. I shared a Bumetanide studies from your blog with my doctor and convinced him to give it a try. But since he doesn’t have experience prescribing it for Autism, he wanted to start slow with 0.5MG Bumetanide 2x per day.

    What are your thoughts starting at 0.5mg 2x day? I’m curious if I would notice any changes at that low dosage given that I'm an adult 178cm 80kg.

    Since this is new for both me and my doctor, your guidance on what changes to expect and when I might see those changes would be extremely helpful in determining the right dosage.

    ReplyDelete
    Replies
    1. I would start with 1mg once a day.

      See how dramatic or not the diuresis is. People do react very differently. I think for most people the diuresis is quite disruptive and I doubt you would want it twice a day.

      In someone of your size and with severe autism, I think 2mg once a day is likely best. In your case I doubt this is needed.

      Delete
    2. I'll give 1mg / day a shot! Since the stimulant I'm on (dexamphetamine) for ADD causes a bit of diuresis as well, I'll monitor whether or not it changes my urination habits. If Bumetanide is something that works for me, would I notice the difference in a day or 2, or a few weeks?

      Delete
    3. The chloride lowering effect of Bumetanide takes about 2 weeks in most people. Before that you just get the diuresis.

      Delete
    4. I had some tests done recently and my chloride was 104 mmol/L (Standard Range 98 - 110 mmol/L) Since my chloride was within a normal range, and if the chloride lowering effect of Bumetanide is what improves symptoms, would that disqualify me from benefiting from Bumetanide?

      Delete
    5. Bumetanide lowers chloride inside neurons, you have measured chloride in your blood. So the result does not tell you anything relevant to bumetanide.

      Delete
  34. Peter, I need some help. How can I improve my kid repetitive behaviors? In the last few months it has gotten a lot worst. Do you have any posts or books that I might read?

    ReplyDelete
    Replies
    1. For some people NAC is very effective, I would try that first.

      Some people find an SSRI is effective.

      Delete
    2. Peter, he is already taking fluoxetine, but it is not working. Do you know another class of SSRI that I can try? About NAC, as my son weights 21kgs, I'm thinking in a total dosage of 1200mg, split twice a day. Would this be enough?

      Delete
    3. The effect of NAC lasts about 4 hours. It best taken 3 or r times a day.

      Delete
  35. Peter,

    Can you please do a post on MiRNA, some how the mtor pathway stops it.

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096907/

    Stephen

    ReplyDelete
  36. Since trialing bumetanide I noticed some very similar trends to my rapamycin trial. I thought you would like this study Peter.

    https://www.biorxiv.org/content/10.1101/2023.03.02.530499v1.abstract

    Rapamycin works on the NKCC1 pathway too.

    ReplyDelete
    Replies
    1. Stephen, that is interesting. We also know that inflammation is a driver of an NKCC1/KCC2 imbalance, so this makes sense.

      I hope your son tolerates bumetanide much better than rapamycin, which should be the case.

      Delete
  37. Peter, can you explain / compare effectiveness of agmatine vs L-arginine supplements as to NO and eNOS? I tried the L arginine myself before giving to my kids and felt its effect (although maybe not the one we are aiming for in them), whereas the Agmatine only comes in powder and is hard to get in them. Thanks, Mira

    ReplyDelete
    Replies
    1. Mira, your body needs arginine to make NO. The issue is where is the NO made and so what function it will have in the body.

      Agmatine is a by-product of arginine, it does not make NO directly, but it does affect the enzymes (iNOS, eNOS, nNOS) that convert arginine to NO.

      Body builder take agmatine so as to make more eNOS which then produces NO in blood vessel, this widens them allowing more blood flow. More blood flow = greater exercise endurance.

      Agmatine is a powder that dissolves very quickly into water. It can be added into pretty much any kind of food or drink.

      Delete
  38. Hi Peter,
    I've been following your blog for many years, it's a real blessing and the perfect place to come and read for us, parents of ASD kids. My boy, 9, has non-regressive autism, is largely non verbal (one word sentence) and has pronounced OCD symptoms (similar to excoriating disorder, but aimed at the environment), hyperactivity and severe gut problems, recurrent vomitting, gastroparesis, etc. The only thing that visibly stopped the hyperactivity and inappropriate laughing and helped him sit for longer periods of time and read his books or watch whole movies was 0.5/kg mg Naltrexone daily, as advised by this paper https://pubmed.ncbi.nlm.nih.gov/16735648/. Lower doses saw the OCD creep back. As for his WGS test results, I've found relevant the fact that he has four pathogenic mutations in the EIF4EBP1, also a de novo mutation in the PIK3R1 gene and multiple other mutations in the STAT3, HTR3a, MAPT and also HLA-DRB1, HLA-DQA1, HLA-A, HLA-B, HLA-C, NRG1, NRG2, SCN4a, CACNA1S genes, amongst many others. We recently tried a course of Azythromycin for immuno-modulation, which saw his OCD reduced further, also his academic interest and focus increased visibly. He responds very well to Ibuprofen, AlkaSeltzer gold, Propranolol, Sytrinol and Cromolyn, but a quite long trial of Bumetanide two years ago did nothing for him. After all trials of various protocols and individual drugs, his gut is still bad, very often food seems to have major difficulty to pass though his digestive tract, no matter how finely tuned his diet is or how many prokinetics he takes. Given your extensive knowledge, I've always wondered what your take on the underlying problem/genetic pathway might be in his case (microglial activation, MTOR activation, perhaps?) and what drugs/cocktail of drugs might work best for his specific genetics and symptoms. He is a smart boy, has self-taught reading, loves music and masters his iPAD like a pro and, unlike what we know about autism, loves being around people. I cannot give up on him. We live in the UK, not the best place to even talk about treatments for autism. Please, if it's not too much to ask, tell me what other medications you thing it might boost his cognition further and help him start talking and develop more skills. Sorry for the long post. And thank you for any advice and ideas you might have to offer.

    ReplyDelete
    Replies
    1. There is a lot of relevant information in the genetic test results. It is quite complex so I will make my answer into the next blog post, so you can have links to further information. It will appear this week.

      Delete
    2. Thank you, Peter. I'm looking forward to read it.

      Delete

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