Brain inflammation, autism, and antivaxers

After I discussed a new paper yesterday purporting to implicate inflammation as a cause of autism, particularly how unimpressive and of unclear significance its findings were and how it was being used and abused by antivaxers to claim that it supported their idea that vaccines cause autism, some of my readers started mentioning another, related paper published this month about inflammation in autism. It was published online first as an accepted but not yet formatted manuscript on October 8 in Annals of Neurology and comes from a group at Harvard led by a pathologist named Matthew Anderson, the Director of Neuropathology at Beth Israel Deaconess Medical Center, whose lab studies the molecular, cellular and neural network mechanisms responsible for disorders of membrane excitability and synaptic transmission in the central nervous system. It’s the same sort of paper as the last one in that it examines the postmortem brains of people with autism and autism spectrum disorder (ASD) and compared them to controls without ASDs and found…inflammation!

The authors note in the introduction:

Autism spectrum disorder (ASD) manifests in early childhood and is diagnosed based on behavioral deficits including impaired social and increased repetitive behaviors and restricted interests. The study of ASD postmortem brain tissue provides insights into the pathologic processes that underlie this disorder currently defined exclusively by behavioral deficits. Using ASD human postmortem brain tissues, investigators have discovered an increase of cytokines, chemokines, growth factors, and activated astroglia and microglia in the cerebral cortex, white matter, and cerebellum in ASD indicating ongoing activity of the innate immune system1-5. Genome-wide transcriptional profiling has revealed an increase in the expression of a diverse array of genes encoding mediators of this activated innate immune response along with an overall decrease in the expression of many neuron-related genes6-8. Here we applied computer vision algorithms to quantify astrocyte-derived round membranous blebs, multifocal perivascular lymphocytic cuffs, and increased perivascular space and collagen; novel neuropathologic features that we found in a large proportion of ASD brains. The results provide the signatures of a cellular immune response, reflected by T-lymphocyte infiltrates and cytotoxic cell injuries (typical of T-lymphocytes) to CSF-brain barrier astrocytes, in ASD compared to control postmortem brains.

As the press release says:

Not previously linked to autism, perivascular lymphocyte cuffing is a well-known indicator of chronic inflammation in the brain. Lymphocyte cuffs in the brain are telltale signs of viral infections or autoimmune disorders. But the pattern Anderson observed did not match any previously documented infection or autoimmune disorder of the brain. In the brains Anderson examined, the cuffs were subtle but distinct. “I’ve seen enough brains to know you shouldn’t see that,” he said.

I can’t help but note that when I see a finding described as so subtle that I often question its physiologic relevance, but we’ll go with Anderson for the moment.

So Anderson’s group was looking for different indications of inflammation in the brain than Theoharides’ group was in the study discussed yesterday. They approached the problem in a similar way, by looking at postmortem brains. The difference, though, is that Anderson also included adult brains. Overall, they examined brains from the Autism Tissue Program and Autism BrainNet brain banking programs, as well as from the neuropathology archives of the Bett Israel Deaconess Medical Center, for a total of 25 cases of ASD and 30 controls. Their exclusion criteria included evidence of neurodegenerative disease, central nervous system (CNS) infection, or other neuropsychiatric disorder where ASD was absent. For control cases, a further exclusion criterion was a known family member with ASD.

Perusing Table 1, which lists the cases and controls by features including age, sex, brain weight, and cause of death, I was struck by the lack of effort on the authors’ part to summarize the information to compare the ASD cases to controls to determine if the two groups were comparable. I realize that beggars can’t be choosers when it comes to brain specimens, particularly brain specimens of patients with ASD who died and whose bodies or brains were donated to science, but it’s hard to tell how comparable the groups are. I did a few quickie calculations, and they were reasonably comparable, with the controls being older, but not statistically significantly so, but it would have been nice to have this information summarized. Another good thing about the study is that the technicians and pathologists evaluating the tissue sections were blinded to diagnosis.

Anderson and company took the ASD brain sections and compared them to the controls after subjecting them to histology and immunohistology, and looked for lymphocytes as an indication of inflammation. They then used automated lymphocyte detection and an automated segmentation procedure. The specific stain was hematoxylin and eosin (H&E) plus Luxol Fast Blue (H&E+LFB). Blocks containing cerebral cortex were subjected to automated lymphocyte quantification, and blocks with the highest lymphocyte counts were selected for staining with primary antibodies to CD3, CD20, CD4, and CD8 using standard immunohistochemistry (IHC). These are markers for various immune cells, and I list them so that the pathologists reading this can comment freely. For some reason, the authors also stained each case with the largest perivascular space (space around blood vessels) for staining with glial markers. Basically, the authors were looking for “perivascular cuffs” of lymphcytes, a sign of chronic inflammation in the brain.

Anderson then did this:

All available H&E+LFB stained slides were reviewed with a standard bright field microscope and photographs were taken using a 60x objective (600x total magnification; TIFF-format; resolution 2448×1920 pixels) using a 5- megapixel CCD camera (Model DP27, Olympus). On every H&E+LFB-stained section from every ASD and control case, while blinded to the diagnosis, we photographed three blood vessels with the most abundant perivascular lymphocytes in each of the following brain compartments: grey matter, white matter, or leptomeninges. Fields containing single vessels (luminal diameter range 15-500μm) involved by the highest density of lymphocytes were selected from ASD and control cases using a 40x objective. This resulted in a collection of images with identical field of view sizes.

So, basically, sections with the most lymphocytes overall were selected and then of those sections the investigators looked at the blood vessels with the most lymphocytes overall were examined. This double selection makes me wonder a bit right there if there was any sort of bias introduced into the study, although I can see the rationale that focal areas of lymphocyte infiltration might be significant. In any event, the authors found multiple foci of lymphocytic cuffs with increased numbers of lymphocytes in 65% of ASD cases compared to control brains. They emphasize that they found this in males and females, across all ages, in white and grey matter and leptomeninges, and in most brain regions. Looking at their data and figures, I see that they found around two- to five-fold increased numbers of lymphocytes per vessel in all brain regions other than the medulla.

An interesting way to look at the data is to plot it this way, in a cascading plot with box-and whiskers plots showing the median, upper and lower quartile, and upper/lower quartiles ± 1.5 x (interquartile range), with the black cutoff line representing the lymphocyte count of 23/vessel that gives the highest sensitivity for ASD versus control groups:

Brain inflammation

What you can see is that, yes, by and large the ASD cases have higher lymphocyte counts per vessel, but that there’s a lot of overlap. Most of the rest of the figures show the same thing, overall differences of a similar magnitude with about a third of ASD cases not showing increased lymphocytes per vessel and considerable overlap between the two groups. A second finding of the paper is that the perivascular cuffs examined were made up of killer T-cells, immune cells that normally attack and destroy infected, cancerous, or dying cells, although they can attack normal cells in autoimmune diseases. Also found were increased cellular debris in the perivascular spaces, suggesting that the T-lymphocytes were attacking cells in that space and were associated with membranous blebs (also known as apoptotic bodies), structures associated with programmed cell death. Noting that astrocyte-derived membranous blebs haven’t been observed in other CNS disorders, Anderson speculated that they might have been generated by a targeted attack of astrocyte processes by cytotoxic (killer) T-lymphocytes at CNS blood-brain boundaries.

Anderson further speculates:

We suggest the invading CD8+ lymphocytes in ASD may have T cell receptors that selectively target epitope(s) presented by MHC-expressing astrocytes localized at the glia limitans where CD8+ T-cell cytotoxic effectors such as granzyme B may be locally released to generate these GFAP+ astrocyte membranous blebs in ASD. An MRI study has revealed dilated Virchow-Robin spaces in the centrum semiovale white matter in 7 of 16 subjects with ASD13; a possible correlate to the dilated perivascular spaces we identified in white matter. Astrocyte debris released into the Virchow- Robin CSF spaces and leptomeningeal fibrosis (collagen deposition) could contribute to other brain pathologic processes such the obstruct of CSF absorption consistent with the evidence of increased extra-axial cerebrospinal fluid in high-risk infants that develop ASD14. The astrocyte-targeted damage could have direct or indirect (cytokine-mediated) immune effects on the ability of astrocytes to provide metabolic support to axons causing action potential transmission failures15 as one explanation for the long-range functional connectivity deficits documented in ASD16.

And concludes:

Our study provides signature features of this T-lymphocyte immune subtype of ASD in postmortem cases and identifies astrocyte debris as a potential source of CSF or serum biomarkers for clinical diagnosis and monitoring of the pathology in living patients. Finally, with biomarkers that define the T-lymphocyte immune subtype of ASD, the efficacy of T-lymphocyte-targeted immunotherapies on biomarker levels and behavioral symptoms could be tested.

Maybe, but what does this all mean? Well, of course antivaxers think they know what it means, and what they think it means is highly predictable. They thinks that this is slam-dunk evidence that vaccines done it when it comes to autism:

The association between vaccination and autism was first reported by medical historian Harris L. Coulter and Barbara Loe Fisher, co-founder of the National Vaccine Information Center, in their 1985 book DPT: A Shot in the Dark (Harcourt Brace Jovanovich). Among the case history descriptions of DPT vaccine injury and death were cases of healthy children who suffered brain inflammation and brain damage after DPT vaccinations and were diagnosed with autism.

Of course, because if inflammation is involved in any condition or disease, then to antivaxers vaccines must be able to cause that condition or disease. There’s just one problem. Large scale well-designed epidemiological studies have repeatedly failed to find an association between vaccination and autism. Actually, there are two problems in that, as I mentioned yesterday, even though there is evidence of chronic inflammation in the brains of many autistic people in postmortem samples (for which this study just provides more evidence), it’s still very much up in the air whether this inflammation described in studies like this is causative for ASDs or epiphenomenon (i.e., a secondary effect or byproduct that arises from but does not causally influence a process). This study changes this no more than the study I discussed yesterday.

Of course, with the epidemiological evidence being what it is, namely negative for an association between vaccination and autism, even assuming that Anderson’s speculation that autism might be an autoimmune disease is correct and the inflammation being observed is not an epiphenomeon, then we know one thing with a high degree of certainty: Vaccines don’t cause it. (Seriously, there isn’t even a hint of a whiff of a signal in the literature looking for correlation between vaccines and autism.) That would mean that, even if autism has an autoimmune component to its pathophysiology, it’s not due to vaccines. That doesn’t stop antivaxers from twisting studies like this to fit their agenda, though, and I expect that they will continue to do so. That is, of course, one of the easiest predictions to make, because twisting science to suit their agenda is what they do.