Does rubella (german measles) cause autism?

Rubella rash @ NHS Choices

"Rubella might still cause autism, even in vaccinated populations."

That was one of the points raised in the '2015 reappraisal' document published by Jill Hutton [1] (open-access) covering a topic that has quite a long history with autism in mind (see here).

Rubella, also called german measles, a previously common childhood disease characterised by a rash, high temperature and cold-like symptoms, has in many parts of the world almost been entirely eradicated as a consequence of vaccination and other health measures. Here in Blighty (UK), our public health agency has even recently announced that it will soon halt screening for rubella in pregnant women as a result of the decline of the disease (see here). That screening was carried out following some potentially pretty serious complications to the unborn child known to be associated with maternal contraction of the disease. Sounds familiar doesn't it?

Anyhow, Hutton takes readers through the main points of the research linking rubella or rather congenital rubella syndrome (CRS) and autism down the years. Stella Chess, the first person to talk about rubella (CRS) and autism [2] (at least in the peer-reviewed domain) gets a pretty big shout-out throughout the article, alongside the overlap between CRS and autism from various different perspectives (behavioural, physiological, genetic). Drawing also on literature around the topic of vaccination and autism (see here), discussions also turn to the quite important group of children with autism who do not seem to display the appropriate immunological response to rubella infection/vaccination as per that discussed by Libbey and colleagues [3] for example. The idea being that antibody titers normally showing whether a vaccination has 'worked' in terms of conferring protection against a disease, were lower or none existent in some children on the autism spectrum and potentially indicates some type of immune dysfunction. Similar things have been talked about in other research papers (see here).

Hutton also discusses an important feature of the literature intersecting with migration and autism (see here) and how some: "Foreign born mothers (from the developing world, without vaccinations) are less likely to be immune or again more likely to be susceptible to rubella" and what potential effect this might have on cases of CRS and autism. During the times I've discussed the topic of autism and migration, I've tended to labour the point about how different foods, different exposures and in particular, different levels of vitamin D *might* be part and parcel of the effect noted in cases (see here). The Hutton discussions have been food for thought for me particularly in light of other findings [4].

"The thought that vaccination has wiped out rubella is falsely reassuring and has managed to wipe out most rubella research, but unfortunately rubella lingers." I was also interested in this statement and how, on the back of those previous findings about how a certain kind of immune system might react, or rather not react, to vaccination (assuming complete vaccine coverage), there may be quite a bit more scope for further research with at least some autism in mind. I know this has the potential to take us back into some quite heated discussions but if we are learning anything about the plural autisms (see here) it is that exposure to viral infection in particular, seems to have some important links to at least some types of autism (see here and see here).

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[1] Hutton J. Does Rubella Cause Autism: A 2015 Reappraisal? Front. Hum. Neurosci. 2016. 1 Feb.

[2] Chess S. Autism in children with congenital rubella. J Autism Child Schizophr. 1971 Jan-Mar;1(1):33-47.

[3] Libbey JE. et al. Are there altered antibody responses to measles, mumps, or rubella viruses in autism? J Neurovirol. 2007 Jun;13(3):252-9.

[4] Bahta L. & Ashkir A. Addressing MMR Vaccine Resistance in Minnesota's Somali Community. Minn Med. 2015 Oct;98(10):33-6.

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Hutton, J. (2016). Does Rubella Cause Autism: A 2015 Reappraisal? Frontiers in Human Neuroscience, 10 DOI: 10.3389/fnhum.2016.00025

Herpes simplex virus (HSV) infection and cognitive ability

So: "our findings indicate that infection with HSV-1 [Herpes simplex virus] is associated with reduced cognitive functioning in healthy individuals."

That was the conclusion reached in the study by Eyal Fruchter and colleagues [1] who based on a "representative, random sample of 612 soldiers before active duty in the Israeli military (Israeli defense force — IDF)" looked at cognitive functioning and language abilities as a function of HSV status ("62.2% HSV positive (n = 381) and 38.8% HSV negative (n = 231)").

They observed some key differences between the HSV positive and negative groups whereby IQ scores were on average lower for the seropositive group compared with the seronegative group. This difference also stretched to language scores too and stayed pertinent as and when various psychopathology (anxiety, depression) were controlled for. The authors plant their findings within the context of schizophrenia and that their results indicate "that many research findings seemingly characteristic of schizophrenia are related to the association between HSV exposure and cognitive functioning in general, and are not illness specific."

This is an interesting branch of research. I was unaware that HSV-1 seropositivity was potentially correlated to cognitive functions but a quick trawl through some of the other literature in this area does indeed reveal that this is not the first time that such an association has been made [2] (with appropriate caveats). Even some big names in schizophrenia research (Faith Dickerson & Robert Yolken) have discussed HSV-1 and cognitive functions, within the context of schizophrenia [3].

There are some potentially pretty stark implications from such findings. That there may be a 'link' between cognitive and intellectual functions and infectious agents is a primary one (see here). That processes such as inflammation (and immune functions) might also play a role is another (see here). Whilst further research is required to put some scientific flesh on the bones of the potential biological / genetic processes involved in such a relationship, the tantalising question is whether 'treating' such viral infections (if treatment is available) might have a knock-on effect for intellectual functions?

Music: Mumford & Sons - Ditmas.

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[1] Fruchter E. et al. The impact of Herpes simplex virus type 1 on cognitive impairments in young, healthy individuals — A historical prospective study. Schizophrenia Research. 2015. Sept 8.

[2] Tarter KD. et al. Persistent viral pathogens and cognitive impairment across the life course in the third national health and nutrition examination survey. J Infect Dis. 2014 Mar;209(6):837-44.

[3] Thomas P. et al. Exposure to herpes simplex virus, type 1 and reduced cognitive function. J Psychiatr Res. 2013 Nov;47(11):1680-5.

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Fruchter, E., Goldberg, S., Fenchel, D., Grotto, I., Ginat, K., & Weiser, M. (2015). The impact of Herpes simplex virus type 1 on cognitive impairments in young, healthy individuals — A historical prospective study Schizophrenia Research DOI: 10.1016/j.schres.2015.08.036

Viral exposure and autism

A whole slew of articles published by Ivan Gentile and colleagues based at the University of Naples (Italy) brought me to writing this post looking at some of the literature on viral exposures and autism. Viruses, in case you didn't know, are some of nature's survivors, infecting host cells and reproducing, onwards hopeful of finding more (un)willing cells/organisms to infect. Humankind have developed various biological defence mechanisms against the viral (and bacterial) onslaught that we all face as part of daily life, part of which is the production of antibodies. Antibodies are all about identification, action and memory and form the basis for why we vaccinate against various disease causing viruses. Sometimes antibodies also take part in a process called neutralisation which is all about rendering a virus ineffective when it comes to infectivity.

A founder of virology @ Wikipedia 

I've talked previously on this blog about viruses and their possible connection to some cases of autism (see here for example). I've also discussed how traces of the viruses of yesteryear (many, many yesteryears) can still be found in our genome and how such fossil viruses may, in some cases, still impact on our health and wellbeing (see here and see here). But enough of all this idle chatter...

The specific papers under discussion today include:

• Prevalence and Titre of Antibodies to Cytomegalovirus and Epstein-Barr Virus in Patients with Autism Spectrum Disorder [1]
• Exposure to Varicella Zoster Virus Is Higher in Children with Autism Spectrum Disorder than in Healthy Controls. Results from a Case-control Study [2]
• Prevalence of Herpes Simplex Virus 1 and 2 Antibodies in Patients with Autism Spectrum Disorders [3]

Let's call them paper 1, paper 2 and paper 3 respectively for convenience.

All were published in the journal In Vivo and follow some history looking at viral infections and autism by this research group [4] including those most contentious of viral infections when it comes to autism: measles, mumps and rubella [5]. I might also add that some speculations from this group looking at linking genetic predisposition, vitamin D deficiency and infection potentially correlating with a "a deranged immune response" with some autism in mind [6] might not be as outlandish as once thought. Indeed, that review paper [6] is probably one of the best I've read in a long time drawing on the available data on immune function and autism bearing in mind the emerging vitamin D story (see here).

Anyhow:

• Papers 1-3 all relied on the same participant groups, that is: 54 children diagnosed with an autism spectrum disorder (ASD) and 46 asymptomatic controls. 
• Exposure (seropositivity) rates and antibody titer levels to Cytomegalovirus (CMV) and Epstein-Barr Virus (EBV) (paper 1), Varicella Zoster Virus (VZV) (paper 2) and Herpes Simplex Virus 1 (HSV1) and Herpes Simplex Virus 2 (HSV2) (paper 3) were measured and compared between groups.
• Paper 3 detailing the findings on HSV1 and HSV2 concluded: "Seropositivity rate and levels of anti-HSV1/2 were not dissimilar between cases and controls". In other words, nothing to see there in this cohort.
• Paper 1 looking at CMV and EBV came to a similar conclusion as that of paper 3, although the authors note: "considering only patients with ASD, those seropositive for CMV tended to test worse to the major severity scales than the seronegative ones". With my recent interest in CMV and autism (see here), I'm intrigued...
• Paper 2 provides something of a more 'positive' result with it's analysis of VZV, the virus linked to chickenpox and shingles, in connection to the autism grouping. Authors concluded: "The exposure rate and titer of anti-VZV antibodies were significantly higher in children with ASD compared to controls (59% vs. 39% and 694 mIU/ml vs. 94 mIU/ml, respectively)". Further: "exposure to VZV was found to be independently associated with ASD".

We do have to be a little bit careful when it comes to these studies on the basis of their small participant numbers and the applicability of results to other groups whether in age or geography. As per some previous chatter about the other Gentile paper on MMR antibodies and autism (see here) not every study agreed with their findings [7]. Likewise, these latest results say nothing about 'causation' in terms of autistic presentation outside what is already suspected with something like CMV and autism in mind [8]. Correlation is not the same as causation, as if you needed telling.

That all being said I do think there is more to do in this area. There is some research history when it comes viral infection and autism as per the review by Libbey and colleagues [9] and in amongst that literature is mention of varicella [10] including "cases of autism associated with postnatal varicella encephalitis" [11]. The quite stark disparity in mean antibody titers to VZV between autism and control groups suggests that something might be afoot outside of just some healthy immunity to something like chicken pox.

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[1] Gentile I. et al. Prevalence and Titre of Antibodies to Cytomegalovirus and Epstein-Barr Virus in Patients with Autism Spectrum Disorder. In Vivo. 2014 07-08;28(4):621-626.

[2] Gentile I. et al. Exposure to Varicella Zoster Virus Is Higher in Children with Autism Spectrum Disorder than in Healthy Controls. Results from a Case-control Study.  In Vivo. 2014 07-08;28(4):627-631.

[3] Gentile I. et al. Prevalence of Herpes Simplex Virus 1 and 2 Antibodies in Patients with Autism Spectrum Disorders. In Vivo. 2014 07-08;28(4):667-671.

[4] Gentile I. et al. Prevalence of HHV-6 and HHV-8 antibodies in patients with autism spectrum disorders. In Vivo. 2013 Nov-Dec;27(6):843-9.

[5] Gentile I. et al. Response to measles-mumps-rubella vaccine in children with autism spectrum disorders. In Vivo. 2013 May-Jun;27(3):377-82.

[6] Gentile I. et al. Etiopathogenesis of autism spectrum disorders: fitting the pieces of the puzzle together. Med Hypotheses. 2013 Jul;81(1):26-35.

[7] Singh VK. et al. Abnormal measles-mumps-rubella antibodies and CNS autoimmunity in children with autism. J Biomed Sci. 2002 Jul-Aug;9(4):359-64.

[8] Sakamoto A. et al. Retrospective diagnosis of congenital cytomegalovirus infection in children with autism spectrum disorder but no other major neurologic deficit. Brain Dev. 2014 Apr 22. pii: S0387-7604(14)00094-1.

[9] Libbey JE. et al. Autistic disorder and viral infections. J Neurovirol. 2005 Feb;11(1):1-10.

[10] Deykin EY. & MacMahon B. Viral exposure and autism. Am J Epidemiol. 1979 Jun;109(6):628-38

[11] Knobloch H. & Pasamanick B. Some etiologic and prognostic factors in early infantile autism and psychosis. Pediatrics. 1975 Feb;55(2):182-91.

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Gentile I, Zappulo E, Bonavolta R, Maresca R, Messana T, Buonomo AR, Portella G, Sorrentino R, Settimi A, Pascotto A, Borgia G, & Bravaccio C (2014). Prevalence and Titre of Antibodies to Cytomegalovirus and Epstein-Barr Virus in Patients with Autism Spectrum Disorder. In vivo (Athens, Greece), 28 (4), 621-626 PMID: 24982232


Gentile I, Zappulo E, Bonavolta R, Maresca R, Riccio MP, Buonomo AR, Portella G, Settimi A, Pascotto A, Borgia G, & Bravaccio C (2014). Exposure to Varicella Zoster Virus Is Higher in Children with Autism Spectrum Disorder than in Healthy Controls. Results from a Case-control Study. In vivo (Athens, Greece), 28 (4), 627-631 PMID: 24982233


Gentile I, Zappulo E, Bonavolta R, Maresca R, Riccio MP, Buonomo AR, Portella G, Vallefuoco L, Settimi A, Pascotto A, Borgia G, & Bravaccio C (2014). Prevalence of Herpes Simplex Virus 1 and 2 Antibodies in Patients with Autism Spectrum Disorders. In vivo (Athens, Greece), 28 (4), 667-671 PMID: 24982239

HERVs and ADHD

HERVs. Human endogenous retroviruses. The remnants of our evolutionary struggle with the viruses of the times of our distant ancestors, now part and parcel of our modern-day genome. Yes, genetically, we are all part virus to various extents [so embrace your viral self].

HMS Beagle @ Wikipedia

I've talked HERVs on a few occasions on this blog, in relation both to the autism spectrum conditions (see here) and myalgic encephalomyelitis (ME) (see here).

Without getting too technical, the crux of those posts was to suggest that although those bits of virus in our genomes are not necessarily 'active viruses' (i.e. not able to produce infectious virus or replicate) they may have implications for things like autoimmunity given that HERV proteins are supposedly recognised as 'self' by the almighty MHC [1] and seem to be able to provoke autoimmunity (in mice) [2]. That and the fact that the expression of HERVs may be kept in check by epigenetic means (methylation) [3] and 'hypomethylation [of DNA] = more genomic instability' [4] so, theoretically under certain circumstances could mean HERVs get a chance to start expressing (something). Or that's the theory (I think)...

With all that in mind, I move to the paper by Balestrieri and colleagues [5] (yes, the same group who completed the HERV paper with autism in mind) who discussed some very preliminary data on the expression of certain families of HERVs. Indeed, they reported: "The expression levels of HERV-H are significantly higher in patients with ADHD [attention-deficit hyperactivity disorder] compared to healthy controls".

I can't pretend to know all the ins-and-outs of how one goes about assaying for the expression of HERVs - "expression of retroviral mRNAs from the three HERV families was evaluated in peripheral blood mononuclear cells (PBMCs)" apparently. It was however interesting to see that HERV-H 'over-expression' was "significantly higher in patients with ADHD compared to healthy controls". HERV-H was also the same family reported to be 'more abundantly expressed' in cases of autism.

A search of some of the literature covering HERV-H reveals that it is a gamma-retrovirus (yes, similar to those letters X-M-R-V and that de-discovery issue). In terms of associations and roles, it "contributes to pluripotency in human cells" according to the paper by Santoni and colleagues [6] and their finding of high levels of HERV-H RNA in human embryonic stem cells. The HERV-H family have also been suggested to have immunosuppressive properties [7].

HERV-H has been linked to conditions such as multiple sclerosis as per papers like this one by Christensen [8] which also hinted at how other viruses may 'interact' with HERVs; in that paper concluding that: "retroviruses and herpes viruses have complex interactions". That being said, not all results have arrived at the same conclusion.

I was also particularly interested to read the paper by Shuvarikov and colleagues [9] who seemed to suggest "HERV-H elements as a mechanism of deletion formation", as in genetic deletions. I might be making mountains out of molehills but their case report that HERV-H elements seemed to flank "recurrent, 3.4-Mb, de novo deletions of 3q13.2-q13.31" could potentially lead down some very important paths as intimated in a previous post. The fact that they mention autism as being part and parcel of some of their participant group description is likewise intriguing.  As I've indicated in other posts on this blog where the term 'de novo' has been used, the uncertainty or chance finding of de novo now, might not be so in X numbers of years time. And the future may already be here [10] with schizophrenia in mind.

I'm going to stop there with this quite heavy going post and the chatter about HERVs and ADHD (or autism or anything else). Science is to quite a large extent still feeling it's way around the HERVs and their role - if any - in health and wellbeing. The reported link between the expression of HERVs and a condition as complicated as ADHD needs a lot more work on it before anyone can arrive at any firm conclusions. That being said, I find this to be a fascinating area of science which really does add a new layer of complexity to the whole genetics-environment relationship.

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[1] Lavie L. et al. CpG Methylation Directly Regulates Transcriptional Activity of the Human Endogenous Retrovirus Family HERV-K(HML-2). J. Virol. 2005; 79: 876-883

[2] Perron H. et al. Human Endogenous Retrovirus Protein Activates Innate Immunity and Promotes Experimental Allergic Encephalomyelitis in Mice. PLoS ONE 8(12): e80128. doi:10.1371/journal.pone.0080128

[3] Wilson AS. et al. DNA hypomethylation and human diseases. Biochimica et Biophysica Acta. 2007; 1775: 138–162.

[4] Tugnet N. et al. Human Endogenous Retroviruses (HERVs) and Autoimmune Rheumatic Disease: Is There a Link? Open Rheumatol J. 2013; 7: 13–21.

[5] Balestrieri E. et al. Human endogenous retroviruses and ADHD. World J Biol Psychiatry. 2013 Nov 28. [Epub ahead of print]

[6] Santoni FA. et al. HERV-H RNA is abundant in human embryonic stem cells and a precise marker for pluripotency. Retrovirology. 2012; 9: 111.

[7] Mangeney M. et al. The full-length envelope of an HERV-H human endogenous retrovirus has immunosuppressive properties. J General Virology. 2001; 82: 2515-2518.

[8] Christensen T. Association of human endogenous retroviruses with multiple sclerosis and possible interactions with herpes viruses. Rev Med Virol. 2005 May-Jun;15(3):179-211.

[9] Shuvarikov A. et al. Recurrent HERV-H-Mediated 3q13.2-q13.31 Deletions Cause a Syndrome of Hypotonia and Motor, Language, and Cognitive Delays. Hum Mutat. 2013 Oct;34(10):1415-23.

[10] Bundo M. et al. Increased L1 Retrotransposition in the Neuronal Genome in Schizophrenia. Neuron. 2014. 2 Jan.

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Balestrieri E, Pitzianti M, Matteucci C, D'Agati E, Sorrentino R, Baratta A, Caterina R, Zenobi R, Curatolo P, Garaci E, Sinibaldi-Vallebona P, & Pasini A (2013). Human endogenous retroviruses and ADHD. The world journal of biological psychiatry : the official journal of the World Federation of Societies of Biological Psychiatry PMID: 24286278

Maternal immune activation and monkeys continued

I sure do hope that all those fictional stories of apes turning the tables on humans don't ever come true. Indeed should you ever hear a Charlton Heston-esque character uttering 'You cut up his brain, you bloody baboon!' you might want to look to those in the field of monkey research as being the first ones with their brains on the chopping block in a sort of tit-for-tat style.

I jest of course. But the theme of monkey research runs strong in today's post as I introduce the paper by Melissa Bauman and colleagues* adding yet further evidence to the possible connection between maternal immune activation (let's call it MIA for short) and offspring behavioural development and characteristics.

For those with a close eye on autism and schizophrenia research, you'll probably already have heard of the work of Paul Patterson and colleagues (see here) and that coming out of the MIND Institute (see here) on how mothers' immune function during pregnancy might influence the risk of offspring presenting with said conditions. Indeed, with no clinical advice given or intended, the extended work looking into things like the use of suramin (see here) on offspring of mouse models with MIA in mind is also worthy of attention (note: the stress is on mouse models not human models).

For those who follow this blog, you might remember that I've already talked about some of the other work of Melissa Bauman and colleagues and the concept of maternal autoantibody-related autism (MAR). The emphasis there was on how monkeys have been introduced into MIA research and so perhaps relieving the burden on our poor Murine friends. If you happen to be a rat however, be warned... it looks like autism research is moving its eye of Sauron on to you.

The latest paper from Bauman (which also included Paul Patterson on the authorship list) differed from their MIA paper** insofar as instead of transplanting purified IgG brain reactive antibodies derived from mums of children diagnosed with an autism spectrum disorder (ASD) into monkeys, they used an artificial 'viral mimic'*** to stimulate the maternal immune system. The mimic was administered to pregnant monkeys either early or late gestation and compared with a non-mimic control group of rhesus monkeys.

As per the previous study, offspring behaviour was analysed over a 2-year period and some notable differences were reported: "MIA yields offspring with abnormal repetitive behaviors, communication, and social interactions". Readers might see a few similarities between the areas reported and the main topic of this blogsite.

This is interesting data of that there is no doubt. Allowing for the relatively small groups of animals included for study (13 experimental animals and 11 controls) and the important fact that monkeys are monkeys and not human beings with all their complexities, one might be inclined to think that MIA is certainly a field making advances.

With my own research hat on, bearing in mind MIA is most definitely not my field of expertise, I do wonder whether we might be hearing more from this group on something reported (but still awaiting peer review publication) by Prof. Patterson not so long ago: leaky gut being present in the offspring of MIA mother mice (see here). Ah yes, leaky gut (gut hyperpermeability if you will). The same leaky gut in cases of autism which has very recently been confirmed by the research tour-de-force that is Laura de Magistris and colleagues (see here). I'd be really quite interested to see if they looked at gut permeability in those MIA offspring monkeys too....

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* Bauman MD. et al. Activation of the Maternal Immune System During Pregnancy Alters Behavioral Development of Rhesus Monkey Offspring. Biol Psychiatry. 2013 Sep 4. pii: S0006-3223(13)00673-2. doi: 10.1016/j.biopsych.2013.06.025.

** Bauman MD. et al. Maternal antibodies from mothers of children with autism alter brain growth and social behavior development in the rhesus monkey. Transl Psychiatry. 2013 Jul 9;3:e278. doi: 10.1038/tp.2013.47.

*** Caskey M. et al. Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans. J Exp Med. 2011 Nov 21;208(12):2357-66. doi: 10.1084/jem.20111171.

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Bauman MD, Iosif AM, Smith SE, Bregere C, Amaral DG, & Patterson PH (2013). Activation of the Maternal Immune System During Pregnancy Alters Behavioral Development of Rhesus Monkey Offspring. Biological psychiatry PMID: 24011823

An anti-viral for chronic fatigue syndrome?

The recent publication of the trial results by Jose Montoya and colleagues* looking at the possibility that the anti-viral drug valganciclovir (see here for some description) might provide clinical benefit for some patients diagnosed with chronic fatigue syndrome (CFS) is the source material for today's post. The ClinicalTrial.gov entry for the study is here.

Virology Founder @ Wikipedia 

Indeed, when reading the Wikipedia entry for how the valganciclovir - lets call it VGCV to save my poor typing hands - trial was long awaited on the back of some rather more preliminary observations (see here** and here***), one gets the idea that the recent Montoya paper might eventually turn out to be something pretty important. That being said, I'm going to turn to my old caveat on this blog about not providing medical or clinical advice.

The value-added on the recent trial was that it was methodological, a lot more comprehensive that the previous trial by the author: now being double-blind and placebo-controlled and following participants over a period of 6 months. The authors also used a suite of measures to ascertain physical and mental fatigue: the "Multidimensional Fatigue Inventory (MFI-20) and Fatigue Severity Scale (FSS)" alongside "monocyte and neutrophil counts and cytokine levels".

They found a few differences between VGCV and placebo in their group, some of them significant and some of them not so. Importantly - long quote coming up - "statistically significant differences in trajectories between groups were observed in MFI-20 mental fatigue subscore (P=0.039), FSS score (P=0.006), and cognitive function (P=0.025). VGCV patients experienced these improvements within the first 3 months and maintained that benefit over the remaining 9 months." Interesting too that among the biological results there were signs of a shift in immune function towards Th1 in the VGCV group (see here for a description) among other things.

OK, a couple of steps back first. This was a study based on quite a well-defined group of people with CFS who also presented with signs of an immune response (IgG antibodies) against human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV). I understand that these viruses or rather exposure to these viruses have been discussed with CFS in mind previously (see here**** and here***** for example) outside of the historical viral reference (PVFS) although there still remains some 'discussion' about their role and relationship to CFS if and when detected.

To add to that viral - immune link, I note also that similar anti-viral preparations have also been examined with CFS in mind as per this study by Lerner and colleagues****** who used valaciclovir and even earlier this study******* using ganciclovir. Whilst methodologically sound, one might also reasonably expect that further research be undertaken following on from the Montoya and other results with a larger patient cohort.

I appreciate that to some, viral infections and CFS is still a little bit of a thorny issue in light of the quite recent XMRV story (see here for the drama) and even earlier some chatter about the use of transfer factor********. There is, therefore, always the possibility that the latest Montoya research might unfortunately suffer a sort of death by association as a consequence of any perceived link, despite the fact that as far as I can see they steered clear of anything murine leukemia virus-related virus in their work. Indeed as an aside, I should also point out the very interesting work being done on HERVs and myalgic encephalomyelitis (ME) which may or may not be relevant to the Montoya investigation too.

That also there is still some historical debate about the nature of CFS and the use of things like graded exercise and CBT as front-line treatment options (of which I am providing no opinion) is also another potential stumbling block to moving work like that of the Montoya paper out of the research domain and into something a little more accessible to specific patient groups. My view, as I've indicated in quite a few other posts on the subject of CFS, is that we are probably talking about a heterogeneous condition (possibly even a spectrum of conditions manifesting common symptoms. Sound familiar?) which appears to be multi-factorial in nature and pathology (see here and here and here for example). It's therefore highly likely that as Montoya and colleagues indicated "VGCV may have clinical benefit in a subset of CFS patients" as might other intervention options. The trick like Montoya has done, is to search out potential markers for what will work for who.

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* Montoya JG. et al. Randomized clinical trial to evaluate the efficacy and safety of valganciclovir in a subset of patients with chronic fatigue syndrome. J Med Virol. 2013 Aug 19. doi: 10.1002/jmv.23713.

** Watt T. et al. Response to valganciclovir in chronic fatigue syndrome patients with human herpesvirus 6 and Epstein-Barr virus IgG antibody titers. J Med Virol. 2012 Dec;84(12):1967-74. doi: 10.1002/jmv.23411.

*** Kogelnik AM. et al. Use of valganciclovir in patients with elevated antibody titers against Human Herpesvirus-6 (HHV-6) and Epstein-Barr Virus (EBV) who were experiencing central nervous system dysfunction including long-standing fatigue. J Clin Virol. 2006 Dec;37 Suppl 1:S33-8.

**** Burbelo PD. et al. No serological evidence for a role of HHV-6 infection in chronic fatigue syndrome. Am J Transl Res. 2012;4(4):443-51.

***** Lerner AM. et al. IgM serum antibodies to Epstein-Barr virus are uniquely present in a subset of patients with the chronic fatigue syndrome. In Vivo. 2004 Mar-Apr;18(2):101-6.

****** Lerner AM. et al. Valacyclovir treatment in Epstein-Barr virus subset chronic fatigue syndrome: thirty-six months follow-up. In Vivo. 2007 Sep-Oct;21(5):707-13.

******* Lerner AM. et al. A small, randomized, placebo-controlled trial of the use of antiviral therapy for patients with chronic fatigue syndrome. Clin Infect Dis. 2001 Jun 1;32(11):1657-8.

******** Ablashi DV. et al. Use of anti HHV-6 transfer factor for the treatment of two patients with chronic fatigue syndrome (CFS). Two case reports. Biotherapy. 1996;9(1-3):81-6.

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Montoya JG (2013). Randomized clinical trial to evaluate the efficacy and safety of valganciclovir in a subset of patients with chronic fatigue syndrome J Med Virol DOI: 10.1002/jmv.23713

Lyme and soda: hold the autism risk?

I've talked about the mighty tick previously on this blog and some speculation on how a tick harbouring the bacteria Borrelia burgdorferi (or a close relation) bites and transmits said bacteria to humans which can lead to Lyme disease and whether this might be implicated in some cases of autism.

 Tickety boo @ Wikipedia  

The suggestion from that post was that whilst the data was speculative and relatively sparse at that time on whether Lyme disease is common in cases of autism or indeed could 'cause' autism, there might be some room to test one or two hypotheses regarding a possible link between the two conditions.

Two years on, I'm happy to report that someone has finally looked at the possibility of a connection in the form of a letter by Mary Ajamian and colleagues* published in the journal JAMA. The results: no link between Lyme disease and autism in a sizeable cohort of children/young adults with autism or indeed Lyme disease and control participants.

The ins-and-outs of the study have been quite widely reported, for example with headlines like: 'Autism-Lyme Correlation Debunked' and 'Autism And Lyme Disease Link Is Bunk, Study Says'. Using a two-tiered assay based on initial screening for antibodies to B.burgdorferi (IgG and IgM) and Western blotting for antibodies in suspect cases, the authors were systematically able to test for signs of infection and found pretty much nothing.

Interestingly, the authors are quoted as saying that their study, whilst well-powered and based on the US CDC advice for screening for Lyme disease, might not necessarily spell the end of any association between Lyme disease and autism. To quote from this news source: "The researchers also pointed out that their analysis did not address the question of whether Lyme disease might cause autism-like behavioral deficits". That for example earlier infection during infancy or even in-utero might be linked to cases of autism was not part and parcel of the Ajamian study. Certainly with all the recent speculation turning to the earliest time of life being linked to autism risk - think folate and valproate for example - I don't think we are in a position to yet discount such possibilities. That and the renewed focus on the tetracyclines with autism in mind (used to treat Lyme disease).

But....

The Ajamian study must be seen as compelling evidence that no current infection pertinent to Lyme disease exists in cases of autism or controls, or at least the cases that were under investigation. I know infection is still quite a hot potato when it comes to autism risk (remembering this study by Hornig and colleagues** and all that XMRV overspill into autism spectrum disorders) bearing in mind the continued speculation on all those possible in-utero exposures whether viral or bacterial. I would bring you back to that most classical of autism risk factors - congenital rubella as per the study by Chess and colleagues*** - as evidence that external agents might be associated with autism onset and how the wider search for potentially linked pathogens should not be tarnished by the latest findings.

Update (10/05/13) And just a few days later... Lack of serum antibodies against Borrelia burgdorferi in children with autism by Burbelo and colleagues****.

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* Ajamian M. et al. Serologic markers of Lyme Disease in children with autism. JAMA. 2013; 309: 1771-1773.

** Hornig M. et al. Lack of association between measles virus vaccine and autism with enteropathy: a case-control study. PLoS ONE. 2008; 3: e3140.

*** Chess S. et al. Behavioral consequences of congenital rubella. J Pediatr. 1978; 93: 699-703.

**** Burbelo PD. et al. Lack of serum antibodies against Borrelia burgdorferi in children with autism. Clin Vaccine Immunol. May 2013.

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Mary Ajamian, Barry E. Kosofsky, Gary P. Wormser, Anjali M. Rajadhyaksha, & Armin Alaedini (2013). Serologic Markers of Lyme Disease in Children With Autism JAMA, 309 (17), 1771-1773

Autism, maternal immune activated mice and suramin

Avid followers of the autism research circuit must have noticed the increasing tide of studies looking at a possible role for maternal immune activation (MIA) in relation to risk of offspring autism spectrum disorder (ASD). It's a topic I've covered more than once on this blog; predominantly in relation to the work of people like Paul Patterson and his colleagues (see here), observations on things like C-reactive protein (see here) and the various ways to experimentally mimic such MIA in the mouse model of autism / schizophrenia / other for example (see here).

Squeakers @ Wikipedia  

So it is in this post that I'm serving a double helping of the MIA model of autism as per the publication of studies from Jared Schwartzer and colleagues from the MIND Institute* (open-access) and Robert Naviaux and colleagues** (open-access).

Both studies looked at the effects of artificial induction of MIA in the mouse model following poly I:C use as an immunostimulant. Thereafter the two studies went their separate ways as Schwartzer looked at the variable of mouse strain on the after-effects of MIA on offspring and Naviaux looked at the role of purinergic signaling.

I'll say right now that I am neither qualified nor experienced enough to go into these papers with any great detail. So I won't; instead a brief overview of each - bearing in mind their open-access status - and some interesting factoids which have already been mentioned in the autism research peer-reviewed domain which might tie into results.

The work of Schwartzer and colleagues basically "indicate[s] the need to consider how genetic predisposition may exacerbate or protect against the effects of environmental insults in the etiology of ASD". In other words, based on a mouse model looking at different strains of mouse, the specific genetic make-up of that mouse model might impact on offspring presentation after an artificial MIA event.

In their case they looked at the C57BL/6J and BTBR T+tf/J inbred mouse strains and concluded that the dangermouse that is the BTBR strain combined with the poly I:C stressor seemed to "be synergistic resulting in greater behavioral impairment than from either factor alone" when compared with the C57BL/6J mouse strain. Some interesting variables are noted including elevations in cytokines like IL-6 (see here) and IL-17 (see here) in the BTBR offspring mice compared to C57BL/6J mice alongside some sex specific behavioural differences. All in all, some very interesting observations; and on that sex-specific notion, not completely at odds with other work in this area (see here).

The work of Naviaux and colleagues - summarised quite well here - has definitely taken the interest of the media as per headlines such as 'New drug that may help reverse autism' or should that be 'Century old drug could beat autism'. I'm confused. The long-and-short of it is that based on the analysis of the MIA mouse model - C57BL/6J mice - there was a suggestion that "hyperpurinergia is a fundamental and treatable feature of the multisystem abnormalities in the poly(IC) mouse model of autism spectrum disorders". Treatable via "antipurinergic therapy (APT)" which in this study was via the drug suramin. The observant reader should immediately be comparing Schwartzer and Naviaux and the MIA mouse models chosen and results obtained.

Anyhow, Naviaux et al continue in their observations on how MIA affected offspring mice and how the administration of suramin seemed to have some pretty wide-ranging effects on offspring mice. Alongside various behavioural effects on social and coordination issues, suramin administration was reported to show important effects such as "the preservation of cerebellar Purkinje cells", which as I discussed in a recent post, have more than a token link to cases of autism. "Suramin treatment strongly increased the expression of the nicotinic acetylcholine receptor subunit α7 (nAchRα7) in cerebral synaptosomes of MIA animals" was another potentially important finding in view of other work in this area. In all, "16 multisystem features of this model were either corrected or improved by suramin treatment".

Impressive stuff I hear you say. Indeed all the more impressive given that the authors on purpose did not start suramin treatment until 6 weeks because they "wished to test the hypothesis that many of the autism-like features of the MIA model were treatable after they appear". And apparently there is more to come according to the authors, with the promise of human trials of suramin...

But just before you pop down to your local doctor or pharmacist to ask for suramin (off-label), it might be worth pointing out a few things. Mice. Yep, this was a study of mice and as per the Schwartzer study, not necessarily the best and only mouse model of autism from an MIA point of view. Indeed if I needed to go back to the BTBR mouse and its overlap with autism, I might also recall some work looking at that most forgotten of autism research parameters, sulphate (sulfate) and findings related to the BTBR model (see here). Mice are not humans and suramin is to be added to a growing list of mouse findings with an autism slant (see here and here).

That the US National Cancer Institute holds an entry for suramin should also give you some idea as to what uses the drug has and why bearing in mind it was injected into the study mice. Alongside its anti-parasitic effects related to things like sleeping sickness, the activity of suramin has been linked to its blocking of various growth factor binding which might yet hold some clue to other effects of the drug outside of competitive inhibiting of purinergic signalling (see here and here). As with most medicines, there are other effects to keep in mind which might also tie into results. And then there are the reported side-effects...

I'm not by any means trying to belittle the Naviaux results of suramin in the MIA mouse model of autism so please do not take this post as such. I am very keen to see some replication studies done in other mouse and other animal models, just to see if the results stack up before progressing to human trials with the all-important focus on 'first do no harm' and whether other meds have similar actions. As such I'll keep my eye open for suramin and autism and perhaps post some updates.

In the meantime, the maternal immune activated hypothesis grinds forward...

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* Schwartzer JJ. et al. Maternal immune activation and strain specific interactions in the development of autism-like behaviors in mice. Translational Psychiatry. 2013; 3: e240.

** Naviaux RK. et al. Antipurinergic therapy corrects the autism-like features in the poly(IC) mouse model. PLoS ONE. 2013; 8: e57380.

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Schwartzer JJ, Careaga M, Onore CE, Rushakoff JA, Berman RF, & Ashwood P (2013). Maternal immune activation and strain specific interactions in the development of autism-like behaviors in mice. Translational psychiatry, 3 PMID: 23481627

Naviaux, R., Zolkipli, Z., Wang, L., Nakayama, T., Naviaux, J., Le, T., Schuchbauer, M., Rogac, M., Tang, Q., Dugan, L., & Powell, S. (2013). Antipurinergic Therapy Corrects the Autism-Like Features in the Poly(IC) Mouse Model PLoS ONE, 8 (3) DOI: 10.1371/journal.pone.0057380

Lipopolysaccharide and autism research

A word that I'm just coming to grips with at the moment forms the subject of this post: lipopolysaccharide, and how LPS is starting to become more and more widely used in research into autism and other developmental / psychiatric conditions. In particular, with reference to some possible involvement from maternal immune activation and risk of various behaviourally-defined conditions in offspring.

What is LPS?

Happy Days @ Wikipedia  

The paper by Raetz & Whitfield* (open-access) provides quite a comprehensive summary of LPS, the hows and whys, but a more concise version goes something like this:

• To use a music analogy, LPS is the cover sleeve for the vinyl album (what's that Grandad?) which is quite a few gram-negative bacteria. 
• LPS is made up of various chemical components; lipid A seems to be an important part of LPS, given that this is the stuff which seems to have quite a lot of the toxicity (endotoxin) effects of gram-negative bacteria attached. One might imagine that with the bacterial connection, those trillions of beasties which inhabit us, or rather our deepest, darkest recesses, might very well produce LPS to some extent.

LPS and immune activation

LPS is turning into quite the immune activation weapon of choice when it comes to animal research on conditions like autism and schizophrenia. Administration of LPS - mimicking gram-negative bacterial infection - is a great way of stimulating the innate immune system as per its effects on macrophage activation (yes, those bigger eaters of the immune system) via the Toll-like receptor 4 (TLR-4) - myeloid differentiation factor 2 (MD-2) complex (see here) and bringing into play all those not-so-lovely pro-inflammatory cytokines. LPS administration also seems to have a few other effects too as per this paper by Suh and colleagues** (open-access) on what happens to various amino acid chemistry when LPS is added. I'm sure there's a lot more also that it does.

With autism research in mind?

Quite a few animals have seen their fair share of LPS in the name of autism and related conditions research with some very interesting observations having been recorded:

• Willette and colleagues*** based on a LPS model of maternal immune activation, found that offspring rhesus monkeys showed more "behavioural disturbance" and brain enlargement when compared with controls. 
• Baharnoori and colleagues**** (open-access) concluded that offspring of LPS immune stimulated mice also showed some interesting changes to dopaminergic chemistry.
• Nouel and colleagues***** reported an effect from prenatal LPS exposure in terms of reduced levels of glutamic acid decarboxylase 67 (GAD67) and reelin in the rat model. Both GAD67 and reelin have been the topic of previous blog posts: GAD in connection to the neurotransmitters glutamate and GABA (see here) and reelin in relation to some interesting research on organophosphates (OPs) (see here).
• Finally, Xu and colleagues****** presented data suggestive that LPS administration might also affect levels of neurotrophin-3 (NT-3), involved in neurogenesis (and not a million miles away from an old favourite, BDNF). 

When applying LPS to media like PBMC provided by people with autism, there have also been some important results:

• Dr Harumi Jyonouchi (a researcher previously discussed on this blog) reported an "excessive innate immune responses in a number of ASD children" following LPS administration in this paper*******. TNF-alpha production (see this post) was of particular interest.
• Further, Dr Jyonouchi and colleagues in this paper******** went on to suggest that the response of adding LPS to PBMCs from participants with autism might also differ as a consequence of whether a gastrointestinal (GI) element was evident alongside autism.

I hope you can see why I'm so interested in LPS as a research tool when it comes to autism. The whole maternal immune activation area of autism research is definitely in the ascendancy as exemplified by the recent inflammation - offspring autism risk paper by Brown and colleagues********* discussed in this post. Indeed for science to even attempt to recreate anything approaching the conditions that *might* be linked to offspring autism with immune function in mind, LPS is a valuable tool alongside other agents such as polyinosinic:polycytidylic acid (poly I:C) highlighted in this paper by Paul Patterson and colleagues********** (open-access).

To finish, how about some Adamski (and Seal)?

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* Raetz CR. & Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem. 2002; 71: 635–700.

** Suh JH. et al. A new metabolomic assay to examine inflammation and redox pathways following LPS challenge. Journal of Inflammation 2012, 9:37

*** Willette AA. et al. Brain enlargement and increased behavioral and cytokine reactivity in infant monkeys following acute prenatal endotoxemia. Behav Brain Res. 2011; 219: 108-115.

**** Baharnoori M. et al. Effect of maternal lipopolysaccharide administration on the development of dopaminergic receptors and transporter in the rat offspring. PLoS One. 2013; 8: e54439.

***** Nouel D. et al. Prenatal exposure to bacterial endotoxin reduces the number of GAD67- and reelin-immunoreactive neurons in the hippocampus of rat offspring. Eur Neuropsychopharmacol. 2012; 22: 300-307.

****** Xu M. et al. Aberrant cerebellar neurotrophin-3 expression induced by lipopolysaccharide exposure during brain development. Cerebellum. January 2013.

******* Jyonouchi H. et al. Proinflammatory and regulatory cytokine production associated with innate and adaptive immune responses in children with autism spectrum disorders and developmental regression. J Neuroimmunol. 2001; 120: 170-179.

******** Jyonouchi H. et al. Dysregulated innate immune responses in young children with autism spectrum disorders: their relationship to gastrointestinal symptoms and dietary intervention. Neuropsychobiology. 2005; 51: 77-85.

********* Brown AS. et al. Elevated maternal C-reactive protein and autism in a national birth cohort. Molecular Psychiatry. January 2013.

GcMAF, nagalase and autism

I think it might be worth starting this blog post with (a) reference to my very well-trodden caveat of not making any medical recommendations on this blog and (b) a little bit of a description of some of the key terms connected with the paper by Jeff Bradstreet and colleagues* (open-access) on nagalase, GcMAF and autism bearing in mind my amateur status in this area. I might also add, don't shoot the messenger.

I'm the big eater @ Wikipedia  

Macrophages. It all begins with monocytes, white blood cells produced in bone marrow from hematopoietic stem cells. Monocytes grow into different types of macrophages.

Macrophages, known as the big eaters of the immune system, are present in every cell of the body and include microglia (in the brain).

As part of their big eating duties, macrophages enjoy dining out on the odd invading pathogen or cell programmed for destruction as well as telling other immune cells what to do. A sort of Mr Creosote if you will (without the bucket). If you want the Star Wars version of macrophages (I'm not kidding) ... here you go (open-access).

Macrophages are activated by Gc-MAF (Gc Macrophage Activating Factor). The production of Gc-MAF is affected by nagalase (alpha-N-acetylgalactosaminidase) encoded by the gene NAGA. Nagalase affects the Gc protein (vitamin D3 binding protein) which has a knock-on effect blocking the production of Gc-MAF. The more nagalase activity, the less Gc-MAF is a rough-and-ready way to look at it. Less Gc-MAF equates as less macrophage activation according to this logic and some potential onward effects for immune function.

Why is it important? Well in cancer research, there is some preliminary chatter that tumors might be able to affect Gc-MAF function by way of altering nagalase activity (open-access)**. Such is the effect of increased nagalase and depressed Gc-MAF function that this has been put forward as a potential explanation of why cancers are able to 'avoid' the immune system and so develop unchecked. There is also some very preliminary evidence that giving supplemental Gc-MAF as an injection might affect cancer growth in animal models*** and human participants**** although this is still an area of some controversy given that one lab seems to be producing all the research.

So what's the logic of this area with autism in mind? I can't claim to be able to provide a definitive answer but one suggestion from press releases such as this one, are the reports of high levels of nagalase activity to be present in quite a few of the cohorts with autism looked at. Remember, nagalase negatively affects levels of Gc-MAF so potentially disrupting the activation of macrophages. Outside of malignant cells, there is a suggestion that elevated nagalase activity might be part and parcel of issues with immune function in cases of autism onwards to things like the presence of some kind of viral activity. Indeed this last point on viruses and nagalase I assume comes from other results on the use of Gc-MAF in the clearance of HIV infection***** bearing in mind replication is still required for this area of work.

So eventually we get back to the paper from Bradstreet and colleagues and in more detail:

• Described as a chart review, 40 participants with autism who sought testing for nagalase activity, pre- and post assessment of nagalase following Gc-MAF injections were followed.
• Diagnosis of participants was determined by having already received a DSM-IV diagnosis of autism independent of the study together with some in-house assessments on the severity of presentation.
• Blood draws signalled the start of the nagalase activity assessment which was shipped to a lab already versed in looking for the enzymatic activity. 
• Gc-MAF was injected on a weekly basis covering an average of 14 injections to get those macrophages stimulated, and nagalase activity assessed again.
• Results: nagalase activity was generally higher in the autism group than the various reference ranges cited by the assaying laboratory.
• Nagalase activity levels dropped in quite a few participants following Gc-MAF administration (24 of 40 decreased to within laboratory reference ranges) and "uncontrolled observations of GcMAF therapy indicated substantial improvements in language, socialization and cognition". Before we get too carried away though, lets remember those words "uncontrolled observations". 
• Importantly (very importantly) no significant side-effects were reported, bearing in mind reports of elevated body temperature occurred post infusion and words like "By the second month, no patients experienced significant febrile events" were used.
• The authors conclude that more research is required in this area.

OK. With the science hat on, one reiterates that this was a very, very preliminary case review and although Gc-MAF was "checked for sterility in-house and externally by the UK Health Protection Agency" apparently, this is still a compound under investigation and is still very experimental. I've not specifically made mention of Gc-MAF on this blog before this post. That being said, I have talked about nagalase in relation to some speculations on the now de-discovered work on XMRV and chronic fatigue syndrome (CFS).

There are some obvious questions raised from the findings reported by Bradstreet. So, assuming all that Gc-MAF does and how it apparently does it, the whole 'underactive immunity' side of autism comes into play. Indeed I'm immediately drawn back to the work by Harumi Jyonouchi and colleagues on SPAD and immunodeficiency detected in their cohort and the possible link with gastrointestinal (GI) dysfunction. That and the low IgA findings also observed on more than one occasion in cases of autism. Of course balancing all that with other findings indicative of other issues with immune function in cases of autism such as an overactive immune system and the whole autoimmunity side of things. I should perhaps also stress that I am not equating autism with HIV or cancer or anything else based on the description of these findings.

Perhaps just as important is the whole viral infection link being implied in some cases of autism by this work. I know this starts to take us into some quite uncomfortable territory with autism in mind as per the study by Mady Hornig and colleagues****** (including virus hunter Ian Lipkin) on a (mostly) lack of measles virus in reply to studies like the one from Kawashima and colleagues*******. I'm not really in a position to offer an expert opinion as to whether this is proof positive that specific viruses are or aren't involved in autism, over an above the multitude of viruses everyone comes across in a lifetime, albeit with an immune response in full working order and the focus being on autisms not autism. Think also back to that most classical autism-viral connection which looked at rubella******** quite a few years back. And then all those ancient remnants of viruses which we all carry in our genome and have been recently looked at with autism in mind and whether there are any connections to be made or not.

Irrespective of any controversy this might unearth - which I assume it probably will - the Bradstreet results are peer-reviewed results and hence worthy of further independent analysis. Perhaps Prof. Lipkin might once again step up to this task?

[Update: you may also want to have a look at the second time Gc-MAF has cropped up on the autism  research circuit too.]

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* Bradstreet JJ. et al. Initial observations of elevated alpha-N-acetylgalactosaminidase activity associated with autism and observed reductions from GC protein—macrophage activating factor injections. Autism Insights. 2012. 4: 31-38.

** Korbelik M. et al. The value of serum alpha-N-acetylgalactosaminidase measurement for the assessment of tumour response to radio- and photodynamic therapy. Br J Cancer. 1998; 77: 1009-1014.

*** Yamamoto N. & Nataparaju VR. Immunotherapy of BALB/c mice bearing Ehrlich ascites tumor with vitamin D-binding protein-derived macrophage activating factor. Cancer Res. 1997 Jun 1;57(11):2187-92.

**** Yamamoto N. et al. Immunotherapy of metastatic breast cancer patients with vitamin D-binding protein-derived macrophage activating factor (GcMAF). Int J Cancer. 2008; 122: 461-467.

***** Yamamoto N. et al. Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF). J Med Virol. 2009; 81: 16-26.

****** Hornig M. et al. Lack of association between measles virus vaccine and autism with enteropathy: a case-control study. PLoS ONE. 2008; 3: e3140.

******* Kawshima H. et al. Detection and sequencing of measles virus from peripheral mononuclear cells from patients with inflammatory bowel disease and autism. Dig Dis Sci. 2000; 45: 723-729.

******** Chess S. Follow-up report on autism in congenital rubella. J Autism Child Schizophr. 1977; 7: 69-81.

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James Jeffrey Bradstreet, Emar Vogelaar, & Lynda Thyer (2012). Initial observations of elevated alpha-N-acetylgalactosaminidase activity associated with autism and observed reductions from GC protein—macrophage activating factor injections Autism Insights