passionless Droning about autism

Seeing Patterns or Chasing Phantoms, or Is There A Biologically Plausible Developmental Programming Pathway Toward Impaired Synaptic Pruning In Autism?

Posted on: December 26, 2011


Hello friends –

Lately I’ve found myself reading papers and knowing and owning several of the references; tragically I can’t tell if I’m reading the right research and am onto something, or I am chasing phantoms and my web of pubmed alerts and reading interests are funneling my reference list into a narrowing echo chamber of sorts.   With that warning in mind, we can proceed to poking around several papers, only some of which mention autism per se.  Along the way, we will see evidence supporting the possibility of a biologically plausible mechanism of developmental programming of the neuroimmune environment, a sequence of events that may lead to impaired synaptic pruning in (some cases of?) autism.

By now, everyone has seen/read/heard about one form or another of the ‘a massive asteroid is going to destroy the world’ story.  One of the common survival strategies from an asteroid strike involves altering the path of the asteroid so that it misses the Earth.  The thoughtful analysis of this problem allows for the physics based reality of the problem, moving an asteroid out of an extinction based trajectory involves just a little work when the asteroid is ten thousand gazillion miles away, but a lot more work when it is only a gazillion miles away.  Upon careful evaluation living organisms display similar behavior, relatively minor disturbances in early life can alter the developmental trajectory, while that same disturbance later in life is unable to materially affect the organism beyond a transient effect.   The accumulated evidence that early life experiences can shape the adult outcome is nearly impossible to dispute with any remaining intellectual honesty, the question is instead, is how large is the effect in autism?

This analogy adequately symbolizes one of the more beautiful and terrifying concepts I’ve come across researching autism, that of ‘developmental programming’, which I blogged some about here, but essentially is the idea that there are critical timeframes during which environmental impacts can have long term persistent effects on a wide range of outcomes.  The most robustly replicated findings involve changes to metabolic profiles in response to abnormal prenatal nutritional environments, but there is also evidence of various other effects, including neurological, and reputable speculation, that autism, may in fact, be in part, a disorder of developmental programming.

Secondarily, there has long been speculation of problems in the removal of ‘excess’ synapses, i.e., ‘synaptic pruning’ in the autism population.   This culling of synapses begins in fetal life continuing throughout adolescence and the repeated observations of increased head circumference during infancy as a risk factor for autism has resulted in the idea that altered synaptic pruning maybe involved in autism.

In the last month or so several rather serendipitously themed papers have been published with tantalizing clues about some of the finer grained mechanisms of synaptic pruning, the possibility of impaired synaptic pruning in the autism population, and a known risk factor for autism that models a developmental programming event sequence that may tie them together.

First off, we have Synaptic pruning by microglia is necessary for normal brain development, (Paolicelli et all) with a very straightforward title, that has this dynamite in the abstract: (snipped for length)

These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.

This paper is short, but pretty cool, and very nice from a new territory perspective.  It also speaks directly towards one of the increasingly hilarious obfuscations you will sometimes see raised in online discussions about immunological findings in autism, namely, that we can’t know if the state of chronic inflammation in the CNS observed in autism is harmful or beneficial.   [hint: It might not be causative, but it isn’t beneficial.]

Here’s is a snippet from the Introduction:

Time-lapse imaging has shown that microglia processes are highly motile even in the uninjured brain and that they make frequent, but transient contact with synapses. This and other observations have led to the hypothesis that microglia monitor synaptic function and are involved in synapse maturation or elimination.  Moreover, neurons during this period up-regulate the expression of the chemokine fractalkine, Cx3cl1, whose receptor in the central nervous system is exclusively expressed by microglia and is essential for microglia migration. If, in fact, microglia are involved in scavenging synapses, then this activity is likely to be particularly important during synaptic maturation when synaptic turnover is highest.

Nice.  A time dependent participation by microglia in the critical process of optimization of neuron numbers, a process we are still very much groping our way in the dark towards untangling.  The researchers focused in on a particular molecular target, a chemical messenger of the immune system, fractalkine, and found that without fractalkine, the process of synaptic turnover was impaired.

A couple of tests were performed, first immunohistochemistry (i.e., exceedingly clever manipulation of antibodies to determine the presence or absence of proteins in very specific locations) which demonstrated that microglia were, in fact, ‘engulfing synaptic material’ in animals during periods of synaptic maturation.

Secondly, so called ‘knock out mice’ (i.e., genetically engineered mice constructed without the ability to make a specific protein, in this case, fractalkine) were used evaluate for changes in synaptic form and function based on a lack of fractalkine.  Changes in dendritic spine density were observed in the knock out mice group, with much higher densities in a very specific type of neuron during the second and third postnatal week of life.  The authors indicate this is a key timeframe in synaptic pruning, and state their findings are “suggesting a transient deficient synaptic pruning in Cx3cr1 knockout mice “.  The effect of not having fractalkine on spine density was time dependent as shown below.

Several other measurements were taken, including synaptic firing frequencies, which also implicated an increased surface area for synapses on dendritic spines, consistent with impaired pruning.  Time dependent effects on synaptic efficiency and seizure susceptibility were also found, which the led the authors to conclude that the findings were “consistent with a delay in brain circuit development at the whole animal level.”

For additional evidence of fractalkine participation in synaptic maintenance, we can look to the opposite direction, where researchers evaluating neuron loss in an Alzheimers model reported “Knockout of the microglial chemokine receptor Cx3cr1, which is critical in neuron-microglia communication, prevented neuron loss”.  Taken together, the conclusion that fractalkine processing is involved with neuron maintenance is highly likely, and correspondingly, highly unlikely to be a set of spurious findings.

There’s a couple paragraphs on potential mechanisms by which fractalkine could be interacting with microglia to achieve this effect, with the authors claiming that their data and other data generally supports a model wherein microglia were not effectively recruited to appropriate locations in the brain due to a lack of fractalkine, or, a ‘transient reduction in microglia surveillance.’

The conclusion is a good layman level wrap up that speaks toward the Interconnectedness of the brain and the immune system:

In conclusion, we show that microglia engulf and eliminate synapses during development. In mice lacking Cx3cr1, a chemokine receptor expressed by microglia in the brain, microglia numbers were transiently reduced in the developing brain and synaptic pruning was delayed. Deficient synaptic pruning resulted in an excess of dendritic spines and immature synapses and was associated with a persistence of electrophysiological and pharmacological hallmarks of immature brain circuitry. Genetic variation in Cx3cr1 along with environmental pathogens that impact microglia function may contribute to susceptibility to developmental disorders associated with altered synapse number. Understanding  microglia-mediated synaptic pruning is likely to lead to a better understanding of synaptic homeostasis and an appreciation of interactions between the brain and immune system

That’s all pretty cool, but there was precious little discussion of autism, except in the general sense of a ‘developmental disorder associated with altered synapse number’.   [But the references do speak to autism, the first reference provided, Dendritic Spines in Fragile X Mice displays a significant relationship to autism, and it describes how another flavor of knock out mice, this time designed to mimic Fragile-X, exhibit a ‘developmental delay in the downregulation of spine turnover and in the transition from immature to mature spine subtypes.’  Go figure!]

The other reason Paolicelli is of particular interest to the autism discussion is one of the major players in this study, the microglia (i.e., the resident immune cells of the CNS), have been found to be ‘chronically activated’ in the autism brain by direct  measurement in two studies (here, and here, [and by me, here]), and tons of other studies have shown indirect evidence of an ongoing state of immunological alertness in the autism brain.

Considering this is a brand new paper, I do not believe that there are any studies illuminating the results of a state of chronic activation of microglia on the process of synaptic pruning per se.  I will, however, go on the record that such an effect is very, very likely, and the logical leap is microscopically small that there will be some detrimental impact to such a state.  The inverse argument, a scenario wherein there could be a state of chronic microglial activation that does not interfere with microglia participation in the synaptic pruning requires logical acrobatics worthy of Cirque Du Soleil.  I am open to evidence, however.

So, from Paolicelli, we know that a ‘transient reduction in microglial surveillance’ induced by a reduction in the ability to production fractalkine can result in a condition ‘consistent with a delay in brain circuit development at the whole animal level’.

Next up, we have a paper that was all over the JerkNet in the days and weeks following its release, Neuron number and size in prefrontal cortex of children with autism.  This is a cool study, and likely a very important paper, but I must say that a lot of the online commentary exhibits an irrational exuberance towards one part of the findings.   Here is part of the abstract.

Children with autism had 67% more neurons in the PFC (mean, 1.94 billion; 95% CI, 1.57-2.31) compared with control children (1.16 billion; 95% CI, 0.90-1.42; P = .002), including 79% more in DL-PFC (1.57 billion; 95% CI, 1.20-1.94 in autism cases vs 0.88 billion; 95% CI, 0.66-1.10 in controls; P = .003) and 29% more in M-PFC (0.36 billion; 95% CI, 0.33-0.40 in autism cases vs 0.28 billion; 95% CI, 0.23-0.34 in controls; P = .009). Brain weight in the autistic cases differed from normative mean weight for age by a mean of 17.6% (95% CI, 10.2%-25.0%; P = .001), while brains in controls differed by a mean of 0.2% (95% CI, -8.7% to 9.1%; P = .96). Plots of counts by weight showed autistic children had both greater total prefrontal neuron counts and brain weight for age than control children.  [PFC == prefrontal cortex]

Essentially the authors used a variety of mechanisms to measure neuron number in a specific area of the brain, the prefrontal cortex, and found large variations (increases) in the autism group.   The prefrontal cortex is thought to be involved in ‘planning complex coginitive behaviors’, and ‘moderating correct social behavior’, among others, so this was a smart place to look.

The implicit hype on the internet is that this firmly indicates a ‘prenatal cause’ to autism, but if you read the paper, read what Courchense has said, and read recent literature, you know that the simplicity of this as a singular prenatal cause of autism is long broad strokes, and short on appreciation of the subtlety that textures reality.

A link @ LBRB sent me to the team at The Thinking Person’s Guide To Autism, who had a very nice transcription of a talk given by Courchesne at IMFAR 2011.  Here is a snipet that started my wheels turning.

What we see in autism is either an excess proliferation, producing an overabundance of neuron numbers, or the excess might be due to a reduced ability to undergo naturally occurring cell death. Or it could be both. We don’t know which and our data don’t speak to that, although our data do suggest that it’s probably both.

Finally, our evidence shows that across time, there’s a prolonged period of apoptosis, removal and remodeling of circuits. In order to get back to where neuron numbers are supposed to be, it takes a very long time for the autistic brain. In the normal developing brain, this takes just a few months. In autism, it’s a couple of decades.

[Note how well this fits within the model described by Paolicelli, i.e., “consistent with a delay in brain circuit development at the whole animal level”.  ]

I would highly recommend anyone who has read this far to go read the entire post @ TPGTA sometime.

As far as synaptic pruning goes, here is the associated segment of the paper:

Apoptotic mechanisms during the third trimester and early postnatal life normally remove subplate neurons, which comprise about half the neurons produced in the second trimester. A failure of that key early developmental process could also create a pathological excess of cortical neurons. A failure of subplate apoptosis might additionally indicate abnormal development of the subplate itself. The subplate plays a critical role in the maturation of layer 4 inhibitory functioning as well as in the early stages of thalamocortical and corticocortical connectivity development.inhibitory functioning and defects of functional and structural connectivity are characteristic of autism, but the causes have remained elusive.

Nearly half of the neurons in the area studied are expected to be removed through pruning, a process that extends well after birth.  That is something that you didn’t see referenced in too many places trumpeting this study as ‘proof’ that autism was caused by disturbances in the prenatal environment.  I’m not coming down on the prenatal environment as a critical timeframe for autism pathogensesis, just the difficult to defend underlying notion that this is the only time the environment should be evaluated, or the idea that if something is initiated prenatally other timeframes are therefore, unimportant.

So, I’d read that microglia were actively involved in proper synaptic pruning, contingent on utilization of fractalkine, and then read that impaired synaptic apoptotic mechanisms could be participating in autism, with a consequence of an over abundance of neurons.

Then, I got myself a copy of Microglia and Memory: Modulation by Early-Life Infection, which is another study in a growing body of evidence that immune challenges early in life can have unpredictable physiological consequences.  (This is another very cool paper with Staci Bilbo as an author, whom I think is seriously onto something.)  This study, in particular, focused on interactions microglia and formation of memories.   Here is the abstract:

The proinflammatory cytokine interleukin-1ß (IL-1ß) is critical for normal hippocampus (HP)-dependent cognition, whereas high levels can disrupt memory and are implicated in neurodegeneration. However, the cellular source of IL-1ß during learning has not been shown, and little is known about the risk factors leading to cytokine dysregulation within the HP. We have reported that neonatal bacterial infection in rats leads to marked HP-dependent memory deficits in adulthood. However, deficits are only observed if unmasked by a subsequent immune challenge [lipopolysaccharide (LPS)] around the time of learning. These data implicate a long-term change within the immune system that, upon activation with the “second hit,” LPS, acutely impacts the neural processes underlying memory. Indeed, inhibiting brain IL-1ß before the LPS challenge prevents memory impairment in neonatally infected (NI) rats. We aimed to determine the cellular source of IL-1ß during normal learning and thereby lend insight into the mechanism by which this cytokine is enduringly altered by early-life infection. We show for the first time that CD11b+ enriched cells are the source of IL-1ß during normal HP-dependent learning. CD11b+ cells from NI rats are functionally sensitized within the adult HP and produce exaggerated IL-1ß ex vivo compared with controls. However, an exaggerated IL-1ß response in vivo requires LPS before learning. Moreover, preventing microglial activation during learning prevents memory impairment in NI rats, even following an LPS challenge. Thus, early-life events can significantly modulate normal learning-dependent cytokine activity within the HP, via a specific, enduring impact on brain microglial function.

Briefly, the authors infected rats four days after birth with e-coli, and then challenged them with LPS in adulthood to simulate the immune system to evaluate if memory formation was affected.   As further evidence of an immune mediated effect, prevention of microglial activation in adulthood was sufficient to attenuate the effect.  Clearly the effect on memory formation was based on the immune system.  (Note:  Most of the studies I’ve read would indicate [i.e., educated guess] that a four day old rat is brain developmentally similar to the third trimester of a human fetus.)  While a terrifying and beautiful expression of developmental programming in its own right, there isn’t much to speak towards synaptic pruning in this paper, except maybe, potentially, one part of their findings.

In our study, CX3CL1 did not differ by group, whereas its receptor was decreased basally in NI rats, implicating a change at the level of microglia.

This is where things get either highly coincidental, or connected.  CX3CL1 is another name for fractalkine, i.e., animals that were infected in early life had decreased expression of the receptor for fractalkine compared to placebo animals, i.e., fractalkine is the same chemical messenger found to be integral in the process of synaptic pruning in Synaptic pruning by microglia is necessary for normal brain development!  From a functionality standpoint, having less receptor is very similar to having less fractalkine; as the animals in Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease tell us.

If, if synaptic apoptotic processes are impaired in autism, perhaps this is one mechanism of action. The timeline would involve a prenatal immune challenge, which causes a persistent decrease fractalkine receptor expression, which in turn, causes a consequent impairment in synaptic pruning through interference in microglial targeting.  There is near universal agreement that immune disturbances in utero are capable of altering developmental trajectory undesirably, and here, in an animal model, we have evidence that infections are capable of reducing availability of receptors of ligands known to play a critical role in synaptic pruning, the absence of which leads to conditions which are “consistent with a delay in brain circuit development at the whole animal level”. 

Only time, and more research, will tell if this is a pattern, a phantom, or a little of both.

–          pD


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15 Responses to "Seeing Patterns or Chasing Phantoms, or Is There A Biologically Plausible Developmental Programming Pathway Toward Impaired Synaptic Pruning In Autism?"

Greetings pD

I’ve gone over your post a couple of times and I reckon I need to do so another few times. Some of the latest research findings in autism and other mental health issues are profound, extremely complex and are changing the way we think about the biology.

I certainly agree with “I can’t tell if I’m reading the right research and am onto something, or I am chasing phantoms and my web of pubmed alerts and reading interests are funneling my reference list into a narrowing echo chamber of sorts. ”

I also think that ‘poking around’ should be just that any certitude in autism research is well …. difficult. I therefore draw your attention to this. (Apologies if you are already aware if this but better to make a mistake than never have had the knowledge)

NIMH’s Top 10 Research Advances of 2011

(Things just got even more confusing.)

http://www.nimh.nih.gov/about/director/2011/index-12-2011.shtml

1. Students in “Genetics 101” learn that messenger RNA precisely mirrors the DNA sequence from which it was transcribed.

However, recent studies suggest a far more complex transmission of information. NIMH-funded researchers compared corresponding RNA and DNA sequences in 27 individuals, and found more than 10,000 sequence sites where the RNA and DNA of the same individual did not match

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2. Another study presents what may be the most extraordinary discovery of 2011: somatic ‘retrotransposition’ can alter brain tissue (2).

Retrotransposons are mobile genetic elements that can copy and insert themselves within a genome causing mutations in dividing cells. Although these insertions rarely lead to harmful effects when they occur in germ line cells (sperm and egg), they are frequently harmful if they occur in somatic cells, such as neurons.

While nearly all studies of the genetics of mental illness have focused on germ line DNA, this new discovery suggests that DNA variation occurring in the developing brain could contribute to mental illness, just as mutations in mature tissues contribute to cancer. These surprising findings suggest a whole new frontier for the biology of mental illness.
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3. Transcriptome: Developing Brains Have Unique Molecular Signatures.

Messenger RNAs, or transcripts, are intermediate products that carry the message from DNA, the genetic blueprint, to create proteins, and ultimately, the many different cell types throughout the brain. Each gene can make several transcripts, which are expressed in patterns unique to each of us.

To better understand how these patterns of gene expression influence the developing brain, NIMH supported the first map of how RNA expression changes across the life span through two parallel studies of postmortem brains, ranging in age from two weeks after conception to 80 years old (3, 4).

The researchers found that nearly 90% of genes are expressed differently during prenatal development, infancy, and childhood. While each of these stages has a distinct transcriptional identity, the fetal brain looks like a different organ compared to the postnatal brain, with 60% of genes expressed differently and 83% of transcripts processed to make unique proteins.

Many of the genetic variations associated with mental illness appear to have a specific effect on the form of the gene expressed uniquely during fetal life.

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De Novo Genetic Variants.

This year scientists looking at families with only one case of autism found that up to eight percent of cases in these families were the result of de novo (unique to the person affected) copy-number variants—stretches of DNA that were either multiplied or truncated (9, 10).

Analysis of the gene regions affected by these variants implicated a network of genes involved in the development of synapses and neuronal function (11).

Another study, focusing specifically on sequences of DNA that code for protein, yielded other de novo genetic changes in one-case families (12).

While providing information on genetic contributors to a significant fraction of sporadic autism cases, the work also reveals gene regions for future investigation and ultimately, information on functional changes underlying autism that will offer clues to therapy.

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…and I saved the final one till last because you are a fan of the white fluffy mouse.

Epigenomics: How Experience Alters Behavior.

In any one individual, patterns of gene expression vary widely among cells, leading to a diversity of cell types and functions, even though the cells all have the same DNA sequence.

Epigenetic processes—heritable changes in gene expression that are not related to DNA sequence—help explain this diversity. Research suggests that epigenetics may also be a sort of programming language through which experience can have lasting effects on behavior, not only in an individual over a lifetime, but across generations.

This effect was demonstrated in a 2011 study of male mice exposed to social defeat—repeated bullying by another aggressive male (13). The bullied males developed behavior resembling depression, and in subtle ways, so did their offspring. This was true even though contact between mother and bullied father was brief and took place well before the birth of the young, suggesting that epigenetic mechanisms played a role.

Understanding the nature of epigenetic changes opens possibilities for therapy; scientists also showed this year that they could reverse the silencing of a gene involved in a rare neurodevelopmental disorder, a proof of concept for interventions targeting epigenetic processes (14).

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Science rolls inexorably forward in 2012 … until the Mayan calendar runs out.

Hi Balckheart –

Thanks for the links. Very interesting. Now if only I had the time to try to get a grasp on some of that stuff!

– pD

I’ve just found your blog and wanted you to know how much I appreciate the work you’re doing. I look forward to reading more.

Hi Carly –

Thank you for stopping by my blog and the kind words.

– pD

Hi pD

“What we see in autism is either an excess proliferation, producing an overabundance of neuron numbers, or the excess might be due to a reduced ability to undergo naturally occurring cell death. Or it could be both. We don’t know which and our data don’t speak to that, although our data do suggest that it’s probably both.

Finally, our evidence shows that across time, there’s a prolonged period of apoptosis, removal and remodeling of circuits. In order to get back to where neuron numbers are supposed to be, it takes a very long time for the autistic brain. In the normal developing brain, this takes just a few months. In autism, it’s a couple of decades.”

This seems to directly relate to the gene profiling studies undertaken by various researchers – most particularly reference 3. (Google for full text version does not link well) Expression Profiling which indicated one of the most ‘expressed genes in autism found in their profiling was –
Expressi­on Profiling of Autism Candidate Genes during Human Brain Developmen­t Implicates Central Immune Signaling Pathways

Ziats and Renart from the National Institutes of Health

*Identifie­d a subset of highly expressed ASD-candid­ate genes from which interactom­e networks were derived.

*Strikingl­y, immune signaling through NFκB, Tnf, and Jnk was central to ASD networks at multiple levels of our analysis

*Cell-type specific expression suggested glia—in addition to neurons—de­serve considerat­ion. This work provides integrated genomic evidence that ASD-implic­ated genes may converge on central cytokine signaling pathways.

* Most prominent transcript­ome changes are related to neuro-immu­ne disturbanc­es. In the Garbett et al study, the most significan­t functional pathway implicated was NFκB

The highly expresses genes identified were –

Table 3. GO enrichment analysis of highly expressed Autism genes.

GO:0002682 Regulation of Immune System Process
GO:0006915 Apoptosis
GO:0012501 Programmed Cell Death
GO:0031347 Regulation of Defense response

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NFkB has been shown to be associated with a range of ‘features’ associated with ASD.

Neuro-infl­ammation

http://www­.neuro.jhm­i.edu/neur­oimmunopat­h/autism_f­aqs.htm

Glial and neuronal cell function

http://www.ncbi.n­lm.nih.gov­/pubmed/91­85306

Inflammato­ry Bowel Disease

http://www­.ncbi.nlm.­nih.gov/pu­bmed/17023­960

NF-κB and Eczema

http://www­.ncbi.nlm.­nih.gov/pu­bmed/17346­428

http://www­.jci.org/a­rticles/vi­ew/21060

Immune Diseases – Rheumattoi­d Arthritis

http://www­.jrheum.or­g/content/­34/10/1976­.abstract

Cortex Structural Plasticity

http://www­.jneurosci­.org/conte­nt/31/32/1­1697.short

Neuronal Migration

http://www­.sciencedi­rect.com/s­cience/art­icle/pii/S­0898656810­003049

Purkinje Cell developmen­t

http://www­.sciencedi­rect.com/s­cience/art­icle/pii/S­0896627310­006239

http://onl­inelibrary­.wiley.com­/doi/10.10­02/jnr.220­56/full

Reptotoive Behaviour

Repetitive behavior and increased activity in mice with Purkinje cell loss.

http://onl­inelibrary­.wiley.com­/doi/10.11­11/j.1460-­9568.2009.­07073.x/fu­ll

Sleep Disturbanc­es

http://www.ncbi.n­lm.nih.gov­/pubmed/17­013605

http://www­.ncbi.nlm.­nih.gov/pu­bmed/21478­225

Rett Syndrome

http://www­.sciencedi­rect.com/s­cience/art­icle/pii/S­0888754310­001990

Anti-depre­ssants and Autism

http://www.ncbi.n­lm.nih.gov­/pubmed/11­122343

Valproic Acid

http://www.ncbi.n­lm.nih.gov­/pubmed/21­722408”

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1. Aberrant NF-KappaB Expression in AutismSpec­trumCondit­ion: AMechanism for Neuroinfla­mmation 2011

http://www­.ncbi.nlm.­nih.gov/pm­c/articles­/PMC309871­3/

Researcher­s from University of St Andrew’s and University of Cambridge reported on measuremen­ts of NF-κB in human post-morte­m samples of orbitofron­tal cortex tissue.

They found

*”NF-κB is aberrantly expressed in the orbitofron­tal cortex as indicated by measuremen­ts on post-morte­m tissue from ASC patients, and particular­ly in highly activated microglia.

* This region is a locus of abnormal function in ASC that underlies the abnormal developmen­t of social and cognitive skills”

2. NF-kB: a crucial transcript­ion factor for glial and neuronal cell function

1997 early research.

http://www­.cell.com/­trends/neu­rosciences­/abstract/­S0166-2236%2896%2901­035-1

3. Expression Profiling of Autism Candidate Genes during Human Brain Developmen­t Implicates Central Immune Signaling Pathways

http://www­.plosone.o­rg/article­/info%3Adoi%2F1­0.1371%2Fj­ournal.pon­e.0024691

4. NF-κB and Autism A Study of Nuclear Transcript­ion Factor-Kap­pa B in Childhood Autism

http://www­.plosone.o­rg/article­/info%3Adoi%2F1­0.1371%2Fj­ournal.pon­e.0019488

Indian researcher­s reported the following in their study …

* “Elevated amounts of NF-κB in children with autism can strengthen the conceptual frameworks of the role of innate immunity and ROS in the etiopathol­­ogy of this condition.

* Children with autism could be in a “hyper arousal” state of NF-κB due to the constant effect of environmen­­tal stressors – even fear is known to upregulate NF-κB . Children with autism may have an altered threshold to fearful stimuli.

* identifyin­­g agents that increase NF-κB in children and regulating these triggers, would go a long way in preventing a certain sub sect of regressive autism.”

More interesting Research on NFkB

Gene finding could yield improved smallpox vaccine

Scientists studying vaccinia virus, a close relative of smallpox, have discovered that a gene necessary for virus replication also plays a key role in turning off inflammation, a crucial antiviral immune response of host cells.

http://www.outsourcing-pharma.com/Preclinical-Research/Gene-finding-could-yield-improved-smallpox-vaccine

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Evaluation of Plant Polysaccharides and Phyto-Compounds on Inflammation-Associated Tumorigenesis by Using Melanoma DNA Vaccine Model

Adjuvant can be used to enhance the immunogenicity of antigen and improve the efficacy of vaccine. Potent adjuvant action is often correlated with NF-kB activation, as exemplified by the case of MPL or LPS. However, NF-kB activation is thought to play an important role in the development and/or maintenance of many types of cancer….

These results suggest that balance the expression level of NF-kB is important in tumorigenesis and vaccination.

http://en.wikipedia.org/wiki/Lipopolysaccharide

*Lipopolysaccharides (LPS), also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals.

*Bordetella pertussis is a Gram-negative bacteria

* Immune response (See pic)

LPS function has been under experimental research for several years due to its role in activating many transcription factors. LPS challenge also produces many types of mediators involved in septic shock. Humans are much more sensitive to LPS than other animals (e.g., mice). A dose of 1 µg/kg induces shock in humans, but mice will tolerate a dose up to a thousand times higher.

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http://mcb.illinois.edu/faculty/profile/1199

Joanna L. Shisler
Associate Professor of Microbiology

Research Topics

The conserved eukaryotic transcription factor nuclear factor-κB (NF-κB) stimulates the expression of proteins that are critical for immune functions.

If activated during a virus infection, the NF-κB response can limit viral pathogenesis and resolve the infection. Consequently, many successful viruses employ strategies to inhibit this response.

***Our investigation of NF-κB activity during poxvirus infection has revealed a novel layer of complexity for this virus/host cell interaction. In our published studies, we demonstrated that the NF-κB response is inhibited when cells are infected with wild-type vaccinia virus, but that it is strongly activated through at least two routes by a highly attenuated vaccinia virus.

We recently identified the vaccinia K1L and M2L gene products as NF-κB inhibitory proteins. We assume that poxviruses express these products to evade immune responses, and survive in the host for longer periods of time. Currently, we are delineating the molecular function of the K1L and M2L viral proteins. It is equally interesting to determine how attenuated poxviruses activate NF-κB. We have found that NF-κB activation depends upon early protein synthesis, and we are currently identifying the proteins responsible for this phenotype.

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NFκB is differentially activated in macrophages from
J774A.1 cell line infected with vaccine or virulent
strains of Brucella abortus

http://www.academicjournals.org/ajmr/…/Cervantes-Flores%20et%20al.pdf

* Brucella is a genus of Gram-negative bacteria. (See LPS / Pertussis)

One of the major functions of NF-kB is its key involvement in
inducing an effective immune/inflammatory response against viral and bacterial infections. The importance of NF-kB role in initiating a potent inflammatory response 942 Afr. J. Microbiol. Res. cannot be better signified than recognizing that the kB consensus sequence is found in the promoter/enhancer regions of more than 50 diverse genes whose expression is known to be crucial in driving an inflammatory
response (Majdalawieh and Hyo-Sung, 2010). Inducible genes that are known to be transactivated by NF-kB include, but are not limited to, IL-1β, IL-6, IL-8, TNFα, IFNγ, MCP-1, iNOS, COX-2, intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion
molecule-1 (VCAM-1) (Baeuerle and Henkel, 1994; Baldwin, 1996; Kopp and Ghosh, 1995).

In this report we studied the cytokine gene expression and demonstrated that infection by the virulent Brucella abortus 2308 strain releases an inactive transcriptional homodimer of NF-kB and did not induce activation of pro-inflammatory cytokine genes; neither did
it induce expression of IFN-g, TNF-k nor of the iNOS enzyme. In contrast, B. abortus RB51 vaccine strain releases an active transcriptional heterodimer of NF-kB and stimulates the pro-inflammatory response by the activation of IL-12 and IFN-g as well as iNOS genes

—————————————-

Imagine an ASD mouse model study combined with NFkB expression assessment.

—————————————-

Characterization of NF-kappa(k)B activity and the effects of its inhibition by NEMO binding domain peptide in spontaneously occurring canine cancer

http://research.vet.upenn.edu/TranslationalResearch/TargetingNFkappakBactivityincaninecancer/tabid/2230/Default.aspx

NF-kappa(k)B is a highly evolutionary conserved family of transcription factors that play important roles in the regulation of genes involved in immune responses, inflammation, stress responses, cellular proliferation, differentiation and apoptosis.

However, the role of NF-kappa(k)B in oncogenesis has only been recognized recently. NF-kappa(k)B activation is highly regulated and in the inactive state, NF-kappa(k)B proteins are sequestered in the cytoplasm through their association with the inhibitory IkB and p100 proteins. Impaired regulation of NF-kappa(k)B activation can lead to loss of its highly controlled inducibility and constitutive activity.

This results in aberrant expression of anti-apoptotic and pro-survival genes and genes that are involved in cell cycle control and migration, processes that occur in the initiation and progression of cancer.

Indeed, NF-kappa(k)B is constitutively active in many solid tumors, where it contributes to malignant cell proliferation, growth and survival. NF-kappa(k)B also regulates the expression of a number of genes that are important in the process of tumor metastases and constitutive activation of NF-kappa(k)B has been reported in highly metastatic tumor cell lines.

Finally, recent studies have indicated that constitutive NF-kappa(k)B activation in malignant cells is responsible for their observed chemoresistance.

——————————————-

Is there a relationship between cancer and ASD ?

——————————————-

How stress influences the immune response.

http://www.direct-ms.org/sites/default/files/Stress%20and%20immunity.pdf

In response to a stressor, physiological changes are set into motion to help an individual cope with the stressor. However, chronic activation of these stress responses, which include the hypothalamic–pituitary–adrenal axis and the sympathetic–adrenal–medullary axis, results in
chronic production of glucocorticoid hormones and catecholamines.
Glucocorticoid receptors expressed on a variety of immune cells bind cortisol and interfere with the function of NF-kB, which regulates the activity of cytokine-producing immune cells. Adrenergic receptors
bind epinephrine and norepinephrine and activate the cAMP response element binding protein, inducing the transcription of genes encoding for a variety of cytokines. The changes in gene expression mediated by
glucocorticoid hormones and catecholamines can dysregulate immune function.

There is now good evidence (in animal and human studies) that the magnitude of stress-associated immune dysregulation is large
enough to have health implications.

————————————-
Active NF-kB signalling is a prerequisite for influenza virus infection

vir.sgmjournals.org/content/85/8/2347.full.pdf

Influenza virus still poses a major threat to human health. Despite widespread vaccination programmes and the development of drugs targeting essential viral proteins, the extremely high mutation rate of influenza virus still leads to the emergence of new pathogenic virus strains. Therefore, it has been suggested that cellular cofactors that are essential for influenza virus infection might be better targets for antiviral therapy. It has previously been reported that influenza virus efficiently infects Epstein–Barr virus-immortalized B cells, whereas Burkitt’s lymphoma cells are virtually resistant to infection. Using this cellular system, it has been shown here that an active NF-kB signalling pathway is a general prerequisite for influenza virus infection
of human cells. Cells with low NF-kB activity were resistant to influenza virus infection, but became susceptible upon activation of NF-kB. In addition, blocking of NF-kB activation severely
impaired influenza virus infection of otherwise highly susceptible cells, including the human lung carcinoma cell lines A549 and U1752 and primary human cells. On the other hand, infection
with vaccinia virus was not dependent on an active NF-kB signalling pathway, demonstrating the specificity of this pathway for influenza virus infection. These results might be of major importance for both the development of new antiviral therapies and the understanding
of influenza virus biology.

——————————————-

Characteristics of mycobacterial infection in patients with immunodeficiency and nuclear factorekB essential modulator mutation, with or without ectodermal dysplasia

http://www.bragid.org.br/download/micobacteriuminfection.pdf

Hypomorphic mutations of the nuclear factor kB essential modulator gene cause ectodermal dysplasia and immunodeficiency. Affected patients have increased susceptibility to mycobacterial disease including cutaneous manifestations. We describe clinical and histopathologic characteristics of 5 patients with nuclear
factor kB essential modulator gene mutations and mycobacterial infections, two of whom had mycobacterial cutaneous infections. ( J Am Acad Dermatol 2004;51:718-22.)

——————————————-

Hostile takeovers: viral appropriation of the NF-kB pathway

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC199181/

Transcriptional regulators of the NF-kB/IkB family promote the expression of well over 100 target genes, the majority of which participate in the host immune response (1). These proteins include a multitude of cytokines and chemokines, receptors required for immune recognition, proteins involved in antigen presentation, and adhesion receptors involved in transmigration across blood vessels walls. Because of this extensive role in immune action, NF-kB has been termed the central mediator of the immune response. Gene knockout and other studies establish roles for NF-kB in the ontogeny of the immune system but also demonstrate that NF-kB participates at multiple steps during oncogenesis (2) and the regulation of programmed cell death (3).
For several reasons, the NF-kB pathway provides an attractive target to viral pathogens. Activation of NF-kB is a rapid, immediate early (IE) event that occurs within minutes after exposure to a relevant inducer, does not require de novo protein synthesis, and results in a strong transcriptional stimulation of several early viral as well as cellular genes. In this review, we will describe strategies that viruses have evolved to modulate the NF-kB pathway, to enhance viral replication, host cell survival, and evasion of immune responses. Activation of NF-kB constitutes an obvious target because many of its target genes — growth factors, cytokines and their receptors, and proto-oncogenes — profoundly influence the host cell cycle. In addition, some viruses exploit the antiapoptotic properties of NF-kB to evade the host defense mechanisms that limit replication by killing infected cells, or conversely to trigger apoptosis as a mechanism to increase virus spread.

“What does this mean to a theory of Autism pathology ?

There are two prevailing theories – From Riat et al suggest a third

* “one suggests exogenous (external) factor(s) stimulate neuro-infl­ammation during development

* autoimmune activation causes ASD pathology.

* “mutations described in ASD result in aberrant signaling regulation of immune cells during neurodevel­opment. This could result in cell-auton­omous (independe­nt) activation and/or improper response to otherwise nominal stimuli, such as occurs in the autoinflam­matory syndromes

Understanding Autoinflammatory Diseases

http://www­.niams.nih­.gov/healt­h_info/aut­oinflammat­ory/defaul­t.asp

—————————————————————————–

What triggers NFkB ?

It seems quite a few things but in the case of autism it is noted above an associatio­n with anti-depre­ssants , valproic acid.

Also of note is stress.

http://www­.springerl­ink.com/co­ntent/7y9u­5kkvjxybyy­49/

…and of particular note virus and bacterial infection. Including the following …

Measles Virus

http://www­.sciencedi­rect.com/s­cience/art­icle/pii/S­0042682201­911742

Measles is a highly contagious viral disease that remains the leading vaccine-preventable cause of child mortality worldwide. Deaths from measles are due largely to an increased susceptibility to secondary bacterial and viral infections, attributed to a prolonged state of immune suppression. Several abnormalities of the immune system have been described, including changes in lymphocyte number and function, shifts in cytokine responses, immunomodulatory effects of interleukin-10, down regulation of interleukin-12, impaired antigen presentation, and altered interferon α/β signaling pathways.

Although the current vaccine is very effective, knowledge of the molecular basis of the immune responses to measles virus could contribute to the development of a safer, more immunogenic measles vaccine.

However, the safety of new measles vaccines must be carefully investigated, as two measles vaccines have resulted in unintended immunologic consequences: atypical measles following administration of the formalin-inactivated measles vaccine and increased mortality in girls following administration of high-titer measles vaccines.

http://www­.ncbi.nlm.­nih.gov/pm­c/articles­/PMC229211­0/

* The genetic mechanisms underlying measles virus modulation of host immunity in response to vaccination are poorly defined and are under active research.

* TLR3 and measles vaccine immunity are particularly intriguing as TLR3 has been previously identified as a prime target for laboratory adapted, but not wild-type measles virus strains in the generation of host immunity [9]

* Tanabe et al [9] reported that laboratory adapted and vaccine strains of measles virus, including Edmonston, up-regulate the expression of TLR3 in human dendritic cells via enhanced IFN-β secretion. The 500bp region upstream of exon 1 is characterized as a measles virus-responsive segment in the TLR3 gene. This region contains the NF-κB and STAT (family of eukaryotic transcription factors that mediate the response to a large number of cytokines and growth factors) binding sites

* Overall, consistent with the limited literature on the role of TLRs in measles vaccine-induced immunity, we found specific SNPs in the coding and regulatory regions of TLRs, specifically TLR3, that were significantly associated with variations in antibody and cellular immune responses to measles vaccination.

* These findings are consistent with the fact that multiple genetic variations in candidate immune response genes contribute to the complex architecture of the immune response, and are important in understanding the immunogenetics of vaccine response. We assessed associations between a large number of SNPs and several different measures of immune response and, therefore, multiple testing issues are a legitimate concern.

* However, we found a higher number of significant associations than we would expect by chance alone (29 vs. 20 significant results assuming independent hypothesis tests and a type I error rate of 0.03) indicating a possible genetic component to measles immune response.

http://jvi­.asm.org/c­ontent/85/­7/3162.ful­l

* Here we show that the immunosuppressive measles virus (MV; Morbillivirus genus, Paramyxoviridae) has evolved multiple functions to interfere with canonical NF-κB signaling in epithelial cells.

* MV V appears to prevent NF-κB-dependent gene expression by retaining p65 in the cytoplasm. These findings reveal NF-κB as a key target of MV and stress the importance of the V protein as the major viral immune-modulatory factor.

http://jgv­.sgmjourna­ls.org/con­tent/83/5/­1157.short

* The influence of measles virus (MV) infection on gene expression by human peripheral blood mononuclear cells (PBMCs) was examined with cDNA microarrays.

* In the present study, a total of 17 genes was found to be upregulated by MV infection. The Edmonston strain grew better in the PBMC cultures than the wild-type MV, and the Edmonston strain was a stronger inducer of the upregulated host cell genes than the wild-type virus.

* The anti-apoptotic B cell lymphoma 3 (Bcl-3) protein and the transcription factor NF-κB p52 subunit were upregulated in infected PBMCs both at the mRNA and at the protein level.

* Several genes of the interferon system including that for interferon regulatory factor 7 were upregulated by MV. The genes for a number of chaperones, transcription factors and other proteins of the endoplasmic reticulum stress response were also upregulated.

* These included the gene for the pro-apoptotic and growth arrest-inducing CHOP/GADD153 protein. Thus, the present study demonstrated the activation by MV of cellular mechanisms and pathways that may play a role in the pathogenesis of measles.

Bordetella pertussis

http://www­.plosone.o­rg/article­/info:doi%2F10.­1371%2Fjou­rnal.pone.­0003825

block NF-kappaB activation­, and perhaps lead to a compromise­d immune response to this bacterial pathogen.

http://www­.ncbi.nlm.­nih.gov/pu­bmed/11254­631

Rubella

http://www.ncbi.n­lm.nih.gov­/pubmed/10­364491

Hepatitis Virus

http://www­.ncbi.nlm.­nih.gov/pm­c/articles­/PMC193325­9/”

Hi Blackheart –

Tragically, I don’t see myself having time to go through too many of these with the detail that I would like to. There is some interesting stuff in there, however.

Do you have a blog where you try to coalesce this type of material. It’s not an easy task, there is so much and it is difficult to know what to focus on with so much data.

If I get a chance, I’ll note some thing I find of particular interest, but I’m working on another post and the (stupid) real world is really kicking me in the nuts these days.

– pD

Sorry pD I’ll get myself organised … I suppose the frustration of having all this information is if you took it to say a ‘skeptik’ website …well.

“What does this mean to a theory of Autism pathology ?

There are two prevailing theories –

* “one suggests exogenous (external) factor(s) stimulate neuro-infl­ammation during development

* autoimmune activation causes ASD pathology.

* “mutations described in ASD result in aberrant signaling regulation of immune cells during neurodevel­opment. This could result in cell-auton­omous (independe­nt) activation and/or improper response to otherwise nominal stimuli, such as occurs in the autoinflam­matory syndromes

http://www­.niams.nih­.gov/healt­h_info/aut­oinflammat­ory/defaul­t.asp

What triggers NFkB ?

It seems quite a few things but in the case of autism it is noted above an associatio­n with anti-depre­ssants , valproic acid.

Also of note is stress (Which I think most parents here will note)

http://www­.springerl­ink.com/co­ntent/7y9u­5kkvjxybyy­49/

…and of particular note virus and bacterial infection. Including the following …

Measles Virus

http://www­.sciencedi­rect.com/s­cience/art­icle/pii/S­0042682201­911742

Measles is a highly contagious viral disease that remains the leading vaccine-preventable cause of child mortality worldwide. Deaths from measles are due largely to an increased susceptibility to secondary bacterial and viral infections, attributed to a prolonged state of immune suppression. Several abnormalities of the immune system have been described, including changes in lymphocyte number and function, shifts in cytokine responses, immunomodulatory effects of interleukin-10, down regulation of interleukin-12, impaired antigen presentation, and altered interferon α/β signaling pathways. Although the current vaccine is very effective, knowledge of the molecular basis of the immune responses to measles virus could contribute to the development of a safer, more immunogenic measles vaccine. However, the safety of new measles vaccines must be carefully investigated, as two measles vaccines have resulted in unintended immunologic consequences: atypical measles following administration of the formalin-inactivated measles vaccine and increased mortality in girls following administration of high-titer measles vaccines.

http://www­.ncbi.nlm.­nih.gov/pm­c/articles­/PMC229211­0/

* The genetic mechanisms underlying measles virus modulation of host immunity in response to vaccination are poorly defined and are under active research.

* TLR3 and measles vaccine immunity are particularly intriguing as TLR3 has been previously identified as a prime target for laboratory adapted, but not wild-type measles virus strains in the generation of host immunity [9]

* Tanabe et al [9] reported that laboratory adapted and vaccine strains of measles virus, including Edmonston, up-regulate the expression of TLR3 in human dendritic cells via enhanced IFN-β secretion. The 500bp region upstream of exon 1 is characterized as a measles virus-responsive segment in the TLR3 gene. This region contains the NF-κB and STAT (family of eukaryotic transcription factors that mediate the response to a large number of cytokines and growth factors) binding sites

* Overall, consistent with the limited literature on the role of TLRs in measles vaccine-induced immunity, we found specific SNPs in the coding and regulatory regions of TLRs, specifically TLR3, that were significantly associated with variations in antibody and cellular immune responses to measles vaccination.

These findings are consistent with the fact that multiple genetic variations in candidate immune response genes contribute to the complex architecture of the immune response, and are important in understanding the immunogenetics of vaccine response. We assessed associations between a large number of SNPs and several different measures of immune response and, therefore, multiple testing issues are a legitimate concern.

However, we found a higher number of significant associations than we would expect by chance alone (29 vs. 20 significant results assuming independent hypothesis tests and a type I error rate of 0.03) indicating a possible genetic component to measles immune response.

http://jvi­.asm.org/c­ontent/85/­7/3162.ful­l

Here we show that the immunosuppressive measles virus (MV; Morbillivirus genus, Paramyxoviridae) has evolved multiple functions to interfere with canonical NF-κB signaling in epithelial cells.

MV V appears to prevent NF-κB-dependent gene expression by retaining p65 in the cytoplasm. These findings reveal NF-κB as a key target of MV and stress the importance of the V protein as the major viral immune-modulatory factor.

http://jgv­.sgmjourna­ls.org/con­tent/83/5/­1157.short

The influence of measles virus (MV) infection on gene expression by human peripheral blood mononuclear cells (PBMCs) was examined with cDNA microarrays.

In the present study, a total of 17 genes was found to be upregulated by MV infection. The Edmonston strain grew better in the PBMC cultures than the wild-type MV, and the Edmonston strain was a stronger inducer of the upregulated host cell genes than the wild-type virus.

The anti-apoptotic B cell lymphoma 3 (Bcl-3) protein and the transcription factor NF-κB p52 subunit were upregulated in infected PBMCs both at the mRNA and at the protein level.

Several genes of the interferon system including that for interferon regulatory factor 7 were upregulated by MV. The genes for a number of chaperones, transcription factors and other proteins of the endoplasmic reticulum stress response were also upregulated.

These included the gene for the pro-apoptotic and growth arrest-inducing CHOP/GADD153 protein. Thus, the present study demonstrated the activation by MV of cellular mechanisms and pathways that may play a role in the pathogenesis of measles.

Bordetella pertussis

http://www­.plosone.o­rg/article­/info:doi%2F10.­1371%2Fjou­rnal.pone.­0003825

block NF-kappaB activation­, and perhaps lead to a compromise­d immune response to this bacterial pathogen.

http://www­.ncbi.nlm.­nih.gov/pu­bmed/11254­631

Rubella

http://www.ncbi.n­lm.nih.gov­/pubmed/10­364491

Hepatitis Virus

http://www­.ncbi.nlm.­nih.gov/pm­c/articles­/PMC193325­9/”

More fluffy white (rats) …

Translational Investigation

Pediatric Research (2012); 71 1, 46–53. doi:10.1038/pr.2011.11
Effects of Bifidobacterium breve on inflammatory gene expression in neonatal and weaning rat intestine

Yoshikazu Ohtsuka1, Takako Ikegami2, Hirohisa Izumi3, Mariko Namura3, Tomomi Ikeda2, Tamaki Ikuse1, Yosuke Baba1, Takahiro Kudo1, Ryuyo Suzuki1 and Toshiaki Shimizu1

Department of Pediatrics and Adolescence Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan

To examine the immune-modulatory effects of probiotics during early infancy, Bifidobacterium breve M-16V (B. breve) was administered to rat pups during the newborn or weaning period, and the expression of inflammatory genes was investigated using a cDNA microarray and real-time PCR.
Results:

After B. breve administration, significant increases in the numbers of Bifidobacterium in both the cecum and colon were confirmed during the newborn period. The numbers of upregulated and downregulated genes were greater during the weaning period than in the newborn period and were greatest in the colon, with fewer genes altered in the small intestine and the fewest in the spleen. The expression of inflammation-related genes, including lipoprotein lipase (Lpl), glutathione peroxidase 2 (Gpx2), and lipopolysaccharide-binding protein (Lbp), was significantly reduced in the colon during the newborn period. In weaning rat pups, the expression of CD3d, a cell surface receptor–linked signaling molecule, was significantly enhanced in the colon; however, the expression of co-stimulatory molecules was not enhanced.
Discussion:

Our findings support a possible role for B. breve in mediating anti-inflammatory and antiallergic reactions by modulating the expression of inflammatory molecules during the newborn period and by regulating the expression of co-stimulatory molecules during the weaning period.

There is historical precedent for early prenatal environmental exposure to some type of teratogenic insult, particularly viral. Pregnant women with German Measles were 200x more likely to have autistic offspring if they harbored the virus in critical periods.

http://raggette.blogspot.com/2011/12/congenital-rubella-syndrome.html

I don’t know whether I am seeing patterns or chasing phantoms, but you know what they say about history…

[…] Synaptic pruning by microglia is necessary for normal brain development, (discussed on this blog, here), which provided evidence of microglial involvement in the ‘pruning’ of synapses, an important […]

[…] Oh hell yeah.  (* concepts and papers discussed on this blog, here) […]

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