Considering the Scaffolds of Interconnectedness, “Environmental enrichment alters glial antigen expression and neuroimmune function in the adult rat hippocampus” and How Seeing The Obvious Can Still Be A Pleasant Surprise
Posted on: March 9, 2012
- In: Antigens | Autism | BDNF | Beautiful Complexity | Bilbo | Brain | Developmentall Programming | Environmental Enrichment | Glial Priming | IL-6 | Immunology | Inflammation | Intriguing | LPS | Microglia | Researchers | Tnf-Alpha
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I’ve been thinking a lot lately about the beauty and trials of the tightly coupled systems, the interconnected pathways that keep popping up when pubmed tells me something that might be of interest on journey autism. One theme bubbling to the top of my thoughts is that there is a large set of inputs capable of tweaking the areas we see altered in autism; broken isn’t necessarily appropriate, but the research increasingly tells us that a delicately balanced set of connected processes is readily changed, and the way that the physics work out, there is no way to change just one thing when you have a polygamous marriage of chemical systems.
Imagine a orchestra where all of the musicians were physically bound to one or more of their counterparts, a system of wires, pulleys, springs and levers such that the musicians are actually participating in the playing of each other, not soccer players doing synchronized flips so much as a set of violin-em-cello-em robots, connected to play their instruments in unison, wind them up and create a symphony. Different orchestras might have a tighter wire from one member to another, or an older spring, but when they worked together, you could tell what composition they were playing. In this analogy, you cannot have the drummers start beating harder and faster without also changing how hard the French horn players blow. The situation only gets more complicated if some of our musicians were connected to several other musicians simultaneously. There would still be music if the cellist couldn’t keep a steady rhythm, but it would be different music, not just a different cello.
The communication between a lot of our “systems”, immune, endocrine, stress response and central nervous systems are a lot like musicians in the orchestra, interdependent and intimately connected.
The funny thing is, this same message is being blared to me, and to you, all the time, damn near every time you turn on the TV, but it is hidden in plain sight by legislatively mandated doublespeak. Consider how many advertisements each of us have seen for pharmaceutical drugs where the number of complications and contra-indicated conditions far, far exceed the number of desired effects?
Here is a list of common side effects of Viagra:
Diarrhea, dizziness, flushing, headache, heartburn, stuffy nose, upset stomach
So right off the bat, besides what we are looking for, we can see it is common to expect Viagra to also affect your GI system, immune system, and/ or brain function. These are the types of things that are “common”. (One wonders how Viagra would sell if it always caused headaches and diarrhea, and sometimes transiently ameliorated erectile dysfunction? ) A list of ‘severe’ side effects includes memory loss and a sudden decrease in hearing or vision. Even after decades of work by a lot of exceptionally smart people and hundreds of billions of dollars, the interlocked complexity of our bodies are continuing to prove very difficult to adjust in only the way we’d like, and seemingly minor perturbations in one area can pop up in very unpredictable fashion in other functions.
Trying to put my mind around the implications of this in regards to autism often leaves me with a sense of being profoundly humbled and woefully underprepared, not unlike a lot of my experiences with autism in the real world. Secondarily, again with great similarity to personal experience, I (eventually) come to the (re-)realization that we should rejoice in opportunities to be challenged and learning more about something makes us richer in ways more important than dollars.
A superb example of all of this and more landed in my inbox the other day, Environmental enrichment alters glial antigen expression and neuroimmune function in the adult rat hippocampus (Williamson et all). [Also on this paper, blog favorite, Staci Bilbo]
Williamson reported that animals given a so called ‘enriched environment’ exhibited significantly decreased immune responses in certain portions of the brain following immune challenge, with reduced levels of several chemokines and cytokines in the hippocampus in the treatment group. (A previous discussion about environmental enrichment on this blog can be found here) In this instance, the treatment group got to spend twelve hours a day in a different area, a housing unit with “a running wheel, a PVC tube and various small objects and toys”, while the control group of animals stayed in their drab, Soviet era proletariat cages all day and all night long. Here is the abstract:
Neurogenesis is a well-characterized phenomenon within the dentate gyrus (DG) of the adult hippocampus. Environmental enrichment (EE) in rodents increases neurogenesis, enhances cognition, and promotes recovery from injury. However, little is known about the effects of EE on glia (astrocytes and microglia). Given their importance in neural repair, we predicted that EE would modulate glial phenotype and/or function within the hippocampus. Adult male rats were housed either 12h/day in an enriched environment or in a standard home cage. Rats were injected with BrdU at 1week, and after 7weeks, half of the rats from each housing group were injected with lipopolysaccharide (LPS), and cytokine and chemokine expression was assessed within the periphery, hippocampus and cortex. Enriched rats had a markedly blunted pro-inflammatory response to LPS within the hippocampus. Specifically, expression of the chemokines Ccl2, Ccl3 and Cxcl2, several members of the tumor necrosis factor (TNF) family, and the pro-inflammatory cytokine IL-1ß were all significantly decreased following LPS administration in EE rats compared to controls. EE did not impact the inflammatory response to LPS in the cortex. Moreover, EE significantly increased both astrocyte (GFAP+) and microglia (Iba1+) antigen expression within the DG, but not in the CA1, CA3, or cortex. Measures of neurogenesis were not impacted by EE (BrdU and DCX staining), although hippocampal BDNF mRNA was significantly increased by EE. This study demonstrates the importance of environmental factors on the function of the immune system specifically within the brain, which can have profound effects on neural function.
Total interconnectedness kick ass!
Considering the wide ranging and predominantly ‘rather-not-have-than-have’ properties of ‘extra’ TNF-alpha and IL-1beta in the CNS, this is a pretty interesting finding. Not only that, animals ‘protected’ through environmental enrichment also showed increased levels of growth factors known to be altered in autism, again in the hippocampus. In a very real and measurable sense, it was possible to shuffle the neuroimmune cocktail of the brain by changing things like the availability of quality leisure time. As we have seen in other areas, altering the chemical milieu of immunomodulatory factors in the brain isn’t trivial, and is increasingly associated with a variety of conditions classically diagnosed through the study of behaviors.
It should be noted that there were unexpected, and generally negative findings from this study, namely, a relative lack of biomarkers indicative of increased neurogenesis in the environmental enrichment group; something that I think took the authors by a bit of surprise.
There is a short discussion on the possibilities on why the findings of differential neuroimmune responses were found only in the hippocampus, with reference being made to previous studies indicating that this area of the brain has been found to be more susceptible to a variety of insults.
There were some other findings that struck me as particularly intriguing; something that has been hinted at previously in other studies (or transcripts), but not yet well described, likely due to the fact that the area is still largely unknown to us. Specifically, the authors reported a state of glial activation, somewhat the opposite of what they expected.
The data instead suggest that EE changes the phenotype of glia, altering their activation and attenuating their pro-inflammatory response to peripheral LPS, although this remains to be directly tested. Interestingly, the blunted neuroinflammatory response within the DG of EE rats occurring concomitant with the increase in classical glial ‘‘activation’’ markers runs counter to our initial prediction. However, we believe these data simply highlight the fact that little is known about the function of these markers. Moreover, there is a growing literature that distinguishes classical versus alternative activation states in microglia, the latter of which is associated more strongly with repair (Colton, 2009; Colton and Wilcock, 2010).
And
Thus, it is possible that EE shifts microglia into an alternatively activated phenotype, an intriguing possibility that we are currently exploring.
(Totally sweet!)
The authors discuss the fact that their findings were highly spatially specific within the brain, involved a subset of cytokines and chemokines, and environmental enrichment did not seem to affect immune response in the periphery.
The immune response within the hippocampi of EE rats was markedly attenuated for a subset of cytokines and chemokines measured in our study. Importantly, not all measured immune molecules were blunted in the hippocampi of EE rats. Furthermore, the immune response was similar for each housing group in the parietal cortex as well as in the periphery. Within the hippocampus, however, EE rats had an attenuated response of interleukin-1b (IL-1b), the TNF family of genes, and several chemokines involved in the recruitment of leukocytes and monocytes. These families of genes indicate an altered hippocampal milieu in EE rats that may be less pro-inflammatory, more neuroprotective and less permeable to peripheral infiltrating immune cells.
There is a short discussion on the existing knowledge concerning IL-B and TNF-alpha in normal and pathological conditions, and how these findings are consistent with other findings involving environmental enrichment and cognition.
Tumor necrosis factor alpha (TNFa) is well characterized for its roles in inflammation and host defense, sepsis and, most intriguing for this study, apoptosis cascades (for review, see Hehlgans and Pfeffer, 2005). The observed attenuation after an immune challenge of TNFa and several associated genes in EE rats compared to HC controls indicates a potential enduring change in the hippocampal microenvironment of enriched rats, such that one mechanism by which EE may increase neuroprotection following insults to the CNS (Briones et al., 2011; Goldberg et al., 2011; Young et al., 1999) is via altered TNF tone and function, increasing the likelihood of cell survival by reducing apoptotic signaling. In addition to attenuated IL-1b and TNF responses, EE rats showed blunted responses for several chemokines known to influence the recruitment of circulating monocytes and leukocytes to the CNS.
Finally, the authors conclude how their findings add to the literature on environmental enrichment and brain function.
In summary, environmental enrichment is a relatively simple manipulation that results in robust beneficial outcomes for the brain. While previous studies have shown a role in post-insult rehabilitation for EE, our study provides evidence that enrichment need not follow the insult in order to be beneficial. Indeed, neuroinflammatory disease states might be attenuated or delayed in their onset in the face of ongoing EE. The translational reach of this manipulation remains to be explored, but in animal models of neuroinflammation, EE may provide a simple preventative measure for negative outcomes.
The bottom line is that a fuller rat life experience resulted in different neuroimmune profiles, findings with some consistency with previous observations that an enriched rat house resulted in improved behavioral manifestations of cognitive performance. The qualities of these different neuroimmune profiles are also consistent with chemical profiles associated with positive outcomes in several conditions.
There is a deceivingly startling realization hidden in these finding, startling because it reveals the malleable nature of the seemingly different, but basic systems interacting and deceptive because it is so obvious. How many of us have known someone who deteriorated upon entering a nursing home, or even retiring from working? How many of us have kept their children inside for a week due to weather and watched their children go crazy after the already inferior indoor entertainment options are long exhausted? Those changes in emotion, in behaviors and function, just like the findings from this study, are founded by chemistry.
But seeing evidence that relatively simple environmental modifications can rejigger the molecular atmosphere of the brain is still more than a little awe inspiring. Knowing there is machinery underneath the hood is a little different than observing the cogs of cognition swell , shrink, or slow down; nothing less than a deeper understanding of the chemical basis of thought. And that is pretty cool.
- pD
Extremely Long Overdue Clinical Findings “Impaired Carbohydrate Digestion and Transport and Mucosal Dysbiosis in the Intestines of Children with Autism and Gastrointestinal Disturbances”, and The Swan Song Of A Tragically Overused Autism Canard
Posted on: January 9, 2012
Hello friends –
My son was a ‘gut kid’. The irony is, for a while, because we were first time parents, we didn’t even know. My son was flagged for evaluation for autism around a year of age and we met with the autism center people several times between his first and third birthdays, with his official diagnosis coming just after he turned three. My wife came home from one of the early meetings convinced that his evaluators didn’t know the first thing about our son, autism, or anything else, and that in fact, they might be insane.
‘Do you know what those idiots asked me today?’
‘What?’
‘What his shits look like. My kid can’t talk and they want to ask me about his diapers!’
‘Who fucking cares?‘
We wound up caring, a lot. It turns out, this wasn’t a stupid question, it just seemed like one to us. The answer to their question was that our son was having at least four or six very messy diapers a day, his stools were never firm logs that look like they came from an spherical filter, but always, always more liquid than solid, and frequently contained chunks of identifiable food. But from our viewpoint, within the context of a child who was not speaking, hurting himself, and never looked at anyone, the idea that we should be worrying about his shit was the stupidest thing we’d ever heard.
But. When we started paying attention, starting reading, and started meeting more people with children with autism, our incredulity waned. We tried GF/CF and probiotics. We paid for lab tests to analyze the bacterial populations in his intestines. We experienced a life saving miracle with anti-fungal agents wherein my son essentially stopped hurting himself over the course of weeks after persistently banging his head dozens of times a day for six months. For nearly a year we removed all complex carbohydrates from our son’s diet, an intervention that makes GFCF feel like a Sunday afternoon after college but before kids and autism. We saw changes in our son based on how his GI tract was performing. For our son, for us, we knew that by some mechanism, what got put in his mouth, and what happened along the way was tightly coupled with how our son felt and behaved.
This is why my vision with spots of rage when I see the ideas of GI and dietary involvement with autism mocked by pseudo-skeptics so rampantly on the Internet. I cannot stand the thought of a single child continuing to suffer the way I watched my son suffer because they were told that there was no basis of GI interaction in autism. That thought hurts.
Those biases stated, we are now, finally, starting to see research indicating that in some cases of autism, there are very real, non imaginary differences in GI function.
A few months ago, Impaired Carbohydrate Digestion and Transport and Mucosal Dysbiosis in the Intestines of Children with Autism and Gastrointestinal Disturbances was published [full, dense, but very cool paper available online]. Here is the abstract.
Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism.
I’ll admit it. From the outside, from the don’t-have-a-kid-with-autism-and-GI-problems perspective, that is some dense and seemingly bland stuff. Essentially children with GI distress and children with GI distress and autism were compared and it was found that there were distinctly qualitative differences regarding the GI function in the groups. This is validation of what a lot of us have been saying for a long time, that the GI problems our kids were experiencing weren’t coincidental to the autism, but somehow related.
For anyone who has been paying attention to the details of the autism-gut debate, these are dynamite findings. These observations are the death knell for the overused, oversimplified notion that the GI connection to autism was a function of some kids having autism, some kids having GI distress, and that therefore, some kids with autism also have GI distress. This research tells us that the reality is not so simple.
This study is the view from the microscope as opposed to the telescope, and took care not to study just anyone with an autism diagnosis, but those with an autism diagnosis and GI distress, problems so severe that invasive procedures to obtain tissue samples from the GI tract was warranted. This is a critically important facet of the study design in my opinion, a lot of the negative findings in this arena have been epidemiological, and cast the widest possible net, capturing everyone with autism and comparing them with a sample of everyone else. This is a great strength of the paper; for too long everyone has acknowledged the heterogeneous nature of autism, but few studies have tried to understand differences at a phenotype level. This paper is different.
As evidence of the non-random population, the autism patient group had a regression incidence of over eighty percent, and nearly as many of the children in both groups were reported to have food allergies.
The details of the findings in the paper get deep pretty fast, but at a high level there were differences found in proteins involved with the digestion of carbohydrates and changes in bacterial populations between the groups, with some differences found with regard to specific locations in the intestine. Based on these findings, the authors speculate that alterations in carbohydrate processing could result in abnormal bacterial populations by way of altered microbial food availability in parts of the gut.
Based on these findings, we propose a model whereby deficiencies in disaccharidases and hexose transporters alter the milieu of carbohydrates in the distal small intestine (ileum) and proximal large intestine (cecum), resulting in the supply of additional growth substrates for bacteria. These changes manifest in significant and specific compositional changes in the microbiota of AUT-GI children (see Figure 7A–C).
The authors discuss a potential feedback loop of effects of intestinal microbes and nutritional processing, and of the known and potential effects of altered bacterial populations.
Additionally, intestinal microbes can influence the expression of disaccharidases and transporters [59] through the influence of pathogen-associated molecular patterns (PAMPs) and butyrate (a byproduct of bacterial fermentation) on CDX2 expression and activity [60], [61], [62], [63]. In this regard, the observation that CDX2 was decreased in AUT-GI children with increased levels of Betaproteobacteria may be important.
Whatever the underlying mechanisms, reduced capacity for digestion and transport of carbohydrates can have profound effects. Within the intestine, malabsorbed carbohydrates can lead to osmotic diarrhea [64]; non-absorbed sugars may also serve as substrates for intestinal microflora that produce fatty acids and gases (methane, hydrogen, and carbon dioxide), promoting additional GI symptoms such as bloating and flatulence [65].
This is very similar to the underlying theory of the Specific Carbohydrate Diet, impaired carbohydrate digestion promotes bacterial imbalances in the intestine by altered food availability, leading to gastrointestinal distress.
Because of the varied nature of the protein imbalances found and absence of the common alleles associated with such conditions, the authors report that it is unlikely that the underlying cause of the imbalances is genetically based.
In our study, 93.3% of AUT-GI children had decreased mRNA levels for at least one of the three disaccharidases (SI, MGAM, or LCT). In addition, we found decreased levels of mRNA for two important hexose transporters, SGLT1 and GLUT2. Congenital defects in these enzymes and transporters are extremely rare [40], [41], and even the common variant for adult-type hypolactasia was not responsible for reduced LCT expression in AUT-GI children in this cohort. Therefore, it is unlikely that the combined deficiency of disaccharidases (maldigestion) and transporters (malabsorption) are indicative of a primary malabsorption resulting from multiple congenital or acquired defects in each of these genes.
There are a couple of ideas presented on what might be causing the altered disaccharide transporter levels, with food composition intake, immune or hormonal irregularities, and bacterial populations and their associated fermentation byproducts listed as possible candidates. This study did not attempt to determine if any of these things were actually responsible, but an upcoming paper will also detail qualitative differences in expression of genes involved with inflammation in the autism group.
Regarding bacterial populations found, there were several differences identified by bacterial classification and location as well as some associations with onset of autistic behaviors and GI distress.
Pyrosequencing analysis of mucoepithelial bacteria revealed significant multicomponent dysbiosis in AUT-GI children, including decreased levels of Bacteroidetes, an increase in the Firmicute/Bacteroidete ratio, increased cumulative levels of Firmicutes and Proteobacteria, and increased levels of bacteria in the class Betaproteobacteria.
Stratification of AUT-GI children based on the timing of GI symptom development relative to autism onset revealed that the levels of Clostridiales and cumulative levels of Lachnospiraceae and Ruminococcaceae were significantly higher in AUT-GI children for whom GI symptoms developed before or at the same time as the onset of autism symptoms compared to AUT-GI children for whom GI symptoms developed after the onset of autism and compared to Control-GI children. However, we cannot discern whether changes in Clostridiales occurred before the onset of autism in this subgroup. We can only conclude that increased levels of Clostridiales members in biopsies taken after the development of both GI symptoms and autism are associated with the timing of GI onset relative to autism onset in this cohort. Although the reason for this association remains unclear, this finding may suggest that the timing of GI onset relative to autism is an important variable to consider in the design of future prospective studies investigating the microbiota of children with autism.
I am in love with the appreciation of the subtlety on display at the end, it may not be sufficient to simply categorize by GI and non GI autism, but also by the temporal relationship to onset of behavioral symptoms. It makes for a messy outlook going forward, but one based on pragmatism as far as coming to valid conclusions.
As is appropriate, the authors end with an admission that we are still largely groping in the dusk about how the microbiome interacts with our tightly coupled systems, but give a variety of reasons to believe that what we do know makes system wide effects reasonable and a relationship with autism plausible.
Metabolic interactions between intestinal microflora and their hosts are only beginning to be understood. Nonetheless, there is already abundant evidence that microflora can have system-wide effects [76], [77], [78], [79], [80], [81], [82], [83] and influence immune responses, brain development and behavior [24], [25], [26], [84], [85].
No kidding!
It should be noted that this paper is a child of a 2010 IMFAR abstract, Intestinal Inflammation, Impaired Carbohydrate Metabolism and Transport, and Microbial Dysbiosis in Autism. If I understand correctly, another paper is being prepared regarding the findings of intestinal inflammation that will be complimentary to Impaired Carbohydrate Digestion and Transport and Mucosal Dysbiosis in the Intestines of Children with Autism and Gastrointestinal Disturbances. I’ll try to post something when it is published.
This study was small, with only twenty two participants, largely as a result of the difficulty in obtaining tissue specimens. While this does give us cause for caution, it is important to note that this research does not exist in a vacuum, but rather, as a much larger set of research that tell us that the relationship between GI complaints and autism is more than the inceptions of DAN doctors. Previously, Gastrointestinal abnormalities in children with autistic disorder, performed similar biochemistry and reported broadly consistent carbohydrate digestion problems, ‘Low intestinal carbohydrate digestive enzyme activity was reported in 21 children (58.3%), although there was no abnormality found in pancreatic function.’ Several other papers analyzing fecal samples have reported altered bacterial populations, including Low relative abundances of the mucolytic bacterium Akkermansia muciniphila and Bifidobacterium spp. in feces of children with autism, Gastrointestinal flora and gastrointestinal status in children with autism–comparisons to typical children and correlation with autism severity, Fecal lactoferrin and Clostridium spp. in stools of autistic children, and Pyrosequencing study of fecal microflora of autistic and control children, among others.
If the findings from this latest paper are spurious finding based on sample size problems, a lot of other studies are coincidentally finding the same type of thing in the wrong way. Does anyone think that is likely?
I entered the autism world and online autism debate from a place of seeing with my own eyes the failures of a toddlers GI function and the difficult to overstate changes in that toddler alongside improvements in his GI health. On one of the first autism blogs on which I participated I got into a discussion (argument?) with a blogger who I came to respect very much, but has since moved on. I described the fact that my son had six or more diarrhea stools, a day, every day, for months on end, and that when we added dietary changes, probiotics, and later antifungal agents, the changes to his GI function were profound and impossible to misinterpret. He told me something along the lines that humans were susceptible to illusions and sleight of hand, and I thought, ‘as if not knowing the difference between diarrhea and a log was along the lines of figuring out where the jack of spades went!’. I couldn’t believe, could not fucking believe, someone would try to convince me that I had imagined my sons problems, and associated recovery. This wasn’t a sugar pill study where I was asked if my child acted more or less hyperactive, this was a matter of asking myself, ‘How many diarrhea diapers did I change today? Six? Or Zero?’ [Repeat once a day for 180 days.]
I doubt this is necessary, but just in case, I will go on the record to state that it is easy, very easy, to tell the difference between a condition of chronic diarrhea and normal GI function. There might not be a more simpleminded determination to make on Planet Earth or indeed, our perceptible universe. This is a situation that is susceptible to placebo effects only in the most elaborate imaginations of people who have never experienced chronic GI problems.
From that time on, with nearly zero exceptions, I have become a little less shocked, but a little more saddened by the doublethink style skepticism applied to GI distress and autism in nearly every single conversation I have ever seen on the Internet. I’ve put some time thinking toward this, why so many otherwise intelligent people house such extreme hostility on a relationship between GI function and autism. I believe that the Wakefield / MMR autism debacle is at the heart of this disconnect; his ill fated and now retracted paper that launched a thousand Internet scribbles has seemingly forever tied GI complaints and autism to bad science.
It doesn’t have to be this way. As a community, the vaccine wars and kissing cousin prevalence question has done a lot to fracture us, and very little to unite us. That is a sad statement, and nothing makes it more unfortunate than the fact that it does not have to be this way. Wakefield can be wrong about the MMR and there can still be very real differences in GI function in some cases of autism. We can respectfully disagree about how well our existing prevalence studies inform us on the incidence of autism without also needing to accept a world view where every child with autism has raging bowel problems.
We should have the intellectual honesty to admit that there is nothing inherently dangerous about acknowledging what the data tells us; GI function seems to be abnormal in a subset of children with autism, and the underlying features of that GI distress are qualitatively different than what is found in ‘normal’ children.
- pD
Seeing Patterns or Chasing Phantoms, or Is There A Biologically Plausible Developmental Programming Pathway Toward Impaired Synaptic Pruning In Autism?
Posted on: December 26, 2011
Hello friends –
Lately I’ve found myself reading papers and knowing and owning several of the references; tragically I can’t tell if I’m reading the right research and am onto something, or I am chasing phantoms and my web of pubmed alerts and reading interests are funneling my reference list into a narrowing echo chamber of sorts. With that warning in mind, we can proceed to poking around several papers, only some of which mention autism per se. Along the way, we will see evidence supporting the possibility of a biologically plausible mechanism of developmental programming of the neuroimmune environment, a sequence of events that may lead to impaired synaptic pruning in (some cases of?) autism.
By now, everyone has seen/read/heard about one form or another of the ‘a massive asteroid is going to destroy the world’ story. One of the common survival strategies from an asteroid strike involves altering the path of the asteroid so that it misses the Earth. The thoughtful analysis of this problem allows for the physics based reality of the problem, moving an asteroid out of an extinction based trajectory involves just a little work when the asteroid is ten thousand gazillion miles away, but a lot more work when it is only a gazillion miles away. Upon careful evaluation living organisms display similar behavior, relatively minor disturbances in early life can alter the developmental trajectory, while that same disturbance later in life is unable to materially affect the organism beyond a transient effect. The accumulated evidence that early life experiences can shape the adult outcome is nearly impossible to dispute with any remaining intellectual honesty, the question is instead, is how large is the effect in autism?
This analogy adequately symbolizes one of the more beautiful and terrifying concepts I’ve come across researching autism, that of ‘developmental programming’, which I blogged some about here, but essentially is the idea that there are critical timeframes during which environmental impacts can have long term persistent effects on a wide range of outcomes. The most robustly replicated findings involve changes to metabolic profiles in response to abnormal prenatal nutritional environments, but there is also evidence of various other effects, including neurological, and reputable speculation, that autism, may in fact, be in part, a disorder of developmental programming.
Secondarily, there has long been speculation of problems in the removal of ‘excess’ synapses, i.e., ‘synaptic pruning’ in the autism population. This culling of synapses begins in fetal life continuing throughout adolescence and the repeated observations of increased head circumference during infancy as a risk factor for autism has resulted in the idea that altered synaptic pruning maybe involved in autism.
In the last month or so several rather serendipitously themed papers have been published with tantalizing clues about some of the finer grained mechanisms of synaptic pruning, the possibility of impaired synaptic pruning in the autism population, and a known risk factor for autism that models a developmental programming event sequence that may tie them together.
First off, we have Synaptic pruning by microglia is necessary for normal brain development, (Paolicelli et all) with a very straightforward title, that has this dynamite in the abstract: (snipped for length)
These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.
This paper is short, but pretty cool, and very nice from a new territory perspective. It also speaks directly towards one of the increasingly hilarious obfuscations you will sometimes see raised in online discussions about immunological findings in autism, namely, that we can’t know if the state of chronic inflammation in the CNS observed in autism is harmful or beneficial. [hint: It might not be causative, but it isn’t beneficial.]
Here’s is a snippet from the Introduction:
Time-lapse imaging has shown that microglia processes are highly motile even in the uninjured brain and that they make frequent, but transient contact with synapses. This and other observations have led to the hypothesis that microglia monitor synaptic function and are involved in synapse maturation or elimination. Moreover, neurons during this period up-regulate the expression of the chemokine fractalkine, Cx3cl1, whose receptor in the central nervous system is exclusively expressed by microglia and is essential for microglia migration. If, in fact, microglia are involved in scavenging synapses, then this activity is likely to be particularly important during synaptic maturation when synaptic turnover is highest.
Nice. A time dependent participation by microglia in the critical process of optimization of neuron numbers, a process we are still very much groping our way in the dark towards untangling. The researchers focused in on a particular molecular target, a chemical messenger of the immune system, fractalkine, and found that without fractalkine, the process of synaptic turnover was impaired.
A couple of tests were performed, first immunohistochemistry (i.e., exceedingly clever manipulation of antibodies to determine the presence or absence of proteins in very specific locations) which demonstrated that microglia were, in fact, ‘engulfing synaptic material’ in animals during periods of synaptic maturation.
Secondly, so called ‘knock out mice’ (i.e., genetically engineered mice constructed without the ability to make a specific protein, in this case, fractalkine) were used evaluate for changes in synaptic form and function based on a lack of fractalkine. Changes in dendritic spine density were observed in the knock out mice group, with much higher densities in a very specific type of neuron during the second and third postnatal week of life. The authors indicate this is a key timeframe in synaptic pruning, and state their findings are “suggesting a transient deficient synaptic pruning in Cx3cr1 knockout mice “. The effect of not having fractalkine on spine density was time dependent as shown below.
Several other measurements were taken, including synaptic firing frequencies, which also implicated an increased surface area for synapses on dendritic spines, consistent with impaired pruning. Time dependent effects on synaptic efficiency and seizure susceptibility were also found, which the led the authors to conclude that the findings were “consistent with a delay in brain circuit development at the whole animal level.”
For additional evidence of fractalkine participation in synaptic maintenance, we can look to the opposite direction, where researchers evaluating neuron loss in an Alzheimers model reported “Knockout of the microglial chemokine receptor Cx3cr1, which is critical in neuron-microglia communication, prevented neuron loss”. Taken together, the conclusion that fractalkine processing is involved with neuron maintenance is highly likely, and correspondingly, highly unlikely to be a set of spurious findings.
There’s a couple paragraphs on potential mechanisms by which fractalkine could be interacting with microglia to achieve this effect, with the authors claiming that their data and other data generally supports a model wherein microglia were not effectively recruited to appropriate locations in the brain due to a lack of fractalkine, or, a ‘transient reduction in microglia surveillance.’
The conclusion is a good layman level wrap up that speaks toward the Interconnectedness of the brain and the immune system:
In conclusion, we show that microglia engulf and eliminate synapses during development. In mice lacking Cx3cr1, a chemokine receptor expressed by microglia in the brain, microglia numbers were transiently reduced in the developing brain and synaptic pruning was delayed. Deficient synaptic pruning resulted in an excess of dendritic spines and immature synapses and was associated with a persistence of electrophysiological and pharmacological hallmarks of immature brain circuitry. Genetic variation in Cx3cr1 along with environmental pathogens that impact microglia function may contribute to susceptibility to developmental disorders associated with altered synapse number. Understanding microglia-mediated synaptic pruning is likely to lead to a better understanding of synaptic homeostasis and an appreciation of interactions between the brain and immune system
That’s all pretty cool, but there was precious little discussion of autism, except in the general sense of a ‘developmental disorder associated with altered synapse number’. [But the references do speak to autism, the first reference provided, Dendritic Spines in Fragile X Mice displays a significant relationship to autism, and it describes how another flavor of knock out mice, this time designed to mimic Fragile-X, exhibit a ‘developmental delay in the downregulation of spine turnover and in the transition from immature to mature spine subtypes.’ Go figure!]
The other reason Paolicelli is of particular interest to the autism discussion is one of the major players in this study, the microglia (i.e., the resident immune cells of the CNS), have been found to be ‘chronically activated’ in the autism brain by direct measurement in two studies (here, and here, [and by me, here]), and tons of other studies have shown indirect evidence of an ongoing state of immunological alertness in the autism brain.
Considering this is a brand new paper, I do not believe that there are any studies illuminating the results of a state of chronic activation of microglia on the process of synaptic pruning per se. I will, however, go on the record that such an effect is very, very likely, and the logical leap is microscopically small that there will be some detrimental impact to such a state. The inverse argument, a scenario wherein there could be a state of chronic microglial activation that does not interfere with microglia participation in the synaptic pruning requires logical acrobatics worthy of Cirque Du Soleil. I am open to evidence, however.
So, from Paolicelli, we know that a ‘transient reduction in microglial surveillance’ induced by a reduction in the ability to production fractalkine can result in a condition ‘consistent with a delay in brain circuit development at the whole animal level’.
Next up, we have a paper that was all over the JerkNet in the days and weeks following its release, Neuron number and size in prefrontal cortex of children with autism. This is a cool study, and likely a very important paper, but I must say that a lot of the online commentary exhibits an irrational exuberance towards one part of the findings. Here is part of the abstract.
Children with autism had 67% more neurons in the PFC (mean, 1.94 billion; 95% CI, 1.57-2.31) compared with control children (1.16 billion; 95% CI, 0.90-1.42; P = .002), including 79% more in DL-PFC (1.57 billion; 95% CI, 1.20-1.94 in autism cases vs 0.88 billion; 95% CI, 0.66-1.10 in controls; P = .003) and 29% more in M-PFC (0.36 billion; 95% CI, 0.33-0.40 in autism cases vs 0.28 billion; 95% CI, 0.23-0.34 in controls; P = .009). Brain weight in the autistic cases differed from normative mean weight for age by a mean of 17.6% (95% CI, 10.2%-25.0%; P = .001), while brains in controls differed by a mean of 0.2% (95% CI, -8.7% to 9.1%; P = .96). Plots of counts by weight showed autistic children had both greater total prefrontal neuron counts and brain weight for age than control children. [PFC == prefrontal cortex]
Essentially the authors used a variety of mechanisms to measure neuron number in a specific area of the brain, the prefrontal cortex, and found large variations (increases) in the autism group. The prefrontal cortex is thought to be involved in ‘planning complex coginitive behaviors’, and ‘moderating correct social behavior’, among others, so this was a smart place to look.
The implicit hype on the internet is that this firmly indicates a ‘prenatal cause’ to autism, but if you read the paper, read what Courchense has said, and read recent literature, you know that the simplicity of this as a singular prenatal cause of autism is long broad strokes, and short on appreciation of the subtlety that textures reality.
A link @ LBRB sent me to the team at The Thinking Person’s Guide To Autism, who had a very nice transcription of a talk given by Courchesne at IMFAR 2011. Here is a snipet that started my wheels turning.
What we see in autism is either an excess proliferation, producing an overabundance of neuron numbers, or the excess might be due to a reduced ability to undergo naturally occurring cell death. Or it could be both. We don’t know which and our data don’t speak to that, although our data do suggest that it’s probably both.
Finally, our evidence shows that across time, there’s a prolonged period of apoptosis, removal and remodeling of circuits. In order to get back to where neuron numbers are supposed to be, it takes a very long time for the autistic brain. In the normal developing brain, this takes just a few months. In autism, it’s a couple of decades.
[Note how well this fits within the model described by Paolicelli, i.e., “consistent with a delay in brain circuit development at the whole animal level”. ]
I would highly recommend anyone who has read this far to go read the entire post @ TPGTA sometime.
As far as synaptic pruning goes, here is the associated segment of the paper:
Apoptotic mechanisms during the third trimester and early postnatal life normally remove subplate neurons, which comprise about half the neurons produced in the second trimester. A failure of that key early developmental process could also create a pathological excess of cortical neurons. A failure of subplate apoptosis might additionally indicate abnormal development of the subplate itself. The subplate plays a critical role in the maturation of layer 4 inhibitory functioning as well as in the early stages of thalamocortical and corticocortical connectivity development.inhibitory functioning and defects of functional and structural connectivity are characteristic of autism, but the causes have remained elusive.
Nearly half of the neurons in the area studied are expected to be removed through pruning, a process that extends well after birth. That is something that you didn’t see referenced in too many places trumpeting this study as ‘proof’ that autism was caused by disturbances in the prenatal environment. I’m not coming down on the prenatal environment as a critical timeframe for autism pathogensesis, just the difficult to defend underlying notion that this is the only time the environment should be evaluated, or the idea that if something is initiated prenatally other timeframes are therefore, unimportant.
So, I’d read that microglia were actively involved in proper synaptic pruning, contingent on utilization of fractalkine, and then read that impaired synaptic apoptotic mechanisms could be participating in autism, with a consequence of an over abundance of neurons.
Then, I got myself a copy of Microglia and Memory: Modulation by Early-Life Infection, which is another study in a growing body of evidence that immune challenges early in life can have unpredictable physiological consequences. (This is another very cool paper with Staci Bilbo as an author, whom I think is seriously onto something.) This study, in particular, focused on interactions microglia and formation of memories. Here is the abstract:
The proinflammatory cytokine interleukin-1ß (IL-1ß) is critical for normal hippocampus (HP)-dependent cognition, whereas high levels can disrupt memory and are implicated in neurodegeneration. However, the cellular source of IL-1ß during learning has not been shown, and little is known about the risk factors leading to cytokine dysregulation within the HP. We have reported that neonatal bacterial infection in rats leads to marked HP-dependent memory deficits in adulthood. However, deficits are only observed if unmasked by a subsequent immune challenge [lipopolysaccharide (LPS)] around the time of learning. These data implicate a long-term change within the immune system that, upon activation with the “second hit,” LPS, acutely impacts the neural processes underlying memory. Indeed, inhibiting brain IL-1ß before the LPS challenge prevents memory impairment in neonatally infected (NI) rats. We aimed to determine the cellular source of IL-1ß during normal learning and thereby lend insight into the mechanism by which this cytokine is enduringly altered by early-life infection. We show for the first time that CD11b+ enriched cells are the source of IL-1ß during normal HP-dependent learning. CD11b+ cells from NI rats are functionally sensitized within the adult HP and produce exaggerated IL-1ß ex vivo compared with controls. However, an exaggerated IL-1ß response in vivo requires LPS before learning. Moreover, preventing microglial activation during learning prevents memory impairment in NI rats, even following an LPS challenge. Thus, early-life events can significantly modulate normal learning-dependent cytokine activity within the HP, via a specific, enduring impact on brain microglial function.
Briefly, the authors infected rats four days after birth with e-coli, and then challenged them with LPS in adulthood to simulate the immune system to evaluate if memory formation was affected. As further evidence of an immune mediated effect, prevention of microglial activation in adulthood was sufficient to attenuate the effect. Clearly the effect on memory formation was based on the immune system. (Note: Most of the studies I’ve read would indicate [i.e., educated guess] that a four day old rat is brain developmentally similar to the third trimester of a human fetus.) While a terrifying and beautiful expression of developmental programming in its own right, there isn’t much to speak towards synaptic pruning in this paper, except maybe, potentially, one part of their findings.
In our study, CX3CL1 did not differ by group, whereas its receptor was decreased basally in NI rats, implicating a change at the level of microglia.
This is where things get either highly coincidental, or connected. CX3CL1 is another name for fractalkine, i.e., animals that were infected in early life had decreased expression of the receptor for fractalkine compared to placebo animals, i.e., fractalkine is the same chemical messenger found to be integral in the process of synaptic pruning in Synaptic pruning by microglia is necessary for normal brain development! From a functionality standpoint, having less receptor is very similar to having less fractalkine; as the animals in Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease tell us.
If, if synaptic apoptotic processes are impaired in autism, perhaps this is one mechanism of action. The timeline would involve a prenatal immune challenge, which causes a persistent decrease fractalkine receptor expression, which in turn, causes a consequent impairment in synaptic pruning through interference in microglial targeting. There is near universal agreement that immune disturbances in utero are capable of altering developmental trajectory undesirably, and here, in an animal model, we have evidence that infections are capable of reducing availability of receptors of ligands known to play a critical role in synaptic pruning, the absence of which leads to conditions which are “consistent with a delay in brain circuit development at the whole animal level”.
Only time, and more research, will tell if this is a pattern, a phantom, or a little of both.
- pD
The Fairytale of a Static Rate of Autism Part 4: Troubling Realities Acknowledged, The Incredible Shrinking Gods of the Gaps, and Otherwise Rational People Using ‘Small’ As An Empirical Measure To Answer A Critical Question
Posted on: August 19, 2011
Hello friends –
These have been rough times for the people who are heavily invested in the kissing cousin theories of autism as a predominantly genetic disorder and the static, or near static rate of autism. The California twin study that is old news by the time I get this finished showed much different rates of genetic participation than previously believed. These findings exposed the underlying frailty of gene-based causation theories, namely that some of the most widely referenced studies in the autism literature, studies used repeatedly as a basis for the notion that autism was ‘the most highly heritable neurodevelopmental disorder’, were, in fact, relatively underpowered, and suffered from serious temporal and methodological shortcomings.
By contrast, the California study looked at two hundred twin pairs, a lot more twins than any previous study and actually performed autism diagnostics on all of the participating children, whereas other studies relied on medical records. Performing dedicated ADOS diagnosis prospectively on the children allowed the researchers to discern between autism and PDD-NOS, something that not all previous studies were not able to perform, if for no other reason than the DSM-IV wasn’t even released when several of the most often cited studies were published. This is from the Comment section of the California twin study:
The results suggest that environmental factors common to twins explain about 55% of the liability to autism. Although genetic factors also play an important role, they are of substantially lower magnitude than estimates from prior twin studies of autism. Nearly identical estimates emerged for ASD, suggesting that ASD presents the same liability spectrum as strict autism.
This is on top of the fact that there is a quiet, but growing acknowledgement of the fact that literally decades of genetic studies have failed to be able to explain more than a fraction of autism cases despite sequencing of tens of thousands of genomes. This is a very similar situation to a great number of other disorders which we thought we would cure once the human genome was decoded. [Note: That isn’t to say that we haven’t learned a lot from sequencing the genome, just that we didn’t quite get what we thought we were going to get.]
This ‘double hit’, so to speak, has reached a critical mass such that health officials are making politically shrewd, but refreshingly realistic statements, and dare I say, a sliver of common sense may be about to infiltrate the discussion about autism prevalence. For example, as pointed out by Sullivan, Tom Insel, head of the National Institute of Mental Health keeps a blog where he recently blogged ‘Autism Spring’, which included this nugget within the context of continued failure of genetic studies to explain any substantial part of autism, “It is quite possible that these heritability estimates were too high. . .” Ouch. (I would recommend the entire blog posting by Mr. Insel.)
The high heritability estimates, and implicit genetically-mediated cause of autism, are foundational pillars of the argument that autism rates have not changed over time. Though overused, or used wrongly in many instances, there is a kernel of dispassionate reality behind the statement, ‘there is no such thing as a genetic epidemic’. Without the crutch of exceedingly high heritability to rely on, the notion of a stable rate of autism loses the only hard science (read: replicable, biologically-plausible), i.e.,genetics, it ever had, and must place complete reliance on the softer sciences (read: unquantifiable, ‘greater awareness’), i.e.,sociology. This is great news if you love impossible to verify estimates of prevalence and anecdotes about crazy uncle George who would have been diagnosed with autism forty years ago. However, if you think we should be relying less on psychologists and cultural anthropologists to answer critical questions, and rely more on hard science, this means that the old narrative on autism prevalence holds even less allure than it did in the past, for those of you who thought this was possible.
Before Kid Autism came around, I would occasionally read discussion boards on the creationism versus evolution ‘debate’. One thing that I noticed was that the creationists would often employ a ‘God of the Gaps’-style argument: anything that couldn’t be explained by science (yet), or anything necessary to support whatever fanciful construct had been erected to protect biblical creation fables, was ascribed to the work of God. That’s one thing you have to give to God, he (or she!) can handle it all; it didn’t matter what primitive logical test biblical creation was failing to pass, the golden parachute clause was always that God could have just made things that way. It was a nifty out on the part of the creationists, kind of like a get out of jail free card. The autism prevalence discussion has been working just like this, and the funny part is that the people that are always claiming to have the intellectual high ground, the supposed skeptics, are playing the part of the creationists! Zing!
Here is how it works:
Concerned Parent: It sure does seem like there is more autism than there used to be, what with there being X in a thousand kids with it! That’s much, much more than even ten years ago! My brothers, sisters and I all knew kids with mental retardation and Down’s syndrome, but we just don’t remember kids like we see today.
Supposed Skeptic: It is diagnostic substitution and ‘greater awareness’; autism incidence has been stable. The DSM was changed which resulted in more children being labeled.
Concerned Parent: It sure does seem like there’s more autism than there used to be. Now there are Y kids in a thousand having autism! Why does my son’s preschool teacher keep insisting something is changing?
Supposed Skeptic: It is diagnostic substitution and ‘greater awareness’; autism incidence has been stable. The DSM was changed which resulted in more children being labeled.
Concerned Parent: What the hell? Now there are Z kids in a thousand having autism! When are those genetic studies going to figure autism out, anyway?
Supposed Skeptic: It is diagnostic substitution and ‘greater awareness’; autism incidence has been stable. When does the new DSM come out again?
(Replace X/Y/Z with any progressively larger numbers.)
It doesn’t matter what prevalence number is thrown about–even the astronomical one in thirty-eight figure bandied about for South Korean children didn’t cause so much as a raised eyebrow; the autism equivalent of God of the Gaps, greater awareness and loosening of diagnostic criteria can handle any amount of increase gracefully. It is the equivalent of an uber-absorbent autism paper towel, capable of soaking up any number of new children with a diagnosis; there is, literally, no amount of an increase that the God of the Gaps can’t handle.
If, instead the question was posed like this, ‘How much of the apparent increase in autism is real?’, the answer was always, ‘Zero’, regardless of what the current rates of autism were when you asked the question
Then a funny thing happened, a series of studies from several researchers showed a consistent trend of older parents giving rise to more children with autism than younger parents. There were differences between the studies on just how much of an effect an older parent had, but the overall direction of association was clear. In this instance, there was also the luxury of a plausible biological mechanism that involved the mediator in favor, genetics. The idea is that advancing age in the parent meant more years for gametes to get knocked by a random cosmic zap or other environmental nastygram and this disturbance created genetic problems down the line for the offspring, a theory I think is probably pretty good. Once a couple of these studies started to pile up, there was a small shift in the narrative regarding autism prevalence; after all, nobody could bother to try to deny that parents were getting older compared to past generations. Here is how it looked:
Concerned Parent: What the hell? Now there are X kids in a thousand having autism!
Supposed Skeptic: Greater awareness and diagnostic substitution are primarily responsible for our observations of increased autism, although, ‘a real, small increase’ cannot be ruled out.
And with that, there was a little less autism prevalence for the God of the Gaps to handle. It never seemed to bother anyone that implicit in this argument is an impossible to quantify concept ‘small increase’. If you were to ask someone what rate of autism ‘a small increase’ amounted to with more precision, the answer is whatever amount rises to the level of autism minus the difficult to quantify effect of older parents. That is some lazy stuff.
Here are some examples of prominent online skeptics discussing the possibility of a true rise in autism. See if you can detect a pattern.
Here is Stephen Novella pushing The Fairytale in 2009:
While a real small increase cannot be ruled out by the data, the observed increase in diagnostic rates can be explained based upon increased surveillance and a broadening of the definition – in fact autism is now referred to as autism spectrum disorder.
[Here we see the notion that everything can be explained by the God of the Gaps.]
Here is an example of Orac toying around with this filibuster just the other day, in August of 2011:
True, the studies aren’t so bulletproof that they don’t completely rule out a small real increase in autism/ASD prevalence, but they do pretty authoritatively close the door on their being an autism “epidemic.”
These aren’t the only examples, far from it. Check it out:
It should be noted that the data cannot rule out a small true increase in autism prevalence. (Stephen Novella in 2008)
If the true prevalence rate of autism and ASDs has increased, it has not increased by very much. (David Gorski, 2010)
We should have the curiosity to wonder, what, exactly, does small mean in these contexts? What percentage size increase should we consider small enough to hide within the data? Five percent? Ten percent? What does ‘small’ mean, numerically, within a range? Is a ten to twenty percent rise in autism rates reason for us to take comfort in the fact that the effect of greater awareness is real? At what level does the percentage of ‘real’ autism increase mandate more than superficial lip service, more than a paragraph about ‘gene-environment interactions’ at the end of a two-thousand word blog post that takes pride in the intellectual chops of outthinking Jenny McCarthy? You won’t get anyone to answer this question; they can’t, because they don’t really know what they mean when they say, ‘small’, other than, ‘it can’t be vaccination’.
How do we know the amount of this increase must, in fact, even be ‘small’? This becomes especially problematic when we consider the smackdown that the canard of autism as ‘among the most heritable neurological conditions’ has taken as of late. If the high heritability estimates of autism are incorrect, yet so often repeated as gospel, why should we also assign confidence to the idea that the increase is trivial? Isn’t one argument the foundation of the other? Did either really have quality data behind them?
This is a terrible, awful, horrible, completely fucking idiotic way to address a question as important as whether or not a generation of children is fundamentally different. We cannot afford the ramifications of being wrong on this, but we seem to find ourselves in an epidemic of otherwise intelligent people willing to accept the pontifications of cultural anthropologists and the feebleness of social scientists on this critical question. I am not arguing against the realities of diagnostic switching and greater awareness affecting autism diagnosis rates. But we can understand that while they are a factor, we must also admit that we have little more than a rudimentary understanding of these impacts, and when we consider the implications of being incorrect, the potential disaster of a very real, not ‘small’ increase in the number of children with autism, we shouldn’t be overselling our knowledge for the sake of expedient arrival at a comforting conclusion. We should be doing the opposite.
If we can’t have the robustly defendable values on autism rates right now, that’s fine, because that is the reality, but we should at least have the courage to acknowledge this truth. This is the nature of still learning about something, which we are obviously doing in terms of autism, but in that situation, we don’t have the currency of scientific debate, decent data, to be saying with authority that any true increase in autism is small.
Unfortunately for the purveyors of The Fairytale, things are going to get a lot worse. The problem is that we are starting to identify extremely common, in some cases, recently more common, environmental influences that subtly increase the risk of autism. These are further problems for a genetic dominant model and effectively mandate that the ‘small increase’ is going to have to start getting bigger as a measurement, with a correlated decrease in the amount of autism that cultural shuffling can be held responsible for. Will anyone notice?
By way of example, we now have several studies that link the seasons of gestation with neurodevelopmental disorders including autism and schizophrenia; i.e., Season of birth in Danish children with language disorder born in the 1958-1976 period, Month of conception and risk of autism, or Variation in season of birth in singleton and multiple births concordant for autism spectrum disorders, which includes in the abstract, “The presence of seasonal trends in ASD singletons and concordant multiple births suggests a role for non-heritable factors operating during the pre- or perinatal period, even among cases with a genetic susceptibility.” Right! As I looked up some of these titles, I found that the evidence for this type of relationship has been well known for a long time; schizophrenia, in particular has a lot of studies in this regard, i.e., Seasonality of births in schizophrenia and bipolar disorder: a review of the literature, which is a review of over 250 studies that show an effect, and I also found Birth seasonality in developmentally disabled children, which includes children with autism and was published in 1989, which is like 1889 in autism research years.
Our seasons have remained constant (but probably won’t stay too constant for much longer. . . ), but this still throws a whole barrel of monkey wrenches into the meme of a disorder primarily mediated through genetics.
More damning for the Fairytale are some studies presented at this year’s IMFAR, and some others just published, that tell us that abnormal immune profiles during pregnancy appear to provide slightly increased risk for autism, roughly doubling the chance of a child receiving a diagnosis. The groovy part is that the studies utilized both direct and indirect measurements of an activated immune system to draw similar conclusions, a sort of biomarker / phenotype crossfire.
From the direct measurement end, we have Cytokine Levels In Amniotic Fluid : a Marker of Maternal Immune Activation In Autism?, which reports that mothers with the highest decile of tnf-alpha levels in the amniotic fluid had about a one and a half times increased risk for autism in their children. This makes a lot of sense considering the robustness of animal models of an acute inflammatory response during pregnancy and its impact on behavior.
Another study, this one from the MIND Institute in California (which I love), is Increased mid-gestational IFN-gamma, IL-4, and IL-5 in women giving birth to a child with autism: a case-control study (full paper). They found that in pregnant mothers, increased levels of IFN-gamma led to a roughly 50% increased risk of an autism diagnosis. Here is a snipet:
The profile of elevated serum IFN-γ, IL-4 and IL-5 was more common in women who gave birth to a child subsequently diagnosed with ASD. An alternative profile of increased IL-2, IL-4 and IL-6 was more common for women who gave birth to a child subsequently diagnosed with DD without autism.
This study took a lot of measurements, and goes to great lengths to explicitly call for additional analysis into the phenomena. IFN-gamma is typically considered pro-inflammatory, while IL-4 and IL-5 are considered regulatory cytokines. In order to determine if these findings were chance or not, the researchers determined if there was a correlation between the levels of IFN-gamma, IL-4, and IL-5, which they reported with very robust results. Less clear is what might be causing these profiles, or how, precisely, they might give rise to an increased risk of autism. The interconnectedness of the brain and the immune systemwould be a good place to start looking for an answer to the last question though.
What about indirect measurements? It just so happens, another paper was published at IMFAR this year that observed the flip side of the coin, conditions associated with altered cytokine profiles in the mother and this study also found an increased risk of autism. The Role of Maternal Diabetes and Related Conditions In Autism and Other Developmental Delays, studied a thousand children and the presence of diabetes, hypertension, and obesity in their mothers in regards to the risk of a childhood autism diagnosis. The findings indicate that having a mother with one or more of those conditions roughly doubles the chances of autism in the offspring. Obesity, in particular, has an intriguing animal model Enduring consequences of maternal obesity for brain inflammation and behavior of offspring, a crazy study that I blogged about when it was published. A variety of auto immune disorders in the parents have been associated with an autism diagnosis in several studies.
The obesity data is particularly troublesome for the idea of a ‘small’ increase in autism, just like parents have been getting older, parents have also been getting fatter, waaaay fatter, (and more likely to have diabetes) the last few decades. There isn’t any squirming out of these facts. If, indeed, being obese or carrying associated metabolic profiles is associated with an increased risk of autism, ‘small’ is getting ready to absorb a big chunk of real increase. But is there any clinical data to support this possible relationship, do we have any way to link obesity data with this autism data from the perspective of harder figures?
It further turns out, there are some very simple to navigate logical jumps between the above studies. Remembering that our clinical measurements indicated that increased INF-gamma, IL-4, and IL-5 from the plasma of the mothers was associated with increased risk, we can see very similar patterns in Increased levels of both Th1 and Th2 cytokines in subjects with metabolic syndrome (CURES-103). Here is part of the abstract, with my emphasis.
Metabolic syndrome (MS) is a cluster of metabolic abnormalities associated with obesity, insulin resistance (IR), dyslipidemia, and hypertension in which inflammation plays an important role. Few studies have addressed the role played by T cell-derived cytokines in MS. The aim of the tudy was to look at the T-helper (Th) 1 (interleukin [IL]-12, IL-2, and interferon-gamma [IFN-gamma]) and Th2 (IL-4, IL-5, and IL-13) cytokines in MS in the high-risk Asian Indian population.
Both Th1 and Th2 cytokines showed up-regulation in MS. IL-12 (5.40 pg/mL in MS vs. 3.24 pg/mL in non-MS; P < 0.01), IFN-gamma (6.8 pg/mL in MS vs. 4.7 pg/mL in non-MS; P < 0.05), IL-4 (0.61 pg/mL in MS vs. 0.34 pg/mL in non-MS; P < 0.001), IL-5 (4.39 pg/mL in MS vs. 2.36 pg/mL in non-MS; P < 0.001), and IL-13 (3.42 pg in MS vs. 2.72 pg/mL in non-MS; P < 0.01) were significantly increased in subjects with MS compared with those without. Both Th1 and Th2 cytokines showed a significant association with fasting plasma glucose level even after adjusting for age and gender. The Th1 and Th2 cytokines also showed a negative association with adiponectin and a positive association with the homeostasis model of assessment of IR and high-sensitivity C-reactive protein.
Check that shit out! Seriously, check that out; increased IFN-gamma, IL-4, and IL-5 in the ‘metabolic syndrome’ group, comprised of people with, among other things, obesity, insulin resistance, and hypertension; the same increased cytokines and risk factors found to increase the risk of autism.
If we look to studies that have measured for TNF-alpha in the amniotic fluid during pregnancy, we quickly find, Second-trimester amniotic fluid proinflammatory cytokine levels in normal and overweight women
There were significant differences in amniotic fluid CRP and TNF-alpha levels among the studied groups: CRP, 0.018 (+/-0.010), 0.019 (+/-0.013), and 0.035 (+/-0.028) mg/dL (P=.007); and TNF-alpha, 3.98 (+/-1.63), 3.53 (+/-1.38), and 5.46 (+/-1.69) pg/mL (P=.003), for lean, overweight, and obese women, respectively. Both proinflammatory mediators increased in women with obesity compared with both overweight and normal women (P=.01 and P=.008 for CRP; P=.003 and P=.01 for TNF-alpha, respectively). There were significant correlations between maternal BMI and amniotic fluid CRP (r=0.396; P=.001), TNF-alpha (r=0.357; P=.003) and resistin (r=0.353; P=.003).
Nice.
What we are really looking at are five studies the findings of which speak directly to one another; a link to metabolic syndrome during pregnancy and increased IFN-gamma, IL-4, and IL-5, a link to obesity and hypertension in pregnant mothers and autism risk, and an increased risk of autism in mothers wherein IFN-gamma, IL-4, and IL-5 were found to be increased outside of placenta. Further, we have a link between amniotic fluid levels of TNF-alpha and metabolic syndrome, metabolic syndrome in mothers and autism risk, and increased risk from increased tnf-alpha in the amniotic fluid.
As I have said previously, one thing that I have learned during this journey is that when we look at a problem in different ways and see the same thing, it speaks well towards validity of the observations. What we see above is a tough set of data to overcome; we need several types of studies looking at the relationship between metabolic syndrome, immune profiles during pregnancy, and autism from different angles to have reached the same wrong conclusion, something that is increasingly unlikely. We are in an epidemic of obesity and the associated endocrine mish mash of metabolic syndrome, there simply isn’t any diagnostic fuzziness on this. It is happening all around us. Even though the total increase in risk is relatively small, the sheer quantity of people experiencing this condition of risk mandates that the numbers game looks favorable towards a real increase in autism. If we acknowledge this, how can we continue to have faith in the concept that any true increase in the autism rates must be ‘small’?
Is the next argument going to be that besides increased parental age, and heavier or more diabetic mothers, the rest of the autism increase is the result of diagnostic three card monte? (Just how much is the rest, anyways?)
And even though these studies, and likely more in the future, expose the crystal delicate backbone of the ‘small true increase’ argument, I have great pessimism that the people so enamored with invoking this phrase will ever acknowledge its shifting size, much less the implications of being wrong on such a grand scale.
- pD
The Fairytale of a Static Rate of Autism Part III – Prevalence Hookups or What if They Threw An Autism Epidemic And Nobody Cared?
Posted on: May 30, 2011
- In: Autism | Hilarious | Prevalence | The Fairytale | Willing Disbelief
- 8 Comments
Hello friends –
The osmotic pressure of cool people and pop culture tells me that what we used to call one night stands are now called ‘hookups’, casual sexual encounters as convenient that don’t necessarily mean people are dating, but some release can be found, and everyone moves on with their lives until the next time. This reminds me a lot of how people that ought to know better have been treating autism prevalence studies lately. The results are useful in cementing an already reached conclusion, but ultimately, the findings are only used as isolated ejaculations of the same ideological tweets. Last week’s hookup doesn’t mean anything come this Saturday night, and there is absolutely no reason, no reason, anyone should be troubled to compare this weeks findings used to trumped a static rate of autism with last weeks findings. What we are witnessing is the equivalent of a scientific one night stand, and anyone who doesn’t think the scientific method should be framed for the sake of expediency ought to be furious.
These posts can oftentimes take me a long while to complete, so dating my start point a bit, about two weeks ago, the NHS study from England came out that described a near 1% prevalence of ‘autism’ in adults. The ‘findings’ from this study actually came to light and received attention in the autism community over a year ago, but the real publication happened in May 2011, so there you are.
About a week ago, the Korea ‘study’ on autism came out; it hit the web with a large footprint, and amazingly, described an atmospheric autism ‘prevalence’ of autism of near 2.5%, with 1 in 38 (!!!!) Korean children ‘estimated’ to be on the autism spectrum. If it has not happened already, this study and ‘conclusions’ will soon became part of the autism lexicon; an uber-Kevlar argument, impervious to any concerns involving the possibility of an actual increase in the number of children with autism.
Both of these studies share very similar methodologies; essentially a lot of people were screened through a questionnaire, a subset of people with ‘high’ scores on the questionnaire were subsequently retested with standard tools for assessing autism. Based on how well the questionnaire did at predicting autism spectrum diagnosis, an extrapolation, with various ‘corrections’, was made as towards how many people in the general public are on the spectrum. In both studies, the overwhelming majority of people ‘estimated’ with autism were previously undiagnosed and were not receiving any services.
Here’s the thing that is driving me up the wall crazy, apeshit mystified and enraged. Nobody cared. Let’s look again at what these studies found and see if we can detect anything of potential interest in their conclusions when compared between one another.
Nobody, and I mean nobody, took these two studies as evidence of an autism epidemic, despite the fact that here we have two supposedly (?) well designed studies that found entire spectrum sized differences in the number of children and adults with autism! You could literally drive the old spectrum through the hole in the new spectrum! If both of these two studies are meaningful, if both have accurately captured autism in their respective target populations, we have no choice but to admit that the epidemic is real, and we have proof that children have an autism spectrum disorder two and a half times more frequently than adults. There is an epidemic of autism in our children; or at least, in Korean children!
Did anyone see those headlines that I somehow missed? Did the online skeptical community acknowledge that we now finally have some solid evidence that indeed, autism rates are higher in children than adults, and somehow I failed to see those conversations?
Here’s what really confuses me. Some of the same people, same ‘skeptics’, and same news organizations breathlessly reported both of these findings without, apparently, understanding their implications alongside one another. For example, in 2009, here’s a post from Stephen Novella at Science Based Medicine that touched on the England study that includes this nugget:
They found a consistent prevalence of 1% in all age groups they surveyed. This is remarkable for two reasons – first, they found the exact same 1% figure as the CDC US survey (assuming the CDC data is more accurate than the phone survey published in Pediatrics). This supports the conclusion that the 1% figure may be close to the true prevalence of ASD in the population.
Second, the NHS study found that the prevalence of autism was the same in all age groups, strongly suggesting that true ASD incidence has not been increasing over recent decades and supporting the increased surveillance and definition hypothesis.
Check out how ‘remarkable’ Mr. Novella thinks the 1% matchup between English adults and American children is in terms of making the case for a static rate of autism. This is a guy whose posts outside the autism realm I tend to enjoy in many instances, he is clearly a superior intellect, and applies a very skeptical eye towards his non-autism posts. My presumption is that he was well aware that the NHS study actually diagnosed a grand total of 19 adults, and had good reasons, which he declined to illuminate in that post, for why this relatively low number of results was immune to significant confounding problems, which is why it provided such ‘remarkable’ evidence ‘strongly suggesting that true ASD incidence has not been increasing’.
Then, in May 2011, Mr. Novella posted Autism Prevalence Higher than Thought, concerning the Korea study. Here is a snippet from the conclusions:
This study adds an interesting data point to the whole picture of ASD. If correct, then the theoretically upper limit of ASD prevalence is about 2.6% of the population, more than twice the previous estimate. It also indicates that when you undergo a program of thorough searching, you will find more diagnoses.
What is going on here? The England study, which found a prevalence of 1%, the study that previously was found to be remarkable evidence of a static rate of autism was exactly the same type of study, wide-scale screening for likely candidates within the general population, followed by targeted autism assessment of people with high scores, and backwards extrapolation. Does anyone think that the Korea study was that much more thorough than the England study? If a study came out tomorrow that reported 5%, or 10% prevalance, would we simply assign this to a even more strenously executed methodology? Is there any evidence that we might use to suspect a 5% prevalance reported next week in Columbia is faulty that could not also be applied against Korea?
For what reason should we, now, believe that the England study of adults was so fatally flawed that it missed more than one autistic adult for every one it found? Surely a study capable of missing more than half of the autistic adults had some type of warning signs back in 2009 that might indicate that the evidence might be less than remarkable, maybe questionable, or that, in fact, it might be a Fairytale?
Am I cynical to suggest that what really made the England study such remarkably ‘strong evidence’ of a static rate of autism was that, at the time, it had findings within the statistical range of existing CDC numbers in children? Was the online and media love affair with the England NHS study little more than prevalence hookup? Have I reached the theoretical limit of jadedness?
There really isn’t a way to reconcile these two findings without either accepting a two and a half times increase in autism in children versus adults, a sort of epidemic-lite, or accepting that one or both of the studies suffer from serious flaws. But if we start accepting that the studies might have serious problems, we shouldn’t be saying they are ‘strong evidence’ of anything, except, perhaps, the difficult to overstate problems of autism prevalence studies. Of course, it is a different ballgame if you are relieved of the intellectual responsibility of actually trying to reconcile the two findings; if you allow yourself the prevalence doublethink that England has meaningful data, and so does Korea, and that the rate of autism isn’t increasing, then, no harm, no foul Big Brother.
One prevalence study that didn’t get the booty call was Brief Report: Prevalence of Pervasive Developmental Disorder in Brazil: A Pilot Study, which came out in February, 2011; just three months before Korea. Methodology wise, this study is a kissing cousin to Korea and England, a screening was performed in the general population, and assessments were subsequently performed and then statistical extrapolations were performed to reach a prevalence rate. Let’s see what these values look like up against each other, and see if we can detect a pattern.
Can anyone see a pattern here?
Now the skeptic might tell you that the Brazil study was a lot smaller, which is true; the initial screening of children only contained a little less than 1,500 children. But it hardly matters; just to get to the level of English adults ‘found’, they would have had to miss two children for every child they found, and to approach Korea values, they needed to have missed almost nine children for every child actually diagnosed. Does anyone think this is reality? Why would prospective screening and backwards extrapolation be so accurate in one population, and so wildly inaccurate in another population? The Brazil and England study used versions of the same screening questionnaire!
I understand that being partially funded by Autism Speaks, and having a ‘cultural anthropologist’ with a book on the subject of autism carries some weight in the press conference area; so that might explain why one study got press, and another didn’t. Forgetting the press issue, where are the calls that we should try throwing four thousand Brazilian genomes at a sequencer to see what in their genetic makeup appears to be protecting them from autism so effectively? Why aren’t these studies meaningful evidence of some environmental force acting to create wildly different rates of autism in these different populations?
I would note that the press releases, media regurgitations, and skeptical viewpoints nearly all contained the boilerplate note that more studies are needed. Consider, however, if our need for ‘more study’ is so extensive, if we place so little confidence in our methodologies that papers published within months of each other, with nearly identical study methods, find literally nine times higher rates of autism in one population aren’t a warning sign of an real difference in incidence, what this ought to be telling us is that all of our prevalence data are crapshoots, at best. We shouldn’t get to pick and choose which studies we think are meaningful because they happen to meet comforting quotas, or discard those that fail to support those palliative notions.
It is tempting to look at the Brazil study and evaluate for design or implementation problems that could cause such startlingly low rates of autism; the authors go into some discussion about the reasons their findings might seem so low. Complicating matters along this line, however, is that the Brazil and Korea studies, shared a researcher, the relatively well known psychiatrist with a large pubmed autism prevalence footprint, Eric Fombonne. It occurred to me that it might be a fun experiment to see how reliable Mr. Fombonne has been regarding autism prevalence.
[Click on the image to get a bigger view / stupid wordpress template] Note that I have omitted review papers, or papers that had no abstracts, but it doesn’t really help. (How could it?)
All of these findings were wholly or partially authored by the same person. Is there anything more damning for the state of autism prevalence research than this person continues to be considered a source of reliable information?
I used to live with a fun dude in college; he went to engineering school and went on to work at a manufacturing facility near our town. One of the funniest things he told me about engineering was this quote:
Dilution is the solution to pollution!
In other words, if you have a hundred pounds of diethyl-pthylate-poisonate to dispose of, ship in a hundred thousand gallons of water, and start pumping; if you have two hundred pounds to eject, ship in two hundred thousand gallons of water. This is what is happening to the definition of autism, the quirky element, the ‘broad autistic phenotype’ is seeping into these studies. After dozens, or hundreds of prevalence studies we are ultimately left with as many portraits of different entities as envisioned by the researcher and width of spectrum de jour. The upshot of this, however, is that it makes no sense to try to compare these studies.
In the meantime, we are told time and time again that even though our common sense, our memories of childhood, and the repeated lamentations from every person who has worked with children for the last few decades, all of which are warning us that something is different; all of these things are all supposedly subject to an array of biases so strong that we cannot trust them to reach any conclusions. Only through carefully planned, objective analysis can we reach any conclusions on autism incidence. The results of this choreographed investigation looks like this:
Does anyone really think there aren’t some pretty serious biases operating here? If we cannot use common sense to try to reconcile the picture above, what can we use? If trusting common sense is dangerous to valid conclusions, so is trusting this.
If anyone really thought that Korea and Brazil were measuring the same condition, a condition that until very, very recently has been considered lifelong and severely debilitating, the two wildly different findings would be cause for alarm, undeniable evidence of a massive environmental force influencing the development of autism in some populations. But no one thinks this, no one cares, and that is because; no one really believes these studies are measuring the same thing. But admitting this is dangerous to too many, it is the implicit acknowledgement of just how little we understand, how beholden our policies and research prioritizations are guided by the softest of science and scientists, and ultimately, how frequently we’ve been sold a narrative with the scientifically defendable value of a set of monetized South Florida mortgages.
Such is the way of the prevalence hookup, transiently entertaining, but without meaning from week to week. Until we can find a way past this, past reliance on the shifting sands of behavioral assessments that can vary from researcher to researcher (or by the same researcher!), we can perform all of the ‘thorough investigations’ that we can afford and repeat the ‘findings’ that support our meme until we are blue in the face. None of it will mean a goddamned thing, though we may lose a generation of children while we bounce from one set of findings to another, feeling pleased with the ones that make doom seem unlikely, and discarding the ones that should be cause for great alarm.
-pD
The Interconnectedness of the Brain, Behavior, and Immunology and the Difficult to Overstate Flaccidity of The Correlation Is Not Causation Argument
Posted on: May 12, 2011
Hello friends –
I’ve gotten into a lot of discussions online about the vaccines and autism; generally with very poor, if not nonexistent, evidence of having changed any opinions, but relatively strong evidence ( p > .001) that persisting in making my arguments can get you called ‘an antivaccine loon’, ‘idiot’, someone who engages in ‘Gish Gallop’, or the worst insult I’ve received so far, ‘anti-science’. While I am really torn on the vaccine issue, I am certain that both peripheries of this debate are at least somewhat wrong in the conclusions that they’ve drawn from the available evidence. I do believe that lots of parents have witnessed a very real change in their children post vaccination, and I also don’t believe for a single second that vaccines are the cause of an epidemic of autism. It’s a mess and I’ve been poking around the Internet almost five years into journey autism and from my eyes, it hasn’t improved any in the past half decade. This is very sad.
That being said, while I do think we need to have a rational and dispassionate discussion about what our existing vaccine studies can and cannot tell us about autism, I’m really concerned about the fact that the vaccine wars seem to have inoculated otherwise intelligent people from any semblance of intellectual curiosity regarding the immunological findings in the autism realm. That’s a problem, because there are lots of things other than vaccines that can modify the immune response, various environmental agents and cultural changes that are relatively new, and ignoring immunological findings in autism because they happen to intersect with the function of vaccination is a huge, massive, supernova sized disservice to what history will view us poorly on, refusing to perform honest evaluation due to fear and the comfort of willful ignorance.
Here, in this post, I will make the case that this lack of curiosity on immunological findings in autism is either born of a lack of understanding on how much we know about the ties between the immune system and the brain, or alternatively, originates from a deep seated desire to avoid honest interactions. This isn’t to make the case that vaccines can cause autism, or even that the immunological disturbances observed in autism are causative, but rather that an obstinate refusal to consider these as possibilities is the sign of someone who cannot, or will not accept, the biological plausibility of immunologically driven behaviors despite a constellation of evidence.
One of the things that jumps out to me why the autism population might be a subgroup of the population susceptible to changes as a result of immune dysfunction (and thus, potentially adversely affected as a result of vaccination), is the sheer volume of evidence we now have available to us indicating an altered immune response, and indeed, an ongoing state of inflammation within the brain in the autism population, and most strikingly, repeated observations of a correlation between the degree of immune dysregulation as a propensity of an inflammatory state, and the severity of autism behaviors. Again and again we’ve seen that as markers indicative of an inflammatory state increase, so too, do severity of autism behaviors. Not only that, but there are instances wherein the decrease of components known to regulate the immune response decrease, autistic behaviors are more severe. Subtle shifts in either the start or the resolution of the immune response seems to affect autistic behavior severity in the same way. I know coincidences happen all the time, but that doesn’t mean that everything is a coincidence.
We also have a large number of studies that tell us that in vitro, similar levels of stimulation with a variety of agents cause exaggerated or dysregulated production of immune markers in the autism population.
A large percentage of the time that I mention these findings, usually within discussions with an origin in vaccination, someone decides to educate me on one of the most rudimentary scientific axioms:
Correlation does not equal causation.
It must be stated, the above statement is absolutely true. Unfortunately for the people for whom this accurate, but simplistic catchphrase comprises the entirety of their argument, it completely ignores a wealth of research that tells us in very unambiguous terms that there is incontrovertible evidence that crosstalk between the immune system and central nervous system can modify behavior. The research indicating a relationship between immune dysregulation and autism does not exist in a vacuum, but rather, is only a tiny fragment of evidence, mostly accumulated within the last few years, that tells us that the paradigm of the past decades, that of the brain as a immune privileged organ without communication to the immune system, is as antiquated as refrigerator moms and a one in ten thousand prevalence.
From a common sense, why didn’t I think of that standpoint, the best example of the interaction between the brain and the immune response is the old standard, just plain old getting sick. You live in the dirty world, you pick up a pathogen, you get sick, and suddenly you get lethargic and you start to run a fever. But is it the pathogen itself that is actually making you feel like staying in bed all day?
What is being learned is that it is not necessarily the microbial invader that is causing you to get tired and feel sore, but rather, that your decreased energy levels are centrally mediated through your brain, and the triggers for your brain to start a fever include molecules our bodies use for a wide range of communications, including immune based messaging, cytokines. Some of the most common cytokines in the research to follow include IL-6, IL-1B, and TNF-Alpha; so called ‘pro-inflammatory’ cytokines. Researchers have been plugging away at just how the immune response is capable of modifying behaviors, i.e., inducing, sickness behavior for a while now, at least in terms of autism research. From 1998, we have Molecular basis of sickness behavior:
Peripheral and central injections of lipopolysaccharide (LPS), a cytokine inducer, and recombinant proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) induce sickness behavior in the form of reduced food intake and decreased social activities. Mechanisms of the behavioral effects of cytokines have been the subject of much investigation during the last 3 years. At the behavioral level, the profound depressing effects of cytokines on behavior are the expression of a highly organized motivational state. At the molecular level, sickness behavior is mediated by an inducible brain cytokine compartment that is activated by peripheral cytokines via neural afferent pathways. Centrally produced cytokines act on brain cytokine receptors that are similar to those characterized on peripheral immune and nonimmune cells, as demonstrated by pharmacologic experiments using cytokine receptor antagonists, neutralizing antibodies to specific subtypes of cytokine receptors, and gene targeting techniques. Evidence exists that different components of sickness behavior are mediated by different cytokines and that the relative importance of these cytokines is not the same in the peripheral and central cytokine compartments.
The first sentence in this abstract references a practice that is extremely common in studying the immune system, intentionally invoking a robust immune response by exposing either animals, or cells in vitro, to the components that comprise the cell wall of certain types of bacteria; lipopolysaccharide, or LPS. LPS could be considered a sort of bacterial fingerprint, a pattern that our immune systems, and the immune system of almost everything, has evolved to recognize, and correspondingly initiates an immune response.
Because this is a conversation that frequently has an origin in vaccination, essentially the act of faking an infection, it is salient to remember that the animals or cell cultures aren’t really getting sick when exposed to LPS; there is no pathology associated with whatever type of bacteria might be housed within a cell membrane containing LPS. Usually, when the body is exposed to a gram negative bacteria, and the consequent LPS exposure, there are also effects of the bacteria that interact with the organism, but by only incorporating the alert signal for a bacterial invader, we can gain insight into the effect of the immune response itself; there isn’t anything else to cause any changes. This means that similarly to LPS administration, straight administration of these pro-inflammatory cytokines are similar to the result of getting sick with a pathogen, at least as far as the immune response is concerned.
In the above instance, administration of LPS, or simply cytokines, had been shown to be capable of causing reduced food intake and ‘decreased social activities’.
Later in 1998, Central administration of rat IL-6 induces HPA activation and fever but not sickness behavior in rats (full version), was published wherein the authors report that central administration (i.e., directly into the CNS), of cytokines in isolation (IL-6) or in combination (IL-6 + IL-1B) were capable of inducing altered HPA activation, fevers, and sickness behaviors. Effects of peripheral administration of recombinant human interleukin-1 beta on feeding behavior of the rat was published a few years later, and observed that peripheral administration (i.e., not the CNS) of IL-1B could affect how much a rat ate, with sucrose ingestion being consistently altered during periods of sickness.
Jumping ahead a few years, a review paper Expression and regulation of interleukin-1 receptors in the brain. Role in cytokines-induced sickness behavior reviewed how cytokines participate in sickness behavior, Interleukin-6 and leptin mediate lipopolysaccharide-induced fever and sickness behavior examined the interactions of IL-6 and leptin in sickness behavior, and Behavioral and physiological effects of a single injection of rat interferon-alpha on male Sprague-Dawley rats: a long-term evaluation reported “these data suggest that a single IFN-alpha exposure may elicit long-term behavioral disruptions”.
Much more recently, Sickness-related odor communication signals as determinants of social behavior in rat: a role for inflammatory processes more elegantly found that behavior was modified by LPS exposure, and that this effect was neutralized by concurrent administration of the anti-inflammatory cytokine, IL-10. Similarly, Inhibition of peripheral TNF can block the malaise associated with CNS inflammatory diseases observed another distinct means by which interfering with the immune response could attenuate the effect of faux sickness, in part, concluding, “Thus behavioral changes induced by CNS lesions may result from peripheral expression of cytokines that can be targeted with drugs which do not need to cross the blood-brain barrier to be efficacious.” In other words, what is happening in the periphery, outside of the protective boundaries of the blood brain barrier, can none the less manipulate behaviors that are controlled by the brain.
There are tons, tons more studies like this, but the point should be clear by now; it is accepted that you can achieve some of the same behaviors the come alongside illness, such as fever and lethargy, without the presence of an actual bacteria or virus; all you need is for your brain to think that you are sick.
While it must be acknowledged that the behavioral disturbances observed in autism are a lot different than feeling the need to watch TV all day, these types of studies were among the first clues that the traditional view of the CNS as a separate entity within the gated community of the blood brain barrier needed revision.
Measuring how much sugar water a rat drank is great stuff, but the reality is that we have conservatively a gazillion studies telling us that disorders that manifest behaviorally have strong, strong ties to the immune system; and once we begin to understand the vast scope of these findings, the utter frailty of “correlation does not equal causation” becomes painfully clear to the intellectually honest observer.
The big problem I found myself with in crafting this posting was that the sheer volume of studies available really makes a complete illustration of the literature impossible; I started looking and pubmed nearly puked trying to return to me a listing of all of the things I wanted to summarize. So here is some of the best of the best; to keep things interesting, I thought I’d only include findings from 2007 or later as a mechanism to show just how nascent our understanding of the connections between the brain and the immune system really are.
Initially, we can start with a condition that nearly everyone agrees is diagnosed based on behavior, depression. It turns out, the number of findings establishing a link between immune system markers and depression is wide and deep.
Here’s a great one, Elevated macrophage migration inhibitory factor (MIF) is associated with depressive symptoms, blunted cortisol reactivity to acute stress, and lowered morning cortisol, which reports, that MIF can modify HPA axis function and is tied to depression; a particularly compelling finding considering well documented alterations in HPA axis metabolites in autism, and the fact that increased MIF has also been found in the autism population, and as levels increased, so too did autism severity.
Here is part of the abstract for Inflammation and Its Discontents: The Role of Cytokines in the Pathophysiology of Major Depression (full paper)
Patients with major depression have been found to exhibit increased peripheral blood inflammatory biomarkers, including inflammatory cytokines, which have been shown to access the brain and interact with virtually every pathophysiologic domain known to be involved in depression, including neurotransmitter metabolism, neuroendocrine function, and neural plasticity. Indeed, activation of inflammatory pathways within the brain is believed to contribute to a confluence of decreased neurotrophic support and altered glutamate release/reuptake, as well as oxidative stress, leading to excitotoxicity and loss of glial elements, consistent with neuropathologic findings that characterize depressive disorders.
Somewhere along the way, researchers discovered that some anti-depressants can exert anti-inflammatory effects, for examples of these findings we could look to Fluoxetine and citalopram exhibit potent antiinflammatory activity in human and murine models of rheumatoid arthritis and inhibit toll-like receptors, or Plasma cytokine profiles in depressed patients who fail to respond to selective serotonin reuptake inhibitor therapy, which concludes in part, “Suppression of proinflammatory cytokines does not occur in depressed patients who fail to respond to SSRIs and is necessary for clinical recovery”.
In Investigating the inflammatory phenotype of major depression: focus on cytokines and polyunsaturated fatty acids, the authors report that, “The findings of this study provide further support for the view that major depression is associated with a pro-inflammatory phenotype which at least partially persists when patients become normothymic.” A nice review of the evidence of immunological participation in depression can be found in The concept of depression as a dysfunction of the immune system (full paper).
Moving forward, we can look to schizophrenia, we have similar findings, including Serum levels of IL-6, IL-10 and TNF-a in patients with bipolar disorder and schizophrenia: differences in pro- and anti-inflammatory balance, which observed an imbalanced baseline cytokine profile in the schizophrenic group; findings very similar in form with An activated set point of T-cell and monocyte inflammatory networks in recent-onset schizophrenia patients involves both pro- and anti-inflammatory forces. Similarly, the findings from Dysregulation of chemo-cytokine production in schizophrenic patients versus healthy controls, (full paper) which states, in part:
Growing evidence suggests that specific cytokines and chemokines play a role in signalling the brain to produce neurochemical, neuroendocrine, neuroimmune and behavioural changes. A relationship between inflammation and schizophrenia was supported by abnormal cytokines production, abnormal concentrations of cytokines and cytokine receptors in the blood and cerebrospinal fluid in schizophrenia
Their findings include differentially increased and decreased production of chemokines and cytokines as a result of LPS stimulations in the case group. Of particular note, a similarly dysregulated immune profile of cytokine and chemokine generation has been found in the autism population in several studies.
We also have several trials of immunomodulatory drugs in the schizophrenic arena that further implicate the immune system in pathology, including Adjuvant aspirin therapy reduces symptoms of schizophrenia spectrum disorders: results from a randomized, double-blind, placebo-controlled trial, a ‘gold standard’ trial which found that, “Aspirin given as adjuvant therapy to regular antipsychotic treatment reduces the symptoms of schizophrenia spectrum disorders. The reduction is more pronounced in those with the more altered immune function. Inflammation may constitute a potential new target for antipsychotic drug development”. A similar clinical trial, Celecoxib as adjunctive therapy in schizophrenia: a double-blind, randomized and placebo-controlled trial , another gold standard trial, which also had findings in the same vein, “Although both protocols significantly decreased the score of the positive, negative and general psychopathological symptoms over the trial period, the combination of risperidone and celecoxib showed a significant superiority over risperidone alone in the treatment of positive symptoms, general psychopathology symptoms as well as PANSS total scores.” [Celecoxib is a cox-2 inhibitor; i.e., anti-inflammatory, i.e., immunomodulatory]
What about bi-polar disorder? More of the same, including, The activation of monocyte and T cell networks in patients with bipolar disorder, or Elevation of cerebrospinal fluid interleukin-1ß in bipolar disorder, which reports, in part, “Our findings show an altered brain cytokine profile associated with the manifestation of recent manic/hypomanic episodes in patients with bipolar disorder. Although the causality remains to be established, these findings may suggest a pathophysiological role for IL-1ß in bipolar disorder.”. These studies were published in April and March, 2011, respectively.
Brain tissue from persons with bi-polar disorder also showed increased levels of excitotoxicity and neuroinflammation in Increased excitotoxicity and neuroinflammatory markers in postmortem frontal cortex from bipolar disorder patients (full version), and authors report differential cytokine profiles depending on state of mania, depression, or remission in Comparison of cytokine levels in depressed, manic and euthymic patients with bipolar disorder.
Another disorder based solely around behavior, Tourette syndrome, has increasingly unsurprising findings. Polymorphisms of interleukin 1 gene IL1RN are associated with Tourette syndrome reports “The odds ratio for developing Tourette syndrome in individuals with the IL1RN( *)1 allele, compared with IL1RN( *)2, was 7.65.” (!!!) , and Elevated expression of MCP-1, IL-2 and PTPR-N in basal ganglia of Tourette syndrome cases is yet another example of observations of CNS based immune participation in a disorder that is diagnosed by behavior.
There are also some reviews that perform a cross talk of sorts between disorders; i.e., The mononuclear phagocyte system and its cytokine inflammatory networks in schizophrenia and bipolar disorder, or Immune system to brain signaling: Neuropsychopharmacological implications, published in May 2011, which has this abstract:
There has been an explosion in our knowledge of the pathways and mechanisms by which the immune system can influence the brain and behavior. In the context of inflammation, pro-inflammatory cytokines can access the central nervous system and interact with a cytokine network in the brain to influence virtually every aspect of brain function relevant to behavior including neurotransmitter metabolism, neuroendocrine function, synaptic plasticity, and neurocircuits that regulate mood, motor activity, motivation, anxiety and alarm. Behavioral consequences of these effects of the immune system on the brain include depression, anxiety, fatigue, psychomotor slowing, anorexia, cognitive dysfunction and sleep impairment; symptoms that overlap with those which characterize neuropsychiatric disorders, especially depression. Pathways that appear to be especially important in immune system effects on the brain include the cytokine signaling molecules, p38 mitogen-activated protein kinase and nuclear factor kappa B; indoleamine 2,3 dioxygenase and its downstream metabolites, kynurenine, quinolinic acid and kynurenic acid; the neurotransmitters, serotonin, dopamine and glutamate; and neurocircuits involving the basal ganglia and anterior cingulate cortex. A series of vulnerability factors including aging and obesity as well as chronic stress also appears to interact with immune to brain signaling to exacerbate immunologic contributions to neuropsychiatric disease. The elucidation of the mechanisms by which the immune system influences behavior yields a host of targets for potential therapeutic development as well as informing strategies for the prevention of neuropsychiatric disease in at risk populations.
All of the conditions above, depression, schizophrenia, bi-polar, and tourettes are diagnosed behaviorally; it is only in the last few years that the medical dimension of these disorders were even understood to exist. None of the studies that I referenced above are more than five years old; the idea that behavioral disorders were so closely entangled with the immune system is very, very new. It should be noted that I intentionally left out disorders that also have reams of evidence of immune participation, but which are more degenerative in nature; i.e., Alzheimer’s, ALS, Parkinson’s. When discussing autism, I also left out studies involving aberrant presence of auto-antibodies, of which there are many.
One of the things that I have learned in trying to refine my thought processes during my time on the Internet is that rarely does a single study tell us much about a condition; but the converse also holds true, if we have many studies with different methodologies or measurement end points, but they all reach similar conclusions, then the likely-hood that the findings are accurate is much, much greater. All of the studies I have listed above tell us something similar; that the immune system is clearly, unmistakably playing a part in a lot of conditions classically considered neurological and diagnosed behaviorally. It isn’t enough to nitpick flaws in a single one of the studies in order for ‘correlation does not equal causation’ to make meaningful headway into the implications of these studies; instead, all of the studies above, and lots more, have to be wrong in the same way if we would like to return to a place where we can keep our heads in the sand, hoping for coincidences and bleating out catchphrases in the face of clinical findings. That isn’t going to happen. Given this reality, we should not and cannot ignore the growing evidence of immune abnormalities in the autism population, no matter how inconvenient following that trail of evidence might become.
-pD
