passionless Droning about autism

Archive for the ‘Immunology’ Category

Hello friends –

I’ve been referencing this paper in some discussions online for a while; I’ve read it, and in fact, while working on another project, got the opportunity to speak with one of the authors of the paper.  It’s a very cool paper with a lot of information in it, some of which, could be considered inconvenient findings.  Here is the abstract:

Immune transcriptome alterations in the temporal cortex of subjects with autism

Autism is a severe disorder that involves both genetic and environmental factors. Expression profiling of the superior temporal gyrus of six autistic subjects and matched controls revealed increased transcript levels of many immune system related genes. We also noticed changes in transcripts related to cell communication, differentiation, cell cycle regulation and chaperone systems. Critical expression changes were confirmed by qPCR (BCL6, CHI3L1, CYR61, IFI16, IFITM3, MAP2K3, PTDSR, RFX4, SPP1, RELN, NOTCH2, RIT1, SFN, GADD45B, HSPA6, HSPB8and SERPINH1). Overall, these expression patterns appear to be more associated with the late recovery phase of autoimmune brain disorders, than with the innate immune response characteristic of neurodegenerative diseases. Moreover, a variance-based analysis revealed much greater transcript variability in brains from autistic subjects compared to the control group, suggesting that these genes may represent autism susceptibility genes and should be assessed in follow-up genetic studies.

(emphasis is mine) [Full paper freely available from that link]

I am particularly intrigued by the second bolded sentence regarding the “these expression patterns appear to be more associated with the later recovery phase of autoimmune brain disorders, than the innate immune response characteristic of neurodegenerative diseases”.  I’ve had it put to me previously that we should not necessarily implicate neuroinflammation in autism, the argument being that even though we had evidence of chronically activated microglia, what we do not seem to have evidence for is actual damage to the brain, and ergo, the neuroinflammation may actually be a byproduct of having autism, as opposed to playing a causative role, or that in fact, the neuroinflammation might even be beneficial.  There have been some other places where the claim has been made that because our profile of neuroinflammation doesn’t match more classically recognized neurodegenerative disorders (i.e., MS/Alzheimer’s/Parkinson’s), that therefore, certain environmental agents need not be fully investigated as a potential contributor to autism.  This is the first time that I am aware that someone has attempted to classify the neuroinflammatory pattern observed in autism not only as distinctly different from classical neurodegenerative diseases, but to also go so far as to provide a more refined example.

From the Introduction:

In order to better understand the molecular changes associated with ASD, we assessed the transcriptome of the temporal cortex of postmortem brains from autistic subjects and compared it to matched healthy controls. This assessment was performed using oligonucleotide DNA microarrays on six autistic-control pairs. While the sample size is limited by the availability of high-quality RNA from postmortem subjects with ASD, this sample size is sufficient to uncover robust and relatively uniform changes that may be characteristic of the majority of subjects. Our study revealed a dramatic increase in the expression of immune system-related genes. Furthermore, transcripts of genes involved in cell communication, differentiation, cell cycle regulation and cell death were also profoundly affected. Many of the genes altered in the temporal cortex of autistic subjects are part of the cytokine signaling/regulatory pathway, suggesting that a dysreactive immune process is a critical driver of the observed ASD-related transcriptome profile.

I was initially very skeptical about this, with a sample set so small, wasn’t it difficult to ascertain if their findings were by chance or not?  It turns out, the answer depends on the type of datapoint you are evaluating against.  A powerful tool in use by the researchers is a recent addition to the genetic analysis research suite, not only the ability to scan for thousands of gene activity levels simultaneously, but the use of known gene networks to identify if among those thousands of results, related genes are being expressed differentially.  This is important for some amazingly robust findings presented later in the paper, so lets sidetrack a little bit.  Here is a nice overview of the process being used:

Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles

Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation.  A common approach involves focusing on a handful of genes at the top and bottom of L (i.e., those showing the largest difference) to discern telltale biological clues. This approach has a few major limitations.

(i) After correcting for multiple hypotheses testing, no individual gene may meet the threshold for statistical significance, because the relevant biological differences are modest relative to the noise inherent to the microarray technology.

(ii) Alternatively, one may be left with a long list of statistically significant genes without any unifying biological theme. Interpretation can be daunting and ad hoc, being dependent on a biologist’s area of expertise.

(iii) Single-gene analysis may miss important effects on pathways. Cellular processes often affect sets of genes acting in concert. An increase of 20% in all genes encoding members of a metabolic pathway may dramatically alter the flux through the pathway and may be more important than a 20-fold increase in a single gene.

(iv) When different groups study the same biological system, the list of statistically significant genes from the two studies may show distressingly little overlap (3).

So, back to Garbett, not only did the authors find a great number of genes overexpressed in the autism group (and a smaller number, underexpressed), when they threw their thousands of results of individual genes into the GSEA, what came back was that several genetic pathways were very significantly altered, many of them immune mediated. This is a big step in understanding in my opinion.  I believe we have likely come full circle on our understanding of very high penetrance genes that might be driving towards a developmental trajectory of autism; i.e., Rhett, Fragile-X.  But using this technique we can determine if entire biological pathways are altered by measuring the output of genes.  Specifically the point made in bullet (iii) stands out to me; having a twenty fold increase in a single gene might not be too big a deal if the other participants in the proteins function cannot be altered by twenty fold as a result due to other rate limiting constraints; but if we can see related sets of genes with similar expression profiles, we can get a much better picture of the biological results of different expression.

The methods get dense pretty quickly, but are worth a shot to show how thorough the researchers were to insure that their findings were likely to be signficant.  Essentially they performed three different statistical tests against their results of differentially expressed genes and broke their results down into genes that passed all three tests, two of three test, or one of three tests.  Furthermore, a selected twenty genes were targeted with qPCR validation, which in all cases showed the expected directionality; i.e., if the expression was increased in the transcriptome analysis, qPCR analysis confirmed the increased expression.To provide another benchmark, they tested for other genes known to be associated with autism, REELN, and GFAP and found results consistent with other papers.

Having determined a large number of differentially expressed genes, the authors then went to try to analyze the known function of these genes.

These classifications were performed on a selected gene set that is differentially expressed between AUT and CONT subjects; based on the success of our qPCR validation, we decided to perform this analysis using transcripts that both reported an |ALR>1| and that reached p<0.05 in at least 2/3 statistical significance comparisons. Of 221 such transcripts, 186 had increased expression in AUT compared to CONT, while only 35 genes showed reduced expression in the AUT samples. We subjected these transcripts to an extensive literature search and observed that 72 out of 193 (37.3%) annotated and differentially expressed transcripts were either immune system related or cytokine responsive transcripts (Supplemental Material 2). Following this first classification, we were able to more precisely sub-classify these 72 annotated genes into three major functional subcategories, which overlap to a different degree; 1) cell communication and motility, 2) cell fate and differentiation, and 3) chaperones (Figure 3). The deregulation of these gene pathways might indicate that the profound molecular differences observed in the temporal cortex of autistic subjects possibly originate from an inability to attenuate a cytokine activation signal.

That last sentence packs a lot of punch for a couple of reasons.  It would seem to be consistent with their statements regarding a “late recovery phase” of an autoimmune disorder; i.e., an immune response was initiated at some point in the past, but has yet to be completely silenced.  This also isn’t the first time that the idea of problems regulating an immune response (i.e., the inability to attenuate a cytokine activation signal) has been suggested from clinical findings, for example, in Decreased transforming Growth Factor Beta1 in Autism: A Potential Link Between Immune Dysregulation and Impairment in Clinical Behavioral Outcomes, the authors found an inverse correlation between TGF-Beta1 and autism behavioral severity:

Given that a major role of TGFβ1 is to control inflammation, the negative correlations observed for TGFβ1 and behaviors may suggest that there is increased inflammation and/or ongoing inflammatory processes in subjects that exhibit higher (worse) behavioral scores.

As such, TGFβ has often been considered as one of the crucial regulators within the immune system and a key mediator in the development of autoimmune and systemic inflammation.

In summary, this study demonstrates that there is a significant reduction in TGFβ1 levels in the plasma of young children who have ASD compared with typically developing children and with non-ASD developmentally delayed controls who were frequency-matched on age. Such immune dysregulation may predispose to the development of autoimmunity and/or adverse neuroimmune interactions that could occur during critical windows in development.

[full paper from the link]

The theme of a critical window of development and enduring consequences of insults during that window is one that is getting more and more attention recently; this is an area that is going to get more and more attention as time goes by, and eventually, as the clinical data continues to pile up, meaningless taglines aren’t going to be enough to keep us from dispassionately evaluating our actions.

The Discussion section is particularly nice, I’ll try not to just quote the entire thing.  Here are the really juicy parts.

The results of our study suggest that 1) in autism, transcript induction events greatly outnumbers transcript repression processes; 2) the neocortical transcriptome of autistic individuals is characterized by a strong immune response; 3) the transcription of genes related to cell communication, differentiation and cell cycle regulation is altered, putatively in an immune system-dependent manner, and 4) transcriptome variability is increased among autistic subjects, as compared to matched controls. Furthermore, our study also provides additional support for previously reported involvement of MET, GAD1, GFAP, RELN and other genes in the pathophysiology of autism. While the findings were obtained on a limited sample size, the statistical power, together with the previously reported postmortem data by other investigators suggest that the observed gene expression changes are likely to be critically related to the pathophysiology seen in the brain of the majority of ASD patients.

There is some description of studies using gene expression testing in the autism realm where the authors ultimately conclude that technical and methodological differences between the studies make them difficult to tie together coherently.  There is another small section re-iterating the findings that were similar to single gene studies; i.e., REELN, MET, and GAD genes.

The most prominent expression changes in our dataset are clearly related to neuroimmune disturbances in the cortical tissue of autistic subjects. The idea of brain inflammatory changes in autism is not novel; epidemiological, (DeLong et al., 1981; Yamashita et al., 2003; Libbey et al., 2005) serological studies (Vargas et al., 2005; Ashwood et al., 2006) and postmortem studies (Pardo et al., 2005; Vargas et al., 2005; Korkmaz et al., 2006) over the last 10 years have provided compelling evidence that immune system response is an essential contributor to the pathophysiology of this disorder (Ashwood et al. 2006). Finally, converging post-mortem assessments and measurements of cytokines in the CSF of autistic children (Vargas et al., 2005), may indicating an ongoing immunological process involving multiple brain regions

Nothing really new here to anyone that is paying attention, but good information for the extremely common, gross oversimplification that ‘immune abnormalities’ have been found in autism, but we don’t have any good reason to think they may be part of the problem.  Of course, this is an argument you’ll see a lot of the time regarding everyone’s favorite environmental agent.

Altered immune system genes are often observed across various brain disorders, albeit there are notable differences between the observed transcriptome patterns. The majority of neuroimmune genes found activated in the autistic brains overlap with mouse genes that are activated during the late recovery or “repair” phase in experimental autoimmune encephalomyelitis (Baranzini et al., 2005). This suggests a presence of an innate immune response in autism. However, the altered IL2RB, TH1TH2, and FAS pathways suggest a simultaneously occurring, T cell-mediated acquired immune response. Based on these combined findings we propose that the expression pattern in the autistic brains resembles a late stage autoimmune event rather than an acute autoimmune response or a non-specific immune activation seen in neurodegenerative diseases. Furthermore, the presence of an acquired immune component could conceivably point toward a potential viral trigger for an early-onset chronic autoimmune process leading to altered neurodevelopment and to persistent immune activation in the brain. Interestingly, recently obtained gene expression signatures of subjects with schizophrenia (Arion et al., 2007) show a partial, but important overlap with the altered neuroimmune genes found here in autism. These commonly observed immune changes may represent a long-lasting consequence of a shared, early life immune challenge, perhaps occurring at different developmental stages and thus affecting different brain regions, or yielding distinct clinical phenotypes due to different underlying premorbid genetic backgrounds.

The last sentence, regarding ‘long-lasting’ consequences of early life immune challenges is one that has a large, and growing body of evidence in the literature that report physiological and behavioral similarities to autism.  We also have recent evidence that hospitilization for viral or bacterial infection during childhood is associated with an autism diagnosis.    There is, of course, a liberal sprinkling of ‘mays’, ‘propose’, and ‘conceivably’ caveats in place here.

Earlier I mentioned that the authors studied gene networks in addition to single gene expressions., and that some of those findings were very significant.  The results of this are found in Table 2.  In one discussion, I had it pointed out to me by ScienceMom that it appeared that some of the networks were not found to be statistically significant (and ergo we should not necessarily assume that immune dysfunction was a participant in autism).   [If you look at Table 2, some networks like a p value of 0000].   I decided to use the data in this paper for another project that isn’t ready yet, but in that process I was able to speak directly with one of the authors of this paper.  I asked him about this, and he told me that this was a function of space limitations; all of the gene networks described were found to be statistically signficant, but in some instances there wasn’t enough space to typeset the p value. In fact, some networks were found to be differentially expressed with a p-value of .000000000000001.  (!!!!!!!!)  That isn’t a value that you see very often.

I recently got a copy of Mitochondrial dysfunction in Autism Spectrum Disorders: cause or effect, which shares an author with this paper, Persico.  In that paper, they reference Immune Transcriptome Alterations In the Temporal Cortex of Subjects With Autism, invoking a potential cascade effect of prenatal immune challenge, inherited calcium transport deficiencies, and resultant mitochondrial dysfunction that could lead to autism.  I’ve generally stayed away from the mitochondria stuff in the discussion realm; even though I think it is probably somewhat important to some children, and critically important to a select few children,  I’ve mostly found that the discussion of mitochondrial issues is comprised of two sets of people talking past one another so as to prove something, or disprove something about everyones favorite environmental agent; but this is a neat paper that I’d like to get to eventually.

– pD

Hello friends –

The abstract for Association of hospitalization for infection in childhood with diagnosis of autism spectrum disorders: a Danish cohort study hit my inbox the other morning.  Here is the abstract

OBJECTIVE: To investigate the association between hospitalization for infection in the perinatal/neonatal period or childhood and the diagnosis of autism spectrum disorders (ASDs). DESIGN: A population-based cohort study. SETTING: Denmark. PARTICIPANTS: All children born in Denmark from January 1, 1980, through December 31, 2002, comprising a total of 1 418 152 children. EXPOSURE: Infection requiring hospitalization. MAIN OUTCOME MEASURE: The adjusted hazard ratio (HR) for ASDs among children hospitalized for infection compared with other children. RESULTS: A total of 7379 children were diagnosed as having ASDs. Children admitted to the hospital for any infectious disease displayed an increased rate of ASD diagnoses (HR, 1.38 [95% confidence interval, 1.31-1.45]). This association was found to be similar for infectious diseases of bacterial and viral origin. Furthermore, children admitted to the hospital for noninfectious disease also displayed an increased rate of ASD diagnoses (HR, 1.76 [95% confidence interval, 1.68-1.86]), and admissions for infection increased the rate of mental retardation (2.18 [2.06-2.31]). CONCLUSIONS: The association between hospitalization for infection and ASDs observed in this study does not suggest causality because a general association is observed across different infection groups. Also, the association is not specific for infection or for ASDs. We discuss a number of noncausal explanatory models

[Emphasis is mine.]

Considering my interest in early life immune activation, and the often difficult to predict, persistent outcomes from a variety of animal models, this study immediately struck me as an interesting one. The authors graciously sent my real world inbox a copy of this paper, as well as a similar one involving maternal infection during pregnancy, which I have yet to read.

Anyways, what strikes me very clearly here is that the authors and I have reached exactly the opposite conclusions towards the potential of a casual link between autism and hospitalization for infection in the perinatal / infancy periods.  They apparently feel that the fact that an association is observed across different infectious agents (i.e., bacterial or viral), that this argues against a causal mechanism.  But, as I have detailed in A Brief History of Early Life Immune Challenges and Why They (Might) Matter, we have an increasing number of animal studies that indicate that spikes in innate immune system cytokines during critical developmental timeframes can have, perverse and often baffling effects that we are only beginning to understand.  Most of this research is brand new, within the past three years, and solely in the realm of animal models.  However, the critical component of these studies that the Denmark study fails to take into consideration is that the innate immune response will be initiated regardless if the stimulant is viral or bacterial in nature. That is to say, the evidence from these studies tells us that the fact that we are observing differences across bacterial or viral pathogens is not necessarily an indication of lack of effect, but rather, could instead point towards a global effect, one that happens in both instances; surges in pro-inflammatory cytokines from the innate immune response.

For an example of some of these animal models, we could look to Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats, which observed a tnf-alpha driven, time dependent mechanism that ‘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.

Another, very similar study, Viral-like brain inflammation during development causes increased seizure susceptibility in adult reports:

Viral infections of the CNS and their accompanying inflammation can cause long-term neurological effects, including increased risk for seizures. To examine the effects of CNS inflammation, we infused polyinosinic:polycytidylic acid, intracerebroventricularly to mimic a viral CNS infection in 14 day-old rats. This caused fever and an increase in the pro-inflammatory cytokine, interleukin (IL)-1beta in the brain. As young adults, these animals were more susceptible to lithium-pilocarpine and pentylenetetrazol-induced seizures and showed memory deficits in fear conditioning. Whereas there was no alteration in adult hippocampal cytokine levels, we found a marked increase in NMDA (NR2A and C) and AMPA (GluR1) glutamate receptor subunit mRNA expression. The increase in seizure susceptibility, glutamate receptor subunits, and hippocampal IL-1beta levels were suppressed by neonatal systemic minocycline. Thus, a novel model of viral CNS inflammation reveals pathophysiological relationships between brain cytokines, glutamate receptors, behaviour and seizures, which can be attenuated by anti-inflammatory agents like minocycline.

If we look closely here, we can see that either viral or bacterial mimics were able to generate similar physiological outcomes, outcomes that have strong correlations to the autism realm, namely increased rates of epilepsy, associations with seizures during infancy, and abnormal EEGs.  But importantly for the decision tree in the case of childhood infections in the studies above, taken together, we can see that it didn’t matter if the trigger was bacterial or viral, just that there was an innate immune response at all. This is further evidenced by the fact that in both instances, different anti-inflammatory agents were capable of attenuating the changes.  Our mechanism of action does not mandate pathogen specific interactions, in many cases, the cut off is whether or not you generate an innate immune response or not, regardless of the specific trigger. Another way of putting this would be, if an immune response for any pathogen were capable of initiating an cascade responsible for development of autistic behaviors, what would a pattern of hospitalization look like?  [Children admitted to the hospital for any infectious disease displayed an increased rate of ASD diagnoses (HR, 1.38 [95% confidence interval, 1.31-1.45]).  This association was found to be similar for infectious diseases of bacterial and viral origin.]

If you ask the wrong question even the right answer might not be useful in understanding a mystery.

All that being said, I have begun to see why Denmark makes such an attractive location for this kind of study.  They have amassed an impressive set of data that could  yield important clues if we can use it wisely.

I also noted that there is a P. Thorsen listed.  I, for one, could care less.

– pD

Hello friends –

So this is a really cool paper by some folks that have a series of interesting stuff:  Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contributions to autism spectrum disorders and a novel autism candidate gene, RORA, whose protein product is reduced in autistic brain.  Here is the abstract:

Autism is currently considered a multigene disorder with epigenetic influences. To investigate the contribution of DNA methylation to autism spectrum disorders, we have recently completed large-scale methylation profiling by CpG island microarray analysis of lymphoblastoid cell lines derived from monozygotic twins discordant for diagnosis of autism and their nonautistic siblings. Methylation profiling revealed many candidate genes differentially methylated between discordant MZ twins as well as between both twins and nonautistic siblings. Bioinformatics analysis of the differentially methylated genes demonstrated enrichment for high-level functions including gene transcription, nervous system development, cell death/survival, and other biological processes implicated in autism. The methylation status of 2 of these candidate genes, BCL-2 and retinoic acid-related orphan receptor alpha (RORA), was further confirmed by bisulfite sequencing and methylation-specific PCR, respectively. Immunohistochemical analyses of tissue arrays containing slices of the cerebellum and frontal cortex of autistic and age- and sex-matched control subjects revealed decreased expression of RORA and BCL-2 proteins in the autistic brain. Our data thus confirm the role of epigenetic regulation of gene expression via differential DNA methylation in idiopathic autism, and furthermore link molecular changes in a peripheral cell model with brain pathobiology in autism.

 [As always, any emphasis is my own.]

This group has published a couple of papers that utilized similar study groups, methodologies, and means to display their findings, all of which I would recommend to anyone interested in learning; specifically, Gene expression profiling of lymphoblastoid cell lines from monozygotic twins discordant in severity of autism reveals differential regulation of neurologically relevant genes [full paper available!], Gene expression profiling differentiates autism case-controls and phenotypic variants of autism spectrum disorders: evidence for circadian rhythm dysfunction in severe autism [full version available!], and Gene expression profiling of lymphoblasts from autistic and nonaffected sib pairs: altered pathways in neuronal development and steroid biosynthesis [full paper available!]. 
There are a couple of things I really like about their methodology and presentation style. 

1) Several studies, including the most recent, included twins with discordant autism severity as study participants as a way to gain insight into the impact of genetic expression, as opposed to genetic structure on autistic behaviors.  The highly cited heritability of autism in twins is used as evidence that the condition is predominantly mediated through genetics, and while no doubt genetic structure is important, by using genetic clones with different manifestations of autism severity, the authors are able to ascertain information about which genes are being affected in twins. 

2) The two stage nature of the study design allows for both large scale analysis of a great number of genes being expressed differentially by genome wide scan, the results of which can be used for highly targeted confirmation by tissue analysis.  Further, the use of cells available in the periphery, lymphobastoid cell lines (LLCs) as measurement points for genetic expression, allows for well thought out investigations of a very rare resource, post morten brain tissue from autistics.  In this instance, different methylation profiles identified from LLCs from blood samples gave the researchers a starting point for what to look for in the brain tissue. 

3) This paper ties together both genetic expression and epigenetics; i.e., not only that genes are being used differently, but it forwards our understandings of the means by which this is happening.  Earlier studies by this group have found differences in genetic expression previously, but hadn’t elucidated on the specific mechanisms of action, in this case, over methylation, and consequent silencing of genetic protein production. 

4) This is the first group of papers I’ve seen that have been using a bioinformatics approach to understanding the pathways affected by their findings; there may be other papers out there in the autism realm, (and almost certainly in others), that have been performing this type of analysis, but I haven’t run into them.  Several of their papers, including the circadian rhythm paper, provide illustrations of associations to biological conditions and pathologies associated with affected networks.  Here is an example from the latest paper.  (Sarcastic apologies for those running at 800 / 600)
 This type of illustration is the death knell for the argument that autism is a condition to be handled by psychologists; there are a couple of similar ones in the paper. 

Considering those points, here are some juicy parts from the paper itself.  From the introduction:

In this study, we use global methylation profiling of discordantly diagnosed monozygotic twins and their nonautistic siblings on CpG island arrays to test the hypothesis that differential gene expression in idiopathic autism is, at least in part, the result of aberrant methylation. Our study reveals distinct methylation differences in multiple genes between the discordant MZ twins as well as common epigenetic differences distinguishing the twins (the undiagnosed twin exhibiting milder autistic traits that are below the threshold for diagnosis) from nonautistic sibling controls.

There are essentially three groups, twins with different autism severity, and non autistic siblings.  One thing that I’m not cerrtain of here is whether or not there were methylation differences found between the twins and their non autistic siblings or not; the text above is a little unclear; i.e., as there are different mechanisms by which genetic expression can be modified besides methylation, this may mean that while there were expression differences found between autism and controls, those differences were not found to be attributed to differential methylation levels.  (?)

From the results:

Network analysis was then performed to examine the relationship between this set of genes and biological processes. As shown in Fig. 1B, many of the associated processes within the network, including synaptic regulation, fetal development, morphogenesis, apoptosis, inflammation, digestion, steroid biosynthesis, and mental deficiency, have been associated with autism. Two genes from this network, BCL-2 and RORA, were selected for further study because of their respective roles in apoptosis and morphogenesis/inflammation. Interestingly, BCL-2 protein has been previously demonstrated to be reduced in the cerebellum and frontal cortex of autistic subjects relative to control subjects (31, 32), but RORA, a nuclear steroid hormone receptor and transcriptional activator that is involved in Purkinje cell differentiation (33) and cerebellar development (34), has never before been implicated in autism. In addition, RORA, a regulator of circadian rhythm (35), is also neuroprotective against inflammation and oxidative stress (36), both of which are increased in autism (37, 38).

Several of the tables are pretty cumbersome to paste in, but do provide more detailed functional level impacts of some of the functions of the differentially methylated genes identified.  Even with the text above, however, we can see a lot of sweet spots being touched on, including several that were identified in previous studies by this group of researchers.  It also illustrates some of the very powerful techniques in use; a broad array of genes were scanned for differential expression, some with different expression and significant roles in processes known to be abnormal in the autism population are identified, and used for further, more pinpointed analysis. 

As noted, Fatemi found reduced BCL-2 in post mortem brain samples in two studies; one of the roles played by BCL-2 is apoptosis, or programmed cell death.  By way of example, here is a study that shows that knockout (or in this case, knockup) mice that overexpress BCL-2 have more Purkinje cells than their non modified counterparts, which states, in part:

Because bcl-2 overexpression has been shown to rescue other neurons from programmed cell death, the increase in Purkinje cell numbers in overexpressing bcl-2 transgenics suggests that Purkinje cells undergo a period of cell death during normal development.

Considering that reductions in Purkinje cells is among the most commonly found brain difference in autism, a reduction in BCL-2 seems appropriate.  The fact that it in this case it was methylation levels leading to a reduction in BCL-2 might also be of interest in regards to the Fairytale Of The Static Rate of Autism; here we have evidence that mechanisms other than genetic structure are leading to decreases in a protein known to protect Purkinje cells from apoptosis.  

I don’t know anything about RORA, but its list of functions make a lot of sense when we consider other findings; a relative dearth of a protein known to protect against neuroinflammation and oxidative stress and a regulatory role in the sleep cycle.

The authors also noticed a dose dependent relationship between expression levels, which in this case represented a silencing of genes and autism severity. 

Quantitative RT-PCR was used to confirm decreased expression of BCL-2 and RORA in autistic samples and to evaluate the effect of a global methylation inhibitor, 5-Aza-2-deoxycytidine, on gene expression. For both BCL-2 and RORA, gene expression was significantly higher (P_0.05) in the unaffected control than autistic co-twins (Fig. 4A). Generally, the diagnosed autistic co-twin (_A) had the lowest level of expression of BCL-2 and RORA, while the milder undiagnosed co-twin (_M) exhibited transcript levels between that observed for unaffected sibling controls and autistic co-twins. This suggests a quantitative relationship between phenotype and gene expression of these 2 genes, although additional studies are required to confirm this observation

Again, this makes plenty of sense if we believe that things like a neuroinflammation, oxidative stress have parts to play in the behavioral manifestation of autism; in this case, get more methylation, and hence, less RORA and BCL-2, which, in turns, makes you more susceptible to neuroinflammation, oxidative stress, and Purkinje cell development abnormalities. 

If we take the predisposition towards problems with inflammation for a closer look, we can find that several other papers, including Grigorenko, Enzo, and Ashwood have all found that a propensity for inflammation, or a propensity towards abnormal regulation of inflammation have correlations with autism severity.  Though potentially inconvenient, this would seem to lend additional evidence for a causal role of immune based pathology in autism, as opposed to autism causing immune abnormalities. 

The discussions section has a lot of good text that is largely a touch up on what we already have here.  Here are some good quotes:

In particular, functional and pathway analyses of the differentially  methylated/expressed genes showed enrichment of genes involved in inflammation and apoptosis, cellulardifferentiation, brain morphogenesis, growth rate, cytokine production, myelination, synaptic regulation, learning, and steroid biosynthesis, all of which have been shown to be altered in ASDs. The candidate genes were prioritized for further analyses by identifying the overlap between the differentially methylated genes and those that had been shown to be differentially expressed in the same set of samples in previous gene expression analyses (18). Pathway analyses of this filtered set of genes thus focused our attention on 2 genes, BCL-2 and RORA, as potential candidate genes for ASDs whose expression may be dysregulated byaberrant methylation.  As shown in Figs. 3 and 4, respectively, RORA was confirmed to be inversely differentially methylated and expressed in LCLs from autistic vs. nonautistic siblings,with expression dependent on methylation, as demonstrated by the absence of methylation in the presence of 5-Aza-2-deoxycytidine. Notably, we also show by immunohistochemical staining of cerebellar and frontal cortex regions of autistic vs. normal brain (Figs. 5, 8), that RORA protein is noticeably reduced in the majority ofthe autistic samples relative to age- and sex-matched controls. This reduction is also specifically demonstrated in Purkinje cells, which are dependent on RORA for both survival and differentiation (Fig. 7). These findings thus link molecular changes identified in a peripheral cell model of ASDs to actual pathological changes in the autistic brain, suggesting that LCLs is an appropriate surrogate for studies on autism.

Finally, this paper generated a lot of press, in part (I think), because somewhere, someone (the authors?), apparently made note of the fact that this type of feature, hypermethylation, is potentially treatable, raising the possibility of palliative avenues.  (Or was this just a function of the fact that it was a finding that wasn’t truly genetic, and thus, ‘fixable’?)  While technically true, I am of the opinion that this is a long ways off; the authors found large numbers of differentially methylated genes; some were also hypomethylated.  The drugs that we know are capable of epigenomic modifications right now, some are used in advanced cancer patients, for example, are not discriminatory in their actions.  What we really would need would be targeted unmethylators that we could use to attach to RORA and BCL-2 genes and specifically free them up to produce more protein.  The same week that this paper came out, another paper was published, entitled Epigenetic approaches to psychiatric disorders which speaks towards this complexity. 

–      pD

I have yet to get my hands on a copy of Plasma cytokine profiles in Fragile X subjects: Is there a role for cytokines in the pathogenesis?, but plan on doing so relatively soon.  Even still, the abstract looks pretty good:

BACKGROUND: Fragile X syndrome (FXS) is a single-gene disorder with a broad spectrum of involvement and a strong association with autism. Altered immune responses have been described in autism and there is potential that in children with FXS and autism, an abnormal immune response may play a role. OBJECTIVES: To delineate specific patterns of cytokine/chemokine profiles in individuals with FXS with and without autism and to compare them with typical developing controls. METHODS: Age matched male subjects were recruited through the M.I.N.D. Institute and included: 19 typically developing controls, 64 subjects with FXS without autism and 40 subjects with FXS and autism. Autism diagnosis was confirmed with ADOS, ADI-R and DSM IV criteria. Plasma was isolated and cytokine and chemokine production was assessed by Luminex multiplex analysis. RESULTS: Preliminary observations indicate significant differences in plasma protein levels of a number of cytokines, including IL-1alpha, and the chemokines; RANTES and IP-10, between the FXS group and the typical developing controls (p<0.01). In addition, significant differences were observed between the FXS group with autism and the FXS without autism for IL-6, Eotaxin, MCP-1 (p<0.04). CONCLUSIONS: In this study, the first of its kind, we report a significantly altered cytokine profile in FXS. The characterization of an immunological profile in FXS with and without autism may help to elucidate if an abnormal immune response may play a role and help to identify mechanisms important in the etiology of autism both with and without FXS.

I love the MIND guys.  Anyways, the quick glance gives me some ideas:

  • The big one here would seem to be that we seem to have additional evidence that immune dysregulation has very specific ties to autism; a pattern not just of immune dysregulation, but indeed, the beginings of an immunological signature, one so precise that given nothing but blood samples, we could beat Vegas odds in selecting which child has Fragile-X, and which child has Fragile-X and autism.   This is also more evidence that an abberant immunological response might be playing a causative role in autistic behavior genesis; just having Fragile-X isn’t enough for you to have this profile, you also have to have autism. 
  • Some of the players there we know about already; IL-6 was found in increased levels in the CNS by  Vargas, and Li , and was found to be generated at higher levels in several studies including Ashwood, Enstrom  , Jyonouchi, and Jyonouchi.  It also has great deal of support for a place in seizure generation.  MCP-1 was also found in Vargas, Eotaxin is also chemotaxic, though I haven’t read anything about it yet.  

 

That’s the abstract view, the whole paper should get here soon.

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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