passionless Droning about autism

Archive for the ‘Biological Plausibility’ Category

Hello friends –

I’ve had a couple of interesting papers land in my pubmed feed the past few weeks that seem to be tangentially touching on something that has been at the back of my mind for a long time; namely, the repeated findings of a state of an ongoing immune response in the CNS of the autism population, coupled with a behavioral state that is either static, or in many cases, showing gradual improvement over time.  [Discussions of ongoing immune response in the brain in autism, here, here, or here].  This is exactly the opposite of what I expected.  Most of the conditions I had generally associated with a state of neuroinflammation, i.e., Alzheimer’s or Parkinson’s show a behavioral profile opposite to autism over time, i.e., a deterioration of skills and cognitive abilities.   The diagnosis for these conditions is never a straight line or a gradual curve upwards, but a dispassionately reliable trajectory of a downward spiral.

This is something that has been really bugging me a lot as a riddle, I’ve mentioned it here in comments, and other places on the Internet.  While outright signs of neuroinflammation are clearly associated with conditions you would rather not have, as opposed to have, we must admit that the available evidence tells us that  we cannot just wave our hands, say ‘neuroinflammation!’, and know much more than the broad strokes.  [Note: In my early days of my AutismNet life, my view was somewhat less nuanced.]  I think that part of what was bothering me is the result of an oversimplified model in my mind’s eye, but I’d formed that model on top of a set of measurements that had empirical precision but underpowered understandings, alongside a more fundamental lack of knowledge.

We know a little more now.

The first paper that really got me thinking along these lines was Synaptic pruning by microglia is necessary for normal brain development, (discussed on this blog, here), which provided evidence of microglial involvement in the ‘pruning’ of synapses, an important step in brain development thought to streamline neural communication by optimizing neuron structure.  This was the first paper I’d read that hinted at microglia participation in ‘normal’ brain function; it was only very recently that microglia were considered to have any role in non pathological states.  Another paper, Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease, also implicated microglia in synaptic pruning.

Then I got myself a copy of The role of microglia at synapses in the healthy CNS: novel insights from recent imaging studies. It is a review of several recent studies on the non-excited life of microglia.

In the healthy brain, quiescent microglia continuously remodel their shape by extending and retracting highly motile processes. Despite a seemingly random sampling of their environment, microglial processes specifically interact with subsets of synaptic structures, as shown by recent imaging studies leading to proposed reciprocal interactions between microglia and synapses under non-pathological conditions. These studies revealed that various modalities of microglial dynamic behavior including their interactions with synaptic elements are regulated by manipulations of neurotransmission, neuronal activity and sensory experience. Conversely, these observations implied an unexpected role for quiescent microglia in the elimination of synaptic structures by specialized mechanisms that include the phagocytosis of axon terminals and dendritic spines. In light of these recent discoveries, microglia are now emerging as important effectors of neuronal circuit reorganization.

This review by Tremblay was published in 2012, evidence of the nascent nature of our available data on microglial involvement in the normal brain environment; Tremblay states that part of the reason this type of finding is so recent is the relative difficulty of measuring microglia in non excited states.  They were the electrons of brain measurements; our previous attempts to measure them were capable of causing them to change morphology.

The roles of ‘resting’ or immunologically quiescent microglia have remained relatively unknown (also see Tremblay et al., 2011). This is largely due to the difficulties of studying microglia in their non-activated state. Microglia respond promptly to any changes occurring in their environment, and therefore experimental ex vivo and in vitro preparations inevitably result in transformation of their normally prevailing behavior.

Nice.

Anyway, some new whizbang technologies (i.e., in vivo two-photon laser scanning microscopy)[?] are allowing researchers to peer into the ho-hum everyday activities of ‘non activated’ microglia, and what they are finding is that the term ‘activated microglia’ might be a bit of a misnomer, microglia have been participating in brain function all along, it is just that our filters were insignificantly powered to detect some of their actions until very recently.   Several studies have shown that so called ‘resting’ microglia are constantly evaluating their environment with protusions that seemed to operate rather quickly in relationship to other types of neurons.

This unexpected behavior suggested that resting or surveillant microglia may continuously survey the brain parenchyma as part of their immune function, which would justify the substantial expenditure of energy required to continuously maintain microglial dynamics in the normal brain, without excluding the possibility of an additional, distinct contribution to normal brain physiology

Several papers are reviewed that utilized a couple of highly technical methods, including double roll your own transgenic mouse models to visualize the interactions of microglia in a non excited state and synapses.  Specific areas of the brain were measured in different studies, microglia were observed transiently engaging with neurons and seemed to target some dendrites for removal.  The authors speculate that this could be a mechanism by which neuronal network maintenance, plasticity, could be affected.

In the mature healthy CNS, neuronal networks are continuously remodeled through the formation, modification and elimination of synaptic structures (see Fortin et al. (2011) for molecular mechanisms of structural plasticity) in relation with behavioral and sensory experience.

And

To determine a possible role of surveillant microglia in the structural remodeling of synaptic structures under normal physiological conditions, Tremblay et al. (2010b) also examined the size changes of spines and terminals before, during and after microglial contacts. Spines contacted by microglial processes during imaging (30–120 min sessions) were found to be smaller initially than those which remained non-contacted. Spines, but not terminals, also underwent transient increases in size during microglial contact, with smaller spines showing the most pronounced changes. Surprisingly, chronic imaging over 2 days further revealed a statistically significant difference in the elimination rate of microglia-contacted spines: spines contacted by microglia were more frequently eliminated than non-contacted spines (24 versus 7%; P  0.05), and in all cases, only the small spines were seen to disappear. These observations suggest that despite an apparently random sampling of the parenchyma, microglial processes specifically target a subset of small, structurally dynamic and transient dendritic spines.

There is also some description of studies that seemed to indicate that the microglial/synapse interactions could be modified through environmental stimulus, two experiments were described involving sensory deprivation and consequent changes in microglia activity.  Other experiments described changes in microglial surveillance as a result of induced changes in neuronal excitability by chemical agonists or antagonists of glutamate receptors.  [Perhaps this is the basis of the curious findings in Neuroprotective function for ramified microglia in hippocampal excitotoxicity?]

In their concluding statements, Tremblay provides a good description of just how little we know, and in a style that I love, poses open questions for the newer rounds of literature to address.

Since the recent studies have barely scratched the surface (of the brain in this case), the modalities of microglial interactions with excitatory and inhibitory synapses throughout the CNS, much as their functional significance and particular cellular and molecular mechanisms still remain undetermined. For example, in which contexts do quiescent microglia directly phagocytose axon terminals and dendritic spines, use other mechanisms such as proteolytic remodeling of the extracellular space, or refrain from intervening?  How do surveillant microglia recognize and respond to the various molecular signals in their environment, including dynamic changes in neurotransmission and neuronal activity at individual synapses? How do these immune cells cooperate with other glial cells, as well as peripheral myeloid cells, in maintaining or shaping neuronal architecture and activity? And, as in the case of microglial memory of past immune challenges (see Bilbo et al., 2012), do surveillant microglia somehow remember their previous behavioral states, the flux of information processing in the brain, or the structural changes of synaptic elements in recent and not so recent windows of intervention?

The last sentence there, I think, is especially salient considered within a context of developmental programming.

So what we’ve learned is that decades after the discovery of microglia cells as the immune regulators in the CNS, they appear to also be participating in more fundamental maintenance of the neural structure of our brains; there is increasing evidence of direct relationships in synaptic and axonal removal as well as roles in neurotransmission and the regulation of excitability.   Is more on the horizon?

But what about autism and our apparent autism paradox of a static or improving behavioral state alongside conditions of immune activation within the CNS?

Well, I have also been thinking about two brain scanning studies that have come out not too long ago, Neuron Number in Children With Autism (Courchesne et all) , which found increased numbers of neurons in the autism cohort, and Differences in White Matter Fiber Tract Development Present From 6 to 24 Months in Infants With Autism (Wolff et all) which found that the autism group showed denser bundled of white matter, so called wiring, between different parts of the brain.  In both of these studies mention is made of the fact that it was possible that their findings, increased cell numbers could be the result of inappropriate removal of excess neurons during development.

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.

and

For example, differences in structural organization prior to a period of experience-dependent development related to social cognition (52–54) may decrease neural plasticity through limitations on environmental input, preventing typical neural specialization (52). These alterations could have a ripple effect through decreasing environmental responsiveness and escalating invariance*, thus canalizing a specific neural trajectory that results in the behavioral phenotype that defines ASDs. In typical development, the selective refinement of neural connections through axonal pruning (55) along with constructive processes such as myelination (56) combine to yield efficient signal transmission among brain regions. One or both of these mechanisms may underlie the widespread differences in white matter fiber pathways observed in the current study. 

* 😦

So, we have growing evidence of microglial participation of neural maintenance alongside growing evidence of impaired maintenance in the autism cohort.

Can our autism paradox be explained by microglia converging in the center of these related lines of thought?  Is the answer to our riddle that the ongoing immune response in the brain is not sufficiently powered, or targeted, to cause increasing loss of abilities, but instead, was enough to keep critical, once in a lifetime chances for brain organization from occurring?  Are increased neuron number and altered white matter tracts the result of microglia not performing the expected maintenance of the brain?  Are the findings from Courchesne and Wolff the opportunity costs of having a microglia activated during decisive developmental timeframes?

That is a pretty neat idea to consider.

Even without the Courchesne and Wolff, the findings that specifically mention impaired network maintenance as possible culprits, the findings of active participation of ‘non-active’ microglia in brain optimization and normal processes is a very problematic finding for another autism canard, the idea that findings of neuroinflammation may not be pathological.  The intellectually honest observer will admit that the crux of this defense lay in vaccine count trial testimony presented by John Hopkin’s researchers after their seminal neuroinflammation paper was published.  Unfortunately, the vigor with which this testimony is trotted out online does not match the frequency with which such ideas actually percolate into the literature.

But with the data from Tremblay, Paolicelli, and others, such an idea becomes even more difficult to defend, we must now speculate on a mechanism by which either microglia could be in an excited state and continue to perform streamlining of the neural structure, or insist that it is possible that microglia were not excited during development, and something else happened to interfere with neuron numbers, and then, subsequently the microglia became chronically activated.

This is unlikely, and unlikelier still when we consider that anyone proposing such a model must do so with enough robustness to overcome a biologically plausible pathway supported by a variety of studies.  And that is only if there was anything underneath the vapor!  Make no mistake, if you ever press someone to actually defend, with literature, the mechanisms by which a state of chronic neuroinflammation might be beneficial in autism, or even the result of something else that also causes autism, no further elucidation of that mechanism is ever forthcoming.  There isn’t anything there.

At some point, it becomes incumbent of people wishing to make an argument that they propose a biologically plausible mechanism if they wish to continue to be taken seriously.  If they cannot, if the literature cannot be probed to make such a case with more empirical support than it might be, the notion so add odds with available evidence should be summarily discarded, unless and until a transcendent set of findings is presented.  There should always be room for more findings in our worldview, but precious limited space for faith in the face of contradictory findings.

–          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 –

I ran into a few abstracts,  read a few papers, and tried to get my way through one really dense paper in the past few weeks that got me thinking about this post.  It’s  all shook up, like pasta primavera in my head, but hopefully something cogent will come out the other end.  (?)

Of the metabolic conditions known to be associated with having a child with autism, hypothyroidism is one that I keep on running into by way of the pubmed alert grapevine.  By way of example, we have two studies that looked for autoimmune conditions in family members which found hypothyroidism to be one of many autoimmune diseases as a risk factor for autism, including,  Familial clustering of autoimmune disorders and evaluation of medical risk factors in autism, and Increased prevalence of familial autoimmunity in probands with pervasive developmental disorders.   This shouldn’t be too surprising, we know that, for example, perinatal hypothyroidism is a leading cause of mental retardation, with similar findings for the condition during pregnancy.  It turns out, it appears that rates of hypothyroidism are slightly increasing, though at this time, the increases are of relatively small proportions, and as such, may be artifacts unrelated to an actual increase in classically recognized hypothyroidism.  In any case, I think it is safe to say that interference with thyroid metabolism is something to be avoided at all costs when possible.

So after having read about that, this paper showed up in my inbox a while ago:

Effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor gene expression in rat hippocampus: Role of DNA methylation and histone acetylation

Thyroid hormones have long been known to play important roles in the development and functions of the central nervous system, however, the precise molecular mechanisms that regulate thyroid hormone-responsive gene expression are not well understood. The present study investigated the role of DNA methylaion and histone acetylation in the effects of perinatal hypothyroidism on regulation of reelin and brain-derived neurotrophic factor (BDNF) gene expression in rat hippocampus. The findings indicated that the activities of DNA methyltransferase (DNMT), methylated reelin and BDNF genes were up-regulated, whereas, the activities of histone acetylases (HAT), the levels of global acetylated histone 3 (H3) and global acetylated histone 4 (H4), and acetylated H3, acetylated H4 at reelin promoter and at BDNF gene promoter for exon II were down-regulated in the hippocampus at the developmental stage of the hypothyroid animals. These results suggest that epigenetic modification of chromatin might underlie the mechanisms of hypothyroidism-induced down-regulation of reelin and BDNF gene expression in developmental rat hippocampus

This gets interesting for autism because reelin, and bdnf levels have been found to be decreased in several studies in the autism population, with direct measurements, genetic expression, mouse knockout based models of autism , and genomic alterations all being implicated.  There have been some negative genetic studies, but considering that it isn’t always the genes you have, but the genes you use, our other available evidence certainly points to BDNF and reelin involvement with some percentage of children with autism, and the association is such that a reduction in reelin or BDNF is a risk factor for developing autism.  It would seem that the paper above might give some insight into the lower level details of the effects of hypothyroidism and subsequent developmental trajectories; modifications of reelin expression; through epigentic mechanisms, no less!.  That’s pretty cool!

Then, I got my hands on a review paper that tries to go into detail as to the functional mechanism by which reelin deficiency could contribute to ASD, Neuroendocrine pathways altered in autism. Special role of reelin.  It is a review that touches on a variety of ways that reelin contributes to neurodevelopment that have findings in the autism realm, including neuronal targeting and migration during brain formation, interactions with the serotonin and GABA systems, testosterone, and oxytocin.   In short, there are plenty of ways that decreased reelin expression can impact development in ways that mirror our some of our observations in autism.

Of the many things that convince me that we are doomed, the proliferation of chemical compounds whose interactions within our bodies we scarcely understand is among them.   In my readings on endocrine disruptors, one thing I found that seemed to be worrying lots of researchers was that some classes of these chemicals are capable of interfering with thyroid metabolism, and in some cases interfering with development of cells known to be associated with autism.    Terrifyingly enough, since I read those papers, several others have come out, including Polybrominated Diphenylether (PBDE) Flame Retardants and Thyroid Hormone during Pregnancy and Mini-review: polybrominated diphenyl ether (PBDE) flame retardants as potential autism risk factors.     At this point, it is important to point out that, as far as I know, there have not been any studies showing that non occupational exposure to PDBEs or other environmental pollutants can lead to classically defined hypothyroidism, at least none that I know of. (?)    Be that as it may, I think it is realistic to assume any interference in thyroid metabolism is a bad thing, and while finding people in the outlier regions of hypo (or hyper) thyroidism gives us information on extreme environments, it would take someone with a lot of misplaced faith to assume that we can safely disturb thyroid metabolism just a little bit, and everything will come out in the wash.

I’ve had the argument made to me in the past that environmental pollutant driven increases in autism lacked biological plausible mechanisms; this argument is almost always made within a context of trying to defend the concept of a static rate of autism.  While the papers I’ve linked to above do not provide conclusive proof that our changing environment is causing more children to be born with autism, they do provide increasing evidence of a pathway from pollutants to ASD, and indeed,  the lack of biological plausibility becomes an increasingly flacid foundation on which to assume that our observations of an increased rate of autism are illusory.   Unfortunately, in my opinion, the focus on vaccines has contributed to the mindset that a static rate of autism (or nowadays, maybe a tiny increase), must be protected at all costs, including some ideas on the application of a precautionary principle that seem outright insane to me (or at least, the exact opposite of what I would consider to be a precautionary path).

One thing is for certain, the number of child bearing women in developing countries with measurable concentrations of chemicals known to interferre with thyroid metabolism nears 100% in the industrialized nation as we eat , drink, breathe and bathe in the microscopic remnants of packaging materials, deteriorating carpet fibers, and baby clothes that are made to be fire resistant.  This is an environment unambiguously different than that encountered by any other generation of infants in the history of mankind.  To believe that we can modify our environment so drastically without having an impact seems incredibly naive to me, or on some days, just plain old stupid.

– 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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