Nystatin in autism - a potent Potassium Channel Kv1.3 blocker (anti-inflammatory) or an antifungal/candida treatment?

Today’s post will go against some people’s understanding of autism and inflammatory bowel disease.

Just as there is a belief that heavy metals are a problem in autism there is another is another belief that candida is involved in autism and indeed inflammatory bowel disease (IBD).  Various types of IBD are highly comorbid with autism, but most people with IBD do not have autism.

The most common treatment for candida is an antifungal medicine called nystatin.  This drug is a cheap and widely available.

But nystatin has another property, it is a highly effective blocker of the potassium channel Kv1.3.

Regular readers will recall that this ion channel is key mediator in the inflammatory process, it is a target in many inflammatory conditions such as IBD and indeed autism.  Those little helminths (TSO) parasites that are being researched for both autism and IBD were found to reduce inflammation by releasing their own Kv1.3 blocker which stops the host (human or animal) from rejecting them.

Immunomodulatory Therapy in Autism - Potassium Channel Kv1.3, Parasitic Worms, and their ShK–related peptides

Candida Albicans Infection in Autism

Abstract: Background: Autism children were reported to have gastrointestinal problems that are more frequent and more severe than in children from the general population. Although many studies demonstrate that GI symptoms are common in autism, the exact percentage suffering from gastrointestinal (GI) problems is not well known, but there is a general consensus that GI problems are common in autism. The observation that antifungal medications improve the behavior of autism children, encourage us to investigate their intestinal colonization with yeasts. Aim of the work: The purpose of this work was to investigate the intestinal colonization with yeasts in autistic patients and to assess the role of yeast as a risk factor to cause autism behavior. Patients and methods: The study included 83 cases diagnosed as autistic children referred from the neuro-pediatric clinic and 25 normal children as a control group. All children under the study came to Phoniatric clinic, during the period from 2010 to 2012, complaining of delayed language development with autistic features. Children in this study were classified into 2 groups; control and study groups. All children were subjected to interview, E.N.T examination, language assessment, Childhood Autistic Rating Score (CARS), stool culture for Candida albicans, complete audiological and psychometric evaluation. Results: There was significant relation between the autistic children and heavy growth of Candida albicans in stool culture. Conclusion: The high rate of Candida albicans intestinal infection in autistic children may be a part of syndrome related to immune system disorders in these patients.

Conclusion: Candida albicans infection may be a part of syndrome related to the immune system and depends on genetic basis of autism, or Candida albicans may be etiological factor lead to excessive ammonia in gut which is responsible of autistic behavior in children. More researches are needed to clarify the exact mechanism by which Candida albicans affects autistic children.

  

In another study the results were not so clear:-

Gastrointestinal flora and gastrointestinal status in children with autism-- comparisons to typical children and correlation with autism severity

This study was done by James Adams (of the Autism Research Institute, former home of DAN).  According to Wikipedia, Adams' research has been described as "a laundry list of autism woo"; I think he is well intentioned.

You would have expected him to find Candida, but he did not. 

Note that they did not find any parasites either, although they did give up testing after the first 20 results were negative (not very scientific, I think).  Regular readers will know that some “holistic doctors” insist that parasites are the cause of autism.

  

Yeast

The presence of yeast was determined by both culture and by microscopic observation. Yeast was only rarely observed by culture in the autism or typical groups, and the difference between the two groups was not significant, as shown in Table ​Table5.5. Yeast was more commonly observed microscopically, but again the difference between the two groups was not significant.

Parasitology

The parasitology test was used on the first 20 autism samples only, which were all negative. It was then decided to do no additional testing on other samples

  

The finding that yeast levels were similar in both the autistic and control group is interesting, as there has been a great deal of speculation that yeast infections are a major problem in autism. Our data indicates that yeast is present at normal levels in the stool of this group of children with autism. A study by Horvath and Perman [21] reported that 43% of children with autism undergoing endoscopies had a positive fungal culture for yeast in their duodenal juice, vs. 23% of age-matched controls with other gastrointestinal problems requiring endoscopies. Since their study involved children with severe enough symptoms to warrant endoscopies, the greater symptom severity may explain some of the difference with our study. Since the survey by the Autism Research Institute of over 25,000 parents' reports that parents find antifungals to be one of the most effective medications for improving behavior [44], our findings are puzzling. It is possible that children with autism are more sensitive to even a normal level of yeast. Also, it is possible that antifungals have other effects, such as reducing inflammation.

  

Which Study to believe?

I have to say that I give more credence to the first study, which is from Egypt.

I think that autism in Egypt is likely to be the “real deal”.  People with severe autism will likely have associated auto-immune/inflammatory conditions and this will include abnormal GI conditions.

Also, the more severe the autism, the more restrictive the diet is likely to be, which will affect what grows inside the intestines.   

   

Ion Channels and Channelopathies

Ion channels are complex, but fortunately there are not that many of them, unlike genes.

A good source of information is provided by École polytechnique fédérale de Lausanne, on the banks of lake Geneva.  On their Channelpedia site you can see a nice entry on the potassium channel Kv1.3.  It may all look rather too complicated, but there under the Scorpion toxin, is a very common drug, Nystatin.

Kv1.3

Interactions

---

PAP-1

MbCD and MbCD/C

Zn

Leukocyte Subunits effect Kv1.3

Cluster at C-terminus

Kv1.3 associates with Kv1.5

Kv1.3 forms heteromeric channels

Scorpion toxin ADWX-1 is a pore blocker of Kv1.3 channel without affecting its kinetics

Nystatin

The concentrations for nystatin and its structural analog, amphotericin B, required to produce half maximal inhibition (IC50) of the current were estimated to be about 3 and 60 microM, respectively. The effects of nystatin on the amplitude and inactivation of Kv1.3 currents were not voltage-dependent. In inside-out patches, tetraethylammonium (TEA) produced a rapid block of Kv1.3 currents upon the onset of a voltage pulse, while the inhibition by nystatin developed slowly. When co-applied with TEA, nystatin potentiated the extent of the TEA-dependent block, and the kinetic effect of nystatin was slowed by TEA. In summary, nystatin, a compound frequently used in perforated patch recordings to preserve intracellular dialyzable components, specifically inhibited the potassium channel Kv1.3 at concentrations well below those required for perforation

KCa3.1 is related to acute immune responses and Kv1.3 is related to chronic immune responses, the combined administration with Kv1.3 and KCa3.1 inhibitors is likely to enhance their effects in autoimmune disorders or graft rejection

We know that Kv1.3 is widely expressed in the brain, but is it expressed in the intestines of people with inflammatory/auto-immune conditions?

We do not have far to look and since we know that ulcerative colitis is comorbid with autism, we can stick with that

Expression of T-cell KV1.3 potassium channel correlates with pro-inflammatory cytokines and disease activity in ulcerative colitis

Abstract

BACKGROUND AND AIMS:

Potassium channels, KV1.3 and KCa3.1, have been suggested to control T-cell activation, proliferation, and cytokine production and may thus constitute targets for anti-inflammatory therapy. Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by excessive T-cell infiltration and cytokine production. It is unknown if KV1.3 and KCa3.1 in the inflamed mucosa are markers of active UC. We hypothesized that KV1.3 and KCa3.1 correlate with disease activity and cytokine production in patients with UC.

METHODS:

Mucosal biopsies were collected from patients with active UC (n=33) and controls (n=15). Protein and mRNA expression of KV1.3 and KCa3.1, immune cell markers, and pro-inflammatory cytokines were determined by quantitative-real-time-polymerase-chain-reaction (qPCR) and immunofluorescence, and correlated with clinical parameters of inflammation. In-vitro cytokine production was measured in human CD3(+) T-cells after pharmacological blockade of KV1.3 and KCa3.1.

RESULTS:

Active UC KV1.3 mRNA expression was increased 5-fold compared to controls. Immunofluorescence analyses revealed that KV1.3 protein was present in inflamed mucosa in 57% of CD4(+) and 23% of CD8(+) T-cells. KV1.3 was virtually absent on infiltrating macrophages. KV1.3 mRNA expression correlated significantly with mRNA expression of pro-inflammatory cytokines TNF-α (R(2)=0.61) and IL-17A (R(2)=0.51), the mayo endoscopic subscore (R(2)=0.13), and histological inflammation (R(2)=0.23). In-vitro blockade of T-cell KV1.3 and KCa3.1 decreased production of IFN-γ, TNF-α, and IL-17A.

CONCLUSIONS:

High levels of KV1.3 in CD4 and CD8 positive T-cells infiltrates are associated with production of pro-inflammatory IL-17A and TNF-α in active UC. KV1.3 may serve as a marker of disease activity and pharmacological blockade might constitute a novel immunosuppressive strategy.

So now we have some evidence that Kv1.3 is involved in the inflammatory response within the intestines of people with inflammatory bowel disease (IBD).

Now we just need to look at what happens when you give Nystatin to people with IBD.

Since we do have to link all this back to Candida, let us look for people with IBD claiming that the problem was all about Candida.

If you google Crohns disease (a type of ulcerative colitis/IBD) you will find numerous reference to the benefit of Nystatin and again the assumption that “yeast overgrowth” is somehow the cause of the disease.  Lots of "holistic" doctors etc.

Why do so many people with autism benefit from Nystatin?

We have seen why some people with GI inflammation should find Nystatin very helpful, it will act locally as an immuno-suppressant.  

By reducing this inflammation there will be a reduction in inflammatory cytokines like IL-6.  But the whole idea of Nystatin being safe for children with autism is that it does not enter the blood stream, in stays inside the intestines.

Leaky Gut

Many people subscribe to the notion of the “leaky gut” in autism.  If indeed the gut was leaky, the Nystatin might leak out.  It would then act as a Kv1.3 blocker elsewhere in the body.  It may, or may not, be able to cross the blood brain barrier.

There is now some scientific evidence to show that  “leaky gut” is a real phenomenon.

In people with ulcerative colitis, of course the gut is leaking.  Blood is coming in and therefore other things can flow the other way.

In healthy people, Nystatin will stay almost entirely where it should, within the intestines.  In people with “leaks” it would seem likely that some will leak out.  In these people we might expect a greater effect.

We do know that inflammatory activity within the gut can transmitted elsewhere in the body via the vagus nerve.  This means that reducing inflammation within the GI will reduce the pro-inflammatory signalling sent around the body via the vagus nerve, even with no "leaky gut".  

This may indeed sound very odd, but very promising results are now being found in treating people with arthritis (an inflammatory condition, where IL-6 plays a key role) using implanted electrical devices that affect the vagus nerve.  Vagus nerve stimulation is not pseudoscience, even though it does sound like it should be.

  

My conclusion

The “father” of ARI and the DAN movement, Dr Bernard Rimland, a research psychologist, suggested that a small proportion of people diagnosed with autism had nothing more than an overgrowth of candida, caused by the frequent use of antibiotics.

It does seem that very many things can lead to “autism” and this diagnosis is now equally applied to people with very mild symptoms and those with debilitating ones.  I imagine that Bernie may indeed have been right; in a small number of people the problem may indeed be yeast.  However, given the relatively large number of people with autism (and IBD) who find Nystatin very helpful, I think the real issue is inflammation and  K_V1.3.  The people who respond to Nystatin would very likely also respond to those TSO helminths, and even Stichodactyla toxin (see later).

One problem with regular use of antifungal medication is that you are going to kill off not just the candida.  A healthy gut is supposed have all sorts of things living in it.   

For me, the conclusion is to go back to the ion channels and look not just for KV1.3 blockers but also KCa3.1.  There are plenty of people doing just this, but not for autism, for example:-

The K+ channels KCa3.1 and Kv1.3 as novel targets for asthma therapy

Kv1.3 blockers do exist and they include:-

·        Curcumin (problem is low bioavailability)

·        Acacetin (rarely studied and mainly used by bodybuilders)

Acacetin attenuates neuroinflammation via regulation the response to LPS stimuli in vitro and in vivo.

Abstract

Under normal conditions in the brain, microglia play roles in homeostasis regulation and defense against injury. However, over-activated microglia secrete proinflammatory and cytotoxic factors that can induce progressive brain disorders, including Alzheimer's disease, Parkinson's disease and ischemia. Therefore, regulation of microglial activation contributes to the suppression of neuronal diseases via neuroinflammatory regulation. In this study, we investigated the effects of acacetin (5,7-dihydroxy-4'-methoxyflavone), which is derived from Robinia pseudoacacia, on neuroinflammation in lipopolysaccharide (LPS)-stimulated BV-2 cells and in animal models of neuroinflammation and ischemia. Acacetin significantly inhibited the release of nitric oxide (NO) and prostaglandin E(2) and the expression of inducible NO synthase and cyclooxygenase-2 in LPS-stimulated BV-2 cells. The compound also reduced proinflammatory cytokines, tumor necrosis factor-α and interleukin-1β, and inhibited the activation of nuclear factor-κB and p38 mitogen-activated protein kinase. In an LPS-induced neuroinflammation mouse model, acacetin significantly suppressed microglial activation. Moreover, acacetin reduced neuronal cell death in an animal model of ischemia. These results suggest that acacetin may act as a potential therapeutic agent for brain diseases involving neuroinflammation.

·        Progesterone (as a hormone, has many other effects)

·        Verapamil (already in the PolyPill)

The most unusual/interesting comes from Cuba:-

Stichodactyla toxin

Stichodactyla toxin (ShK) is a peptide toxin that blocks the voltage-gated potassium channels: K_v1.1, K_v1.3, K_v1.6, K_v3.2 and K_Ca3.1.

Selective Kv1.3 channel blocker as therapeutic for obesity and insulin resistance

In humans, a polymorphism in the Kv1.3 promoter is associated with impaired glucose tolerance and with lower insulin sensitivity (11). These results suggest that selective Kv1.3 blockers might have use in the management of obesity and insulin resistance

Because pancreatic beta cells, which have K_v3.2 channels, are thought to play a role in glucose-dependent firing, ShK, as a K_v3.2 blocker, might be useful in the treatment of type-2 diabetes.

  

You may recall we already saw in this blog the older people taking Verapamil (for heart problems) did not develop type 2 diabetes. According to the table below, ShK toxin is a K_v3.2 blocker in humans, but Verapamil only works in rats.

Since it looks like selective Kv1.3 blockers may prevent/treat obesity, you can expect them to be attractive targets for pharmaceutical companies.  This is a disease of the 21^st century.

The spin-off might later be a cost-effective treatment for inflammatory conditions like IBD and autism.

The clever new arthritis treatments, that could be used in autism, are hugely expensive.

The over-activated immune system, Kv1.3, ASD-IE, Acacetin and sloppy science

One of the people I have met during my investigation into autism, recently pointed out to me that much scientific research cannot be trusted.  He forwarded a study to me showing just how many researchers admit is omitting data that did not suit what they were trying to prove.  I replied that I made a point of checking the credentials of the lead author.  He then replied that it is not the lead researcher who collects the data, he has a little army of PhD students doing this and nobody is checking them.

The study showed it was the younger researchers, eager to prove themselves that were the most likely to “fiddle” the results.  The problem is that by the time you become an “older researcher” you are not the one collecting the data.

Doubts about Sprouts 

One of the people who I keep forgetting to add to my Dean’s list is John Gargus; he is a professor at University of California at Irvine and Director of the Center for Autism Research and Treatment at UCI.  He is also a specialist in the complex field of ion channels and channelopathies.

He was asked to comment about the Johns Hopkins broccoli/Sulforaphane autism trial.

Trial sprouts doubts about broccoli extract for autism

Cruciferous caution: 

Some independent researchers have similar reservations, noting that the control group showed an unusually small placebo response.“You always see a 20 to 25 percent improvement in placebo,” says John Jay Gargus, director of the Center for Autism Research and Translation at the University of California, Irvine. For example, the placebo effect plagued trials of the gut hormone secretin and antidepressants for autism.
“It’s stunning that they’ve managed to have found a placebo that doesn’t give the placebo effect that we see in every other neuropsychiatric drug trial,” Gargus says.

Now as regular readers will know, at least in Monty, aged 11 with ASD, broccoli sprout powder and we assume the Sulforaphane produced by it, does have a near immediate effect.

But as Gargus says, you will always have some people with the placebo appearing to improve.  In an old post I looked at the placebo effect in autism.  It seems that the more involved the trial and hence the more 1:1 attention the child gets, the more the placebo appears to make things better.  In fact it was not the placebo, it was the 1:1 attention that improved the autism.

So, just as we have to be cautious of the placebo effect, we have to be cautious of sloppy science/scientists.  When financial interests are involved you need to be even more cautious.

The other thing I have learnt to be cautious of, is scientists who have spent many years in one very narrow field, often trying to prove their initial hypothesis to be correct.  Their eyes are then closed to everything else.

Autism Flare-ups, Summertime raging and GI issues

We have investigated in depth the fact that in some people with autism their immune system appears to be over-activated, as the result of an allergic response.  What then happens is that their autism “flares-up” and therapies that previously worked, seem to stop doing so.

The conclusion was that the allergy had caused mast cell activation and this triggered the release of pro-inflammatory chemicals (IL-6, histamine etc).  The solution was:-

·        Avoid the allergen (a type of food, or even airborne pollen)
·        Use mast cell stabilizers to minimize degranulation; even common H1 anti-histamines are partially effective
·        Inhibit the potassium ion channel Kv1.3, which seems to mediate the resulting “over-activation” of the immune response.

The good news is that it really does work and not just in Monty.  The bad news is that the optimal therapy uses a prescription drug (Verapamil).

While trawling through the research on novel anti-oxidants, I stumbled upon something that may help those people who cannot access Verapamil.

There is a flavonoid called Acacetin, which is found in asplenioid ferns.  This flavonoid has long been has used for its anti-inflammatory and immunomodulatory effects.  Now it has been shown to block Kv1.3 channels and inhibits human T cell activation.  This is one of the effects of Verapamil (there are others).  Acacetin has also been shown to have anti-cancer properties in prostate cancer cells.

Acacetin Blocks Kv1.3 Channels and Inhibits Human T Cell Activation

Acacetin (5,7-dihydroxy-4'-methoxyflavone)exhibits in vitro and in vivo anticancer activity through the suppression of NF-κB/Akt signaling in prostate cancer cells

Remember the odd therapy used to block Kv1.3, those TSO parasites, I mentioned in previous posts.  My favorite is this one:-

Immunomodulatory Therapy in Autism - Potassium ChannelKv1.3, Parasitic Worms, and their ShK–related peptides

Acacetin is available as a supplement.  So if you think blocking Kv1.3 might help and cannot access Verapamil or TSO, there are other options.

Indeed, for completeness, there at least two other Kv1.3 blockers that are available.  One is progesterone, the hormone and the other is Curcumin.

  

A nongenomic mechanism for progesterone-mediated immunosuppression: inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes.  Curcumin serves as a human kv1.3 blocker to inhibit effector memory T lymphocyte activities.

  

You may recall that Progesterone was found to be highly neuro-protective and for this reason was trialed for use in the ER, immediately after a traumatic brain injury.  It was shown to save lives.  In autism, we previously found that some people, at the high functioning end, find they feel better when they apply progesterone cream, i.e. transdermal route.

Curcumin has been used for centuries as a drug.

I have not tried them, but I will continue to use Verapamil.  Acacetin, Progesterone and Curcumin share some, but not all of each other’s effects.

Progesterone, in common with Verapamil, affects both potassium and calcium channels.

There are many different potassium and calcium channels and you would hope to find a selective channel blocker and hence affect only the ones you need to.

ASD-IS  (Inflammatory Subtype)

I came across a promising study on Paul Whiteley’s blog.  It is a study of a sub-type of autism characterized by fluctuating behavioral symptoms following immune insults.  In the trial group the children all had GI problems, some had enterocolitis or esophagitis.  The entire group had been noticed by teachers/therapists to lose cognitive skills following immune insults.

Cytokine profilesby peripheral blood monocytes are associated with changes in behavioral symptoms following immune insults in a subset of ASD subjects: an inflammatory subtype?

Regular readers of this blog will see lots of familiar points.  This appears to be exactly the same thing as my “over-activated immune response”.

Now this study comprised children who had Non-IgE mediated allergies.  This does matter because classic allergies are called IgE-mediated and they result in little cells called mast cells getting activated and then releasing IL-6 and histamine in the blood supply.

From Wikipedia we have a summary:-

Conditions caused by food allergies are classified into 3 groups according to the mechanism of the allergic response:

1.     IgE-mediated (classic) – the most common type, occurs shortly after eating and may involve anaphalaxis.

2.     Non-IgE mediated – characterized by an immune response not involving immunoglobulin E; may occur some hours after eating, complicating diagnosis.

3.     IgE and/or non-IgE-mediated – a hybrid of the above two types.

Treating allergy is a “fuzzy” area and, depending on which country you live in, some aspects are seen as science and others pseudo-science. 

Perhaps we should see it as an important, but emerging field of science.

I am not an allergist/immunologist, so I have to look things up.

Since in the trial the children had Non-IgE mediated allergies, we can then look to see whether mast cell activation is relevant.

NON-IgE MEDIATED FOOD ALLERGY 

Mast cell and eosinophil activation is an important component of the non-IgE-mediated response

The authors of the autism study believe that the research subjects with allergy did not have mast cell activation, because they had NON-IgE mediated allergies.

Since I am not an allergist, all I can say is the author of the above paper from the Royal Free & University College School of Medicine in London thinks that mast cell activation is an important component of the non-IgE-mediated response.

Anyway, make your own mind up and continue to see what the study found.

The study looked at children with autism and allergy, whose autism flares up and affects (impairs) their cognitive function.  This group is ASD-IS (Inflammatory Subtype)

ASD-IS children: ASD-IS children are defined as those with a history of fluctuating behavioral symptoms following immune insults (mainly microbial infection). Symptoms must have been documented by individuals other than parents, such as teachers/therapists, a minimum of three times. In addition, a history of persistent GI symptoms, often diagnosed as non-IgE mediated food allergy (NFA - see next section for diagnostic criteria), is required. Among the ASD-IS subjects, 14/24 subjects were diagnosed with food protein induced enterocolitis syndrome (FPIES), a severe form of NFA, prior to enrollment in this study, and two ASD-IS subjects were diagnosed with eosinophilic esophagitis (EoE) on the basis of biopsy results. These ASD-IS subjects reported to have had loss of once-acquired cognitive skills based on the reports of teachers, therapists and/or previous records of developmental assessment.

We defined ‘flares’ as worsening behavioral symptoms following immune insults, despite the resolution of acute conditions such as viral syndrome (that is, the resolution of other infectious symptoms if associated with a microbial infection, lack of fever, and no other acute physical symptoms associated with immune insults). Most of the immune insults in this study were clinically judged to be microbial infection (mainly viral syndrome). In ASD-IS children, we obtained samples at least once in the ‘flare’ and ‘non-flare’ states. Changes in behavioral symptoms by parental reports were confirmed by reports from

teachers and other caregivers.

The authors have an entirely different hypothesis to mine.

But I find their data remarkably similar to what I see being caused by a pollen allergy in my son - summertime autism flare-up and regression.  This is why there were so many posts about the inflammatory cytokine IL-6 and how to minimize it.

Have the authors stumbled upon exactly the same phenomenon as I did?  I very much think so.

I have shared my therapy with the authors, but they think that Non-IgE mediated allergies have nothing to do with mast cell activation.   That sounds odd to me.