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Showing posts with label Neuroinflammation. Show all posts
Showing posts with label Neuroinflammation. Show all posts

Wednesday 18 March 2015

The Role of Microglia in the Puzzle of Neuro-inflammation in Autism





Regular readers of this and similar blogs will have noticed that the human body functions in quite irrational ways.  We know why this is; we are the product of a very slow evolutionary process, rather than being a clean-sheet design like your smart phone or iPad.

As a result, nothing is ever quite as simple as it seems and at times the cleverer you are, the less likely you are to find a medical therapy effective in humans.

Such is the case with autism, inflammation and microglia.

It might seem that you can track back inflammation in autism to its “root cause”, which could appear to be those immune cells in the brain, called microglia.  We know they are “activated” in autism and we know that autism is typified by an “over-activated” immune response.

Working with the assumption that autism is a brain dysfunction, you would assume that the effective therapy should be inside the so-called blood brain barrier (BBB).

You would then just look for a potent drug that could “stabilize” the microglia/immune cells in the brain, to calm things down.  Having achieved this, you would sit back and marvel at the behavioral change and improvement in cognitive function.

This was exactly the thought process a few years ago when the US  National Institute of Mental Health (NIMH) got together with the Johns Hopkins researchers to follow up on their findings of chronic inflammation in the brains of people with autism.  Subsequent, third party, research has also confirmed that the microglial cells are “activated” in autism


Trial Description


There is a subgroup of children with autism that appear to develop typically for a period of time, and then lose skills, or regress. A recent study by Vargas and co-workers at Johns Hopkins has demonstrated that the regressive subtype of autism is associated with chronic brain neuroinflammation as exemplified by activation of microglia and astroglia and the abnormal production of inflammatory cytokines and growth factors assayed in both tissue samples (brain banks) and CS. The authors remarked that these responses were similar to those seen in some neurodegenerative disorders such as amyotrophic lateral sclerosis, and that chronic microglia activation appears to be responsible for a sustained neuroinflammatory response that facilitates the production of multiple neurotoxic mediators. Chronic neuroglial activation could be the result of an abnormal persistence of a fetal development pattern. In this scenario neuroglial activation could play a role in initiating and in maintaining the pathology. Alternatively, neuroglial activation may only be a secondary response to the initiating causal factor(s) and not a direct effector of injury. Since neuroglial activation requires the nuclear translocation of the pro-inflammatory transcription factor NF-kappa B, and since inhibitors of NF-kappa-B with good CNS penetrance are available, the role of neuroinflammation in initiating and sustaining the autistic condition can be probed.
The antibiotic minocycline is a powerful inhibitor of microglial activation, apparently through blockade of NF-kappa-B nuclear translocation. Minocycline is neuroprotective in mouse models of amyotrophic lateral sclerosis (ALS) and Huntington's disease and has been recently shown to stabilize the course of Huntington's disease in humans over a 2-year period.
To evaluate the possibility of benefit in autistic children, we propose to conduct an open-label trial of the anti-inflammatory antibiotic minocycline, an agent that reduces inflammation by blocking the nuclear translocation of the proinflammatory transcription factor NF-kappa-B. Minocycline is Food and Drug Administration (FDA)-approved for treatment of a variety of infections and has been widely used for the treatment of adolescent acne. Minocycline is currently in phase III trials for the treatment of Huntington's disease and amyotrophic lateral sclerosis.
This proposal is for an initial 6-month, single-arm, off label, open-label study (with a 3 month extension phase offered to responders) that will evaluate dose safety and efficacy of minocycline in 10 children, ages 3 to 12 years, with a primary diagnosis of autism and a history of developmental regression. The subjects will be evaluated by a diagnostic/behavioral assessment, and the extent of neuroinflammation judged by CSF cytokine/chemokine profiles before and after the 6-month treatment. Subjects will also be given 0.6 mg/kg vitamin B6 twice a day as a prophylactic for possible minocycline induced nausea and vomiting. If the results of this feasibility study are encouraging, we expect to conduct a double-blind, placebo-controlled trial of minocycline therapy.


Nothing happens fast in the world of autism and so this six month study of 10 people (who completed the actual trial) was conceived in 2006, was actually concluded in 2013.  Here is the resulting paper:-
  


Conclusions
Changes in the pre- and post-treatment profiles of BDNF in CSF and blood, HGF in CSF and CXCL8 (IL-8) in serum, suggest that minocycline may have effects in the CNS by modulating the production of neurotrophic growth factors. However, in this small group of children, no clinical improvements were observed during or after the six months of minocycline administration.

Unfortunately, this study showed that a treatment, known to effectively stabilize microglial cells, had no positive effect on autism and actually seemed in some cases to make it worse.

We can conclude from this that stabilizing the microglia will not be the “holy grail” for treating autism.  Rather, the activated microglia is just one part of a complex, and only partially understood process.


Microglia as the Immunostat 

In a recent post we saw how Rodney Johnson referred to the microglia as the “immunostat” of the body.  Like the thermostat on the wall in your home central heating system.



This is indeed an interesting analogy and might explain some of what is going on.

We saw in Johnson’s paper all the ways that the immune system outside the blood brain barrier (BBB) was able to communicate with the microglia.  We should assume that this communication works both ways; something that is usually overlooked.

In a perfectly functioning body, as in a perfectly functioning house, the immunostat/thermostat gives a good indication of the actual state/temperature, as well as the one you intended.  So if you set your room thermostat to 72 Fahrenheit / 22 Celsius  you expect the actual temperature to be 72 Fahrenheit / 22 Celsius.

However, in the real world things do not work like this.

We live in a house with very large south facing windows, a big fireplace, underfloor heating in some places and European-style hot water radiators (in the US they do have them).  So we have at least four sources of heat.  In spite of having clever German electronics to control our heating system, the thermostat in the centre of the house, by itself, is not adequate.

Something similar is happening in body and brain of people with autism, just replace temperature with inflammation.

Just as my house has multiple systems resulting in heating, the human body has numerous processes leading to “inflammation”.  Some of these inflammatory processes are interconnected and some are not.  The net result at any one time can be measured by looking at various cytokine levels, gene expression, microglial activation and numerous other things; there is no single measurable thing called “inflammation”.

There will never be a single wonder anti-inflammatory treatment.

The activated state of the microglia rather than being the ultimate target for intervention may just be a reflection of inflammation elsewhere in the body, or alternatively it may be just the result of oxidative stress in the brain.

Just like after a few years you may need to replace your wall thermostat, because it is giving false data, the clever immunostat, that may be the microglia, could have been disrupted by all that oxidative stress in the brain.  It might even be sending its proinflammatory signal in reverse, back across the BBB, to the rest of the body. Not such a crazy idea?


The future of anti-inflammatory interventions

The NIMH and Johns Hopkins would naturally be disappointed by the results of their study; but it was a study well worth doing.  Hopefully they will pursue other avenues of thought.

We already know that there are numerous ways to achieve a degree of immuno-modulatory change and that in some types of autism there can be a profound behavioral impact.

These range from simple Ibuprofen, to steroids like Prednisone; not to mention those Kv1.3 blockers and ShK-peptides.  These will likely all affect the microglia, but it is not their main mode of action.


Insights

As is often the case, there are useful insights that you can learn from a “failed” trial.

I would imagine that an autistic person with ulcerative colitis would also have activated microglia. Treating that person with minocycline should have some stabilizing influence on the microglia, but without resolving the ulcerative colitis, the pro-inflammatory signals continue to be sent around the body.

Turning down the thermostat in my house, when I have a big log fire blazing, has no effect on the temperature. 

The microglia in the brain of people with autism probably should not be activated; we really need to know why they are activated.

If you can work on the numerous processes/pathways leading to “inflammation” you would most likely also achieve some deactivation of the microglia.

Therefore we should look at things like PPAR gamma which are directly relevant to the pathology of autism, and agonists of PPAR gamma also happen to be “anti-inflammatory” and indeed, in the test tube, some can stabilize microglia.

One, far away, day they will bring those ShK-peptides to the market. 

In the meantime, my current targets are Tangeretin and Nobiletin, flavonoids found in tangerines.


For the scientists among you:-

In addition to being a PPAR gamma agonist, Tangeretin is also a known P2Y2 receptor antagonist.  Both properties are potentially useful.

PPAR gamma has been covered in this blog already.  P2 receptors are a class of Purinergic receptor.  Within the field of purinergic signalling, these receptors have been implicated in learning and memory, locomotor and feeding behavior, and sleep. 

Suramin is used in research as a broad-spectrum antagonist of P2 receptors.

It is Suramin that Robert Naviaux, at UC San Diego, has been researching as a potent autism therapy.  He has shown it effective in mouse models, but the problem is that it is not safe for long term use in humans.  Regular readers should note that, yet again, an anti-parasite drug has been found to have an effect in autism.  Parasites do not cause autism, but understanding them better would be a potential advantage.

Why Suramin, a Century-Old, Anti-Parasitic Drug May Hold the Key to Understanding Autism


Dr. Robert Naviaux's recent finding suggests reversible metabolic syndrome could be at core of autism



The full paper is below:-




In particular, P2Y11 is a regulator of immune response.  There are big gaps in the science and I have no idea if tangeretin affects P2Y11.











Thursday 26 February 2015

Inflammation Leading to Cognitive Dysfunction


Today’s post highlights a paper with some very concise insights into how microglial cells become “activated” resulting in the “exaggerated inflammatory response” that many people with autism experience on a daily basis.  

This is very relevant to treatment, which is not usually the objective of much autism research.

I recall reading a comment from John’s Hopkins about neuroinflammation/activated microglia in autism; they commented that no known therapy currently exists and that, of course, common NSAIDs like ibuprofen will not be effective.  But NSAIDs are effective.

As we see in today’s paper, there a least 4 indirect cytokine-dependent pathways leading to the microglia, plus one direct one.
NSAIDs most definitely can reduce cytokine signaling and thus, indirectly, reduce microglial activation.

The ideal therapy would act directly at the microglia, and as Johns Hopkins pointed out, that does not yet exist with today's drugs.  If you read the research on various natural flavonoids you will see that “in vitro” there are known substances with anti-neuroinflammatory effects on microglial activation.  The recurring “problem” with such substances is low bioavailability and inability to cross the blood brain barrier.


Back to Today’s Paper

It was a conference paper at the 114th Abbott Nutrition Research Conference - Cognition and Nutrition



The paper is not about autism, it is about more general cognitive dysfunction.  It is from mainstream science (I checked).

It explains how inflammation anywhere in the body can be translated across the BBB (Blood Brain Barrier) to activate the microglia.  This of course allows you to think of ways to counter these mechanisms.

It also raises the issue of whether or not anti-inflammatory agents really need to cross the BBB.  While you might think that ability to cross the BBB is a perquisite to mitigate the activated microglia, this may not be the case.  Much can be achieved outside the BBB, and we should not rule out substances that cannot cross the BBB.

Very many known anti-inflammatory substances do not cross the BBB.   

  



extracts from the above paper ...








Example – Influenza and Cognition

Neurological and cognitive effects associated with influenza infection have been reported throughout history.

The simplest explanation for these neurocognitive effects is that influenza virus makes its way to the brain, where it is detected by neurons.

However, most influenza strains, including those responsible for pandemics, are considered non-neurotropic, neurological symptoms associated with influenza infection are not a result of direct viral invasion into the CNS.

Moreover, neurons do not have receptors to detect viruses (or other pathogens) directly.

Cells of the immune system do, however, as the immune system’s primary responsibility is to recognize infectious pathogens and contend with them. For example, sentinel immune cells such as monocytes and macrophages are equipped with toll-like receptors (TLR) that recognize unique molecules associated with groups of pathogens (i.e., pathogen-associated molecular patterns). Stimulation of TLRs that recognize viruses (TLR3 and TLR7) and bacteria (TLR4) on immune sentinel cells can have profound neurological and cognitive effects, suggesting the immune system conveys a message to the brain after detecting an infectious agent. This message is cytokine based.

Macrophages and monocytes produce inflammatory cytokines (e.g., interleukin [IL]-1β, IL-6, and tumor necrosis factor-α [TNF-α]) that facilitate communication between the periphery and brain.


Cytokine-dependent Pathways to the Brain

Several cytokine-dependent pathways that enable the peripheral immune system to transcend the blood-brain barrier have been dissected.

Inflammatory cytokines present in blood can be actively transported into the brain.
But there are also four indirect pathways:-

1.     Cytokines produced in the periphery need not enter the brain to elicit neurocognitive changes. This is because inflammatory stimuli in the periphery can induce microglial cells to produce a similar repertoire of inflammatory cytokines. Thus, brain microglia recapitulates the message from the peripheral immune system.

2.     in a second pathway, inflammatory cytokines in the periphery can bind receptors on blood-brain barrier endothelial cells and induce perivascular microglia or macrophages to express cytokines that are released into the brain

3.     In a third pathway, cytokines in the periphery convey a message to the brain via the vagus nerve. After immune challenge, dendritic cells and macrophages that are closely associated with the abdominal vagus have been shown to express IL-1β protein; IL-1 binding sites have been identified in several regions of the vagus as well. When activated by cytokines, the vagus can activate specific neural pathways that are involved in neurocognitive behavior. However, activation of the vagus also stimulates microglia in the brain to produce cytokines via the central adrenergic system 

4.     A fourth pathway provides a slower immune-to-brain signaling mechanism based on volume transmission.  In this method of immune-to-brain communication, production of IL-1β by the brain first occurs in the choroid plexus and circumventricular organs—brain areas devoid of an intact blood-brain barrier. The cytokines then slowly diffuse throughout the brain by volume transmission, along the way activating microglia, neurons, and neural pathways that induce sickness behavior and inhibit cognition.


Can Flavonoids Reduce Neuroinflammation and Inhibit Cognitive Aging?

Flavonoids are naturally occurring polyphenolic compounds present in plants. The major sources of flavonoids in the human diet include fruits, vegetables, tea, wine, and cocoa.  Significant evidence has emerged to indicate that consuming a diet rich in flavonoids may inhibit or reverse cognitive aging

Flavonoids may improve cognition in the aged through a number of physiological mechanisms, including scavenging of reactive oxygen and nitrogen species and interactions with intracellular signaling pathways. Through these physiological mechanisms, flavonoids also impart an anti-inflammatory effect that may improve cognition. This seems likely for the flavone luteolin, which is most prominent in parsley, celery, and green peppers.
Whereas luteolin inhibits several transcription factors that mediate inflammatory genes (e.g., nuclear factor kappa B [NF-κB]and activator protein 1 [AP-1]), it is a potent activator of nuclear factor erythroid 2-related factor 2 (Nrf2), which induces the expression of genes encoding antioxidant enzymes. A recent study of old healthy mice found improved learning and memory and reduced expression of inflammatory genes in the hippocampus when luteolin was included in the diet. Thus, dietary luteolin may improve cognitive function in the aged by reducing brain microglial cell activity.
Hence, the flavonoid luteolin is a naturally occurring immune modulator that may be effective in reducing inflammatory microglia in the senescent brain.

Conclusion
In light of the recent evidence suggesting microglial cells become dysregulated due to aging and cause neuroinflammation, which can disrupt neural structure and function, it is an interesting prospect to think dietary flavonoids and other bioactives can be used to constrain microglia. But how can flavonoids impart this anti-inflammatory effect? Although in vitro studies clearly indicate that several flavonoids can act directly on microglial cells to restrict the inflammatory response, results from in vivo studies thus far do not prove that dietary flavonoids access the brain to interact with microglia in a meaningful way. This is a complicated question to dissect because flavonoids reduce inflammation in the periphery and microglia seem to act like an “immunostat,” detecting and responding to signals emerging from immune-to-brain signaling pathways. Thus, whether dietary flavonoids enter the brain and impart an anti-inflammatory effect on microglia is an interesting question but one that is more theoretical than practical because what is most important is how the immunostat is adjusted, whether that is via a direct or indirect route. However, because flavonoids are detectable in the brain they most likely affect microglia both directly and by dampening immune-to-brain signaling.



Interesting Natural Substances

In no particular order, these are several very interesting flavonoids/carotenoids.  In the lab, they all do some remarkable things.

In humans, they also do some interesting things; how helpful they might be in autism remains to be seen.

Being “natural” does not mean they are good for you and have no side-effects.

Some of the following are very widely used and so you can establish if there are issues with long term use.  It also makes them accessible.


Quercetin (found in many fruits, numerous interesting effects)


and two Quercetin-related flavonoids:-

Kaempferol (widely used in traditional medicine)

Myricetin (has good and bad effects)



Lycopene  (from tomatoes, potent anti-cancer, does not cross the BBB)

  
Luteolin(in many vegetables, like broccoli) 

Apigenin (from chamomile, stimulates neurogenesis, PAM of GABAA, block NDMA receptors, antagonist of opioid receptors …)


Tangeretin (from tangerines, does cross the BBB, has potent effects in vitro)


Nobiletin (from tangerines)

Hesperidin (from tangerines)


Naringin (from Grapefruit, contraindicated with many prescription drugs)


Epicatechin/Catechin  (the chocolate/cocoa flavonoids, do cross the BBB, well researched)








Tuesday 13 January 2015

Cytokines from the Eruption of Permanent Teeth causing Flare-ups in Autism




A recent post looked again at inflammation in autism and some possible therapies to try.  Over Christmas and New Year, Monty, aged 11 with ASD, had occasional outbursts, more typical of his summertime raging, which was later solved using allergy /mast cell therapies.

At least it did let me establish whether Verapamil was a universal “cure” for SIB.  It is not.  It works great for allergy-driven aggressive behaviors, but had no effect on these ones.

Christmas is often a stressful period for many people with, or without, autism; but Monty likes presents and he loves food.

Having pulled out a wobbly tooth on Boxing Day and noticed an apparent behavior change, I thought that perhaps the loss of milk teeth and development of permanent teeth might cause an effect similar to that of his mild pollen allergy.  Monty, in common with many people with autism, has a high pain threshold.  While teething causes well known problems in babies, most children have minimal problems when their milk teeth are replaced by their permanent ones.

I just wondered if perhaps the underlying biological mechanism might provide an inflammatory insult to the highly inflammation-sensitive autistic brain.

Just as histamine provokes a release of inflammatory cytokines like IL-6, perhaps losing your milk teeth does something similar.


Ibuprofen experiment

I decided that I would buy some Ibuprofen, the least problematic NSAID.   A day or two later, Monty declared that another tooth was wobbly and needed to be pulled out.  This tooth was, and remains, well and truly attached.

So I decided that in advance of another, potentially stressful, Christmas event, I would give 10 ml of Ibuprofen.  I did not give it in response to any comment about pain.

It did indeed seem to work.


Skiing

A few days later we were in the Alps for skiing.

Monty can ski, but we always give him a 1:1 instructor.  On the first day, without Ibuprofen, he got agitated during the queuing at the bottom of the beginners’ ski lift.  The instructor thought it was the loud booming music.  It was clear that by the end of the lesson, it was no fun at all.

The following days, I gave 10 ml of Ibuprofen, 20 minutes before the lesson started.  He had a great time, going up by cable car to the top of the mountain and skiing along the blue/red slopes and coming down in a neighboring resort a couple of hours later.  Even a change of instructor on one day, passed without issue.

It might not be scientific proof of the effectiveness of Ibuprofen, but it was enough for me.


The Science

Since this is a scientific blog, arriving home I did some checking on the biology of what happens when you lose your milk teeth.

There is more written about “teething” when you first get your milk teeth, but there is information about “root resorption” of milk teeth and “eruption” of the permanent teeth.  The process is indeed modulated by inflammatory cytokines and transcription factors.

These cytokines will then circulate around the body and cross the blood brain barrier.





Abstract

PURPOSE:
The aim of this study was to investigate whether there are increased levels of the inflammatory cytokines IL-1beta, IL-8, and TNF alpha in the gingival crevicular fluid (GCF) of erupting primary teeth. This increase could explain such clinical manifestations as fever, diarrhea, increased crying, and sleeping and eating disturbances that occur at this time.

METHODS:

Sixteen healthy children aged 5 to 14 months (mean=9.8 months) were examined twice a week over 5 months. Gingival crevicular fluid samples were taken from erupting teeth. As a control, GCF was collected from the same teeth 1 month later. Cytokine production was measured by ELISA. Signs and clinical symptoms were listed. Pearson correlation coefficients were used in the comparisons described below. A paired t test was used to analyze the same variable at different times.

RESULTS:

Fifty teeth of the 16 children were studied. GCF samples were collected from 21 of these teeth. Statistically significant differences (P<.05) were found with regard to the occurrence of fever, behavioral problems, and coughing during the teething period and the control period. During the control period, 72% of the children did not exhibit any clinical manifestations, whereas during the teething period only 22% of the children did not exhibit any clinical manifestations. The study revealed high levels of inflammatory cytokines during the teething period, with a statistically significant difference in TNF alpha levels (P<.05) between the teething period and the control period. Correlations were found between cytokine levels and some of the clinical symptoms of teething: IL-1beta and TNF alpha were correlated with fever and sleep disturbances; IL-beta and IL-8 were correlated with gastrointestinal disturbances; IL-1beta was correlated with appetite disturbances.

CONCLUSIONS:

Cytokines appear in the GCF of erupting primary teeth. The cytokine levels are correlated to some symptoms of teething.



Mechanism of Human Tooth Eruption: Review Article Including a New Theory for Future Studies on the Eruption Process



Physiologic root resorption in primary teeth: molecular and histological events


Root resorption is a physiologic event for the primary teeth. It is still unclear whether odontoclasts, the cells which resorb the dental hard tissue, are different from the osteoclasts, the cells that resorb bone. Root resorption seems to be initiated and regulated by the stellate reticulum and the dental follicle of the underlying permanent tooth via the secretion of stimulatory molecules, i.e. cytokines and transcription factors. The primary root resorption process is regulated in a manner similar to bone remodeling, involving the same receptor ligand system known as RANK/RANKL (receptor activator of nuclear factor-kappa B/ RANK Ligand). Primary teeth without a permanent successor eventually exfoliate as well, but our current understanding on the underlying mechanism is slim. The literature is also vague on how resorption of the pulp and periodontal ligament of the primary teeth occurs. Knowledge on the mechanisms involved in the physiologic root resorption process may enable us to delay or even inhibit exfoliation of primary teeth in those cases that the permanent successor teeth are not present and thus preservation of the primary teeth is desirable. (J. Oral Sci. 49, 1-12, 2007)


Nonsteroidal anti-inflammatory drugs (NSAIDS), such as ibuprofen, work by inhibiting the enzyme COX which converts arachidonic acid to prostaglandin H2 (PGH2). PGH2, in turn, is converted by other enzymes to several other prostaglandins ,which are mediators of pain, inflammation, and fever.


Prostaglandin E synthase


Prostaglandin E2 (PGE2) is generated from the action of prostaglandin E synthases on prostaglandin H2 (PGH2).

PGE2 has various known effects, but one known effect is to increase the pro-inflammatory cytokine IL-6.  The same one that is increased by histamine released from mast cells during allergic reactions.

Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: possible role of cyclooxygenase-2.


Abstract

Injection of mineral oils such as pristane into the peritoneal cavities of BALB/c mice results in a chronic peritonitis associated with high tissue levels of interleukin 6 (IL-6). Here we show that increased prostaglandin E2 (PGE2) synthesis causes induction of IL-6 and that expression of an inducible cyclooxygenase, Cox-2, may mediate this process. Levels of both PGE2 and IL-6 are elevated in inflammatory exudates from pristane-treated mice compared with lavage samples from untreated mice. The Cox-2 gene is induced in the peritoneal macrophage fraction isolated from the mice. A cause and effect relationship between increased macrophage PGE2 and IL-6 production is shown in vitro. When peritoneal macrophages are activated with an inflammatory stimulus (polymerized albumin), the Cox-2 gene is induced and secretion of PGE2 and IL-6 increases, with elevated PGE2 appearing before IL-6. Cotreatment with 1 microM indomethacin inhibits PGE2 production by the cells and reduces the induction of IL-6 mRNA but has no effect on Cox-2 mRNA, consistent with the fact that the drug inhibits catalytic activity of the cyclooxygenase but does not affect expression of the gene. Addition of exogenous PGE2 to macrophages induces IL-6 protein and mRNA synthesis, indicating that the eicosanoid stimulates IL-6 production at the level of gene expression. PGE2-stimulated IL-6 production is unaffected by addition of indomethacin. Taken together with the earlier finding that indomethacin diminishes the elevation of IL-6 in pristane-treated mice, the results show that PGE2 can induce IL-6 production in vivo and implicate expression of the Cox-2 gene in the regulation of this cytokine


Indomethacin is another NSAID, like Ibuprofen.



Implications

If, as seems likely, many incidents of anxiety, aggression, explosive behavior, or "meltdowns" are made possible by elevated levels of the pro-inflammatory cytokine IL-6, then the occasional use of drugs known to inhibit IL-6 makes a lot of sense.

Ibuprofen is an NSAID and it is known that some people respond much better to certain NSAIDs and suffer side effects from others.   NSAID drugs work by affecting both COX-1 and COX-2.  It appears that desired effect of NSAIDs comes from their effect on COX-2, while the side effects come from changes made to COX-1.  So it is logical that some NSAIDs are better tolerated than others and for some people a different NSAID may be more appropriate.

Other common drugs also lower IL-6;  leukotriene receptor antagonists like Montelukast (Singulair)  being an example.  This drug is used in autism, but a known side-effect in typical people is to worsen behavior, sometimes severely.  There are plenty of reports of Singulair in autism, some good and some bad.  Since almost all drugs have multiple effects, this is not surprising.

Interestingly, one of the drugs in my Polypill, NAC, is also known to reduce IL-6; but it also reduces the “good” anti-inflammatory cytokines like IL-10.  Perhaps this is why NAC is not beneficial to some people with autism?

Occasional use of Ibuprofen at times anticipated to be stressful makes a lot of sense. 


Conclusion

While it is well known that Ibuprofen relieves pain from teething, low level pain is often completely ignored by people with ASD.  The cytokine release associated with the resorption of the milk teeth and the eruption of the permanent tooth appears to be much more problematic.

Ibuprofen, available OTC, limits the production of pain mediators, called prostaglandins, which in turn stimulate production of the inflammatory cytokine IL-6.

Ibuprofen will reduce both pain and the level of cytokines like IL-6.

In earlier extensive posts on mast cell degranulation in autism, I concluded that the resulting elevated levels of IL-6 likely produced behaviors ranging from anxiety, through aggression, all the way to self-injury.




Thursday 18 December 2014

Activated Microglia and Inflammation in Autism






There have been yet more autism studies recently, highlighting neuroinflammation and the role of cells called microglia.  The result is this rather long post; but there is film to watch, if it gets heavy going.

Glia derives from a Greek word for glue. The original thought was that the glial cells “glued” the neurons together.

It turned out that glial cells do very much more and might be better thought of as “resident immune cells”.  They have other functions including synaptic pruning, which appears to have gone awry in autism.  They also form myelin, and when this goes wrong, big problems follow.

Microglia are inside the blood brain barrier and one of their jobs is to swallow up any foreign bodies that should not be there, before they can do damage.  It appears that this process is mainly modulated via potassium channels.  The majority of research focuses on the calcium-activated K+ channels, particularly KCNN4/KCa2 and 3.1, and ATP-sensitive K+ channels (KATP).  Administration of diazoxide, a classic KATP channel activator, is shown to reduce microglial activation and is neuroprotective in a variety of models involving neuroinflammation. 

However, Kv 1.3 and Kv 1.5 are also involved in activated glia.  We have seen in earlier posts, that blocking Kv 1.3 can be effective in autism (remember those TSO worms).



For the scientists among you:-






Synaptic pruning


A very small Acer Palmatum


Synaptic pruning could itself be the subject of an entire blog.  I will just use the analogy of a different kind of pruning.

With ornamental trees, to obtain the perfect form, pruning is very important.  You have to clear away the dead wood and encourage growth in particular areas to achieve the optimal shape.  You need to know when to cut, where to cut and how much to cut.

The human brain develops with far too many synapses and they too need pruning.  The weak ones need to give way for the strong ones to prosper.  Too many synapses lead to poor brain function.  This process is going on from childhood to early adulthood.  Microglia are heavily involved in this pruning process, as you will see in the video shortly.

We know that synaptic pruning is implicated in autism and very likely in its big brother, schizophrenia.




Activation of Microglia

Microglia can be in either a resting or activated state. In the activated state, for no good reason, they can do damage.  They can also react with mast cells to produce more inflammation.

(here is a link for the mast cell followers of Theoharides; they know who they are)




The subject is very complex.  For those with an hour to spare there is an excellent presentation by Beth Stevens from Harvard.  Click on the link below to go to the SFARI website and the video.











By a bizarre coincidence, there is another B Stevens researching glial cells and autism.  This time it is Bruce Stevens, in Florida.

His paper is interesting because he is using a known anti-oxidant (alpha lipoic acid, ALA) to affect brain glial cells.

One of the odd things is that we know in autism there is both oxidative stress and neuro-inflammation; they are a self-perpetuation combination.  There are numerous effective anti-oxidants; almost too many.  There is, however, a paucity of effective, safe, anti-inflammatory drugs.  In fact the best anti-inflammatory drug is probably an anti-oxidant.  So called Reactive Oxygen Species (ROS) are among the biggest causes of neuroinflammation.  With anti-oxidants you can neutralize the ROS, and thereby you take a big bite out of the neuroinflammation.
  

Abstract
Double-stranded RNAs (dsRNA) serve as viral ligands that trigger innate immunity in astrocytes and microglial, as mediated through Toll-like receptor 3 (TLR3) and dsRNA-dependent protein kinase (PKR). Beneficial transient TLR3 and PKR anti-viral signaling can become deleterious when events devolve into inflammation and cytotoxicity. Viral products in the brain cause glial cell dysfunction, and are a putative etiologic factor in neuropsychiatric disorders, notably schizophrenia, bipolar disorder, Parkinson's, and autism spectrum. Alpha-lipoic acid (LA) has been proposed as a possible therapeutic neuroprotectant. The objective of this study was to test our hypothesis that LA can control untoward antiviral mechanisms associated with neural dysfunction. Utilizing rat brain glial cultures (91% astrocytes:9% microglia) treated with PKR- and TLR3-ligand/viral mimetic dsRNA, polyinosinic-polycytidylic acid (polyI:C), we report in vitro glial antiviral signaling and LA reduction of the effects of this signaling. LA blunted the dsRNA-stimulated expression of IFNα/β-inducible genes Mx1, PKR, and TLR3. And in polyI:C treated cells, LA promoted gene expression of rate-limiting steps that benefit healthy neural redox status in glutamateric systems. To this end, LA decreased dsRNA-induced inflammatory signaling by downregulating IL-1β, IL-6, TNFα, iNOS, and CAT2 transcripts. In the presence of polyI:C, LA prevented cultured glial cytotoxicity which was correlated with increased expression of factors known to cooperatively control glutamate/cysteine/glutathione redox cycling, namely glutamate uptake transporter GLAST/EAAT1, γ-glutamyl cysteine ligase catalytic and regulatory subunits, and IL-10. Glutamate exporting transporter subunits 4F2hc and xCT were downregulated by LA in dsRNA-stimulated glia. l-Glutamate net uptake was inhibited by dsRNA, and this was relieved by LA. Glutathione synthetase mRNA levels were unchanged by dsRNA or LA. This study demonstrates the protective effects of LA in astroglial/microglial cultures, and suggests the potential for LA efficacy in virus-induced CNS pathologies, with the caveat that antiviral benefits are concomitantly blunted. It is concluded that LA averts key aspects of TLR3- and PKR-provoked glial dysfunction, and provides rationale for exploring LA in whole animal and human clinical studies to blunt or avert neuropsychiatric disorders

The obvious question is whether other antioxidants have the same effect.  Most likely nobody knows.  I did ask both B Stevens #1 and B Stevens #2 for their thoughts on this – so far no answer.



Brain inflammation a hallmark of autism, according to large-scale analysis


Finally to the subject of this post, the recent Johns Hopkins study that shows inflammation in the autistic brain.


This is the press release from Johns Hopkins so it is quite readable.

While many different combinations of genetic traits can cause autism, brains affected by autism share a pattern of ramped-up immune responses, an analysis of data from autopsied human brains reveals. The study, a collaborative effort between Johns Hopkins and the University of Alabama at Birmingham, included data from 72 autism and control brains. It was published online today in the journal Nature Communications.

There are many different ways of getting autism, but we found that they all have the same downstream effect,” says
Dan Arking, Ph.D., an associate professor in the McKusick-Nathans Institute for Genetic Medicine at the Johns Hopkins University School of Medicine. “What we don’t know is whether this immune response is making things better in the short term and worse in the long term.”

The causes of autism, also known as autistic spectrum disorder, remain largely unknown and are a frequent research topic for geneticists and neuroscientists. But Arking had noticed that for autism, studies of whether and how much genes were being used — known as gene expression — had thus far involved too little data to draw many useful conclusions. That’s because unlike a genetic test, which can be done using nearly any cells in the body, gene expression testing has to be performed on the specific tissue of interest — in this case, brains that could only be obtained through autopsies.

To combat this problem, Arking and his colleagues analyzed gene expression in samples from two different tissue banks, comparing gene expression in people with autism to that in controls without the condition. All told, they analyzed data from 104 brain samples from 72 individuals — the largest data set so far for a study of gene expression in autism.

Previous studies had identified autism-associated abnormalities in cells that support neurons in the brain and spinal cord. In this study, Arking says, the research team was able to narrow in on a specific type of support cell known as a microglial cell, which polices the brain for pathogens and other threats. In the autism brains, the microglia appeared to be perpetually activated, with their genes for inflammation responses turned on. “This type of inflammation is not well understood, but it highlights the lack of current understanding about how innate immunity controls neural circuits,” says Andrew West, Ph.D., an associate professor of neurology at the University of Alabama at Birmingham who was involved in the study.

Arking notes that, given the known genetic contributors to autism, inflammation is unlikely to be its root cause. Rather, he says, “This is a downstream consequence of upstream gene mutation.” The next step, he says, would be to find out whether treating the inflammation could ameliorate symptoms of autism.

The full study is here:-




What I liked about the study was the comment made by Arking, a specialist in genetics, that it did not seem to matter what the genetic cause was, all the brain samples exhibited the same inflammation.  So it does not matter which of millions of possible combinations of genetic dysfunction is present, one key physiological result is shared neuroinflammation.

Take home message:  Treat the neuroinflammation in people with Autism.

The question of course is how.

Since it seems easy to treat oxidative stress, a leading cause of neuroinflammation, we should go to extreme lengths to finish that job. 

I started it with NAC and recently added Sulforaphane/broccoli.  I suspect there are more “low hanging fruit” to be gathered here. Perhaps just an additional supplemental (exogenous) antioxidants, or perhaps something clever like increasing the amount DJ-1, which is needed to support Nrf2 which turns on the anti-oxidant genes. Early 2015 will see my oxidative stress therapy optimized.


Treating Neuroinflammation in Autism

There are lots of possible ways to treat neuroinflammation, some of which we have already covered in this blog.  Sometimes it gets called immunomodulatory therapy.

There are some natural options like quercetin and turmeric.  Turmeric is also possibly chemo-protective:-

“Currently there is no research evidence to show that turmeric or curcumin can prevent or treat cancer but early trials have shown some promising results.”

Cancer Research UK


Interestingly, people who eat a lot of curry (Indians) have a very low incidence of cancer.



1.     Steroids, like Prednisone

These are already used, particularly in regressive autism.  They are potent, but have side effects.

2.     Blockers of Potassium channel Kv1.3

This is a clever approach, since it appears that this potassium channel is involved in mediating the inflammatory response. By blocking these channels the response we have seen that the immune response can be moderated and in some people, there autism moderated.

3.     Activators of Potassium channel KATP

We learned earlier in this post about diazoxide

4.     Other Microglial Ion Channels

The various other potassium, calcium and sodium channels need to be considered.

5.     Ibuprofen

This common painkiller reduces inflammation and is used to reduce inflammation associated with autism secondary to mitochondrial disease.

Do not use acetaminophen/paracetamol/Tylenol.  These will increase oxidative stress, since it depletes GSH and also affect mitochondria.


6.     Leukotriene receptor inhibitors (i.e. montelukast, zafirlukast)

These are interesting because they are used to treat asthma and so are very widely used. They are not steroids and so do not have their side effects.  They are proved to have anti-inflammatory effects.

Montelukast/Zafirlukast is used to reduce inflammation associated with autism secondary to mitochondrial disease.


7.     Pregnenolone

I wrote a post a while back on Pregnenolone, which is interesting, since you do not need a prescription.  But does it work?

Well, after I wrote the post below, the results from a clinical trial in adults with autism was finally published.



Abstract
The objective of this study was to assess the tolerability and efficacy of pregnenolone in reducing irritability in adults with autism spectrum disorder (ASD). This was a pilot, open-label, 12-week trial that included twelve subjects with a mean age of 22.5 ± 5.8 years. Two participants dropped out of the study due to reasons unrelated to adverse effects. Pregnenolone yielded a statistically significant improvement in the primary measure, Aberrant Behavior Checklist (ABC)-Irritability [from 17.4 ± 7.4 at baseline to 11.2 ± 7.0 at 12 weeks (p = 0.028)]. Secondary measures were not statistically significant with the exception of ABC-lethargy (p = 0.046) and total Short Sensory Profile score (p = 0.009). No significant vital sign changes occurred during this study. Pregnenolone was not associated with any severe side effects. Single episodes of tiredness, diarrhea and depressive affect that could be related to pregnenolone were reported. Overall, pregnenolone was modestly effective and well-tolerated in individuals with ASD.

Trial doses were:-

Days 1-14: 100 mg
Week 1 and 2: 200 mg
Week 3 and 4: 350 mg
Week 5 and 6: 400 mg
Week 7 -12: 500 mg

So it was modestly effective, but the doses were huge.  It is a hormone and our endocrinologist did not much approve of the idea.

I will give this idea a miss.


8.     Statins

The current treatment for neuroinflammation in my Polypill is Atorvastatin.

I have already written a great deal about why statins may be effective in some people with autism; just make sure you do not have low cholesterol or mitochondrial disease.

Arthritis is another disease mediated by inflammation:-



To me it is no surprise that statins have therapeutic value in rheumatoid arthritis.


9.     NF-κB inhibitors


Because NF-κB controls many genes involved in inflammation, it is not surprising that NF-κB is found to be chronically active in many inflammatory diseases, such as inflammatory bowel disease, arthritis, sepsis, gastritis, asthma, atherosclerosis and others.

So perhaps NF-κB is for inflammation ,what Nrf2 is for oxidative stress, a force multiplier?

There are very many other inflammatory diseases like rheumatoid arthritis and so it is quite a well-trod path looking for inhibitors of NF-κB.

Before we get into that, a quick check on what we already know from research to schizophrenia (adult-onset autism).


Abstract
BACKGROUND:
Many reports suggest that schizophrenia is associated with the inflammatory response mediated by cytokines, and nuclear factor-kappa B (NF-kappaB) regulates the expression of cytokines. However, it remains unclear whether the interaction between NF-kappaB and cytokines is implicated in schizophrenia and whether the effect of neuroleptics treatment for 4 weeks is associated with the alteration of cytokines.
METHODS:
Sixty-five healthy subjects and 83 first-episode schizophrenic patients who met DSM-IV criteria and who were never treated with neuroleptics previously were included. Serum levels of cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were examined by using sandwich enzyme immunoassay (EIA). Peripheral blood mononuclear cell (PBMC) mRNA expressions of cytokines (IL-1beta, TNF-alpha) and NF-kappaB were detected by using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). NF-kappaB activation was examined by using transcription factor assay kits.
RESULTS:
Schizophrenic patients showed significantly higher serum levels and PBMC mRNA expressions of IL-1beta and TNF-alpha compared with healthy subjects. However, treatment with the neuroleptic risperidone for 4 weeks significantly decreased serum levels and PBMC mRNA expressions of IL-1beta in schizophrenic patients. NF-kappaB activation and PBMC mRNA expression in patients were significantly higher than those in healthy subjects. Furthermore, PBMC mRNA expressions of IL-1beta and TNF-alpha were positively correlated to NF-kappaB activation in both schizophrenic patients and healthy control subjects.
CONCLUSIONS:
Schizophrenic patients showed activation of the cytokine system and immune disturbance. NF-kappaB activation may play a pivotal role in schizophrenia through interaction with cytokines.

It seems fair to conclude that NF-κB inhibitors are well worth investigating.

Interestingly, one of my new “pet” compounds, alpha lipoic acid appears to have another role here:-


Evidence that α-lipoic acid inhibitsNF-κB activation independent of its antioxidant function.


Abstract

OBJECTIVE:

α-Lipoic acid (LA) exerts beneficial effects in cardiovascular diseases though its antioxidant and/or anti-inflammatory functions. It is postulated that the anti-inflammatory function of LA results from its antioxidant function. In this study we tested whether inhibition of NF-κB by LA is dependent on its antioxidant function.

METHODS:

Human umbilical vein endothelial cells (HUVECs) were treated with tumor necrosis factor-α (TNFα) in the presence of various antioxidants, including LA, tiron, apocynin, and tempol. The activation of the nuclear factor-κB (NF-κB) signaling pathway was then analyzed.

RESULTS:

LA, but not other tested antioxidants, inhibited TNFα-induced inhibitor-kappaB-α (IκBα) degradation and VCAM-1 and COX2 expression in HUVECs. Although LA activated the phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in HUVECs, inhibition of Akt by LY294002 did not affect inhibition of TNFα-induced IκBα degradation by LA. In transient co-transfection assays of a constitutively active mutant of IκB kinase-2 (IKK2), IKK2(EE), and a NF-κB luciferase reporter construct, LA dose-dependently inhibited IKK2(EE)-induced NF-κB activation in addition to inhibiting IKK activity in in vitro assays. Consistent with the effect on luciferase expression, LA inhibited IKK2(EE)-induced cyclo-oxygenase-2 (COX2) expression, suggesting that IKK2 inhibition by LA may be a relevant mechanism that explains its anti-inflammatory effects.

CONCLUSIONS:

LA inhibits NF-κB activation through antioxidant-independent and probably IKK-dependent mechanisms.

 


This really makes ALA look very interesting.  It is cheap, widely available and well tolerated.


10.       Low Dose Naltrextone                       

Your local doctor will probably tell you that Low Dose Naltrexone (LDN) is a load of quack nonsense, partly because it is claimed to help so many unrelated disorders.

I would not have questioned that opinion, before I had started by investigation into the biology of the brain and seen how many apparently unrelated conditions are actually interrelated.  This can be established by science, not quackery.

First to note is that tiny doses of some substances do indeed sometimes have effects quite different to large doses.

We saw earlier how a tiny stimulation of the body’s nicotinic receptors produces a different effect to a large dose.

My own experience showed that a tiny, but specific, dose of Clonazepam has a marked effect, whereas conventional medical wisdom would say such a small dose would do absolutely nothing.  In this case, I was just following the clever idea of Professor Catterall, from the University of Washington.

I also found that tiny doses of a TRH analog had a positive effect and quite different to the “regular” dose.

The advocates of LDN suggest it for conditions including Crohn's disease, fibromyalgia and multiple sclerosis (MS).  As I mentioned earlier in this blog, some Fibromyalgia appears to be a condition that was almost autism; perhaps the final hit, in a multiple-hit process failed to occur.  Crohn’s is an immune disease and is a type of inflammatory bowel disease (IBD).  MS is an inflammatory disease in which the insulating covers of nerve cells in the brain and spinal cord are damaged.

Preliminary research suggests LDN may have an effect on inflammation. Naltrexone has an antagonistic effect on Toll-like receptor 4 (TLR4), which are found on microglia, which can modulate the body's response to inflammation. It has been hypothesized that LDN may have anti-inflammatory effects through this pathway.

  

Conclusion

The immediate conclusion is that there are plenty of ways, already existing, that might very well help reduce neuroinflammation in autism.  They just requires a little further thought and investigation.

The broader conclusion here is about the merit of genetic testing.

Undoubtedly, if you could analyze the entire genome in a person with autism and also measure the expression of those suspect genes in the brain, you would gain a great deal of information.  In a few cases, where there is a single gene causing the “autism”, you might well be able to figure out a therapy.

You cannot take brain biopsies from living people.  We did come across that clever Ricardo Dolmetsch, growing brain samples from skin cells.  He has now moved over to the private sector.


So for the moment genetic testing will just generate a vast amount of data, that in many cases will not be of any immediate clinical relevance.

The good news, as pointed out by Dan Arking, from Johns Hopkins, is that many of these numerous, unrelated, genetic dysfunctions end up with the same biological manifestations.

There may be thousands, or even millions of combinations, of genetic dysfunctions that lead to autism with neuro-inflammation.

You can go ahead and treat the neuro-inflammation, without any knowledge of exactly which gene has which SNP (single nucleotide polymorphisms)  or who had what CNV (copy number variant).

For me, the identification of so-called autism genes like PTEN and BCL2 is interesting, as are the single gene causes of autism.  We can then see that a reduced expression of that gene might contribute to autism, caused by multiple gene dysfunction (multiple-hits).  For the great majority of people with ASD, they have had multiple-hits.


I read Ricardo Dolmetsch’s Stanford research into Timothy syndrome, which is caused just by one gene, albeit a very important one.  I considered that perhaps a partial dysfunction might occur, leading to disturbance in the protein expressed by this gene.  I had no idea whether in my son this dysfunction existed, whether it might be caused by a SNP (there are several known ones) or if a dysfunction was caused as a consequence of a metabolic disruption caused by autism, such as oxidative stress or neuroinflammation,  affecting the function of an undamaged gene.

It did not matter; I just carried on and did a little practical test.  This led me to include Verapamil in my Polypill.  No genetic testing was required.

It was suggested to me that genetic testing might help point me in the right direction.  I think it would likely point me in all directions.  We all carry many genetic errors, and most of us thrive regardless, so most genetic errors are irrelevant.

The clever future diagnostic tool is proteomics.

  
Clusters

From now, I will consider autism in terms of a manageable group of clusters.  Once you know, based on symptoms and some measurable biomarkers, which cluster you are in, you would have a good chance of predicting which drugs would be effective.

The underlying genetic causes may, or may not, overlap with other people in that cluster.

Some clusters may overlap. Note the case of siblings with autism, when one is early onset and the other is regressive.  Was the regressive one really symptom free early one? Or, was it just a second hit nudged him “over the edge” and then people noticed?

This would be a practical approach that could be used.  I think when people talk of phenotypes and autisms, they are thinking about very precise biological causes and then it just becomes too complicated to expect your local doctor to ever figure out.

90+% of people quite probably fit into a handful of clusters.  Then you just need a diagnostic flowchart leading to the relevant cluster and then a specific drug toolkit.

My Polypill is the drug toolkit for one cluster; and it is not a rare one.