passionless Droning about autism

Archive for the ‘Mr. Rat’ Category

Hello Friends – There are (at least) two big
classifications of microglia findings in autism, an altered
morphology (i.e., shape and function, or ‘activated’ versus
‘quiescent’), and an increased number (i.e., more), with both
parameters varying with each other and spatially.  In other
words, disparate parts of the brain have different numbers of
microglia in them, and the functional profile of those microglia
also varies from one area to another. 
[Note: There is ongoing
discussion
regarding the appropriate definition of
‘activation’ of microglia, with evidence of (at least) four states
of microglial morphology.] Recently I saw a discussion on the SFARI
site about the fancy in
vivo study of microglia numbers in high functioning males with
autism
.  (I believe I am growing
increasingly jaded, as it occurs to me that radio tracing against
[11C](R)-PK11195) to
show microglial activation is a fancy trick, but one leaving us
open to detecting other stuff too.)  In any case, the findings
are not especially unexpected by now, well not to me anyways, but a
comment at the SFARI site really got me thinking about the chain of
events that could lead to different spatial and
morphological characteristics of microglia.  Perhaps we could
gain insight into the question of what the microglia are doing by
trying to understanding how they got there. Do we have any
biologically plausible models that might educate us on how a
different morphology and distribution of microglia could be
achieved? 
A while ago I got a copy of a few
articles that don’t have autism in them per se, but they kept
coming to the forefront of my mind when I thought about that
question.  The first is Distribution
of microglia in the postnatal murine nigrostriatal
system
,
which had a disease focus on
Parkinson’s, but what really grabbed my attention is what they
learned about the developmental pathway microglia took to populate,
and then depopulate the substantia nigra (SN), a little wedge of
brain involved with motor skills, reward seeking, and addiction.

Interestingly, the SN
has been shown to contain more microglia than
adjacent 
structures. We have analysed
changes in microglia numbers and in microglial morphology in the
postnatal murine nigrostriatal system at various stages ranging
from postnatal day 0 (P0) up to 24 months of age. We
clearly show that the microglia numbers in the SN and in the
striatum dramatically increase from P0 to P15
and
significantly decrease in both areas in 18-month-old and
24-month-old animals.

[Note: There seems to be
some variance in the appropriate ‘rat-to-human-age’ approximations;
especially when trying to do something as
expeditionary as comparing brain development.  We should
extrapolate only with caution.] The part that makes me grin is that
it illustrates our nascent understanding of the process of
microglial colonization into the CNS, the hows,
whens
, wheres, and whys are still
shrouded in mystery. The broadest outlines tell us that microglial
penetration into the brain is a long running, dynamic process; the
microglia are slow infiltrators, gaining access into parts of the
brain in concert with a swath of proliferating and inhibitory
factors, all at a time of once in a lifetime neurodevelopmental
modifications. Regulation
of postnatal forebrain amoeboid microglial cell proliferation and
development by the transcription factor Runx1
paints a
beautiful portrait of functionality.  Runx1 is a chemical
messenger that participates in phenotyopic determination of blood
cell progenitors into mature cells.  The researchers observed
spatial, time dependent expression of Runx1 in the developing
forebrain, and differential levels following injury.

Here we show that the mouse
transcription factor Runx1, a key regulator of myeloid
cell proliferation and differentiation, is expressed in forebrain
amoeboid microglia during the first two postnatal weeks
.
Runx1 expression is then downregulated in ramified microglia. Runx1
inhibits mouse amoeboid microglia proliferation and promotes
progression to the ramified state. We show further that
Runx1 expression is upregulated in microglia following nerve injury
in the adult mouse nervous system.
These findings provide
insight into the regulation of postnatal microglia activation and
maturation to the ramified state and have implications for
microglia biology in the developing and injured brain.

It doesn’t really tell us much about a
persistent change in microglia per se, but it does render a picture
of proliferation and differentiation as an easily
disrupted
symphony.  When we think about the
developing brain, I won’t pretend to have more than a lightyear
close guess at what microglia might be doing
differently between amoeboid and ramified
morphologies in this locale, at this time, but I
very highly doubt there isn’t
a functional impact on microenvironment neurodevelopment; our
developing brains are using opportunities like the Indians used the
buffalo, no waste, no excess, and because balance is important,
everything is important. Moving back to the
question of the plausibility of a pathway to the autism state,
luckily (or unluckily?) the literature is veritably littered with
insults that perturb microglial development, leading to
 persistent changes to microglial morphology, ultimately
percolating up to behavioral changes. Prenatal stress is a bad, bad
thing, and here is a study that finds that extreme mice stress can
persistently alter the mice activation profile of mice microglia.
Prenatal
stress increases the expression of proinflammatory cytokines and
exacerbates the inflammatory response to LPS in the hippocampal
formation of adult male mice
, was just published, and
comes wrapped up with a double hit, and
different resting and stimulated neuroimmune environments.

Under basal conditions,
prenatally stressed animals showed increased expression of
interleukin 1ß and tumor necrosis factor-a (TNF-a) in the
hippocampus and an increased percentage of microglia cells with
reactive morphology in CA1 compared to non-stressed males.
Furthermore, prenatally stressed mice showed increased TNF-a
immunoreactivity in CA1 and increased number of Iba-1
immunoreactive microglia and GFAP-immunoreactive astrocytes in the
dentate gyrus after LPS administration. In contrast, LPS did not
induce such changes in non-stressed animals. These
findings indicate that prenatal stress induces a basal
proinflammatory status in the hippocampal formation during
adulthood that results in an enhanced activation of microglia and
astrocytes in response to a proinflammatory
insult.

Note: I have not read this
paper so I do not know if a qualitative number of microglia, or
just more immune-targeted microglia were found, but likely the
latter. A similar, full free paper, Prenatal
stress causes alterations in the morphology of microglia and the
inflammatory response of the hippocampus of adult female
mice
, found broadly similar results; perturbed resting
and stimulated states in the treatment group.

Prenatal stress, per se,
increased IL1ß mRNA levels in the hippocampus, increased the total
number of Iba1-immunoreactive microglial cells and increased the
proportion of microglial cells with large somas and retracted
cellular processes. In addition, prenatally stressed and
non-stressed animals showed different responses to peripheral
inflammation induced by systemic administration of LPS.
LPS induced a significant increase in mRNA levels of IL-6,
TNF-a and IP10 in the hippocampus of prenatally stressed mice but
not of non-stressed animals.
 

Going back to my
ramblings on glial priming
, it seems that here we have an
example of a type of cross system priming (sweet!), where
disturbing the stress response system changed the immune system;
such is the way of the polyamorous chemical families interacting in
our brain.  It also occurs to me that given the
delicate nature of the developing brain, and the
crazy important
tasks going on in there, we might want to think very
carefully before we ‘induced a significant increase in
mRNA levels of IL-6, TNF-a and IP10 in the hippocampus‘

on subgroups who might be environmentally predisposed to react with
exaggerated vigor.  But what do I know? Of course, the
prenatal immune challenge arena holds a ton of studies on
persistent microglial function, and ‘consequences’.  There are
way too many to list, but a quick overview of some very recent ones
would include: Enduring
consequences of early-life infection on glial and neural cell
genesis within cognitive regions of the brain
, an early
life real infection model with e coli that
concludes, “Taken together, we have provided evidence that
systemic infection with E. coli early in life has significant,
enduring consequences for brain development and subsequent adult
function
.”  (Staci Bilbo!)  This paper was sort
of a quinella, as it showed both changes in immune responsiveness
into adulthood; it also demonstrated the ability
of an immune insult to alter
the developmental trajectory of the
microglia, i.e., E. coli increased the number of newborn
microglia within the hippocampus and PAR compared to controls. The
total number of microglia was also significantly increased in E.
coli-treated pups, with a concomitant decrease in total
proliferation.
Neonatal
lipopolysaccharide exposure induces long-lasting learning
impairment, less anxiety-like response and hippocampal injury in
adult rats
very directly blasted rats with some LPS
immune activation action, and includes, ”Neonatal LPS
exposure also resulted in sustained inflammatory responses in the
P71 rat hippocampus, as indicated by an increased number of
activated microglia and elevation of interleukin-1ß content in the
rat hippocampus.”  
(Sound familiar?) Interleukin-1
receptor antagonist ameliorates neonatal lipopolysaccharide-induced
long-lasting hyperalgesia in the adult rats
took the
extra step of adding a set of animals that got inhibited
inflammatory responses.  Results are increasingly
unsurprising.

Neonatal
administration of an IL-1 receptor antagonist (0.1mg/kg)
significantly attenuated long-lasting hyperalgesia induced by LPS
and reduced the number of activated microglia in the adult rat
brain. These data reveal that neonatal intracerebral LPS
exposure results in long-lasting hyperalgesia and an elevated
number of activated microglia in later life. This effect is similar
to that induced by IL-1ß and can be prevented by an IL-1 receptor
antagonist

I love how (once again) we
can see how interrupting the immune response can have an effect.
Environmental impacts outside of the immune
activation realm may also find a place within the ‘big tent’ of
microglial agitation with consequent developmental impacts. 
The people who made the first big neuroimmune / autism splash at
Johns Hopkins later came out with Neuroinflammation
and behavioral abnormalities after neonatal terbutaline treatment
in rats: implications for autism
, which found that an
agent used to prevent labor in some situations could
produced a robust increase in microglial activation on PN
30 in the cerebral cortex”
in treatment animals. 
The drug in question, terbutaline, has been weakly associated with
increased incidence of autism, i.e., Prenatal
exposure to ß2-adrenergic receptor agonists and risk of autism
spectrum disorders
, and beta2-adrenergic
receptor activation and genetic polymorphisms in autism: data from
dizygotic twins
. And now, in 2013, Beta-adrenergic
receptor activation primes microglia cytokine production
,
displays another example of cross system
priming.

To determine
if ß-AR stimulation is sufficient to prime microglia, rats were
intra-cerebroventricularly administered isoproterenol (ß-AR
agonist) or vehicle and 24h later hippocampal microglia were placed
in culture with media or LPS. Prior isoproterenol treatment
significantly enhanced IL-1ß and IL-6, but not TNF-a production
following LPS stimulation. These data suggest that central
ß-AR stimulation is sufficient to prime microglia cytokine
responses.

In other words, they gave
the rats a drug in the class of terbutline, and subsequently
observed an increased microglia responsiveness in cultured
cells.  What a crazy coincidence. Detecting total
populations
of microglia in adulthood, either regionally
or in the brain as a whole is a little more difficult, the little
buggers are a lot easier to detect when we light them up with neon
green tracers that stick to proteins expressed at ‘activation’
time, and it just doesn’t look like the question has been asked too
many times.  I did, however, find something that has a sort of
chip shot on this analysis, Prenatal
stress alters microglial development and distribution in postnatal
rat brain
, which looked at regional microglia populations
and phenotypes at two time periods following prenatal stress
events.

Prenatal
stress consisting of 20 min of forced swimming occurred on
embryonic days 10–20. On postnatal days 1 and 10, stressed and
control pups were killed. Microglia were identified using Griffonia
 simplicifolia lectin and quantified in the whole encephalon.
In addition, plasma corticosterone was measured in dams at
embryonic day 20, and in pups on postnatal days 1 and 10.
At postnatal day 1, there was an increase in number of
ramified microglia in the parietal, entorhinal and frontal
cortices, septum, basal ganglia, thalamus, medulla oblongata and
internal capsule in the stressed pups as compared to controls, but
also there was a reduction of amoeboid microglia and the total
number of microglia in the corpus callosum.
By postnatal
day 10, there were no differences in the morphologic type or the
distribution of microglia between the prenatal stress and control
groups, except in the corpus callosum; where prenatal stress
decreased the number of ramified microglia. The stress procedure
was effective in producing plasma rise in corticosterone levels of
pregnant rats at embryonic day 20 when compared to same age
controls. Prenatal stress reduced the number of immature
microglia and promoted an accelerated microglial differentiation
into a ramified form.

They did a lot
of clever stuff at analysis time, taking samples from several
locations after birth and ten days later, and
also did some fine grained classification of the
shape of the microglia.  They include spatial and temporal
mappings of four microglial developmental profiles.  It looks
as if prental stress was able to alter the developmental speed of
microglia from one morphology to another in different parts of the
brain.  There was as small section in the discussion that
speculated on what such changes might mean for neurodevelopment.

Given that during the
early postnatal period occur numerous brain developmental processes
(e.g. neurogenesis, myelination, synaptogenesis, astrogliogenesis,
neuronal cell death and blood–brain barrier maturation) [6, 19, 22,
25, 36, 52] it is possible that altered microglial
development induced by in utero stress may affect other
developmental processes either changing microenvironment molecular
constitution or triggering earlier inflammatory changes secondary
to the blood–brain barrier opening induced by prenatal
stress
.  Although punctual, the altered microglial
development might alter extensively the other
neurodevelopmental processes
ensuing perdurable
structural changes
; for example it is possible that the
change in the distribution pattern of microglia in the prenatal
stress group may render vulnerable some neuroanatomic
regions due to the reduction of neurotrophic factors
,
such as the corpus callosum where there is a continuous axonal
growth

No kidding! [There is also some very
interesting notes regarding microglial participation in purkinje
cell death that deserves and entire post. . .] This should be the
point that any rational observer must accept that we several lines
of evidence that early life experiences can persistently alter
microglial function with plausible mechanisms that could affect
neurodevelopment.  Our data concerning total population
numbers in adulthood is a lot more difficult to come by, but I
think this will probably be getting looked at soon enough. Of
course, in any particular individual it is difficult (or
impossible?) to know how they may have arrived at a state of
increased microglial activation, but at the same time, it is not as
if we have no clue on possible pathways to this destination; our
short list of environmental factors includes immune insult, stress,
and chemical agents. If the question is, ‘what are the microglia
doing in the autism population?’, one plausible answer is ‘their
phenotype was persistently altered by an early life event through a
developmental programming model’. As I was mulling all of this
over, two things happened.  First, a maternal CRP
study
came out, and found a pretty strong relationship
between direct measurements of mommy inflammation with increased
risk of baby autism.  The nice part is that they had a
gigantic data set (1.2M births!) to work with thanks to a few
decades of single payer medicine.  (Very
nice!
)

For maternal CRP
levels in the highest quintile, compared with the lowest quintile,
there was a significant, 43% elevated
risk.
This finding suggests that
maternal inflammation may have a significant role in autism, with
possible implications for identifying preventive strategies and
pathogenic mechanisms in autism and other neurodevelopmental
disorders.

Just after that paper came out, I
made some Fred Flintstone style beef ribs.  I ‘primed’ the
meat with a Moroccan inspired spice rub overnight, then
slow, slow, slow cooked them with a
low, low, low heat all day
long
, and blasted away with a date glaze under the
broiler just before go time and they were caveman style
primal fucking awesome
.  The key to arriving there
was the slow cooking. The rib preparation
process got me thinking about our population wide experiment
in replacing infection with inflammation
where we have
traded in death by pathogens or other once fatal ailments in
exchange for a longer life frequently plagued by conditions
associated with higher inflammation.  Our analysis on long
term alterations to microglial proliferation and morphology is
largely comprised of studying acute insults
(sound familiar?), i.e., injection of purified bacterial cell
components known to trigger a robust immune response, ten sessions
of mouse based pregnant forced swimming, or exposure to chemicals
with rare and particular exposure routes in humans.  Mostly I
think this is due to the black swan nature of the developmental programming
model
alongside the very new idea that microglia are
doing jobs other than responding to infections; our models are
crude because of our relative ignorance.  What will we find
when our filters are appropriately powered to detect for chronic,
but subtle insults? It occurs to me that there may be a ribs model
of altered microglial colonization of the fetal brain; it seems
clear that proliferation and differentiation of microglia can
clearly be changed by powerful inputs, but the
chemical messengers that impact that change are closely related (or
the same) as the measurement points in the maternal CRP study.
Could a slow cooking of slightly higher but not acutely
increased
maternal inflammation be participating in the
genesis of autism (in some children) through altering the migration
and proliferation of microglia into the neonatal brain?  Could
the same chemical messengers of inflammation be subtly
priming
the microglia to respond with increased vigor to
insults later in life?  Has our replacement of infection with
inflammation included an unanticipated effect that alters the
developmental pathway of the very cells that help shape our
children’s brains? I don’t think we are (quite) clever enough to
answer these types of questions yet, but we are at least starting
to generate the right kind of data to inform us on how to get
started.  I don’t know what we will find, but the initial data
doesn’t look very good.  In the meantime, I am recommending
you go get some ribs and let them cook all day long.      

pD

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


Hello friends –

I’ve been planning to write something about the idea of environmental enrichment for a while now but other stuff kept on popping up.  At a broad level, researchers are finding that the type of external stimulation an animal is raised or housed in can have dizzyingly unpredictable effects on a range of physiological and behavioral endpoints, many of which are of great interest to the autism community. This is a tough area to dance through in the autism world; the available literature has shades of refrigerator mothers, and TV based causation; yet, the underlying idea of environmental enrichment, that the external environment can affect a person in a very physical way, is something known to the autism community in concrete ways.  What’s more, much of our data in the environmental enrichment realm is nothing less than compelling.  It is exciting to know that we are beginning to have insight into the molecular mechanisms by which the environment can affect the body and brain, and with that insight, just maybe the wisdom to help our children and help ourselves.

From the biomarker side, a couple of neat studies would include Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice,  wherein striking reductions in amyloid proteins were found in knockout mice housed in a stimulating environment compared to those in standard housing.   Or the very recently published,  Complex environment experience rescues impaired neurogenesis, enhances synaptic plasticity, and attenuates neuropathology in familial Alzheimer’s disease-linked APPswe/PS1DeltaE9 mice which hits a lot of keywords with parallels to the autism research world.  There are many, many others including hits like Altered plasticity in hippocampal CA1, but not dentate gyrus, following long-term environmental enrichment, or hilariously named, soundbyte laden,  Hippocampal epigenetic modification at the brain-derived neurotrophic factor gene induced by an enriched environment. The lower level details of these studies and their many ancestors are beyond the scope of what I have time for now, but clearly anything that can be affecting synaptic plasticity, BDNF expression, and neurogenesis should be of interest to the autism community.

If we turn to measurements that go beyond frozen slices of tissue (but do not necessary exclude them), our data regarding behavioral differences in EE housed animals is also robust.  For example, we could look at Environmental enrichment delays the onset of memory deficits and reduces neuropathological hallmarks in a mouse model of Alzheimer-like neurodegeneration, which found that EE housed mice performed significantly better at memory tasks that other mice housed in non stimulatory environments.  Environmental-enrichment-related variations in behavioral, biochemical, and physiologic responses of sprague-dawley and long evans rats concludes by saying, The data support the claim that environmental enrichment may render animals more resilient to challenges.   Ouch.  Forgetting chronic diseases such as Alzheimer’s or Parkinson’s, which get a lot of attention in the EE world, even things like traumatic brain injury or lack of oxygen to the brain seem to show benefits from a stimulating environment, as we can see from studies like Environmental Enrichment Influences BDNF and NR1 Levels in the Hippocampus and Restores Cognitive Impairment in Chronic Cerebral Hypoperfused Rats or Empirical comparison of typical and atypical environmental enrichment paradigms on functional and histological outcome after experimental traumatic brain injury.  The flip side, a ‘de-enriched’ environment has findings along the lines of what you might expect; i.e., Environmental impoverishment and aging alter object recognition, spatial learning, and dentate gyrus astrocytes.  Ouch.

So what is, exactly, an enriched environment?  The methods section from Environmental enrichment reduces Abeta levels and amyloid deposition in transgenic mice says this:

Animal experiments were conducted in accordance with institutional and NIH guidelines. Male offspring of transgenic breeding pairs APPswe × PS1 were separated from their mother at 3 weeks of age (after weaning), genotyped, and housed four males to a cage. Enriched environment was composed of large cages running wheels, colored tunnels, toys, and chewable material. For 1 month, mice were exposed to enriched environment every day for 3 hr and were returned to their original cages for the remaining 21 hr. After 1 month of daily enrichment, mice were introduced to the enriched environment three times a week for an additional 4 months. Mice were sacrificed at age of 6months. Following weaning, a control group of animals was maintained for 5 months in standard housing conditions.

Lets consider the implications of these findings.  Reducing amyloid buildup has been a holy grail of the pharmaceutical companies for a long time now, though it is possible that this plan of attack was based on bad assumptions.  Tens of millions of dollars (or hundreds of  millions) have been thrown at synthetic ways to reduce or eliminate the buildup of amyloid plaque in mice, rats, and recently, people with mixed to poor results.  Even if it turns out that amyloid isn’t causing Alzheimer’s, that doesn’t do anything to change the fact that these researchers were able to make very significant changes to biological systems by setting their rats up in a rat mansion with rat delivered food and a rat tennis court for a few hours a day.   Despite the mixed findings as of late on the effect of brain training in order to stave off dementia, I think most of us have known someone, or Kevin Baconed one degree out to know someone who has seemingly either degenerated with a stagnant environment, or kept on trucking through old age with a more active lifestyle.  Is their environment participating?

So what about autism?  The most extreme and tragic parallels can be seen in studies of children from orphanages, notably in Romania, where children were raised in absolutely destitute surroundings.  A recent study is entitled Stereotypies in children with a history of early institutional care with these findings:

RESULTS: At the baseline assessment prior to placement in foster care (average age of 22 months), more than 60% of children in institutional care exhibited stereotypies. Follow-up assessments at 30 months, 42 months, and 54 months indicated that being placed in families significantly reduced stereotypies, and with earlier and longer placements, reductions became larger. For children in the foster care group, but not in the care as usual group, stereotypies were significantly associated with lower outcomes on measures of language and cognition.

CONCLUSIONS: Stereotypies are prevalent in children with a history of institutional care. A foster care intervention appears to have a beneficial/moderating role on reducing stereotypies, underscoring the need for early placement in home-based care for abandoned children. Children who continue to exhibit stereotypies after foster care placement are significantly more impaired on outcomes of language and cognition than children without stereotypies and thus may be a target for further assessments or interventions.

Another study that looks specifically towards autistic like behaviors in children raised in orphanages is Early adolescent outcomes of institutionally deprived and non-deprived adoptees. III. Quasi-autism.

BACKGROUND: Some young children reared in profoundly depriving institutions have been found to show autistic-like patterns, but the developmental significance of these features is unknown.

METHODS: A randomly selected, age-stratified, sample of 144 children who had experienced an institutional upbringing in Romania and who were adopted by UK families was studied at 4, 6, and 11 years, and compared with a non-institutionalised sample of 52 domestic adoptees. Twenty-eight children, all from Romanian institutions, for whom the possibility of quasi-autism had been raised, were assessed using the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS) at the age of 12 years.

RESULTS: Sixteen children were found to have a quasi-autistic pattern; a rate of 9.2% in the Romanian institution-reared adoptees with an IQ of at least 50 as compared with 0% in the domestic adoptees. There were a further 12 children with some autistic-like features, but for whom the quasi-autism designation was not confirmed. The follow-up of the children showed that a quarter of the children lost their autistic-like features by 11. Disinhibited attachment and poor peer relationships were also present in over half of the children with quasi-autism.

CONCLUSIONS: The findings at age 11/12 years confirmed the reality and clinical significance of the quasi-autistic patterns seen in over 1 in 10 of the children who experienced profound institutional deprivation. Although there were important similarities with ‘ordinary’ autism, the dissimilarities suggest a different meaning.

Similarly depressing findings can be found in places like Institutional rearing and psychiatric disorders in Romanian preschool children, or Placement in foster care enhances quality of attachment among young institutionalized children.   There is a gripping This American Life about a child adopted from Romania.    Please be sure your head is in the right place before listening to part II, which describes a family trying to decide of their very severely autistic son should be placed in residential care.  I ran into this episode on accident one day in the car when I was already feeling bleak and walked out the other end pretty fucked up for a few days; those guys are really good and the narrative can hit very close to home for some.

Calling up images from the dark(er) days of autism and Bettleheim we have an array of studies on the effect of maternal separation and subsequent physiological and behavioral effects that have parallels in autism findings.  For example, here is an abstract from Behavioural and neurochemical consequences of early weaning in rodents

Among all mammalian species, pups are highly dependent on their mother not only for nutrition, but also for physical interaction. Therefore, disruption of the mother-pup interaction changes the physiology and behaviour of pups. We review how maternal separation in the early developmental period brings about changes in the behaviour and neuronal systems of the offspring of rats and mice. Early weaning in mice results in adulthood a persistent increase in anxiety-like and aggressive behaviour. The early-weaned mice also show higher hypothalamic-pituitary-adrenal activity in response to novelty stress. Neurochemically, the early-weaned male mice, but not female mice, show precocious myelination in the amygdala, decreased brain-derived neurotrophic factor protein levels in the hippocampus and prefrontal cortex, and reduced bromodeoxyuridine immunoreactivity in the dentate gyrus. Because higher corticosterone levels are persistently observed up to 48 h when the mice are weaned on postnatal day 14, the exposure of the developing brain to higher corticosterone levels may be one of the effects of early weaning. These results suggest that deprivation of the mother-infant interaction during the late lactating period results in behavioural and neurochemical changes in adulthood and that these stress responses are sexually dimorphic (i.e. the male is more vulnerable to early weaning stress).

The rapidfire analysis tells us that altered HPA-Axis activityBDNF levels, and anxiety all have parallels in autism, along with perhaps the most consistent finding in animal studies that have interest to autism, the problems of being born male and consequent risk factors from nearly everything.   This is a review paper, but there are a gazillion others with titles like Maternal separation disrupts dendritic morphology of neurons in prefrontal cortex, hippocampus, and nucleus accumbens in male rat offspring, Short- and long-term consequences of different early environmental conditions on central immunoreactive oxytocin and arginine vasopressin levels in male rats, or Prolonged maternal separation decreases granule cell number in the dentate gyrus of 3-week-old male rats.

Though I’m pretty sure that this should be clear to everyone, just to be sure, I’m not proposing a refrigerator mother theory of autism. But the data is the data and the logical opposite of an enriched environment is also born out.

So what?  Well, this reminded me of the “Rat Park” studies an Internet friend told me about, wherein researchers seemed to find that animals dosed with opiates for several weeks would voluntarily wean themselves from the drugs if moved to much larger enclosures where they had access to either drugged water or plain water.  The startling thing about the Rat Park studies isn’t so much what was learned about opiate addiction, so much as the broader implications that the existing studies on drug addiction might not be studying the right thing; that instead of testing the effects on opiate availability on rodents, they were testing the effects of opiate availability on chronically depressed rodents.   Following through, it occurs to me that in addition to the bazillion other problems we have moving from rodent to human with anything other than a hopeful educated guess, we must grudgingly admit that the condition the animals were housed in may be affecting a lot of findings.  As if we didn’t have enough confounders already!

But more importantly, these types of findings are beautiful portraits of complexity, the dispassionate hand of nature and the dangers of thinking you understand.

There are so many instances where we have found that as we gain the ability to make more detailed observations, we learn that our existing conclusions were crude facsimiles of reality, and oftentimes, conclusions that had been formed on dangerously unsound foundations.  By way of example, exposure to lead and consequent effects on neurodevelopment.  At one point, lead was used as a pesticide, eventually we figured out that wasn’t such a good idea, but it should be fine in paint and gasoline.  Then we removed it from paint.  Then gasoline.  And just a few years back, the ‘safe’ level of lead was deemed to be zero; and even the tiniest increases in lead were associated with developmental problems.  Of course, this was always the reality, but it was not until we applied filters of sufficient sophistication that our observations were adequately powered to understand the reality.   Are our studies of any number of factors clever enough to discern the changes we’d like to understand when we realize that subtle changes are still changes?

It gets thornier for the autism community in particular.  One thing a lot of our kids aren’t very good at are “complex environmental interactions”, in fact, a lot of our kids are flat out terrible at them.  After a couple of weeks/months/years of soul crushing experiences trying new things out with kid autism, some parents might start to think to themselves that a trip to the zoo, or the museum, or the movie theater or even the super market just isn’t worth it.  The result, while not necessarily an abject environment can start to resemble a single square mile of ocean, indistinguishable from the sea for backwards or forwards; the real world equivalent of a DVD set on repeat play.  I speak from experience, a rule in our household when one of the parents had to leave the other home for a weekend with kid autism was ‘survive, don’t thrive’. If that meant a trip to the same lake, spinning the same DVD, and a meal of the same food, but a relatively meltdown free weekend, that was OK.

We survived, but did we spite ourselves in the process?  Were we reinforcing at a neurochemical level some of the causes of the very behaviors that were causing us to retreat to the middle of the ocean?  We are starting to learn that this might be somewhat of a self fulfilling prophecy; taking a child who already does very poorly in new environments and run him or her through the same things over and over could be exacerbating their ability to handle new environments in a physiological way.  The data is the data.

That being said, there is an upside, a big one; the flip side is that parents have a chance to make real and salient changes in their child by the least controversial methods possible; gentle but repeated exposure to new things.  For some of us, this means a lot of shitty days and late night drinks to get through to the other side.  That’s OK.  It’s worth it.  Steel yourself for a meltdown and turn off the goddamned TV, take kid autism to the zoo, or the bounce house playground, or art festival, or a ‘non-autism’ friends house.  A lot of our children might need a helping hand, a gentle push, or a well meaning shove into the world,  but someone has to do it, and the world isn’t going to get any less complicated while we wait.  When it works out, and you have even a single new experience your child enjoys, that is an enriched environment for you, and enriched environments aren’t just for rats and kids.

– pD

Hello friends –

A new paper  looking for evidence of an ongoing immune reaction in the brain of people with autism landed the other day, Microglial Activation and Increased Microglial Density Observed in the Dorsolateral Prefrontal Cortex in Autism

BACKGROUND: In the neurodevelopmental disorder autism, several neuroimmune abnormalities have been reported. However, it is unknown whether microglial somal volume or density are altered in the cortex and whether any alteration is associated with age or other potential covariates. METHODS: Microglia in sections from the dorsolateral prefrontal cortex of nonmacrencephalic male cases with autism (n = 13) and control cases (n = 9) were visualized via ionized calcium binding adapter molecule 1 immunohistochemistry. In addition to a neuropathological assessment, microglial cell density was stereologically estimated via optical fractionator and average somal volume was quantified via isotropic nucleator. RESULTS: Microglia appeared markedly activated in 5 of 13 cases with autism, including 2 of 3 under age 6, and marginally activated in an additional 4 of 13 cases. Morphological alterations included somal enlargement, process retraction and thickening, and extension of filopodia from processes. Average microglial somal volume was significantly increased in white matter (p = .013), with a trend in gray matter (p = .098). Microglial cell density was increased in gray matter (p = .002). Seizure history did not influence any activation measure. CONCLUSIONS: The activation profile described represents a neuropathological alteration in a sizeable fraction of cases with autism. Given its early presence, microglial activation may play a central role in the pathogenesis of autism in a substantial proportion of patients. Alternatively, activation may represent a response of the innate neuroimmune system to synaptic, neuronal, or neuronal network disturbances, or reflect genetic and/or environmental abnormalities impacting multiple cellular populations.

This is a neat paper,  to my eye not  as comprehensive as the landmark paper on microglial activation, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism Neuroglial Activation andNeuroinflammation in the Brain of Patientswith Autism, but still a very interesting read.  Here are the some areas that caught my eye.   From the introduction:

These results provide evidence for microglial activation in autism but stop short of demonstrating quantifiable microglial abnormalities in the cortex, as well as determining the nature of these abnormalities. Somal volume increases are often observed during microglial activation, reflecting a shift toward an amoeboid morphology that is accompanied by retraction and thickening of processes (13). Microglial density may also increase, reflecting either proliferation of resident microglia or increased trafficking of macrophages across a blood-brain barrier opened in response to signaling by cytokines, chemokines, and other immune mediators (13–16). These results provide evidence for microglial activation in autism but stop short of demonstrating quantifiable microglial abnormalitiesin the cortex, as well as determining the nature of these abnormalities. Somal volume increases are often observed during microglial activation, reflecting a shift toward an amoeboid morphology that is accompanied by retraction and thickening ofprocesses (13). Microglial density may also increase, reflecting either proliferation of resident microglia or increased trafficking of macrophages across a blood-brain barrier opened in response to signaling by cytokines, chemokines, and other immune mediators(13–16).

Tragically, my ongoing google based degree in neurology has yet to cover the chapters on specific brain geography, so the finer points, such as the difference between the middle frontal gyri and the neocortex are lost on me.  None the less, several things jump out at me from what I have managed to understand so far.  The shift to an ‘ameboid morphology’ is one that I’ve run into previously, notably in Early-life programming of later-life brain and behavior: a critical role for the immune system, which is a paper I really need to dedicate an entire post towards, but as applicable here, the general idea is that the microglia undergo structural and functional changes during times of immune response; the ‘ameboid’ morphology is associated with an active immune response.  Regarding increased trafficking of macrophages across the BBB, Vargas 2005  noted chemokines (MCP-1) increases in the CNS, so we do have reason to believe such signalling molecules are present.

The authors went on to look for structural changes in microglia, differences in concentration of microglia, and evaluated  for markers indicative of an acute inflammatory response.  Measurements such as grey and white matter volumes and relationships to microglia structural differences, and correlations with seizure activity were also performed.   There were three specimens from children under the age of six that were analyzed as a subgroup to determine if immune activation was present at early ages.   From the discussion section:

Moderate to strong alterations in Iba-1 positive microglial morphology indicative of activation (13,29) are present in 5 of 13 postmortem cases with autism, and mild alterations are present in an additional 4 of 13 cases. These alterations are reflected in a significant increase in average microglial somal volume in white matter and microglial density in gray matter, as well as a trend in microglial somal volume in gray matter. These observations appear to reflect a relatively frequent occurrence of cortical microglial activation in autism.

Of particular interest are the alterations present in two thirds of our youngest cases, during a period of early brain overgrowth in the disorder. Indeed, neither microglial somal volume nor density showed significant correlation with age in autism, suggesting long running alteration that is in striking contrast with neuronal features examined in the same cases (Morgan et al., unpublished data, 2009). The early presence of microglial activation indicates it may play a central pathogenic role in some patients with autism.

The authors evaluated for IL-1R1 receptor presence, essentially a marker for an inflammatory response, and found that the values did not differ between the autism population and controls, and that in fact the controls trended towards expressing more IL-1R1 than the autism group.  I think this was the opposite of what the authors expected to find.

While Iba-1 staining intensity increases modestly in activated microglia (30), strong staining and fine detail were apparent in Iba-1 positive resting microglia in our samples. Second, there is no increase in microglial colocalization with a receptor, IL-1R1, typically upregulated in acute inflammatory reactions (28). The trend toward an increase in colocalization in control cases may also hint at downregulation of inflammatory signal receptors in a chronically activated system.

I don’t think I’ve seen this type of detail in qualitative measures of the neuroimmune response in autism measured previously, so I definitely appreciate the detail.  Furthermore, from a more speculative standpoint, we may have some thoughts on why we might see this in the autism population specifically that I’ll go into detail below a little bit.

The authors failed to find a relationship between seizure activity and microglial activation, which came as a surprise to me, to tell the truth.  Also discussed was the large degree of heterogeneity in the findings in so far as the type and severity of microglial morphological differences observed.  The potential confounds in the study included an inability to control for medication history, and the cause of death, eight of which were drowning in the autism cases.  There was some discussion of potential causes, including, of course, gene-environment interactions, maternal immune activation, neural antibodies, and the idea that “chronic innate immune system activation might gradually produce autoimmune antibodies via the occasional presentation of brain proteins as antigens”  (!)  There was also this snipet:

Microglial activation might also represent an aberrant event during embryonic monocyte infiltration that may or may not also be reflected in astroglial and neuronal populations (17), given the largely or entirely separate developmental lineage of microglia (13). Alternatively, alterations might reflect an innate neuroimmune response to events in the brain such as excessive early neuron generation or aberrant development of neuronal connectivity.

There is a short discussion of the possible effects of an ongoing microglial immune response, including damage to neural cells, reductions in cells such as Purkinjes, and increases in neurotrophic factors such as BDNF.

This is another illustration of an ongoing immune response in the CNS of the autism population, though in this instance, only some of the treatment group appeared to be affected.  It would have been nice to see if there were correlations between behavioral severity and/or specific behavior types, but it would seem that this information is was not available in sufficient quality for this type of analysis, which is likely going to be an ongoing problem with post mortem studies for some time to come.   I believe that an effort to develop an autism tissue bank is underway, perhaps eventually some of these logistical problems will be easier to address.   The fact that some of the samples were from very young children provides evidence that when present, the neuroinflammatory response is chronic, and indeed, likely lifelong.

Stepping away from the paper proper, I had some thoughts about some of these findings that are difficult to defend with more than a skeletal framework, but have been rattling around my head for a little while.  Before we move forward, let’s be clear on a couple of things:

1) The jump from rodent to human is fraught with complications, most of which I doubt we even understand.

2) We can’t be positive that an activated neuroimmune system is the cause of autistic behaviors, as opposed to a result of having autism.  I still think a very strong argument can be made that an ongoing immune response is ultimately detrimental, even if it cannot be proven to be completely responsible for the behavioral manifestation of autism.

3) At the end of the day, I’m just Some Jerk On The Internet.

Those caveats made, Morgan et all spend a little time on the potential cause of a persistent neuroinflammatory state as referenced above.  One of the ideas, “an aberrant event during embyronic monocyte infiltration that may or may not also be reflected in astroglial and neuronal populations  given the largely or entirely separate developmental lineage of microglia”  struck me as particularly salient  when considered alongside the multitude of data we have concerning the difficult to predict findings regarding an immune insult during critical developmental timeframes.

We now have several papers that dig deeper into the mechanism by which immune interaction during development  seem to have physiological effects with some parallels to autism; specifically, Enduring consequences of early-life infection on glial and neural cell genesis within cognitive regions of the brain (Bland et all), and Early-Life Programming of Later-Life Brain and Behavior: A Critical Role for the Immune System (Bilbo et all) ; both of which share Staci Bilbo as an author and I think she is seriously onto something.  Here is the abstract for Bland et all:

Systemic infection with Escherichia coli on postnatal day (P) 4 in rats results in significantly altered brain cytokine responses and behavioral changes in adulthood, but only in response to a subsequent immune challenge with lipopolysaccharide [LPS]. The basis for these changes may be long-term changes in glial cell function. We assessed glial and neural cell genesis in the hippocampus, parietal cortex (PAR), and pre-frontal cortex (PFC), in neonates just after the infection, as well as in adulthood in response to LPS. E. coli increased the number of newborn microglia within the hippocampus and PAR compared to controls. The total number of microglia was also significantly increased in E. coli-treated pups, with a concomitant decrease in total proliferation. On P33, there were large decreases in numbers of cells coexpressing BrdU and NeuN in all brain regions of E. coli rats compared to controls. In adulthood, basal neurogenesis within the dentate gyrus (DG) did not differ between groups; however, in response to LPS, there was a decrease in neurogenesis in early-infected rats, but an increase in controls to the same challenge. There were also significantly more microglia in the adult DG of early-infected rats, although microglial proliferation in response to LPS was increased in controls. Taken together, we have provided evidence that systemic infection with E. coli early in life has significant, enduring consequences for brain development and subsequent adult function. These changes include marked alterations in glia, as well as influences on neurogenesis in brain regions important for cognition.

Bland et all went on to theorize on the mechanism by which an infection in early life can have such long lasting effects.

We have hypothesized that the basis for this vulnerability may be long-term changes in glial cell function. Microglia are the primary cytokine producers within the brain, and are an excellent candidate for long-term changes, because they are long-lived and can become and remain activated chronically (Town et al., 2005). There is increasing support for the concept of ‘‘glial priming”, in which cells can become sensitized by an insult, challenge, or injury,  such that subsequent responses to a challenge are exaggerated (Perry et al., 2003).

The authors infected some rodents with e-coli on postnatal day four, and then evaluated for microglial function in  adulthood.

We have hypothesized that the basis for early-life infection-induced vulnerability to altered cytokine expression and cognitive deficits in adulthood may be due to long-term changes in glial cell function and/or influences on subsequent neural development. E. coli infection on P4 markedly increased microglial proliferation in the CA regions of the hippocampus and PAR of newborn pups, compared to a PBS injection (Figs. 3 and 4). The total number of microglia, and specifically microglia with an ‘‘active” morphology  (amoeboid, with thick processes), were also increased as a consence of infection. There was a concomitant decrease in non microglial newborn cells (BrdU + only) in the early-infected rats, in the same regions.

Check that shit out! Rodents infected with E-coli during the neonatal period had an increased number of active microglia when compared to rodents that got saline as neonates.   Keep in mind that the backbone of these studies, and studies from other groups indicate that this persistence of effects are not specific to an e-coli infection, but rather, can be triggered by any immune response during critical timeframes.  In fact, at least two studies have employed anti-inflammatory agents, and observed an attenuation of effect regarding seizure susceptibility.

A final snipet from Bland et all Discussion section:

Although the mechanisms remain largely unknown, the ‘‘glial cell priming” hypothesis posits that these cells have the capacity to become chronically sensitized by an inflammatory event within the brain (Perry et al., 2003). We assessed whether glial priming may be a likely factor in the current study by measuring the volume of each counted microglial cell within our stereological analysis. The morphology of primed glial cells is similar to that of ‘‘activated” cells (e.g., amoeboid, phagocytic), but primed glial cells do not chronically produce cytokines and other pro-inflammatory mediators typical of cells in an activated state. There was a striking increase in cell volume within the CA1 region of adult rats infected as neonates (Figs. 2 and 8), the same region in which a marked increase in newborn glia was observed at P6. These data are consistent with the hypothesis that an inflammatory environment early in life may prime the surviving cells long-term, such that they over-respond to a second challenge, which we have demonstrated at the mRNA level in previous studies (Bilbo et al., 2005a, 2007; Bilbo and Schwarz, in press).

The concept of glial priming, close friends with the ‘two hit’ hypothesis (or soon to be, the multi-hit hypothesis?),  has some other very neat studies behind it, the coolest ones I’ve found so far are from a group at Northwestern, and include “hits” such as  Glial activation links early-life seizures and long-term neurologic dysfunction: evidence using a small molecule inhibitor of proinflammatory cytokine upregulationEnhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a ‘two-hit’ seizure model and Minozac treatment prevents increased seizure susceptibility in a mouse “two-hit” model of closed skull traumatic brain injury and electroconvulsive shock-induced seizures.   Also the tragically, hilariously titled, Neonatal lipopolysaccharide and adult stress exposure predisposes rats to anxiety-like behaviour and blunted corticosterone responses: implications for the double-hit hypothesis. (!)  These are potentially very inconvenient findings, the details for which I’ll save for another post.

Moving on to Bilbo et all, though a pure review paper than an experiment, it provides additional detailed theories on the mechanisms behind persistent effects of early life immune challenge.  Here’s the abstract:

The immune system is well characterized for its critical role in host defense. Far beyond this limited role however, there is mounting evidence for the vital role the immune system plays within the brain, in both normal, “homeostatic” processes (e.g., sleep, metabolism, memory), as well as in pathology, when the dysregulation of immune molecules may occur. This recognition is especially critical in the area of brain development. Microglia and astrocytes, the primary immunocompetent cells of the CNS, are involved in every major aspect of brain development and function, including synaptogenesis, apoptosis, and angiogenesis. Cytokines such as tumor necrosis factor (TNF)α, interleukin [IL]-1β, and IL-6 are produced by glia within the CNS, and are implicated in synaptic formation and scaling, long-term potentiation, and neurogenesis. Importantly, cytokines are involved in both injury and repair, and the conditions underlying these distinct outcomes are under intense investigation and debate. Evidence from both animal and human studies implicates the immune system in a number of disorders with known or suspected developmental origins, including schizophrenia, anxiety/depression, and cognitive dysfunction. We review the evidence that infection during the perinatal period of life acts as a vulnerability factor for later-life alterations in cytokine production, and marked changes in cognitive and affective behaviors throughout the remainder of the lifespan. We also discuss the hypothesis that long-term changes in brain glial cell function underlie this vulnerability.

Bilbo et all go on to discuss the potential for time sensitive insults that could result in an altered microglial function.  Anyone that has been paying attention should know that the concept of time dependent effects is, to my mind, the biggest blind spot in our existing research concerning autism and everyones favorite environmental agent.

Is there a sensitive period? Does an immune challenge early in life influence brain and behavior in a way that depends on developmental processes? Since 2000 alone, there have been numerous reports in the animal literature of perinatal immune challenges ranging from early gestation to the juvenile period, and their consequences for adult offspring phenotypes (see Table 1). It is clear that the timing of a challenge is likely a critical factor for later outcomes, impacting the distinct developmental time courses of different brain regions and their underlying mechanisms (e.g., neurotransmitter system development, synapse formation, glial and neural cell genesis, etc; Herlenius and Lagercrantz, 2004; Stead et al., 2006). However, the original question of whether these changes depend on development has been surprisingly little addressed. We have demonstrated that infection on P30 does not result in memory impairments later in life (Bilbo et al., 2006), nor does it induce the long-term changes in glial activation and cytokine expression observed with a P4 infection (Bilbo et al., unpublished data). The factors defining this “sensitive period” are undoubtedly many, as suggested above. However, our working hypothesis is that one primary reason the early postnatal period in rats is a sensitive or critical period for later-life vulnerabilities to immune stimuli, is because the glia themselves are functionally different at this time. Several studies have demonstrated that amoeboid, “macrophage-like”, microglia first appear in the rat brain no earlier than E14, and steadily increase in density until about P7. By P15 they have largely transitioned to a ramified, adult morphology. Thus, the peak in density and amoeboid morphology (and function) occurs within the first postnatal week, with slight variability depending on brain region (Giulian et al., 1988; Wu et al., 1992).  [emphasis theirs]

[Note:  The authors go on to state that this time period is likely developmentally equivalent to the late second, to early third trimester of human fetal development.]

We seem to have a growing abundance of evidence that immune stimulation in utero can have neurological impacts on the fetus that include schizophrenia, and autism.   In some instances, we have specific viral triggers; i.e., the flu or rubella, but  I’d further posit that we have increasing reason to believe that any immune response can have a similar effect.  The Patterson studies involving IL-6 in a rodent model of maternal activation seem to make this point with particular grace, as the use of IL-6 knockout mice attenuated the effect, as did IL-6 antibodies; and direct injection of IL-6 in the absence of actual infection produced similar outcomes.  In animal models designed to study a variety of effects, we have a veritable spectrum of studies that tell us that immune insults during critical developmental timeframes can have lifelong effects on neuroimmune activity, HPA-axis reactions, seizure susceptibility, and ultimately, altered behaviors.  I believe that we are rapidly approaching a point where there will be little question as towards if a robust immune response during development can lead to a developmental trajectory that includes autism, and will instead be faced with attempting to detangle the more subtle, and inconvenient, mechanisms of action, temporal windows of vulnerability, and indeed if there are subgroups of individuals that are predisposed to be more likely to suffer from such an insult.

Another thing that struck me about Morgan was the speculation that an increased presence of IL-1R in controls may have been suggestive of an attempt to muzzle the immune response in the case group; repeated from Morgan “The trend toward an increase in colocalization in control cases may also hint at downregulation of inflammatory signal receptors in a chronically activated system.” In other words, for controls it wasn’t a big deal to be expressing IL-1R in a ‘normal’ fashion, because the immune system is in a state of balance.  Another way of looking at our observations would be to ask the question as towards what has caused the normally self regulating immune system to fail to return to a state of homeostasis?   Ramping up an immune response to fight off pathogens and ratcheting back down to avoid unnecessary problems is something most peoples immune systems do with regularity.  Is the immune system in autism trying to shut down unsuccessfully?

There are clues that the homeostatic mechanisms are trying to restore a balanced system.  For example, in Immune transcriptome alterations in the temporal cortex of subjects with autism, researchers reported that the genetic pathway analysis reveals a pattern that could be consistent with “an inability to attenuate a cytokine activation signal.” Another paper that I need to spend some read in full, Involvement of the PRKCB1 gene in autistic disorder: significant genetic association and reduced neocortical gene expression describes a genetic and expression based study that concludes, in part, that downregulation of PRKCB1 “could represent a compensatory adjustment aimed at limiting an ongoing dysreactive immune process“.

If we look to clinical evidence for a decreased capacity to regulate an immune response, one paper that might help is Decreased transforming growth factor beta1 in autism: a potential link between immune dysregulation and impairment in clinical behavioral outcomes, the authors report an inverse dose relationship between peripheral levels of an important immune  regulator, TGF-Beta1,  and autism severity; i.e., the less TGF-Beta1 in a subject, the worse the autism behaviors [the autism group also, as a whole, had less TGF-Beta1 than the controls].

And then, in between the time that Morgan came out, and I completed this posting, another paper hit my inbox that might provide some clues,a title that is filled to the brim with autism soundbytes, “Effects of mitochondrial dysfunction on the immunological properties of microglia“.  The whole Hannah Poling thing seemed so contrived to me, basically two sets of people trying to argue past each other to reach a predetermined conclusions, and as a result, I’ve largely shied away from digging too deeply into the mitochondrial angle.  This may not be a luxury I have anymore after reading Ferger et all.   For our purposes, lets forget about classically diagnosed and acute mitochonrdrial disease, as Hannah Poling supposedly has, and just acknowledge that we have several studies that show that children with autism seem to have signs of mitochondrial dysfunction, as I understand it, sort of a halfway between normal mitochondrial processing and full blown mitochondrial disorder.  Given that, what does Ferger tell us?  Essentially an in vitro study, the group took microglial cells from mice, exposed some of them to toxins known to interferre with the electron transport chain, and exposed the same cells to either LPS or IL-4 to measure the subsequent immunological response.  What they observed was that the response to LPS was unchanged, but the response to IL-4, was blunted; and pertinently for our case, the IL-4 response is a so called ‘alternative’ immune response, that participates in shutting down the immune response.  From the conclusion of Ferger:

In summary, we have shown that mitochondrial dysfunction in mouse microglial cells inhibit some aspects of alternative activation, whereas classic activation seems to remain unchanged. If, in neurological diseases, microglial cells are also affected by mitochondrial dysfunction, they might not be able to induce a full anti-inflammatory alternative response and thereby exacerbate neuroinflammation. This would be associated with detrimental effects for the CNS since wound healing and attenuation of inflammation would be impaired.

If our model of interest is autism, our findings can begin to fit together with remarkable elegance.  And we haven’t even gone over  our numerous studies that show the flip side of the immunological coin; that children with autism have been shown time and time again to have a tendency towards an exaggerated immune response, and increased baseline pro-inflammatory cytokines when compared with their non diagnosed peers!

Anyways, those are my bonus theoretical pontifications regarding Morgan.

– pD

Hello friends –

My over riding idea set on vaccines is relatively complicated; but at the heart, I just am not confident that we are smart enough to understand all of the potential impacts of aggressively pursuing mass vaccination; it is too drastic a change from how every animal on the planet evolved to be taken as lightly as we seem to be. The indisputable success of vaccination, I fear, has made it difficult to even ask questions about the pragmatism of moving forward with breakneck speed to a place where we think are clever enough to take a shortcut over millions of years of evolution without even bothering to evaluate for unintended consequences.  It isn’t that I think I am smarter than the immunologists who develop vaccines; it is that I think  we as a species, are too dumb to understand the impact of our actions without quality evaluations; detailed analysis that is sorely lacking in the area of vaccines. 

Before moving forward, it should be made clear that for a variety of reasons, the research regarding vaccines and autism that is available to us is has a very tight focus, either thimerosal content or its absence, or MMR. This is a statement of fact. There are no autism studies that evaluate anything else in the vaccination schedule other than these two components. There just aren’t. Anyone who tells you otherwise is either misinformed, or intentionally trying to deceive you for reasons of their own.

A common refrain heard in the debates over vaccinations and autism goes something like this: “We’ve studied it again and again, and every time, we see no association. It is time to move on and spend precious dollars and researcher hours elsewhere.” Also frequently used is the a variation on this quote: “Insanity is repeating the same thing and expecting different results.”

When I hear things like this, it drives me apeshit crazy; especially when it comes from the mouths of scientists, people who (supposedly?) understand the simplest foundation of the scientific principle, that you only learn about what you study. A study evaluating use of thimerosal vaccines and their non thimerosal containing counterparts has no mechanism of gaining insight into the effect cumulative early life immune activations; both vaccines have the exact same mechanism of action in that regard; otherwise, they wouldn’t be vaccines. In an ironic twist, continuing to study thimerosal, but claiming it gives us information regarding vaccination is, in large part, doing the same thing again and again and expecting different results.  Likewise, studying the MMR has been useful, as we have learned much about the MMR; but it gives us precious little insight into the effect of other vaccines, including those given at much, much earlier ages.

If we did twenty studies on cigarettes with tar and those without tar, should the resultant pattern of epidemiological findings give us any reason to believe we should extrapolate our findings outside the realm of the impact of tar in cigarettes?  Or, just because the MMR has been found to not to be associated with autism at eighteen months, should we also assume that other vaccines given at two months are therefore not associated with autism?  This is absolutely analogous to what we are being told regarding our existing set of research. 

That being said, it isn’t really enough to start questioning a massively successful health policy that has saved millions of lives just because some jerk on the Internet doesn’t think we’ve done enough looking into it and our existing studies have yet to tackle every imaginable combination of vaccine schedule, and genetic variation. The argument goes something along the lines of, “Yes, you are technically correct, but without some biologically plausible mechanism of action, we cannot keep on studying something just because there exists a temporal relationship between an increase in the vaccination schedule and an apparent increase in autism diagnosis.”  Without any concept of how more, earlier vaccines could be having an adverse effect that is invisible to our existing studies, there is merit to this argument. 

It is here that the research outlined below helps fill a gap in the discussion.   Anyways, a few weeks ago I was  reference backtracking, and wound up reading a set of research involving rather unexpected findings regarding the result of early life infectious exposure and resultant immune system activation. Looking through those references, it turns out, several research groups have been doing work regarding the effect of early development immune system activation and consequent alterations to a variety of biological realms, including effects on immune system functioning, altered stress responses, seizure susceptibility, and behavioral changes, into adulthood. In fact, many researchers have reported that a transient inflammatory response is capable of creating lifelong differences in exposed animals, if they are exposed during early development. The immune system is a work in progress in the prenatal and early post natal periods, and appears to be highly impressionable, and in some instances, unforgiving in response to disturbances. Note that the vast majority of the research below has only been published in the last couple of years; long, long after we started to aggressively increase the number of vaccines our youngest infants were receiving.

Most of the studies use a relatively standard component for initiating an immune response in the animals, Lipopolysacccharide, or shorthanded, LPS.  All of them deal with observing changes in animals into adulthood after early postnatal activation of the immune system.  Several were able to concurrently determine that the time of the immune activation was the determining factor in the animals persistent changes. 

Once I started trying to create a list of all of the research into this area, it was quickly apparent that it was overly cumbersome to get through, and ran the risk of turning into a stream of consciousness style listing of papers without an over riding set of guide posts as to why they might have implications for our vaccination schedule and autism or other neurological disorders.  With that in mind, I constructed a list of areas that these papers address and speak towards the blindspots in our existing vaccine research. 

  • Studies that took care to answer the question that the immune response  was responsible for differential behavioral or physiological outcomes.   For example, could the same outcome be achieved by administering inflammatory cytokines, as opposed to a bacterial or viral protein analog?  Was there an attempt made to introduce inflammatory inhibitors  that resulted in a negation of effects?  This is an important distinction, as otherwise, the argument could  be made that it was the LPS, as opposed to the resultant immune response that was responsible for the different outcomes. 
  • Studies that evaluated the effect of a time dependent effect on behavioral or physiological outcomes.   For example, did animals have different outcomes if their was an immune challenge at one week, as opposed to one month?   An extremely common refrain in this discussion is ‘the poison makes the dose’, unfortunately, it would seem that this is not necessarily the case.  
  • Studies that had findings that have correlations with known behavioral or physiological findings in autism.  Of course, without any findings that have similarities to autism, this exercise would be largely futile for a blog about autism!

Before getting started, I’d like to be clear that I am not advocating that vaccines cause autism; but rather, that we haven’t studied the issue very well, and that we are gaining experimental evidence that our existing studies are inadequately designed to capture many potential unintended effects.  My belief is that increased vaccination could impart a mild to moderate risk of autism diagnosis in some genetically predisposed individuals, and while I believe  that a true increase in autism prevalence is occurring, that not all of this is caused by vaccination.  A fuller detailing of these views if for another post, but the pertinent part here is that the studies outlined below tell us that we still have a lot to learn about how the immune system operates, especially during early development, and given that, proclamations that the vaccine schedule has been fully evaluated involve large leaps of faith. 

That being said, lets take a look at some of the papers on the subject.  In all instances below, the emphasis provided is my own.  Many abstracts are snipped for space purposes.

Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats

There are critical postnatal periods during which even subtle interventions can have long-lasting effects on adult physiology. We asked whether an immune challenge during early postnatal development can alter neuronal excitability and seizure susceptibility in adults. Postnatal day 14 (P14) male Sprague Dawley rats were injected with the bacterial endotoxin lipopolysaccharide (LPS), and control animals received sterile saline. Three weeks later, extracellular recordings from hippocampal slices revealed enhanced field EPSP slopes after Schaffer collateral stimulation and increased epileptiform burst-firing activity in CA1 after 4-aminopyridine application. Six to 8 weeks after postnatal LPS injection, seizure susceptibility was assessed in response to lithium–pilocarpine, kainic acid, and pentylenetetrazol. Rats treated with LPS showed significantly greater adult seizure susceptibility to all convulsants, as well as increased cytokine release and enhanced neuronal degeneration within the hippocampus after limbic seizures. These persistent increases in seizure susceptibility occurred only when LPS was given during a critical postnatal period (P7 and P14) and not before (P1) or after (P20). This early effect of LPS on adult seizures was blocked by concurrent intracerebroventricular administration of a tumor necrosis factor (TNF) antibody and mimicked by intracerebroventricular injection of rat recombinant TNF. Postnatal LPS injection did not result in permanent changes in microglial (Iba1) activity or hippocampal cytokine [IL-1β (interleukin-1β) and TNF] levels, but caused a slight increase in astrocyte (GFAP) numbers. These novel results indicate that a single LPS injection during a critical postnatal period causes a long-lasting increase in seizure susceptibility that is strongly dependent on TNF.

The most exciting finding of the present study is that a mild inflammatory response evoked by LPS during a critical period of development causes a long-lasting increase in hippocampal excitability in vitro, and enhanced seizure susceptibility to the convulsants LI-PILO, KA, and PTZ in vivo. The latter effect was observed over a range of mildly inflammatory doses of LPS and was only evident if administered during the second postnatal week (P7 and P14), and not before (P1) or after (P20) this time. Importantly, inactivation of the proinflammatory cytokine TNF with an intracerebroventricular TNF antibody blocked the long-term changes to seizure susceptibility induced by LPS, whereas intracerebroventricular administration of rrTNF alone mimicked the effect of LPS on seizure susceptibility. These novel results indicate that a single transient inflammatory episode during development can modify the brain through a TNF-dependant mechanism, making it more susceptible to generate seizures in adulthood.

This paper hits a lot of the sweet spots we defined above; there was a differential effect on depending on when an immune response was initiated, pro inflammatory cytokine administration alone was sufficient to cause the same effect, and furthering the link to the immune response, administration of tnf alpha antibodies negated any effects. 

Of particular interest to our population of children, it is well established that children with autism grow up into adults with of epilepsy at far, far greater rates than their non diagnosed peers. One way to increase the likelihood of having more seizures, it appears, is to get a large dose of tnf alpha in early development.  Having a seizure in the first year of life has been found to be very strongly associated with an autism diagnosis, however, with this type of study it is difficult to detangle the cause and the effect; i.e., are they having seizures because they have autism, or are they getting diagnosed with autism as a result of early life  seizures?   There are some studies on the long term effect of early life seizures that show chronic activation of the brains immune system; and again, the innate inflammatory immune response is implicated in causation. 

Also of particular interest regarding autism is that the driving factor in this case was tnf alpha, a proinflammatory cytokine that has been shown to be elevated in several studies of children with autism. In fact, when researchers use drawn blood to determine immune responses, children with autism have been found to generate far more tnf alpha than controls in response to increasingly common (and scary) pollutants, common dietary boogeymen, and LPS. In other words, children with autism seem predisposed to creating more tnf alpha in response to a variety of environmental factors .  In two studies that analyzed the brains and CSF of children with autism, highly elevated levels of tns alpha were observed. 

 

 

 Here is one with a great title:

 

Long-term alterations in neuroimmune responses after neonatal exposure to lipopolysaccharide.

Fever is an integral part of the host’s defense to infection that is orchestrated by the brain. A reduced febrile response is associated with reduced survival. Consequently, we have asked if early life immune exposure will alter febrile and neurochemical responses to immune stress in adulthood. Fourteen-day-old neonatal male rats were given Escherichia coli lipopolysaccharide (LPS) that caused either fever or hypothermia depending on ambient temperature. Control rats were given pyrogen-free saline. Regardless of the presence of neonatal fever, adult animals that had been neonatally exposed to LPS displayed attenuated fevers in response to intraperitoneal LPS but unaltered responses to intraperitoneal interleukin 1 or intracerebroventricular prostaglandin E2. The characteristic reduction in activity that accompanies fever was unaltered, however, as a function of neonatal LPS exposure. Treatment of neonates with an antigenically dissimilar LPS (Salmonella enteritidis) was equally effective in reducing adult responses to E. coli LPS, indicating an alteration in the innate immune response.In adults treated as neonates with LPS, basal levels of hypothalamic cyclooxygenase 2 (COX-2), determined by semiquantitative Western blot analysis, were significantly elevated compared with controls. In addition, whereas adult controls responded to LPS with the expected induction of COX-2, adults pretreated neonatally with LPS responded to LPS with a reduction in COX-2. Thus, neonatal LPS can alter CNS-mediated inflammatory responses in adult rats.

Here, again, the authors again went to some trouble to provide evidence that the effect was based on the resultant immune response by providing different bacterial proteins.  We also observe long term, persistent alterations in COX-2 levels; essentially an indicator of changes to the immune regulatory system; inhibition of the COX enzymes is how pain relievers such as aspirin and  Vioix work.   Strangely, it seems that the treatment group had higher resting levels of COX-2, but reduced production in response to a challenge.   The authors went on to perform a similar experiment on female rodents, with mixed similarities; the reduced fever response and response to challenge were observed as with the males, but, baseline levels of COX-2 were found to be the same between treatment and control animals.   There are obvious correlations here with the sexual dismorphism in autism diagnosis here, if anyone has any thoughts on sexual dismorphic COX-2 profiles, please send me a comment. 

I have not seen any papers directly measuring COX-2 in autism, but there are a great number on the related regulation of the immune system which show large differences between autism and control subjects.  Polymorphisms in the genes responsible for COX-2 have been found to be highly transmitted in the autism population.  I’ve been having some problems identifying the impact of this particular allele on circulating levels of COX-2.  One study on colorectal cancer seemed to indicate a protective effect from the allele, which would seemingly be at odds with a inflammation promoter.  Anyways, if anyone has any knowledge on this, please send it my direction. 

Also interest here, though I am having problems articulating the precise issue, is that IL1 or prostaglins did not result altered responses.  This, to me, could potentially speak towards a preferential training of the Toll Like Receptors, as opposed to a downstream functional area. 

Neonatal infection-induced memory impairment after lipopolysaccharide in adulthood is prevented via caspase-1 inhibition

We have reported that neonatal infection leads to memory impairment after an immune challenge in adulthood. Here we explored whether events occurring as a result of early infection alter the response to a subsequent immune challenge in adult rats, which may then impair memory.In experiment 1, peripheral infection with Escherichia coli on postnatal day 4 increased cytokines and corticosterone in the periphery, and cytokine and microglial cell marker gene expression in the hippocampus of neonate pups. Next, rats treated neonatally with E. coli or PBS were injected in adulthood with lipopolysaccharide (LPS) or saline and killed 1-24 h later. Microglial cell marker mRNA was elevated in hippocampus in saline controls infected as neonates. Furthermore, LPS induced a greater increase in glial cell marker mRNA in hippocampus of neonatally infected rats, and this increase remained elevated at 24 h versus controls. After LPS, neonatally infected rats exhibited faster increases in interleukin-1beta (IL-1beta) within the hippocampus and cortex and a prolonged response within the cortex. There were no group differences in peripheral cytokines or corticosterone. In experiment 2, rats treated neonatally with E. coli or PBS received as adults either saline or a centrally administered caspase-1 inhibitor, which specifically prevents the synthesis of IL-1beta, 1 h before a learning event and subsequent LPS challenge. Caspase-1 inhibition completely prevented LPS-induced memory impairment in neonatally infected rats. These data implicate IL-1beta in the set of immune/inflammatory events that occur in the brain as a result of neonatal infection, which likely contribute to cognitive alterations in adulthood.

Another instances where the authors used inflammatory inhibitors to obtain evidence that the behavioral outcomes were dependent on the immune response by administering inflammatory inhibitors. And again, we see drastic differences in behavior and immune response between animals that had an immune response early in life, and those that did not. This looks to be part of a multiple paper study, the initial paper can be found here.  Increased levels of IL-1beta have not been observed in children with autism; though some researchers have found that in response to LPS, children with autism produce more IL-1Beta than their non diagnosed counterparts.

Early-life immune challenge: defining a critical window for effects on adult responses to immune challenge

It was titles like this that really got my head spinning early on when I started to realize just how much information there was on this subject.

Many aspects of mammalian physiology are functionally immature at birth and continue to develop throughout at least the first few weeks of life. Animals are therefore vulnerable during this time to environmental influences such as stress and challenges to the immune system that may permanently affect adult function. The adult immune system is uniquely sensitive to immune challenges encountered during the neonatal period, but it is unknown where the critical window for this programming lies. We subjected male Sprague-Dawley rats at postnatal day (P)7, P14, P21, and P28 to either a saline or lipopolysaccharide (LPS) injection and examined them in adulthood for differences in responses to a further LPS injection. Adult febrile and cyclooxygenase-2 responses to LPS were attenuated in rats given LPS at P14 and P21, but not in those treated at P7 or P28, while P7-LPS rats displayed lower adult body weights than those treated at other times. P28-LPS rats also tended to display enhanced anxiety in the elevated plus maze. In further experiments, we examined maternal-pup interactions, looking at the mothers’ preference in two pup-retrieval tasks, and found no differences in maternal attention to LPS-treated pups. We therefore demonstrate a ‘critical window’ for the effects of a neonatal immune challenge on adult febrile responses to inflammation and suggest that there are other critical time points during development for the programming of adult physiology.

If you believe in reincarnation, I wonder just how bad a person you need to be in order to wake up as a Sprague-Dawley rat the next time?  Anyways, here we can see different results depending on the timeframe of immune activation.  And again, we see modifications to immune system regulation and behaviors. 

Early life immune challenge–effects on behavioural indices of adult rat fear and anxiety

Neonatal exposure to an immune challenge has been shown to alter many facets of adult physiology including fever responses to a similar infection. However, there is a paucity of information regarding its effects on adult behavioursMale Sprague-Dawley rats were administered a single injection of the bacterial endotoxin lipopolysaccharide (LPS) at 14 days old and were compared, when they reached adulthood, with neonatally saline-treated controls in several behavioural tests of unconditioned fear and anxiety. There was no effect of the neonatal treatment on performance in either the elevated plus maze, modified Porsolt’s forced swim test or the open field test. However, neonatally LPS-treated rats did show significantly reduced exploration of novel objects introduced to the open field arena, indicating an effect of the neonatal immune challenge on behaviours relating to anxiety in the adult.

Here, we see that early life exposure to endtoxin was seen to alter behaviors, including reduced exploration, indicative of increased anxiety. 

Long-term disorders of behavior in rats induced by administration of tumor necrosis factor during early postnatal ontogenesis

Another great title! In this case, the authors used straight tnf alpha and observed ‘long term disorders of behavior’. For our particular subset of children, who have been shown to create tnf alpha at highly exaggerated rates compared to their non diagnosed peers, the triggering mechanism has particular salience. 

 Early-life infection leads to altered BDNF and IL-1beta mRNA expression in rat hippocampus following learning in adulthood

By this point, the hows of what they did are probably pretty straightforward. Snipped from the abstract:

Taken together, these data indicate that early infection strongly influences the induction of IL-1beta and BDNF within distinct regions of the hippocampus, which likely contribute to observed memory impairments in adulthood.

Early-life exposure to endotoxin alters hypothalamic–pituitary–adrenal function and predisposition to inflammation

This is the oldest paper I have come across so far, published in 2000.

We have investigated whether exposure to Gram-negative bacterial endotoxin in early neonatal life can alter neuroendocrine and immune regulation in adult animals. Exposure of neonatal rats to a low dose of endotoxin resulted in long-term changes in hypothalamic–pituitary–adrenal (HPA) axis activity, with elevated mean plasma corticosterone concentrations that resulted from increased corticosterone pulse frequency and pulse amplitude. In addition to this marked effect on the development of the HPA axis, neonatal endotoxin exposure had long-lasting effects on immune regulation, including increased sensitivity of lymphocytes to stress-induced suppression of proliferation and a remarkable protection from adjuvant-induced arthritis. These findings demonstrate a potent and long-term effect of neonatal exposure to inflammatory stimuli that can program major changes in the development of both neuroendocrine and immunological regulatory mechanisms.

On the basis of our data, it does appear, however, that activation of endocrine and immune systems during neonatal development can program or “reset” functional development of both the endocrine and immune systems. In this respect it is noteworthy that exposure to steroids during immunization schedules in early life can alter the development of immune tolerance, and that animals raised in pathogen-free environments have increased susceptibility to inflammatory disease (20, 29–31). The environment in which a mammal develops is often the environment in which it must survive throughout life, and developmental plasticity must surely be of adaptive advantage. We suggest that “immune environments” during development not only can alter inflammatory and neuroendocrine responses throughout life but also may alter predisposition to stress-related pathologies associated with HPA activation.

There are some other papers out there that have failed to find a relationship between early life immune activation and subsequent HPA axis modulations.

Neonatal inflammation produces selective behavioural deficits and alters N-methyl-D-aspartate receptor subunit mRNA in the adult rat brain

Thus, a single bout of inflammation during development can programme specific and persistent differences in NR mRNA subunit expression in the hippocampus, which could be associated with behavioural and cognitive deficits in adulthood.

The author reports highly variable NMDA expression changes in animals tested over a variety of timeframes. Changes to NMDA receptors have been reported in autism, as well as in animals treated prenatally with valporic acid; which has been shown to greatly increase risk of autism diagnosis.

Neonatal immune challenge exacerbates experimental colitis in adult rats: potential role for TNF-alpha

Four days after TNBS treatment, plasma corticosterone was unaltered in all groups; however, TNF-alpha was significantly increased in adult TNBS-treated rats that had LPS as neonates compared with all other groups. In conclusion, neonatal, but not later, exposure to LPS produces long-term exacerbations in the development of colitis in adults.This change is independent of HPA axis activation 4 days after TNBS treatment but is associated with increased circulating TNF-alpha, suggestive of an exaggerated immune response in adults exposed to neonatal infection

Again, we see tnf alpha implicated as a mediating factor; in this case, animals treated with LPS during development went on to develop much more severe colitis symptoms when drug induced. These changes were only apparent if the immune insult occurred during a specific timeframe.  An increased baseline level of tnf alpha is also something that has been observed in the autism population. 

 Neonatal programming of the rat neuroimmune response: stimulus specific changes elicited by bacterial and viral mimetics 

Here, researchers performed an experiment to determine if immune stimulants other than LPS could generate ‘neonatal programming of the rat neuroimmune response’, so they used PolyIC; a viral protein analog during early life.  What was observed was that animals treated on postnatal day 14 showed attenuated febrile responses into adulthood, coinciding with altered corticosteroid responses.  Concurrent administration of a corticosteroid receptor blocker caused observed abnormalities to dissipate.  Very interestingly, they also observed that a mixed early life, adulthood challenge did not result in the observed differences; i.e., if an animal got a PolyIC immune stimulant in infancy, and an LPS stimulation in adulthood, no changes from saline animals were seen.  To me, this speaks again towards a specific training of the toll like receptors as the detection of viral proteins and consequent immune activation is handled by TLR-3, while the same job duties are handled by TLR-4 for bacterial proteins (i.e., LPS). 

Neonatal bacterial endotoxin challenge interacts with stress in the adult male rat to modify KLH specific antibody production but not KLH stimulated ex vivo cytokine release

While postnatal bacterial infection is capable of inducing a variety of long lasting functional alterations in immune function, the specific physiological pathways responsible for this modification are largely unknown. In the current investigation we explore the hypothesis that early life exposure to endotoxin permanently modifies the function of T helper (Th) cell activity. Therefore we examined Th-cell regulated in vivo humoral and ex vivo cellular responses to keyhole limpet hemocyanin (KLH). Given that stress has been shown to exacerbate some of the immunological alterations exhibited by the neonatally endotoxin challenged adult, we examined the adult’s Th1/Th2 responses to KLH under conditions of no stress, acute stress (2 daysx2 h), and chronic stress (7 daysx2 h). Our results demonstrate that adults neonatally challenged with endotoxin were found to produce significantly less IgG1 following KLH challenge following acute stress (p<0.05). Neonatally endotoxin treated animals exposed to acute stress were also found to produce less IgM than saline or endotoxin treated animals exposed to no-stress or chronic stress. No neonatal treatment group differences observed in the production of INF-gamma or IL-4 in adulthood. In summary, the results from the present study provide little evidence to directly support the hypothesis that neonatal endotoxin exposure significantly alters the Th1/Th2 balance in adulthood

This is a nice touch because the researchers mixed the exposure of acute stress with early life immune activation.  There are many studies on increased levels of biomarkers of stress in autism, and, it just so happens, children with autism have been shown to have decreased levels of IgG1 and IgM when compared to children without a diagnosis.  Other cytokine measurements were unchanged. 

There are other papers available with similar findings, but this set is a large chunk of what is out there.   Our summarization is as follows:

  • 12 studies showing  analyzing the effect of early life immune activation on rodents with findings into adulthood on behavior differences, seizure susceptibility, colitis susceptibility, HPA Axis modifications, and immune system changes. 
  • 1 study observed behavioral results from administration of tnf alpha alone.  (Zubareva OE, 2009)
  • 1 study observed physiological results from administration of tnf alpha alone. (Galic, 2008)
  • 3 studies used inflammatory inhibitors to validate the immune response was responsible for the physiological changes (seizure susceptibility), behavioral changes (memory impairments), and immune function.  (Galic, 2008, Ellis 2006,  Bilbo 2005).
  • 2 studies found that the timing of the immune activation was a mediating factor in causing persistent changes (Spencer, 2006, Galic, 2008).
  • 7 studies found persistent changes to the immune system.  (Boisse, 2004, Spencer 2006, Bilbo 2008, Shanks 2000, Spencer 2007, Ellis 2006, Walker 2009).
  • 1 study finding changes to brain receptor structures.  (Harre 2008).
  • 3 studies finding increased anxiety and / or fear responses.  (Zubareva 2009, Spencer 2006, Spencer 2005

In developing some of these ideas online, I ran into several arguments as to why we these findings have no bearing on our existing research into vaccination taking into consideration the a time dependent effect of immune activation.   Below, as near as I can remember, is a cataloging of these complaints, and my take on their validity, and in what ways the studies above provide information. 

1) Vaccines are already tested for safety and efficacy. 

Technically a true statement, but one that very quietly attempts to substitute safety testing for evaluation of autism.  Most of the safety studies, even those that follow participants for several years, are not designed to capture either neurological outcomes like autism, or more subtle changes such as persistent changes to immune system markers.  For verification of this, all one really needs to do is take a look at what happened in reality, and apply a primitive logical filter.  When it was posited that the MMR might be causing autism in some children, there was a flurry of retrospective studies performed on children who did or did not get the MMR.  Whatever your position on the quality of those studies, the fact is, those studies were necessary only because the existing set of safety and efficacy studies were not sufficient to answer the question of if there was an association with the MMR and autism.   In other words, why bother with retrospective studies if the existing literature already had evidence of no link? 

As for testing of immune system changes, you will be very hard pressed to find studies on the existing vaccine schedule for children that takes into consideration pre and post cytokine or related immunological measurements.  If anyone has any studies that I haven’t seen (which is two), please let me know.   One that I have found, Modulation of the infant immune responses by the first pertussis vaccine administrations has some rather startling findings. 

Many efforts are currently made to prepare combined vaccines against most infectious pathogens, that may be administered early in life to protect infants against infectious diseases as early as possible. However, little is known about the general immune modulation induced by early vaccination. Here, we have analyzed the cytokine secretion profiles of two groups of 6-month-old infants having received as primary immunization either a whole-cell (Pw) or an acellular (Pa) pertussis vaccine in a tetravalent formulation of pertussis–tetanus–diphtheria-poliomyelitis vaccines. Both groups of infants secreted IFN-γ in response to the Bordetella pertussis antigens filamentous haemagglutinin and pertussis toxin, and this response was correlated with antigen-specific IL-12p70 secretion, indicating that both pertussis vaccines induced Th1 cytokines. However, Pa recipients also developed a strong Th2-type cytokine response to the B. pertussis antigens, as noted previously. In addition, they induced Th2-type cytokines to the co-administrated antigen tetanus toxoïd, as well as to the food antigen beta-lactoglobulin. Furthermore, the general cytokine profile of the Pa recipients was strongly Th2-skewed at 6 months, as indicated by the cytokines induced by the mitogen phytohaemagglutinin. These data demonstrate that the cytokine profile of 6-month-old infants is influenced by the type of formulation of the pertussis vaccine they received at 2, 3 and 4 months of life. Large prospective studies would be warranted to evaluate the possible long-term consequences of this early modulation of the cytokine responses in infants.

 

Now this doesn’t mean that DTaP causes autism, but it does tell us that we are largely operating based on our findings of reduced disease and empirical measurements of seriopositivity, as opposed to a true understanding of all of the effects of vaccination; this study was conducted eight years after DTaP was licensed for use.   Clearly our existing set of safety and efficacy tests for DTaP were not sufficiently designed to capture this kind of information.  If anyone tells you that have the slightest fucking clue as to the result of such cytokine shifts in a generation of infants, you are being lied to.  This study also casts a relatively poor light on argument 3. 

Strength of argument: Zero. 

2) The vaccination hypothesis cannot explain X characteristic of autism.  (Where X is a ‘characterization’ of autism, such as improved spatial skills)

What this argument really says is that the person making cannot imagine a way in which characteristic X could be caused by vaccination, and therefore, the hypothesis is invalid.  Of course, of the few accepted causes of autism, such as prenatal exposure to ruebella, there is also no well defined mechanism by which such an event could lead to most of the characteristics that this argument utilizes.  An even biggest problem with this argument is that it mandates that every person with autism has characteristic X, when in fact, autism is characterized in part by large heterogeneousness.  And if we were to expand our premise from, ‘vaccines may cause autism through early life immune activation’, to, ‘autism may modify the behavioral or immune system functioning through early life immune activation’, this argument falls to complete irrelevancy without making our existing set of research any more robust.

Strength of argument: One.

3)  Infants are bombarded with antigens all the time and their immune system is not overwhelmed.  Vaccination is no different. 

Again, this argument starts with a kernel of technical truth; infants are forced to deal wifth a variety of bacterial and viral antigens from the moment they are born.   However, in the first place, the simplest commonsense logical tests tell us that there is a big difference between ‘everyday exposure’ and the contents of a vial.  For starters, your child comes equipped with an array of defense mechanisms to keep bacteria and viruses outside of their bodies, namely the skin, mucous, tears, and gastric acid.  When we use a needle to penetrate the skin and inject the antigens into the tissue, all of these natural defense barriers are immediately bypassed.  Secondly, the antigens in a vaccine aren’t alone; they come with aluminum based salts that are designed to enhance the immediate innate immune response.  Funny enough, the mechanism by which these chemicals achieve their function is still under investigation, but they are absolutely necessaray for a vaccine to initiate a sufficient immune response for the body to develop antibodies.   If we simply evaluate what regulatory agencies tell us; that low (or high) grade fevers are a common side effect of vaccination, between 5% and 30% of the time depending on the vaccine, we are forced to acknowledge that our children do not develop fevers anywhere close to the same frequency.  Or, we can look at the DTP / DTaP study above, where we observed highly differential immune profiles between different vaccines.  If all of the thousands or millions of antigens these children were exposed to in the intervening months were having a meaningful impact, it should have been impossible to identify one group from another; and yet, the different profiles were strikingly clear.   If everyday exposure is equivalent, how can we resolve these seemingly paradoxical findings? 

Strength of argument:  Three.  Even though vaccination is very different, the strawman argument of an ‘overwhelmed immune system’ is nicely defeated by this argument.   In our studies above that dealt with observed immune system alterations, however, it is not an “overwhelming” of anything that was observed, but rather, a persistent modulation with wide ranging effects. 

There are several frequent answers to these counter arguments.

3a) Just because you  sometimes have a fever after vaccination doesn’t mean your immune system isn’t activated the rest of the time. 

Again technically true, but it leaves out the fact that a fever indicates a more robust immune response.  Any effect that is dependent on the relative strength of  immune activation forces us to conclude that  we cannot draw equivalencies between normal immune system activation and what happens when you get a vaccine.  We can consider all of the animals from the studies above for insight; they were all exposed to plenty of bacteria on their own, they were rats after all; and yet, only those that received LPS, PolyIC, or tnf-alpha were seen to have changes.  In other words, if common immune system activation was sufficient to cause differential effects, with all of that background immune activation  there should have been no ability to tell which animals were in the treatment group.  And again, we can refer to the DTP/DTaP study for insight as to our ability to discern vaccine exposure and everyday exposure with measures beyond single pathogen seriopositivity.

3b) The immune response generated by the actual diseases are far more robust than that from vaccination.  Considering this, it is even more important to vaccinate children earlier.

If one of the concerns we have applies to the timing of the immune response, this answer is only sensible if we had a reasonable expectation that an infant  will become infected with diptheria, tetanus, pertussis, hepatitis b, rotavirus, haemophilus influenza, and/or polio by the age of two months, and again at four and six months of age.   While such a situation would no doubt have very poor outcomes for the child in question, the chances of any one of these things happening is very low.  On the other hand, the chances of an infant having an immune response initiated via vaccination at this age is approaching 100%.   Furthermore, this argument is frequently based on duration of response (a measure of bacterial or viral persistence, as opposed to strength of response), but several of the studies above found that a single, transient immune activation was sufficient to cause differences into adulthood. 

4) None of the studies above test vaccination. (sometimes coupled with: they test exposure to LPS)

Whatever the trigger, there is only one innate immune system to generate a response, and the gatekeepers of the immune response, the toll like receptors, are the components responsible for initiating the innate immune response be it by vaccination or wild bacterial/viral exposure.  It should be noted that there are times when both arguments 3 and 4 will be used nearly simultaneously.  For anyone concerned with an over reliance on LPS, we have several studies where viral analogs were used, and others where straight tnf-alpha achieved similar results.   Likewise, we have three studies showing that the use of inflammatory inhibitors resulted in amelioration of effects; strong evidence that the trigger of the immune response is relatively unimportant compared to the immune response itself. 

None the less, this argument has some validity in that it is difficult to compare the immunological strength of the response between dosages of LPS, PolyIC, and tnf-alpha with what happens after standard childhood immunizations.  Unfortunately, the reason such a comparison is impossible to perform is that we have no values to use as comparisons from the end of vaccination.   If someone could provide a study showing pre- and post- cytokine levels after common childhood vaccinations, please post a link.   Even with our studies that tell us that children with autism have a tendency to respond more vigorously to immune stimulants than their non diagnosed peers, this is a large unknown.  It would be very tricky to capture excellent in vivo comparison information here, as it would require injecting infants with LPS in order to gauge the immune response; there are all kinds of problems with that.  Animal models and in vitro may be the only options available. 

Strength of argument:  Five. 

 5) Humans grew up in dirty environments; they were exposed to viruses and bacteria all the time.  What is different about the most recent generation than the thousands of generations past?

 This is a pretty strong argument, after all, in general, conditions in the past were, generally, germier than they are now.  The issue, to my mind, is that even with a dirtier past, our actions have skewed what was once a distribution.  We have taken efforts to insure that every infant gets a robust immune response, and earlier in life; as opposed to what used to be some infants.  In other words, even if there was a fifty percent chance of a two month old having generated a strong immune activation in generations past, the chances are now much closer to one hundred percent.  The same thing happens at four months, and six months.  With the insane well meaning introduction of the Hep-B vaccine at the day of birth, this radical alteration to this distribution is unmistakable.  Part of the problem with gauging this argument is that there seems to be a wide range of ‘average’ infections reported in infants during their first year of life; with ranges from 0 – 12; and even with these, it is impossible to get a measure of the strength of the response.   Our ability to understand what the average was just a few generations ago completely futile. 

There are also large problems with drawing equivalencies between the other components of the environment of previous generations and the current generation. 

Strength of argument:  Seven. 

6) The model is wrong, there is just too much difference between human and rat physiology to be worried. 

The strongest contributor to this argument is uncertainty in our ability to accurately interpret the jump from prenatal to postnatal immune activation between rodents and humans.  But again, this is driven in large part by a relative paucity of information as opposed to a deeper understanding of the differences between the two.  After all, any amount of intellectual honesty tells us that the researchers in the experiments above are not overly concerned over the question as to if rats develop immune system differences into adulthood following early development immune activation; these experiments are being funded and performed because there are things to be learned about human physiology from the results.  To put another way, if researchers and funding agencies were confident that there was no way the same transient inflammatory episodes could have similar effects on people, would any of these studies actually been funded or performed?  The effect size also speaks towards the complexity of  going from rodent to humans. 

Strength of argument: Eight.  There is a real chance that all of the effects observed here in rodent models only have experiments to pre-natal exposures in humans.  Likewise, it is acknowledged that the rodent model is useful in many areas but that  frequency that results that look good in rodents and then poor in people, is very large.  Unfortunately, to my mind, this does not constitute evidence of lack of effect of our vaccination schedule, just one reason why it might not be having an effect.  It is the assumption of no effect, as opposed to the presence of quality analysis.

I’ve never actually had this argument made to me, strangely enough, but it does strike me as a very large question mark. 

Conclusions

The fact that these experimentsare being carried out at all, with the findings being described as novel, should be enough to tell us that for all practical purposes, we still are gaining an understanding of the effects of early life immune activation, some twenty years after we began to aggressively increase the number of vaccines our infants receive.  Just because the effects that were observed are sometimes very subtle does not mean that they cannot have profound ramifications, and if our existing analysis was not designed to capture subtle effects, drawing far reaching conclusions from them is worthless, and indeed, potentially dangerous.

With that in mind, is it possible to have a rational discussion about the possibilities of finding ways to gain more insight into the potential outcomes of earlier and more vaccination without invoking vitriole, charges of scientific illiteracy, the big pharma gambit or accusations of child abuse? 

– pD


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