Resolution of ASD symptoms with fever. Is it real, and if so, what does it mean?

…when you have eliminated the impossible, whatever remains, however improbable, must be the truth. S. Holmes

There is a report that children with ASDs sometimes acutely exhibit improved behaviors during a fever, and that their behavior then returns to baseline after the fever passes.

What can we infer about the mechanisms behind ASDs and ASD symptoms from this?

I only saw this on Friday (12/07), so have mostly missed the blogging about it at Autism Vox and at Prometheus. I have seen the comments there and will attempt to answer some of the questions raised. The more I get into it, the more complicated it becomes and it is very hard to force myself to be simplistic about anything.

I will talk about some of the background in fever therapy, and what I think the acute effects in treating some mental disorders derive from. There are non-acute effects which likely derive from resolution of the infection (when that is what is causing the symptoms such as in neurosyphilis), but the acute effects on mental activities are probably not from that. My conclusion is that the improvement is real, and that it is due to increased basal NO from iNOS expressed during the immune system activation. I think that inducing a fever can only produce transient improvement while the NO level is acutely higher. I think it is very likely that autistic symptoms will be made chronically worse by transient fevers (even if there is acute improvement during each fever). I discuss this in the low NO ratchet section.

I spend a lot of time considering other signaling molecules in addition to NO. The simplest explanation is that the effects are due to NO. Maybe other things are involved too, perhaps as intermediates, but in my opinion, NO is the most likely candidate. I spend a fair amount on what it can't be. For the acute, short term effects, nothing but NO seems credible to me.

I think that my bacteria can produce some improvement too, but unlike immune system stimulation I think the improvement will last as long as there is a biofilm of the bacteria on the skin increasing basal NO levels. How much improvement? If autism is characterized by low basal NO as many symptoms indicate, then there will be some improvement. All disorders characterized by low basal NO will be improved by an increase in basal NO with no threshold. The reason is, because NO is used as a signaling molecule in many feedback regulatory control loops, the basal NO level is necessarily coupled to the basal NO level. When that basal level is perturbed, so are the operating points of those NO mediated control loops. Because NO is already in the active range, any change in NO changes the output of those control loops. If those control loops are in a dysfunctional range because of low basal NO, increasing basal NO will move them into a more functional range.

A long term improvement in basal NO levels may (very likely) have much greater normalization of behaviors by normalizing physiology and allowing normalization of neuronal remodeling and a restoration of a more normal neurodevelopmental path. This remodeling could not occur during the short term effects observed in this study. How much is far too complicated to predict a priori, and is no doubt idiosyncratic for each individual and will depend on many other factors too, including age and how much of the ASD neurological phenotype the individual has (which depends on prior neurodevelopment including in utero).

This article is not to be taken as any type of medical advice. It is provided for informational purposes only. If anyone is interested in doing an actual clinical trial along the lines I suggest, please contact me so it can be done in the proper way with real IRBs and stuff. If you have any questions, or if you have found any errors, please let me know. I have tried to be pretty conservative in introducing new ideas, everything in here is pretty well supported in the literature, perhaps not cited as well as it should be, but I wanted to get this out fast (which is why parts of it are still kind of rough). I cite a lot of stuff, there is a lot more that I haven't cited. I haven't "cherry-picked". All of the stuff I have cited is what I consider to be pretty main stream and pretty much correct. I don't agree with every conclusion in every paper I cite, I am mostly citing the data which they report and which I then use to draw my own conclusions. My conclusion that NO is involved is "robust", that is it doesn't hinge on one or even a few assumptions that the data in the papers is correct. There are many independent lines of research that point to the same conclusion. Virtually all of them would have to be wrong for NO to not be involved.

There have been some comments that people want to use their own anecdotes of feeling and thinking worse while they have a fever to discount this data that some people with ASDs have improved behaviors. Use of anecdotes to discount other people’s data is no better than using anecdotes as being equivalent to data. Even if only a subset of people with ASDs respond this way to a fever, it may still be real, and may be useful in understanding ASDs. In any case, fevers make people feel "sick". They do not cause NTs to have ASD behaviors or the opposite of ASD behaviors. Nausea is one symptom is be made worse by a placebo. I see that as evidence that nausea is due to elevated NO (see my piece on the placebo effect). Nausea during sickness (and during pregnancy) likely relates to this. If your basal NO level is low, and is acutely raised but not to the level where you feel nauseous, then you could experience an improvement in low NO symptoms without feeling "sick".

Physiology is inherently non-linear. Using linear extrapolation from common everyday experience will be completely ineffective in estimating physiological responses in regions of the physiology parameter space which are not linearly related to the normal state. Activation of the immune system is highly non-linear with feedback and hysteresis. Any sort of linear model isn't going to capture what is important.

This result is completely consistent with my low NO hypothesis of ASDs. My explanation for the disparity between how some ASDs and NTs respond to fever is because how a neural network reacts depends on which side of the percolation threshold it is on. NO increases connectivity. If the network is above the percolation threshold, that increased connectivity moves it away from the peak and decreases functionality if it is below it, the change moves it closer and increases functionality. The increase when the network is below is much larger than the decrease when it is above.

First, is it credible?

This study was the result of one author's PhD thesis, at Johns Hopkins University. That is a first rate school, and the other co-authors are first rate scientists from first rate institutions. The journal it is published in is Pediatrics, a first rate peer reviewed journal. There are a number of anecdotal reports in the literature of the effect of fevers on ASDs which they cite. This is a prospective study, not a series of retrospective anecdotes. None of the authors are selling "fever treatments", they are serious researchers, and have written many serious research papers with multiple different collaborators and on multiple different subjects, some relating to autism, some not, published in first rate journals. There is no comparison between this report in a first rate journal with the "mercury causes autism" or the "MMR causes autism" crap. I looked at the publication records of each of the co-authors, and none of them seem like wackos. They haven't published anything wacko before (that I could find), they don't suggest a mechanism, let alone mechanisms that are implausible or that contradict anything that is well known in physiology. They don't cite anything wacko. They seem like credible responsible scientists reporting data as they measured it. The data collectors were the children's parent or guardian, the people who know them best. They would be the most accurate assessors of the child's behavior, provided that confounding factors did not introduce bias. The anecdotal comments of people in blogs expressing skepticism would suggest that any bias would be to expect worse behavior during a fever. It is doubtful that these authors are going to set up clinics to induce fevers to treat autism. They would have disclosed it, it isn't patentable, and no IRB would let them do it. I don’t think that they have an agenda they are trying to push. In any case, a paper like this isn't close to a sufficient basis to justify giving children fevers.

History of Fever Treatments and rationale based on modern understanding

There is a long history of "fever treatments" for mental disorders. Hippocrates mentioned that malaria improved epilepsy. The person who pursued it the most Wagner-Jauregg and is credited with originating it received the Nobel Prize (1927) (though he credits AS Rosenblum who published 'Relation of febrile diseases to the psychoses' in 1876-77 but which wasn't translated into English until 1943). He used malaria, typhoid and recurrent fever. This was before the days of antibiotics and any type of psychopharmacology (except for opiates and cocaine). It was called fever therapy, but it was actually inoculation with a disease that caused fever. They tried a number of different ones, and the one that seemed to work the best was malaria. Malaria due to Plasmodium falciparum is the most severe (and life threatening), and gives repeated bouts of fever due to the life cycle of the parasites as they reproduce. The type of malaria that was most used for fever therapy was the kind that is actually considered the mildest (P malariae) and so is the safest. Safety was probably the major reason it was selected. It worked, and wasn't so life threatening. It does produce fevers every 72 hours. One day of fever, then in 3 days another. It was mostly used against syphilis and the treatments were first done on people with paralysis and nerve damage from syphilis. This was called GPI (generalized paralysis of the insane), and affected mostly men, often of high intelligence, a few percent of people who were infected with syphilis. They had very good success. There were large numbers of well documented improvements. There were also not a few deaths, tens of percent. It was a desperate treatment for a debilitating disease that was certain death. Multiple types of fever producing methods were tried, inducing malaria was pretty clearly demonstrated to work the best. They would infect the patient, let them go through a number of cycles, usually around 10, then cure the malaria with quinine. The induction of malaria was the "standard of care" for neurosyphilis for decades. There was considerable resistance to using only penicillin when it was introduced.

He did try simply inducing fever using killed cultures of bacteria, this did cause remissions in the paralysis, but the syphilis and paralysis did relapse. Experiments with killed bacteria preceded those with active disease agents because while killed bacteria helped some, the results were not satisfactory. Detection of neurosyphilis is quite difficult even today. Recovery from the symptoms of paralysis does not occur instantly upon eradication of infection. Also, infection can remain even after the paralysis symptoms have gone, but the paralysis then returns (usually, but the testing of the time was not sufficient to be completely sure). It isn't exactly clear, but I think the symptoms of paralysis resolved faster with fever therapy than they did with penicillin. Which may have been why treatment with penicillin was followed up with fever therapy for years after penicillin was introduced. I think this relates to the effects of fever on neuronal function and resolution of neurological symptoms of neurosyphilis not being solely due to eradication of the infection (because symptoms would remit and then return if the infection was not cleared).

The diagnosis of the cases of insanity and psychosis were probably not up to modern standards, and some of these cases may have been due to some type of infection (as was neurosyphilis), which resolved after the infection was resolved during the fever. There were some cases of epilepsy that were treated and said to have been cured. The "causes" of many of the disorders that were treated remain mostly unknown. However, this remains true today. There is no known "cause" of psychosis, of schizophrenia, bipolar, of some other mental disorders, and no instrumental or other diagnostic tests other than interview by a clinician. Clinicians 100 years ago didn't have the DSM to consult, but then papers written about fever therapy were not written by work-a-day clinicians but by very senior researchers. There may have been some misdiagnosis, but it is not safe to assume that every successful case was a misdiagnosis. Were the successful cases only from something like neurosyphilis which had an infective cause? Perhaps, but epilepsy and psychosis isn't usually causes by an infection, and virtually all infections either are resolved, or kill the host. Neurosyphilis is a rare chronic bacterial infection. There are a few others, but it is likely unreasonable to assume that all the mental disorders other than neurosyphilis were other type of chronic bacterial diseases (which remain unknown today).

Fever treatment was used for a lot of mental disorders, labeled "insanity" or "psychosis". Precisely what those disorders were is hard to tell from the multi-hand accounts I have access to. What is notable is that some of the recoveries were quite prompt, a day after receiving an injection of tuberculin (a sterile solution containing growth products of tuberculosis) the sister of the patient remarked "What have you done with my sister? she has suddenly become intelligent".

That diverse mental conditions were effectively treated by fever therapy implies that on some level, some of the fundamental pathology in all of these different disorders is "the same", and that fever therapy was working on that common fundamental pathological state, and that restoring that fundamental state to "normal", restored normal function, sometimes extremely rapidly, in less than 1 day. That is too rapid for repair or remodeling of neuroanatomy, but must instead be a modification of a regulatory pathway(s). That a "normalization" occurs implies that the regulatory pathway is intact, it is simply exerting control at a dysfunctional operating point.

White matter hyperintensities and ATP status of the brain

In neurosyphilis MRI imaging, there are enhancements in white matter hyperintensities, (WMH ), called leukoaraiosis, which is also characteristically observed whenever there are abnormalities in brain perfusion and brain metabolism. The precise mechanism of leukoaraiosis is not well understood, however it is a decrease in the local diffusion of hydrogen, usually thought to be water as observed by MRI. My interpretation is that in the white matter (which is mostly axons, called white because of the abundant myelin covering them), a major source of that water movement (and apparent diffusion on MRI) is active axonal transport and not passive diffusion. This would explain the anisotropy of the diffusion also. WMH are observed in just about all of the neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's, Lewy body neuropathies and some others. These are all also characterized by reduced metabolism, and reduced concentrations of ATP or related energy metabolites.

All components of a neuron are made in the cell body, where the nucleus and all the DNA is. Everything then is carried out to the tippy end of the axons by ATP powered motors moving on the actin skeleton. That includes mitochondria which have only a 30 day half life (in rat CNS), it is probably longer in humans, how much longer is unknown. Under conditions of insufficient ATP (as in ischemia, hypoxia), ATP conservation pathways will kick in, and reduce ATP consumption. I think the ultimate "cause" of WMH is low ATP. It is estimated that ~50% of neuron ATP consumption is via the actin skeleton. The ATP conservation pathways would have to address such a major ATP consuming pathway. With less stuff being moved, or being moved slower, there would be less water entrained, less water movement, and so less apparent diffusion of water due to that shear and mixing. WMH does correlate pretty well with ischemia from stroke, in vascular depression, and other neurodegenerative diseases characterized by reduced brain metabolism. This axonal transport has a maximum velocity of a few microns per second. The transport is usually not continuous, but proceeds and then stops. Different cargo is transported at different velocities, but much of that difference is likely due to different times the cargo is stationary. The fastest transport is about 250 mm/day.

Actin does have regions of enhanced water mobility surrounding it, and these regions are affected by ATP levels. The addition of ATP liberates strongly bound water forming weakly bound water. In MRI, this would show up as increased water mobility at higher ATP levels.

WMH are more complicated than just ATP and active axonal transport because they are still observed post mortem, when there is complete ATP depletion and a complete absence of ATP powered motion. Are the axons partially clogged due to accumulation of cargo in "traffic jams"? Axons have a very high aspect ratio, microns in diameter and thousands or tens of thousands of times longer. A small change in cargo velocity would result in accumulation if the change were not exactly uniform across the entire length of the axon over the time scale of the change in velocity. Movement control of cargo, that is regulation of cargo velocity, has to be "local", that is it can only depend on the local conditions of the motor, the cargo, the cytoskeleton, cargo density, and the ATP level (and perhaps other local parameters). Those local parameters may have input from distant signals, but the ultimate transduction into cargo velocity must be locally determined. There are too many different individual pieces of cargo being carried for there to be external non-local control on each one. The axon 100 microns away can't "know" the status of cargo transport other than by local signals transmitted in the axon. Presumably the velocity of cargo can be regulated within some range, but as ATP depletion occurs, that active range can only go down, and eventually transport will stop.

Unless that slowing and eventual stoppage is exactly synchronous, then presumably the cargo would accumulate in slow regions, and could become "jammed", that is become so close packed that the force exerted by the ATP powered motors moving cargo into the "jam" would be insufficient to move the "jam". The shear strength or effective velocity of a granular material is a very sensitive function of the particle-particle spacing. This "jam" would show up as retarded water diffusion because the cargo would be forced into a close enough packing that the essentially motionless hydrogen in proteins becomes a larger fraction of the total hydrogen (proteins plus water).

As the transport along an axon degrades from the "normal" well controlled nearly uniform velocity, it would be expected to degrade non-uniformly, that is each axon would devolve into regions where cargo is sparse plus regions where cargo is "jammed". The effective water diffusivity in regions where cargo is sparse may be a little higher, it is not going to be much higher than normal regions. In contrast, where cargo is "jammed", the apparent hydrogen diffusivity would be expected to be lower. At the "normal" cargo density, cargo moves in both directions, and also moves at different velocities simultaneously. Thus there is lots of free volume for cargo to pass each other. The average cargo density can only change slowly, as cargo is either removed at the cell body or at the tippy ends. The effective diffusivity of water in a volume of cargo will go down as that cargo is segregated into regions of higher and lower density. The loss of effective diffusivity in the regions of highest density will not be made up for by the gain in diffusivity in the regions of lowest density. This starts to happen when the particle-particle spacing becomes less than the diffusion distance of hydrogen during the MRI scan.

A "jam" can only be cleared by withdrawing cargo from the jam at the ends where the jam is adjacent to unjammed regions. A "jam" that is present at death will not be cleared. Do axonal contents become "jammed"? Some axonal components do precipitate and become solids, presumably that occurs when the local concentration exceeds the solubility. Accumulation of inclusions is observed (eventually) in essentially all conditions where WMH are observed, Alzheimer's, Parkinson's, Lewy body neuropathies, amyloidosis, and prion disorders. These are also all conditions where brain metabolism is reduced, in some cases profoundly. They are also observed during seizures. On acute occlusion of cerebral arteries, WMH occur very rapidly, in 2.7 minutes in the rat. It has been suggested that edema is the cause of WMH, however edema does not occurs this quickly, but ATP depletion does.

There may be other changes that result in WMH too. WMH are associated with markers for hypoxia. In any case, the relevance of this diversion into WMH is only to connect WMH to the ATP status of the brain. The association of WMH with low brain ATP is pretty well established, even if the mechanism(s) for that association is not.

There is a report that WMH are associated with lower NOx levels in plasma and also with increased markers of oxidative stress. NOx in plasma is not a good measure of NO. NO concentrations are never much greater than 10 nM/L. NOx is the terminal metabolite, and is present at orders of magnitude higher levels (this report says 70 mM/L, I think that is a misprint, it is more likely to be 70 micromoles/L. Nitrate behaves like chloride, plasma levels of chloride are ~100 mM/L. This report on LPS production of sepsis in rats shows increased NOx to 300 micromolar which is more reasonable.) In this report they show the increase in NOx due to hemorrhagic shock and also due to endotoxin shock. This report shows NOx levels ~50 micromolar in humans. During sepsis NO is greatly increased due to expression of iNOS via NFkB, but the regulation is quite complicated.

NO and superoxide from iNOS are protective against excitotoxic injury, and this protection can be induced via LPS treatment.

Subjects with WMH have reduced density of blood vessels in regions which show hyperintensity. These blood vessels are often tortuous and appear as a tortuous arteriole in a cavity. These images are quite striking. The vessel is quite corkscrew-like, very tortuous and is in an empty cavity, devoid of white matter. These are sites of apoptosis. Presumably, that blood vessel must either be a source of a pro-apoptotic signal, or a sink of an anti-apoptotic signal. It turns out that vessels are sinks for NO, which is an anti-apoptotic signal. However it is not precisely the vessel that is the sink, rather it is the hemoglobin in the red blood cells that is the sink. This is how the tortuosity develops (my hypothesis). It is exactly like the meandering of a stream. The high velocity of the stream on the concave side erodes sediments which deposit in the low velocity region on the convex side. The stream migrates in the direction of the highest erosion rate. Similarly, the blood vessel migrates in the direction where NO is the lowest, where the fluid flow pushes red blood cells up against the vessel wall. With the shortest diffusion path and the highest red blood cell density and velocity, NO is lowest there, and lowest on the outside bend of the vessel. This low NO causes the tissues outside the vessel to regress (via apoptosis) and so the vessel migrates in that direction. Tortuous vessels like this are easily seen in retinopathy (where they accompany WMH) where they are caused by the same mechanism. In retinopathy, often when vessels cross there is observed to be "nicking", that is, a reduction in the diameter of the vessels. This is due to the decreased NO at the site of crossing (my hypothesis) where there is more hemoglobin to act as a sink of NO. This migration and remodeling of blood vessels by local NO levels is part of the normal regulation of capillary spacing (my hypothesis).

The reduced blood vessel density is likely due to a remodeling of the vasculature due to chronic low NO. With O2Hb being the sink for NO, if the level of NO is lower, then less hemoglobin is needed to act as a sink. I think this remodeling of the vasculature is the mechanism behind the lower brain blood flow observed in all of the neurodegenerative disorders characterized by WMH. I think it is also the mechanism for capillary rarefaction in non-neuronal tissues observed in hypertension and other disorders.

In looking more into the details of neurosyphilis, some of the neurodegenerative symptoms also bear some (faint) resemblance to Friedriech's ataxia. Friedreich's ataxia is caused by a mutation in the transcription factor that regulates the production of frataxin, an essential mitochondrial protein which locates to mitochondria, and appears to be essential in the formation and regulation of proteins with iron sulfur clusters (both inside and outside of mitochondria). This is a very large class of enzymes, largely involved in regulation of oxygen consumption for ATP production and for manipulation of oxidizing or reducing equivalents for synthesis of compounds. Deficiencies in frataxin result in mitochondrial dysfunction, iron accumulation, oxidative stress, nerve damage and eventual atrophy and nerve death. It seems that the nerves that are the longest get affected first (just like ALS).

This would be consistent with an insufficient mitochondria biogenesis etiology. Mitochondria biogenesis is triggered by nitric oxide. When there are insufficient mitochondria, they get pushed to higher potentials, where they generate ATP less efficiently (leading to a hypermetabolic state as in ALS, cardiomyopathy, obesity) and generate more superoxide which lowers the NO level more and renders the low NO state permanent (as in chronic fatigue syndrome).

Regulation of ATP concentrations in axons must be "local". That is, it must be regulated within each cell (because cell membranes are not permeable to ATP), but also within each axon because the ATP demand is "local".

In autism, there are reductions in brain concentrations of chemicals associated with brain energy status. There are reductions in T2 relaxation times in gray matter reported for children with autism. There are reports of reduced cerebral blood flow in children with autism. There are reports of neuroinflammation caused by neuroglial activation. Low NO causes the activation of microglia.

In people with autism, these signs point to low NO in the brain, which leads to low ATP via regulation of ATP via sGC. The regulation of sGC by NO and ATP and other phosphates is not simple (although this interpretation has been questioned). Acute sepsis leads to high NO from expression of iNOS, and leads to high ATP concentration. This is not generally appreciated. During acute sepsis, ATP levels are actually higher (if the patient survives) than normal controls. It is my interpretation that the authors of this last report don't appreciate what their own data clearly shows. They show higher ATP levels in skeletal muscle during sepsis than in uninfected controls (p =0.05). ATP is higher because NO is higher. High NO blocks cytochrome c oxidase, so mitochondrial ATP generation is shut down (mostly). This is why septic shock causes cachexia. The body is generating ATP via glycolysis. The mitochondria are shut off by the high ATP, so the body needs to make glucose without using ATP, so it does so by turning the muscles into alanine which the liver can turn into glucose without consuming ATP. All of this glycolysis generates a lot of lactate, which can't be turned back into glucose because the mitochondria are shut down. So the body turns it into fat. That is what septic shock does, it turns muscle into fat. Turning protein into fat and carbohydrate liberates a lot of ammonia. If that is sweated out to the skin, a resident biofilm can turn it into NO/NOx while conserve NOS substrates arginine, NADPH and O2.

Note this ATP measurement during sepsis was in muscle, however the ATP levels of all the cells in the body have to go up and down in sync for physiology to be regulated in a stable way. There has to be a "signal" that communicates the ATP level in cells, so that level can be regulated up and down in sync. This "signal" has to be uncharged to penetrate lipid membranes, and rapidly diffusible to communicate the signal quickly. It is pretty clear that the signal has to be NO. The coupling of NO and ATP via sGC makes perfect sense in this light. NO is the diffusible signal that causes all cell to regulate their ATP levels up and down "in sync". This is especially important in the brain, where everything really does need to operate "in sync" for the brain to function properly.

In ischemic preconditioning, a transient ischemia induces a physiological state which is largely protective of future episodes of ischemia within a certain time frame. Ischemic preconditioning is quite complex, and is not fully understood. I discuss a number of aspects of it in my article on the placebo effect and the one on acute psychosis. Ischemic preconditioning can be mediated by oxidative stress in multiple ways. In the first case, HIF-1α causes expression of the enzymes that mediate glycolysis, increasing capacity for glycolytic ATP production. My interpretation is that acute ischemia, hypoxia, or oxidative stress all trigger reduced ATP, and this reduced ATP triggers the ATP conservation pathways that mediate ischemic preconditioning.

Ischemic precondition is a lower ATP state, but more importantly, it is a lower ATP consuming state. ATP consuming pathways that can be shut off temporarily have been shut off, and this preserves ATP for those pathways that are more essential and cannot be shut off. Ischemic preconditioning is induced in whole organs, comprising multiple different cell types. Presumably the regulation of ATP production and consumption in those diverse cell types is regulated "in sync" for ischemic preconditioning to be effective. For cells to be regulated in sync, there must be signal(s) that passes between them to communicate the need to invoke the ischemic preconditioning pathways. We know that NO is a signal that modulates ATP level (it increases it observed in septic shock), we know that ischemic preconditioning is triggered by ROS, and we know that ROS destroys NO with near diffusion limited kinetics. If diverse cell types are all regulated "in sync", presumably they all use "the same" signaling molecules, and in "the same" control scheme. It is much easier for one (perhaps very complex) control scheme to evolve that covers all cells than for multiple control schemes controlling multiple overlapping cell types to evolve.

So, autism like neurosyphilis and like many neurodegenerative diseases is a state of lower metabolism in the brain, lower blood flow, and lower ATP in the brain. In neurosyphilis there is inflammation, in autism there are also reports of inflammation and oxidative stress. Inflammation and oxidative stress would reduce NO levels and reduce ATP levels.

So what is the mechanism(s) for the acute improvement of people with autism during fever?

Presumably nerve damage during a fever could not "normalize" behaviors (that is improve function from abnormal to more normal). Similarly if the fever caused a repair of nerve damage, that repair would not reverse itself when the fever passed. The fevers in the report are mild, likely not enough to produce damage at all.

Changes in neuroanatomy would not be so acute, and would not be so reversible. Presumably an acute and reversible effect on the function of the brain, has to do with change in the normal acute regulation of that function of the brain, that is, change in the physiological regulation of neural activity, and not a change in neuroanatomy.

If a fever can induce more normal behavior in an ASD child, obviously the brain of that ASD child has the neuroanatomy to support that more normal behavior. It simply needs better acute regulation of that neural activity to produce behaviors that are considered more normal.

Something as important as neural activity has to be under feedback control. While we don't know the details of that feedback control, we do know that it has to have a "setpoint", and mechanisms that sense deviations from that setpoint and which then act to restore what ever physiological processes are involved back to that "setpoint" when there is deviation. That "setpoint" is probably something dynamic and reflects the dynamic needs of the brain region(s) being activated (deactivated) at that particular time.

Dysregulation can either be good regulation around a bad setpoint, or bad regulation around a good setpoint (or some of both). Bad regulation can be simple. Good regulation of something as complex as the brain has to be extremely complex. A change from bad regulation to good regulation is necessarily also complex. For something "simple" to change regulation from bad to good and then back to bad, presumably a small subset of the regulatory pathways are involved. For something "simple", (a fever) to change the regulation of multiple individuals from bad to good, implies that the "bad" regulation is not idiosyncratic, but common to all of the normalized individuals. This implies the dysregulation of neural function in ASDs is good regulation around a bad setpoint and that a fever somehow shifts that setpoint(s).

This is an extremely important conclusion. It supports the idea that the ASDs are a unified set of symptoms from a unified set of causes, and not idiosyncratic with each person having a different set of reasons for their ASD symptoms. ASDs have global characteristic physical symptoms, implying that the shifted setpoint(s) is also global and affects physical as well as mental symptoms.

The brain is composed of neurons, some of which are inches long, and which transmit signals from one part of the brain to another. There is considerable thought that in ASDs, there is reduced connectivity, both constitutive (that is via neuroanatomy), and also functional (that is via the normal regulation by what ever it is that regulates neuronal activity). If the change due to fever is transient, then it is not constitutive, rather it must be functional.

As a neural network doing computations, the brain is a "small world" network with each neuron having more than 10^4 or so connections. Most connections are local (80% less than 2 mm), but a small fraction are long range. When a neuron fires, on average one "downstream" neuron fires. If on average more than one downstream neuron fired, the number of neurons firing would increase exponentially, and soon every nerve in the brain would be firing, leading to a seizure. Similarly, if less than one neuron fired the avalanche of neurons firing would extinguish and neural activity would stop. Thus there is a delicate balance between neurons firing and not firing, and exquisite control of that balance. The regulation of which and how many neurons fire is obviously an important part of the normal regulation of neural activity.

It is this regulation of nerves firing that sets the functional connectivity of the brain, and that functional connectivity is regulated to be in the near percolation threshold. All natural neural networks self-regulate in the near percolation threshold because that is where the network is most sensitive to change. The percolation threshold is a critical point; that is the properties of the network change exponentially with respect to the connectivity in the near percolation region. At the percolation threshold, the sensitivity of the network is infinite, as you go away from the critical point that sensitivity decreases exponentially.

There is a large difference between behavior of a network below and above the percolation threshold. Above the threshold, the network is connected and can function with increased reliability, but with reduced sensitivity. Below the critical point, the network becomes increasingly disconnected and the global functionality of the network rapidly collapses. I think a degree of this disconnection is (sometimes) adaptive in stress, and results in an improved capacity for multi-tasking (but only of simple tasks).

I think it is operation in the sub-critical connectivity region that causes the decreased functionality of the brains of people with autism. To increase the functionality, the connectivity of the brain must be increased. Presumably that is what is happening during the fevers that are causing normalization of behaviors. If someone with connectivity above the critical point got a fever and that increased their connectivity still more, they would experience degraded functionality.

The functionality of the network changes exponentially with respect to connectivity in the near percolation threshold. That is why the brain can be regulated. A slight change in connectivity changes its properties a large amount. This is inherently a highly non-linear process. A linear model is completely inappropriate.

To normalize the activity of an extended object as complex as the brain, presumably the "something" has to act on large parts of it, encompassing whole neurons. While the brain is well vascularized, the blood brain barrier blocks the entry of many compounds in the blood from getting into the CSF and affecting the brain. Virtually all of the compounds that do enter the brain, do so via active transport and are quite well regulated.

A fever is an acute activation of the immune system, preparing the organism to deal with what ever infectious agent has elicited the fever. A number of cytokines are produced, some of which are pro and anti-inflammatory. These cytokines don't normally enter the brain and CSF (very much) and so their presence in a fever would not be expected to result in normalized neural activity. Normal individuals don't normally express cytokines or their receptors at the levels that ASDs express cytokines during a fever, so presumably the normalization of brain function is not due to the cytokine response per se, but rather to a "normal" constituent of "normal" physiology that is transiently upregulated (or down regulated) during an immune stimulation.

There is a very recent review of neurological effects of immune system stimulation. It is mostly focused on the "sickness behaviors" that inflammation and other immune system activation cause and suggests that disorders such as depression may have components that are influenced by the immune system. While the focus is on pathology, they don't discuss any mechanisms that would have the effect of normalizing behaviors.

One effect of cytokines is expression of indoleamine 2,3-dioxygenase which lowers levels of the essential amino acid tryptophan. This does deplete levels of tryptophan, but the time course of that depletion is longer than was the course of fever in this study. Tryptophan is the precursor of serotonin. In cancer patients undergoing cytokine therapy, there is decreased mood associated with reduced serum tryptophan levels. Interferon does cause depression, and that is the major side effect of interferon therapy for cancer, hepatitis. Interferon can also cause delirium, psychosis, and even mania. Cytokines do affect brain neurochemistry. However, the levels of cytokines in non-infected individuals are usually small, and cytokines are usually associated with sickness behaviors, usually depressive symptoms. Cytokine effects have not been clearly linked to any major disease. It would seem unlikely that the cytokines which normally cause "sickness behaviors" in NTs, would result in a normalization of behavior in ASD individuals. Particularly at the mild levels likely in the modest fevers these children had. Most cytokine effects are mediated through transcription. Transcription in neurons occurs in the cell body, and the products have to be carried out to the tippy ends of axons for those axons to be affected by those transcription products.

There are other cells in the brain that are affected by cytokines that are not so extended as neurons. Cytokines might induce more rapid changes in them, however for neurological behaviors to be affected, neurons must be affected. Non-neuronal cells may be affected by cytokines and then communicate that change to neurons via some other signaling molecule. If that signaling molecule is NO, then it is the NO that is normalizing the neuron behavior. Axons are mostly covered in myelin, so any signaling molecule would need to be able to get into the neurons so as to affect them. Myelin is permeable to NO, most everything else would require specific receptors.

So what aspect of a fever is normalizing the regulation of neuronal activity?

Presumably it is "something" that is present at an appropriate level in non-ASD individuals at all times, but is transiently increased (or decreased) in these ASD individuals during their fever. Presumably it is a signaling molecule, because any feedback mechanism is essentially a signaling process.

The paper suggests a number of possible mechanisms which may be involved, although in quite generic terms.

1. neurobiological effects of selected proinflammatory and/or anti-inflammatory cytokines.
2. modification of neuronal and synaptic function secondary to variations in body temperature that influence neural conduction velocities or synaptic transmission,
3. modification of dynamic neural networks as a result of changes in cellular signal
transduction and gene transcription that regulate synapse formation and function,
4. increased production of other stress-related proteins, such as heat-shock proteins,
during fever that might modify energy consumption and mitochondrial activity,
5. stimulation of the hypothalamic-pituitary-adrenal axis leading to modifications
of neurotransmitter production and interaction.

While these mechanisms could well modify the function of the brain, it is difficult to see how the specific effects of a fever (or the immune system activation accompanying a fever) would acutely modify the function of the brain of someone with an ASD through any of these mechanisms to produce an acute normalization of function. For these mechanisms to produce acute normalization, implies that the dysfunction of ASDs is due to a dysfunctional level of such things in the non-fever state in ASD individuals. There is no evidence that any of the levels of any of these parameters are causal in ASDs. There is evidence that all of these systems are perturbed in ASDs (except for temperature perhaps), but that likely results from the coupling of all of physiology together. One aspect of physiology cannot be modified independent of other aspects. Dysregulation of diverse physiological systems in ASDs implies a shifted setpoint(s) in all of those physiological systems.

Could the regulation be improved simply by temperature? Doubtful. Perturbed body temperature isn't reported or observed in ASD individuals, and a change in regulation of neuronal function by a change in body temperature away from normal would not be expected to normalize function. Perturbing the body temperature of normal individuals to lower doesn't result in ASD behaviors. Perturbing temperature away from normal would be expected to decrease fidelity of control, not increase it. Temperature gradients in the brain during a fever are likely greater than during a non-fever simply because control is not expected to be as good. If there were temperature perturbation of normal neuronal function in ASDs, it would likely be due to specific structural or anatomical changes, changes in structures of proteins, in lipid concentrations, in lipid-protein interactions, in lipid raft properties. Different structures for common neuron components, such as ion channels sufficient to perturb temperature dependence of function in such characteristic ways would be pretty obvious in genetic studies. Genetic studies haven't found that, so that likely isn't what is happening.

Fever may modify synapse formation; however the promptness of the improvement and the promptness of the return to baseline function implies that the changes are not due to structural changes in neuroanatomy implied by synapse changes. If new synapses did form, they wouldn't disappear when the fever passed.

Stress related proteins? Stress related proteins are extremely well regulated (as are all stress responses). Psychological stress exacerbates many symptoms of ASDs. Stress related proteins are regulated locally to each cell. In a neuron, they would necessarily be synthesized in the cell body where the DNA is. They would then need to be transported out the axon to the tippy end. For the proteins to be cleared after the fever passes, they would need to be transported back to the cell body for disposal and recycling. Any kind of transcription regulation has this same problem. It takes time for the proteins to be expressed and transported to the ends of the neurons where (presumably) they have their effects. The promptness of the normalization, and the promptness of the reversion after the fever passes argues against transcription mediated mechanisms (at least transcription inside neurons).

It is likely that it is the longest length connections that are normalized. Why? There are many more short range connections, so there is more redundancy in them. Simple neurological effects such as sensory processing have not been reported to be disrupted in ASDs (they are actually improved). The effects that are disrupted are very complex neurological properties, such as behavior, social interactions and communication. The importance of a few long range connection in the brain (as a "small world" network) are most important in determining global properties, less important in determining local properties such as sensory processing.

Pro-and anti-inflammatory cytokines can have large effects on most any tissue, but there isn't much evidence that they are responsible for the acute neuronal regulation of the types of behaviors that are characteristic of ASDs. Immune suppression drugs have not been reported to produce a normalization of ASD behaviors.

No doubt there are many compounds with different concentrations in ASD and non-ASD individuals. However to normalize behaviors, a compound with a changed concentration during a fever must have specific properties and effects. To summarize, the agent responsible for a normalizing of neuronal behavior during a fever in ASD individuals has to have a number of properties:

1. normal CNS signaling molecule
2. active range in both fever and non-fever
3. present in normal CNS, body
4. perturbed levels in ASD CNS, body
5. change in fever results in a more normal level in ASDs
6. passes through blood brain barrier
7. change up and/or down is rapid
8. change not easily induced by other means

CO2 is reportedly decreased during fever (observed as an increase in blood pH due to hyperventilation). Supplemental CO2 does extinguish those febrile seizures. In rats, maximum susceptibility to febrile seizures occurs at an age when the ventilatory response to CO2 is at a minimum. However neither hypercapnea nor hypocapnea produce symptoms characteristic of ASDs, so a resolution of hypercapnea during a fever would not be expected to resolve them. Rett Syndrome is characterized by breathing regulation irregularities, which may well indicate abnormal regulation of breathing by CO2. However other ASDs are not characterized by breathing abnormalities. People with known genetic causes of autism (such as Rett Syndrome) were excluded from this study.

Nitric oxide does have all of these properties. It is lower in autism and ASDs, it is acutely increased in fever. During immune system activation, iNOS is expressed, and NO levels are increased. The production rate of NO can get very high. However, the basal level of NO still does not get above ~10 nM/L (0.3 ppb) which is the EC50 for sGC. If the basal level did get to 10 nM/L, there would be 50% activation of sGC and generalized vasodilation leading to severe hypotension. There can be severe hypotension during septic shock, and that can be sufficient to be fatal, but it isn't that high in a mild fever, and didn't get that high in these ASD individuals who normalized their behavior during their fever (if it did, they would have collapsed from hypotension).

The low NO ratchet

The mechanism for the NO increase is that activation of NFkB causes the expression of iNOS, which generates NO via open loop control, that is, the NO generated is not regulated other than by the amount of iNOS produced and the availability of substrates and the presence of inhibitors. This NO inhibits NFkB and prevents the expression of more iNOS. Thus the level of NO before NFkB activation determines in part the amount of NO after NFkB activation. However it is an inverse regulation. The lower the initial NO level, the higher the iNOS expression and the higher the ultimate NO level. This high NO level then reduces the expression of eNOS and nNOS, lowering the basal NO level when the iNOS is degraded in a day or so.

This behavior of NO physiology is extremely important. When basal NO is low, any immune system activation raises NO levels higher (due to less inhibition of NFkB) than if NO was higher before immune system activation. I hypothesize that this can lead to what I call the low NO ratchet, where activation of the immune system under conditions of low basal NO causes basal NO levels to ratchet lower each time the immune system is activated. I think this is one of the fundamental causes of regressive autism, and also of low basal NO in adults as characterized in chronic fatigue syndrome (CFS). Many people with CFS can identify when they acquired it, and it corresponded with an acute serious infection. Similarly, many parents anecdotally identify the immune reaction of a vaccination as a precipitating event leading to regression. However the large scale epidemiology shows no change in incidence of autism with changes in vaccination. My hypothesis is that in susceptible individuals, any immune system activation is sufficient to activate the "low NO ratchet", a vaccination, or one of the zillions of infections of childhood. It is the low NO ratchet that (I hypothesize) causes Gulf War Syndrome. Receiving multiple immune system activations (vaccinations) during a high stress period (being deployed to a war zone) causes basal NO to ratchet lower with each immune system activation until it saturates and produces chronic fatigue. This takes a few weeks, while the mitochondria turn-over and are not replaced (due to the low NO level from the chronic stress). Once mitochondria numbers are low, the low NO state is perpetuated due to superoxide from too few mitochondria being pushed to higher potentials to supply the same ATP. With continually low NO, mitochondria biogenesis can't occur enough to get back to the level that is "normal".

There are some reports that there are WMH observed in a subset of CFS patients, and some that there are not. The primary symptom of CFS is muscle fatigue, implying insufficient mitochondria in muscle to supply sufficient ATP for continued exertion. Mitochondria biogenesis in both tissues is initiated by NO, but the details of that regulation, including time constants may be quite different. Muscle has storage of ATP equivalents as phosphocreatine in much larger quantities than does the brain, and muscle can derive ATP from glycolysis which neurons cannot. While there should be some similarities, we should not expect identical behaviors.

If an acute fever reduces the symptoms of ASDs, is malaria "protective" against acquisition of an ASD? It would appear not. The epidemiology of ASDs in regions where malaria is endemic is not good, however there was a case series from Tanzania where medical histories of 14 children with autism were examined, and 3 of 14 were reported to definitely occur precipitously with malaria infection, 4 of 14 possibly coincident, including cerebral malaria and one case of Salmonella meningitis. 7 of 14 were reported not associated with a severe infection. The onset of autism in many of these children was many years earlier. Presumably they continued to be exposed to malaria while living where malaria was endemic and this ongoing malaria exposure did not "cure" them of their autism. This is malaria from P. falciparum which is the most severe type, and not the milder form from P malariae. 4 of 14 cases had dysmorphic facial features, and none of them had genetic work-ups, so how comparable these cases are to standard cases where malaria is not endemic is unknown. The autism in some of these cases may be quite different than in the cases in the current fever study (which did exclude known genetic causes). There may have been brain damage due to these very serious infections. The degree of immune system stimulation from cerebral malaria is completely different than the mild immune system stimulation from childhood vaccines. Childhood malaria is a leading cause of death where it is endemic.

The blood brain barrier is completely transparent to NO which has physical diffusion properties close to that of O2. Normally, blood is not a source of NO, but rather O2Hb is the sink for NO. NO is produced by the endothelium by eNOS, and also in nerves by nNOS.

Precisely where immune system activation increases NO production is not well characterized. Mostly it is thought to be generated in immune cells, which are spread throughout the body. Some in the blood, but most in the extravascular space and especially in the lymph nodes and at specific sites of infection where those cells are attracted. The NO they produce locally is important in inducing the hyperemia of inflammation. The supply of O2Hb by that hyperemia keeps the NO level from getting much above ~10 nM/L. Sites of infection do attract immune cells, and the NO they produce is enhanced under conditions of fever. Local concentrations might get very high, but those very high concentrations can't be systemic or there would be systemic vasodilation and systemic hypotension.

Normal brain activation can be monitored using BOLD fMRI. What is measured is the magnetic susceptibility of deoxyhemoglobin and how the concentrations of that change before, during and after neuronal activation. The precise physiology that is going on is mostly unknown. The suggestion is that the increased blood flow is primarily nutritive, however the flow exceeds the nutritive demands to support the increased metabolic load. There has been a suggestion that NO is related to this. I think the idea that NO is related is precisely correct, and that in fact the dominant physiological effect is the release of NO. This NO blocks O2 uptake by cytochrome c oxidase, increasing O2 levels. The NO also activates sGC producing cGMP which relaxes vascular smooth muscle and causes vasodilation and increased flow of O2Hb to the NO affected site. This O2Hb then takes up the NO and converts it into nitrite and nitrate at near diffusion related kinetics. The flow takes the nitrite and nitrate away and the vasodilation subsides. The vasodilation directly relates to the magnitude of the NO signal, and so the blood flow directly relates to the NO signal. Under conditions of low basal NO, the NO signal reaching sGC is less, less cGMP is produced, there is less vasodilation, and less blood flow. I think that low basal NO is the precise reason for the reduced cerebral blood flow observed in autism, and in other neurological disorders characterized by reduced blood flow.

Because the "normal" increased blood flow exceeds the nutritive requirements for the metabolic burst in activity, when that blood flow is reduced, so will be the metabolic burst in activity. Over time, the metabolic rate of that region of the brain will be reduced. This is not "dysfunction", the metabolic rate is being controlled by the "normal" control parameters, the "problem" is that the "setpoint" is bad. This is how low basal NO leads to eventual neurodegeneration.

This NO has a lot of simultaneous effects in the local site. NO regulates long term potentiation. NO binds to heme and inhibits heme enzymes in mitochondria, but also in microsomes where the cytochrome P450 enzymes make a variety of neuroactive compounds such as steroids and prostaglandins. NO destroys superoxide, and so regulates signaling via superoxide. When superoxide is destroyed by NO, peroxynitrite is produced, which can nitrate proteins and affect enzyme activity.

NO diffuses through myelin, and so NO can affect mitochondria (and other things) inside axons that merely pass through the NO affected region. That is, there need not be any synapses or other neural terminations for NO to have effects on axons in the vicinity of the NO release. Nerves that are not connected by a synapse must communicate in some way before a synapse can be formed.

NO regulates synaptic remodeling, including synaptogenesis following injury, no doubt it regulates it before injury too. Many growth factors have effects mediated by NO, they are endocytosed in receptors where nNOS is activated and the combined receptor/growth factor/nNOS complex is transported to endosomes for recycling. NO may modify those endocytosed complexes via nitration.

There are many ways by which NO affects neural activity. I am cutting short the discussion here because I could go on for many pages, and that isn't necessary. There are many mechanisms by which NO regulates neural activity. Which ones are important in which region of the brain is unknown but it is likely that many are important with different levels of importance in many (probably all) regions of the brain, and that those differences may well be idiosyncratic and depend on genetic as well as epigenetic factors.

Many neural mechanisms known to be affected in autism have effects mediated by NO, and are affected in the direction that would be expected if the basal NO level were lower. For example, maternal bonding is mediated through oxytocin and NO. While NOS is inhibited postpartum, ewes do not develop smell memories of their lambs. If NOS is inhibited and NO is supplied they do. Maternal bonding is the primary social behavior in mammals. If that depends on NO, likely many other social behaviors (if not virtually all) do too.

Many organs are known to be programmed in utero in humans, including heart, liver, kidney, vasculature, pancreas, immune system, bone, endocrine system, reproductive system, muscle. Some of those programming mechanisms involve NO. It would be surprising (actually beyond surprising, it would be inconceivable) if the most important organ, the brain were not programmed in utero too. There are some indications of long term programming of the brain, for example the cycle of violence.

Therapeutic considerations.

The objective of any treatment is to restore normal function in the long term without producing adverse effects. That fever produces a transient improvement implies that function is only abnormal due to good regulation around a bad setpoint. To successfully treat this requires shifting the setpoint long term. Simply moving the setpoint temporarily (as in a fever) won't produce long term normalization. Attempting to do this long term (as in a permanent fever) is extremely likely to induce side effects, because physiology will most likely adapt to the perturbation.

In fever therapy, malaria was used to induces multiple bouts of fever in rapid succession (with a 4 day period). The time constant for iNOS expression and degradation is about a day or so. One of the things that maintains low basal NO is reduced mitochondria number, which pushes mitochondria potential to higher levels (to generate the same ATP), which generates more superoxide which perpetuates low NO and retards mitochondria biogenesis. NO from iNOS during a fever would override all of that and produce high NO during mitochondria biogenesis and so increase mitochondria levels. Mitochondria are generated at night during sleep, so multiple cycles of fever as used in fever therapy could result in increased mitochondria numbers and may resolve a condition of insufficient mitochondria. This might be a mechanism by which multiple bouts of fever may have very different effects than single bouts. Single episodes of fever may activate the "low NO ratchet" and worsen chronic low NO. Multiple bouts of fever may activate processes with different time constants (for example mitochondria biogenesis) which may over multiple episodes counter the adverse effects which may occur in single episodes. NO physiology is not well enough understood to predict this a priori, and some individuals may have idiosyncratic responses.

NO physiology is under intense regulation. There are no generally accepted methods for raising NO levels long term. Many pharmacological methods have been tried. There has been no report of a long term successful trial at raising NO systemic NO levels or in ameliorating chronic disorders characterized by low NO through any pharmacological treatment. There are short term methods that have been used, but they only work short term. So far there has always been compensation if the study was extended long enough. Most studies have been too short to show this.

NO can be given in inhalation air, however there are essentially no systemic effects. The NO is converted to nitrite and nitrate by oxyhemoglobin in the lung even before the blood leaves the lung. In air, NO is rapidly oxidized to NO2, which is toxic at ppm levels (NO is non-toxic at hundreds of ppm). Inhaled NO must be continuously monitored for NO2, and can only be done in a hospital setting.

NO can be given intravenously, as actual NO, or as sodium nitroprusside. NO and SNP have very short lifetimes (few minutes at most), and can only be administered in hospital settings.

Organic nitrates are sometimes (erroneously) referred to as "NO donors". They are not NO donors. Organic nitrates such as nitroglycerine are sometimes given, however they produce what is called "nitrate tolerance", where the therapeutic effect are diminished and oxidative stress effects occur. The precise physiological details behind these effects are unknown.

L-arginine is the substrate for nitric oxide synthase, and when given acutely, does transiently raise NO levels. However over time, the increased NO is diminished and long term use of L-arginine doesn't improve low NO mediated diseases such as peripheral artery disease and perhaps may lead to "arginine tolerance". This is thought to be due to upregulation of NOS inhibitors such as asymmetrical dimethyl arginine and arginase which turns arginine into ornithine plus urea. Production of urea via arginase may be a mechanism to increase urea excretion in sweat to increase NO/NOx via biofilm (my hypothesis)

Oxidative stress does lower NO levels by consumption of NO via superoxide. There is some data in the literature that short term administration of antioxidants does transiently resolve some symptoms of oxidative stress and presumably those of low NO. However there is no data to suggest that long term administration of antioxidants has any therapeutic benefit and much to demonstrate that it doesn't. Every long term placebo controlled trial of antioxidants has shown zero or slightly negative effects. A large meta-analysis has shown this quite clearly. It has been suggested that oxidative stress is a regulated parameter of physiology, and that a state of oxidative stress is due to good regulation around a bad setpoint rather than bad regulation around a good setpoint. This is discussed in Chapter 8 of Oxidative Stress: Clinical and Biomedical Implications. This explanation fits the data observed in all the large placebo controlled trials, and also explains the very robust observation that a self-selected diet rich in antioxidants is inversely correlated with the diseases of oxidative stress. The conclusion is that a self-selected diet is part of the control system that physiology uses to regulate the state of oxidative stress that the setpoint calls for. Excess antioxidants must be destroyed at some metabolic cost; in a high oxidative stress metabolic state it is better to not consume them in the first place. In other words, individuals with a high oxidative stress setpoint self-select a diet low in antioxidants. This is why in the JAMA review article consumption of excess antioxidants correlates (slightly) with adverse effects. There is a metabolic cost to destroying those excess antioxidants.

Whole body vibration does produce bone strengthening. The primary mechanism for regulation of bone stiffness is via NO released during bone strain. This NO increases deposition of bone mineral where NO levels are highest, which corresponds to where bone has the lowest stiffness. Whole body vibration in sheep does release NO and reduced an immunogenic hyper-response and airway inflammation in sheep on antigen challenge. Whole body vibration is reported to be beneficial in muscle function and balance in the elderly. That is an effect that could be mediated by NO. However effects on bone density and muscle strength may be mediated through NO generated via vibration in those tissue compartments and by exerting only local effects. There may be no systemic effects, or no effects on the brain. Local vibration can cause neuropathy and also Raynaud's phenomena. Vibration is probably not a good method to try and raise NO levels in the brain. In vibration produced neuropathy, the conduction velocity of peripheral nerves is reduced. A reduction in conduction velocity in the brain would likely cause greater dysfunction due to asynchronous operation.

Meditation does raise NO levels, and systemic increases in NO have been measured. This is thought to be the mechanism for many of the beneficial effects of meditation. While meditation might be effective, teaching autistic children to meditate is likely difficult. I suspect that many "stimming" behaviors are actually things that increase NO levels which is why they are done, particularly during times of stress when more NO is needed. Humming certainly does.

Parasitic intestinal worms are an effective treatment of Crohn's disease and infection with helminthes and also malaria increases production of NOx metabolites in humans, demonstrating that NO production is upregulated via intestinal parasite infection and also during malaria infection. The precise interaction of intestinal parasites and malaria and NO is somewhat controversial. Some suggest increased NO is pathological and exacerbates malaria however for our purposes all we need to know is that malaria increases NO. NO is a very good anti-inflammatory agent (due to its inhibition of NFkB), and the positive effects of intestinal worms on Crohn's disease are likely mediated through NO as well. It has been suggested that the NO produced during infestation with intestinal worms is protective against malaria.

People with malaria induced anemia and people who have been infected with malaria and have recovered do have a higher basal level of NO/NOx and their immune cells have greater NO/NOx production capacity. NO/NOx levels correlate with severity of malaria infection.

However, while NO/NOx in malaria exposed individuals is higher, this high NO/NOx production is not always well correlated with higher NO/NOx production by blood mononuclear cells, suggesting that there is another source of NO/NOx. The NO/NOx production in individuals was stable over time and didn't correlate (in individuals) with their malaria status, suggesting that something else was controlling the NO/NOx level. They found NO/NOx production was much higher in the study subjects from rural PNG than in urban adult controls from northern Australia. This study was very interesting in that they tested the same individuals after their malaria had been cleared with antimalarial drugs (2-4 weeks later), and again (7 weeks after initial screening). They spend some time discussing the implications of a non-correlation of NO/NOx status with parasitemia, and how to rationalize this with earlier studies showing a correlation. I suspect that they haven't accounted for the source that I am studying. This source was not observed in controls from urban areas, and (my hypothesis) it may be a biofilm of ammonia oxidizing bacteria.

Repeated bouts of fever and infection is probably a more "normal" state than the disease free state that people like to maintain and are able to maintain in modern society. In other words, during evolutionary time, humans were not disease free, they evolved with a significant load of parasites and diseases. The NO regulatory systems probably work better under normal conditions of illness than in the abnormal disease free state. Restoring the NO status of the body to a more "natural" state will probably improve NO regulation.

For fever therapy to achieve long term recoveries, it had to restore regulation of neuronal physiology long term, that is even after the malaria was cured with quinine. Modern measurements to show that NO/NOx levels are raised following malaria infection even after that infection has been cleared. I think this strengthens the case for NO/NOx elevations during a fever as being at least part of the mechanism for the short term resolution of behavioral symptoms and the long term resolution following fever therapy with malaria.

Mitochondrial disorders are associated with white matter hyperintensities. I think that long term oxidative stress is a generic factor that can lead to (or exacerbate) essentially any type of neurodegeneration via inhibition of mitochondria biogenesis and reduced ATP levels and reduced transport of essential cell components in axons. I suspect that the paralysis of neurosyphilis is actually due to the low NO from neuroinflammation, rather than from destruction of nerves. If it were from nerve destruction, the paralysis wouldn't be reversible in a day. If the state of oxidative stress is due to insufficient mitochondria, then the high NO state from fever therapy would have to persist long enough for the mitochondria number to be raised sufficiently to drop the mitochondrial potential, reduce the superoxide production, and normalize the basal NO level. This has to occur over the entire length of the axons in the brain. This cannot happen in a short period of time. Mitochondria must be synthesized in the cell body and transported out the axons. Presumably it would take on the order of the normal mitochondria lifetime to attain a new stable steady state. 10 cycles of fever from P malariae would take 40 days. This is probably long enough for a new steady state to be produced. It would be unreasonable to expect recovery in less time. Even if behaviors are normalized, the feedback regulatory system may not be. It is the feedback system that must be normalized for any recovery to persist long term.

Exposure to pyrogens such as lipopolysaccharide (LPS) causes acute expression of iNOS and acutely higher NO levels. LPS also causes LPS tolerance, where there is a refractory period where additional LPS does not elicit as much of a response. That tolerance is due in part to NO generated by iNOS in activated macrophages. I suspect this feedback inhibition is what prevented non-infective fevers from being effective treatments of neurosyphilis. The high NO state was not maintained long enough for the regulatory pathways to compensate and form a new steady state.

Fever therapy was first tested and was in use before the modern era of daily bathing and daily hair washing with detergents and antimicrobial agents. I think it is likely that institutionalized patients were not bathed every day, and so had an abundant biofilm of the bacteria that I am working with. Malaria is characterized by fever, sweating and chills. The periods of sweating would have nourished any biofilm of ammonia oxidizing bacteria and would likely have contributed to any therapeutic effect of increased NO. Similarly the above results on NOx from malaria and intestinal parasites were done on native people living in "the wild", so presumably they had a more natural biofilm of commensal bacteria, but the actual status of their biofilm is unknown. Increased sweating providing more substrate to their biofilm may have been one of the mechanisms for the increased NOx that was observed.

Presumably the ASD children in the fever study did bathe with normal frequency and so did not have a biofilm of ammonia oxidizing bacteria, and any NO they produced during their fever was generated by iNOS.

A more constant (but still regulated) source of NO, from a biofilm of autotrophic ammonia oxidizing bacteria will improve natural regulation of NO physiology. How much more, and will that improvement be enough to achieve the effects of this study of fever in autism is unknown and unpredictable a priori. However, if any symptoms of autism are caused by low basal NO, then raising basal NO levels will improve those symptoms with no threshold.

NO/NOx from a biofilm of the bacteria I am studying is the method that I prefer and would suggest. It is the only method that is under direct physiological control via sweating. Sweating releases ammonia to the biofilm, and the biofilm delivers NO and nitrite in less than a minute. I think this is the reason for non-thermal sweating, as adrenergic sweating during stress, shock, and when a fever breaks. These autotrophic ammonia oxidizing bacteria are common in the environment, are incapable of growth on any media used for isolating pathogens, and have never been implicated in any infection. An "infection" by them may not be possible (even in immunocompromised individuals) because they are obligate aerobes and lack all virulence factors and cannot metabolize or derive energy from animal substrates other than ammonia.

If my hypothesis is correct, that humans evolved to have these bacteria growing on our skin and providing us with regulated levels of NO/NOx, then normal physiology cannot be attained without them. There is no conceivable artificial mechanism that could provide the same regulatory fidelity in terms of time, dose, and delivery mechanism. NO physiology remains mostly unknown. We know that there are thousands of pathways that involve NO. We don't know the complete details of even a single one of them and we know they are all coupled. There is simply no conceivable method for artificially regulating NO to achieve the proper levels for optimum function of a single one of those thousands of pathways let alone for all of them simultaneously.

In summary

I have shown a plausible case for the improved behaviors observed in this study being due to an acute increase in NO due to expression of iNOS. There may be other effects from other immune system compounds, but using actual disease organisms treat autism the way that malaria was used to treat neurosyphilis is a very high risk approach. Unless the immune stimulation is long term (as in parasitic worm infestation), rebound and the "low NO ratchet" effect is to be expected. This could well exacerbate ASD symptoms in the long term, even if there is improvement at each cycle.

Any questions?