Why do some IgG antibodies give protection and others don’t?


The immune system is capable of making many different types of immune responses to the same antigen, some of which will be effective, others less so and still others ineffective. Outcome of a given immune response usually reflects the balance of all these various types of immune responses that constitute it.

IgG antibodies are usually very effective against some types of bacteria such as Polysaccharide encapsulated bacteria – Wikipedia whose examples include pathogens such as Haemophilus influenzae – Wikipedia, Streptococcus pneumoniae – Wikipedia, Neisseria meningitidis – Wikipedia.

Usually extracellular, these bacteria multiply outside of body cells. Such bacteria can be dangerous, even life-threatening if they gain access to internal organs and tissues such as the brain, from their initial portals of entry such as nasal/oral passages. In such situations, presence of sufficient titers of bacterial antigen(s)-specific IgG can stop the spread of these bacteria dead in their tracks by neutralizing not just such antigens, a very important disease-alleviating function when such antigens are toxins, but also the whole bacteria themselves. This is also why transfer of such IgG antibodies can be protective, for example in the form of maternal IgG in the case of Passive immunity – Wikipedia.

OTOH, IgG antibodies can be similarly antigen-specific but just not effective when the source of their antigen is an intracellular organism that might be spreading stealthily from cell to cell through membrane-coated vesicles, for example.

Thus, like any other type of adaptive immune response, IgG antibodies are directly effective if ultimately their effector function can directly reduce the source of their antigen. Since, however, no single entity within the immune system can ‘see’ the ‘whole’ target, be it virus, bacteria, allergen, tumor cell or even transplant tissue, over the course of its evolution, the system appears to have chosen to hedge its bets and allow a broad range of different types of immune responses to develop over the course of a process, with the intent that at least one of them would prove effective, an approach that, given how our species is flourishing in terms of sheer numbers, suggests seems to have worked spectacularly well.



Is the healthy human brain home of a microbiome?

Does the healthy human brain have its own stable microbiota? Thus far, at least one peer-reviewed study (1) appears to suggest ‘healthy’ human brains could have their own share of microbiota.

A simple enough study (1), the authors performed deep sequencing of white matter-derived RNA from 4 HIV patients, 4 other disease controls and 2 cerebral surgical resections from epilepsy patients, and found alpha-proteobacteria in all of them as well as some herpes viruses and bacteriophages. Given none of the brains were technically from ‘healthy’ individuals, results from such a small study are inconclusive and will remain so until replicated or until other studies report finding other micro-organisms in healthy human brains. However, such a task is very much uphill and definitely not for the faint-hearted.

Human gut microbiota is unremarkable because it’s entirely expected. OTOH, brain microbiota is far more controversial simply because microbes are unexpected in such supposedly sterile organs as the healthy brain. Any and every critique applied to human brain microbiota is just as applicable to human gut microbiota since similar methods are used to analyze microbiota anywhere.

After all, microbiota analysis methods have considerable problems in the form of study design flaws, poor data quality and reproducibility, and ambiguous and questionable statistical approaches used for data analysis (2, 3, 4, 5, 6, 7, 8), except such critiques are usually glossed over in human gut microbiota studies even as they would likely be the centerpiece of focus about microbiota found in unexpected places such as the human brain.

Finding microbes in the healthy human brain would thus get parsed using much more stringent critiques that just don’t get applied to human gut microbiota analysis. Unsurprising then that the result of this sole study was easily considered suspect and discredited with the argument that microbiota such as alpha-proteobacteria are often found as contaminants (9). Casting doubt on the method casts doubt on the results.

Rightly or wrongly, the brain is typically privileged as the seat of the super-self. Microbes in the healthy brain necessitates some means of surveillance by the immune system but for a mindset that privileges the brain, both microbes and the immune system are potentially threatening invaders, given their unpredictable capacity for invasion, damage and just plain downright mischief. No surprise then that for long the consensus has been that the brain has systems in place to keep both the immune system and microbes at bay.

One of the clearest examples of such a system is the notion of Immune privilege – Wikipedia, an idea initially articulated by Peter Medawar – Wikipedia, which holds that being exquisitely vulnerable to irreversible inflammatory damage, certain parts of the body such as the brain limit their interaction with the environment beyond, and with the immune system in particular. The Blood–brain barrier – Wikipedia (BBB) embodies immune privilege in the form of a physical barrier preventing the immune system from fully accessing the brain. If the circulating immune system itself is supposed to have limited access to the brain, it’s not surprising that it then follows that microbes in the brain would be considered not a feature of health but of disease.

However, the notion of immune privilege lacks evolutionary coherence. Any part of the body inaccessible or poorly or less accessible to the immune system renders it unprotected. Akin to open sesame to pathogens, such an idea makes no evolutionary sense whatsoever. All the more flabbergasting then that this idea remained an entirely acceptable construct for decades and lingers on even in current thinking, as evidenced by an entire Wikipedia article devoted to it that discusses it wholly at face value, without ever referring to the hugely problematic implication of its evolutionary unworkability.

Steady drumbeat of data in recent years has however provided a couple of countervailing pieces of evidence that suggest the ground may be fast getting cut out from under the bastions holding the old ideas of immune privilege and BBB in place,

  • Numerous studies (see reviews 10, 11, 12, 13, 14), especially in preclinical animal models, have now shown gut microbiota influence many if not most aspects of brain development and function.
  • The 2015 report that contrary to long-held dogma, the (rodent) brain isn’t devoid of lymphatics (15, 16, 17). The so-called BBB may not be an unimpregnable barricade after all.

There remains then only the small (ahem!) matter of reconciling current immune system theory to healthy human brain microbiota as in how could the immune system tolerate them without constant, protracted inflammation? It’s doable if one accepts that any commensal microbiota that applied evolutionary selection pressure would be tolerated through the process of thymic Central tolerance – Wikipedia wedded to antigen-specific Regulatory T cell – Wikipedia development and function.

However, current immunological dogma would consider such an argument heretical since it assumes the immune system ontogenetically learns within each individual’s lifetime to distinguish what is self from what is not. OTOH, microbiota in the healthy human brain is not a problem for those like me who believe immune, especially T cell, development to be a phylogenetically powered process that is being perfected over the evolutionary history of a species. Only time and weight of scientific evidence favoring one or the other view will settle that debate.


1. Branton, William G., et al. “Brain microbial populations in HIV/AIDS: α-proteobacteria predominate independent of host immune status.” PloS one 8.1 (2013): e54673. http://journals.plos.org/plosone…

2. Lozupone, Catherine A., et al. “Meta-analyses of studies of the human microbiota.” Genome research 23.10 (2013): 1704-1714. Meta-analyses of studies of the human microbiota

3. Goodrich, Julia K., et al. “Conducting a microbiome study.” Cell 158.2 (2014): 250-262. http://ac.els-cdn.com/S009286741…

4. McMurdie, Paul J., and Susan Holmes. “Waste not, want not: why rarefying microbiome data is inadmissible.” PLoS computational biology 10.4 (2014): e1003531. http://journals.plos.org/ploscom…

5. Sinha, Rashmi, et al. “The microbiome quality control project: baseline study design and future directions.” Genome biology 16.1 (2015): 276. https://genomebiology.biomedcent…

6. Weiss, Sophie, et al. “Correlation detection strategies in microbial data sets vary widely in sensitivity and precision.” ISME J 10.7 (2016): 1669-1691. https://www.researchgate.net/pro…

7. Boers, Stefan A., Ruud Jansen, and John P. Hays. “Suddenly everyone is a microbiota specialist.” Clinical Microbiology and Infection 22.7 (2016): 581-582. http://www.clinicalmicrobiologya…

8. Bik, Elisabeth M. “Focus: microbiome: the hoops, hopes, and hypes of human microbiome research.” The Yale journal of biology and medicine 89.3 (2016): 363. https://www.ncbi.nlm.nih.gov/pmc…

9. Salter, Susannah J., et al. “Reagent and laboratory contamination can critically impact sequence-based microbiome analyses.” BMC biology 12.1 (2014): 87. https://bmcbiol.biomedcentral.co…

10. Diamond, Betty, et al. “It takes guts to grow a brain.” Bioessays 33.8 (2011): 588-591. https://www.researchgate.net/pro…

11. Al-Asmakh, Maha, et al. “Gut microbial communities modulating brain development and function.” Gut microbes 3.4 (2012): 366-373. http://www.tandfonline.com/doi/p…

12. Collins, Stephen M., Michael Surette, and Premysl Bercik. “The interplay between the intestinal microbiota and the brain.” Nature reviews. Microbiology 10.11 (2012): 735.

13. Tillisch, Kirsten. “The effects of gut microbiota on CNS function in humans.” Gut microbes 5.3 (2014): 404-410. https://www.ncbi.nlm.nih.gov/pmc…

14. Sampson, Timothy R., and Sarkis K. Mazmanian. “Control of brain development, function, and behavior by the microbiome.” Cell host & microbe 17.5 (2015): 565-576. http://ac.els-cdn.com/S193131281…

15. Louveau, Antoine, et al. “Structural and functional features of central nervous system lymphatics.” Nature 523.7560 (2015): 337. https://www.researchgate.net/pro…

16. Tirumalai Kamala’s answer to Why aren’t there lymph nodes in the brain?

17. Tirumalai Kamala’s answer to Why did it take so long to discover that the brain is connected to the immune system?


Each B cell is antigen specific. How many such B cells would be present for a particular antigen in the whole body?

It’s difficult enough to estimate the total number of B cells in the body, let alone the number of B cells specific for any given antigen, i.e., antigen-specific B cell frequency, aka precursor frequency. Additional obstacles include the fact that the pool of cells being analyzed include

  • B cells at various stages of development, especially in the bone marrow.
  • Not just conventional adaptive but also innate B cell subsets.
  • Not just naive (antigen-inexperienced) but also memory B cells.

Though they all express antigen-specific receptors, the B-cell receptor – Wikipedia (BCR), which when secreted is called the antibody, B cells aren’t a monolithic entity. Rather, the classical B (as also T) cell subsets with somatically recombined antigen receptors (V(D)J recombination – Wikipedia) belong to the adaptive immune system, which is characterized by remarkable diversity. Such classical or conventional B cells are B-2 or Follicular B cells. They constitute the bulk of B cells in the lymph nodes, spleen, bone marrow and in circulation.

However, other B cell subsets such as B-1 cell – Wikipedia and Marginal zone B-cell – Wikipedia (MZ B) also secrete antibodies, mostly IgM, some IgG3, usually also termed Natural antibodies – Wikipedia, but they tend to not somatically recombine their antigen receptors, i.e., they retain germline receptors, to not circulate, to not convert into memory cells, and to perform their effector functions without the help of T cells.

Thus, frequency of a given antigen-specific B cell is obviously very different between the ‘innate’ and ‘adaptive’ subsets of B cells. Greater the receptor diversity, lower the frequency of any specificity, simply for a practical reason, there just isn’t enough space in the body to house expanded numbers of each and every antigenic specificity, given that the total B cell pool in the body is estimated to be 1 to 2 X 10^11 (1, 2; also see below from 3).

Frequency of individual B cell specificities in humans are also exceedingly difficult to estimate simply because of difficulty of access to source material. Blood, an obvious choice to sample, is estimated to harbor only ~2% of the total B cell pool (4, also see above from 3), with much higher numbers present in lymph nodes (~28%), spleen (~23%) and red bone marrow (~17%) (3, 4).

Add the additional complexity that humans are estimated to have ~600 to 750 lymph nodes (3, 5, 6, 7) and difficulty of enumerating antigen-specific B cell frequency becomes more than obvious. Further, even a mere ~2% of total body B cell numbers still amounts to ~ 2 to 4 X 10^9 B cells in blood. Reasonably accurate estimates crucially turn on how much needs to be minimally sampled and method(s) used to sequence and quantitate BCRs (see below from 8, emphasis mine).

The consequences of insufficient biological sampling have been investigated previously by Warren and colleagues [26]: they showed that distinct 20 ml blood samples from the same individual captured only a portion of the TCR peripheral blood repertoire (biological undersampling). Furthermore, technological undersampling has been shown to compromise the detection of ‘public’ clones (clones shared across individuals), which are a common target in immune repertoire studies [27,28]. In fact, several studies indicated that there was a positive correlation between sequencing depth and the number of public clones detected [13,29,30]. Thus, the biological conclusiveness of a study benefits from the implementation of biological replicates (test for biological undersampling [26,31,32]) (Figure 1A) and technical replicates (test for technological undersampling [33–36]) (Figure 1A), which may be performed once for each cell population analyzed. It is important to note that biological undersampling can only be meaningfully addressed if sufficient technological sampling has been established [33]. Furthermore, species accumulation and rarefaction analyses may be performed to quantify the extent of (under)sampling [29,33,35,37]

Thus, there is likely to be substantial margin of error in estimates based on blood B cells. As well, antigen-specific B cell frequency is unsurprisingly extremely dynamic, changing with age and unpredictably varying exposure to antigens over time.

For what it’s worth, a commonly bandied about estimate of antigen-specific B cell frequency in the circulating, naive repertoire is one in 10^5 to 10^6. Extrapolating from blood and totally disregarding the contribution of memory and innate B cells to the estimated total B cell number of 1 to 2 X 10^11, that means each B cell specificity could range from 1 to 2 X 10^5 to 10^6 as also a total of 1 to 2 X 10^5 to 10^6 different individual B cell specificities or capacity to bind that many different antigens. Obviously, since memory and innate B cells are indeed part of the total B cell number, actual numbers of naive B cells specific for a particular antigen are likely markedly lower.

Taking such estimates at face value, is that sufficient frequency and diversity, given that over the course of a lifetime an anticipatory defense system such as the B cell has to contend with a potential universe of antigens that is likely orders of magnitude higher? Important at this point to recall that in B cells, the naive or antigen-inexperienced repertoire diversity is bolstered, maybe even more than amply so, by three other cardinal features, namely, clonal proliferation, Cross-reactivity – Wikipedia (which some refer to as polyreactivity) and Somatic hypermutation – Wikipedia (SHM), with that last, SHM, being a unique property of conventional B cells.

  • Clonal proliferation is the capacity of an activated B or T cell to quickly proliferate (some estimates even suggest dividing every 16 to 20 hours). Progeny of each such single B or T cell are their clones having the exact same antigen receptor (BCR or TCR). Thus, at the height of an immune response, say during the acute phase of an infection, frequencies of individual B or T cells could increase to as many as 1 in 10^3 to 1 in 10^4, i.e., a 100 to 1000-fold increase, at least locally. Clonal proliferation helps bolster the sufficiency of antigen-specific B cell frequency.
  • Cross-reactivity (aka polyreactivity) is the capacity for a given BCR (and antibody) to bind more than one antigen. Often but not always, this relates to structural similarity between different antigens. After all, though the antigenic universe is vast, biology still dictates its sequence and structural constraints. Cross-reactivity helps mitigate the potential insufficiency of antigen-specific B cell diversity.
  • SHM is the process by which conventional B cells that bound their specific antigen and presented pieces of it in the MHC (Major histocompatibility complex – Wikipedia) to cognate T cells receive T cell help that drives mutations within the V gene segment of the BCR. This creates BCR (and antibody) variants additional to those generated during primary B cell development by somatic recombination. Thus, though monozygotic (identical) twins have nearly identical primary antibody repertoire (9), meaning it is largely the product of genetic factors, antigenic experience over time, which can be highly individual and variable, leads to a secondary repertoire that can vary substantially between individuals, even identical twins. SHM helps enhance antigen-specific B cell diversity.


1. Morbach, H., et al. “Reference values for B cell subpopulations from infancy to adulthood.” Clinical & Experimental Immunology 162.2 (2010): 271-279. Reference values for B cell subpopulations from infancy to adulthood

2. Greiff, Victor, et al. “Bioinformatic and statistical analysis of adaptive immune repertoires.” Trends in immunology 36.11 (2015): 738-749. https://www.researchgate.net/pro…

3. Apostoaei, A. Iulian, and John R. Trabalka. “Review, Synthesis, and Application of Information on the Human Lymphatic System to Radiation Dosimetry for Chronic Lymphocytic Leukemia.” Inc., Tennessee (2012). https://www.cdc.gov/NIOSH/ocas/p…

4. Georgiou, George, et al. “The promise and challenge of high-throughput sequencing of the antibody repertoire.” Nature biotechnology 32.2 (2014): 158-168. https://www.researchgate.net/pro…

5. Trepel, F. “Number and distribution of lymphocytes in man. A critical analysis.” Klinische Wochenschrift 52.11 (1974): 511-515.

6. Valentin, Jack. “Basic anatomical and physiological data for use in radiological protection: reference values: ICRP Publication 89.” Annals of the ICRP 32.3 (2002): 1-277. http://dspace.elib.ntt.edu.vn/ds…

7. Agur, Anne MR, and Arthur F. Dalley. Grant’s atlas of anatomy. Lippincott Williams & Wilkins, 2009.

8. Greiff, Victor, et al. “Bioinformatic and statistical analysis of adaptive immune repertoires.” Trends in immunology 36.11 (2015): 738-749. https://www.researchgate.net/pro…

9. Glanville, Jacob, et al. “Naive antibody gene-segment frequencies are heritable and unaltered by chronic lymphocyte ablation.” Proceedings of the National Academy of Sciences 108.50 (2011): 20066-20071. http://www.pnas.org/content/108/…


How does PD-L1 checkpoint inhibition selectively target cancer cells but not healthy cells?


, ,

PD-L1 – Wikipedia checkpoint inhibition doesn’t selectively target cancer cells. Rather, targeting PD-L1 with a PD-L1-specific monoclonal antibody (mAb) prevents it from engaging with its ligand, PD-1 (Programmed cell death protein 1 – Wikipedia), a cell-surface molecule largely, though not exclusively, expressed on the surface of immune cells such as T (both CD4 and CD8), B, NK (Natural Killer) T cells, monocytes and some DCs (dendritic cells) (1, 2).

PD-L1 and PD-L2 are expressed on the cell-surface of a much wider variety of cells, being reported not just on T and B cells but also endothelial and epithelial cells, heart, lung, skeletal muscle, placenta, among others (2, 3). PD-L1 became relevant for cancers when multiple studies reported (3)

  • Its high level expression by many types of cancers such as Breast, Cervix, Colon, Esophagus, Liver, Lung, Kidney, Ovary, Pancreas, Skin.
  • Its expression by tumors correlated with poorer patient prognosis.

At the same time, multiple studies also correlated high PD-1 expression levels on Tumor-infiltrating lymphocytes – Wikipedia (TILs) with poor prognosis of cancer patients as well as poor effector function (anti-tumor activity) of such TILs in in vitro studies (3).

High PD-L1 expression on tumor cells is considered a tumor adaptation attempting to thwart effective anti-tumor immune responses by inhibiting PD-1-expressing TILs. Persistent T cell expression of PD-1 is interpreted as a sign of T cell exhaustion, a colorful description signifying the cell is or has become poorly capable of performing its antigen-specific effector functions.

  • In the case of helper CD4 T cells, PD-1 expression implies poor capacity to help B and cytotoxic CD8 T cells perform their effector functions.
  • In the case of cytotoxic CD8 T cells, PD-1 expression implies poor capacity to kill their target cells.

The hope behind PD-L1 or PD-1 blockade is doing so would release from inhibition PD-1-expressing cancer-specific T cells present in the tumor (and maybe even anywhere else in the body), and thus render them capable of attacking and ridding of the tumor since blocking PD-1PD-L1 engagement was found to reverse lymphocyte effector function inhibition, at least in preclinical (mouse model) studies.

Ideally, the most optimal cancer immunotherapy approach would be cancer antigen-specific since they would likely be those with minimal collateral cost. For example, where an immune cell, say a cytotoxic CD8 T cell specific for a cancer cell antigen, bound its target antigen on the surface of a cancer cell and killed it.

Obviously, PD-L1 or PD-1 blockade is a very different process, affecting not just tumor antigen-specific lymphocytes but others as well so it’s not surprising to note then that it specifically and checkpoint inhibitors in general have at least two major drawbacks.

  • They are not antigen-specific in the strict immunological sense, i.e., they do not target an antigen expressed only by the tumor but not by healthy cells. Thus there is scope for off-target effects (4), meaning attack on non-tumor tissue(s) as well. The hope there is that careful application of blockade dose and frequency would help focus the Rx more to cancer cells and help mitigate targeting of healthy tissue cells.
  • Tumor-infiltrating and therefore presumably tumor-specific T cells could express not just PD-1 but multiple cell-surface inhibitory receptors such as LAG3 – Wikipedia (5) and TIM-3 (HAVCR2 – Wikipedia) (6). Blocking PD-1 alone on such T cells might not suffice to reverse their inhibition. May need to block these other inhibitory molecules as well.

PD-L1 blockade also suffers from an additional drawback, namely, the lack of reliable identification, which means lack of reliable targeting. Identification of PD-L1 expressing cells was mired in technical difficulties since antibodies specific for human PD-L1 had a track record of poor validation. This made it hard to accurately and reliably ascertain whether a particular tumor sample expresses PD-L1 or not. This improved only in recent years after technically validated Immunohistochemistry – Wikipedia (IHC) assays using specific anti-human PD-L1 antibody clones such as Dako/BMS clone 28-8, Merck’s mAb clone 22C3, and Ventana (Genentech/Roche) mAb clone SP142 appeared on the scene (7, 8).


1. Ishida, Yasumasa, et al. “Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death.” The EMBO journal 11.11 (1992): 3887. https://www.ncbi.nlm.nih.gov/pmc…

2. Keir, Mary E., et al. “PD-1 and its ligands in tolerance and immunity.” Annu. Rev. Immunol. 26 (2008): 677-704.

3. Ohaegbulam, Kim C., et al. “Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway.” Trends in molecular medicine 21.1 (2015): 24-33. https://pdfs.semanticscholar.org…

4. Fay, André P., et al. “The management of immune-related adverse events associated with immune checkpoint blockade.” Expert Review of Quality of Life in Cancer Care 1.1 (2016): 89-97. http://www.tandfonline.com/doi/p…

5. Matsuzaki, Junko, et al. “Tumor-infiltrating NY-ESO-1–specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer.” Proceedings of the National Academy of Sciences 107.17 (2010): 7875-7880. http://www.pnas.org/content/107/…

6. Du, Wenwen, et al. “TIM-3 as a Target for Cancer Immunotherapy and Mechanisms of Action.” International journal of molecular sciences 18.3 (2017): 645. TIM-3 as a Target for Cancer Immunotherapy and Mechanisms of Action

7. Herbst, Roy S., et al. “Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients.” Nature 515.7528 (2014): 563. http://www.livewell-bioscience.c…

8. Gandini, Sara, Daniela Massi, and Mario Mandalà. “PD-L1 expression in cancer patients receiving anti PD-1/PD-L1 antibodies: A systematic review and meta-analysis.” Critical reviews in oncology/hematology 100 (2016): 88-98. https://www.researchgate.net/pro…


Is there any scientific proof that vaccines cause autism?

This answer briefly summarizes some overarching inferences,

  • Autism – Wikipedia / Autism spectrum – Wikipedia (Autism Spectrum Disorders, ASD) rates started greatly increasing in some countries such as the US and UK since the 1980s even as doctors little understood these conditions and offered little of value to increasingly anxious parents desperately seeking definitive answers. Thus, in such an Autism causation vacuum, Andrew Wakefield – Wikipedia et al’s 1998 Lancet report (1), the first to offer an explanation for the ‘autism epidemic’, became a convenient crutch for many frustrated parents who felt either ignored or condescended to by the medical establishment.
  • However, in ~20 years, there’s surprisingly scant scientific evidence to support the contention that ‘vaccines cause autism’. Surprising because 20 years is a long enough period to be able to bolster the argument with solid data sets.
  • Even taken at face value, many risk factors about Autism/ASD simply cannot be explained by a ‘vaccines cause autism’ notion. The more facts it can explain about a given phenomenon, the stronger a given hypothesis. That is just not the case with the ‘vaccines cause autism’ notion, which is simply inherently scientifically weak.

On a topic so controversial as a potential vaccine(s)-Autism link, it may be best to start by scrutinizing the original data that got this particular idea started. In 1998, Andrew Wakefield and 12 co-authors published a Lancet article on 12 children, claiming they had identified in them evidence of a novel syndrome they called Autistic enterocolitis – Wikipedia (1).

To digress just a bit at first, it’s somewhat surprising that there isn’t yet an agreed-upon consensus on the etiquette regarding scientific papers that have been retracted (2, 3). Specifically, should they continue to be cited in the literature or not? For example, this Wakefield et al paper continues being cited, 85 times already over six months in 2017 according to Google Scholar.

This answer however requires not just citing this paper but also looking at what it actually says since it subsequently served as the launchpad for a purported vaccine(s)-Autism link. While 8 of these 12 children (67%) had received the MMR vaccine by the time of their symptom onset, the authors concluded (see below from 1, emphasis mine),

‘We identify associated gastrointestinal disease and developmental regression in a group of previously normal children, which was generally associated in time with possible environmental triggersWe did not prove an association between measles, mumps, and rubella vaccine and the syndrome described…If there is a causal link between measles, mumps, and rubella vaccine and this syndrome, a rising incidence might be anticipated after the introduction of this vaccine in the UK in 1988. Published evidence is inadequate to show whether there is a change in incidence22 or a link with measles, mumps, and rubella vaccine.23′

Since these authors did suggest ‘a rising incidence [of their newly coined syndrome] might be anticipated after the introduction of this [MMR] vaccine in the UK in 1988‘, if we give them the benefit of the doubt and assume their autistic enterocolitis concords to some extent with Autism, what do epidemiological data show so far? In a nutshell, nothing that supports their supposition. On the contrary, such studies haven’t found a link between vaccines and Autism.

  • A 1999 study of children in North Thames, London, found rising cases of ASD since 1979 without a sharp increase after MMR was introduced in 1988 (4).
  • A 2001 British study found that while Autism rates in 2 to 5 year olds had increased from 8 boys per 10000 to 29, a 3.6-fold increase, from 1988 to 1993, rates of MMR vaccination had remained stable across these birth cohorts, meaning it wasn’t possible to attribute the Autism rate increase to the MMR vaccine (5).

Thus an examination of the original paper that jump-started the vaccines-Autism controversy finds it did not even make such an assertion and that subsequent studies found no evidence of such a link either. OTOH, one detailed review after another has since found that the MMR vaccine

  • Is safe (6, 7).
  • Is unlinked to Autism (8).

The furore, notoriety and controversy about a link between vaccines and Autism begins with this one study of a mere 12 children, only 8 of whom had received the MMR vaccine by the time of their symptom onset, and it turns out the study didn’t even make that claim. Also more accurately, the paper explores not a link between vaccines and Autism in general but rather one specifically between the MMR vaccine and autistic enterocolitis, a syndrome that isn’t listed in medical textbooks.

So, how did a link between vaccines and Autism even get made? Turns out to have been a subsequent interpretation (3), perhaps helped along by an immediate press conference when this paper was published followed by copious contemporaneous sensationalist front-page coverage by several British newspapers (9) of a kind that suggests (3) many couldn’t even be bothered to read what was actually in the paper.

Subsequent uncovering of undisclosed conflicts of interest behind Wakefield’s study followed by predictable establishment backlash against him then cast him in the potent ‘martyr’ mode, which further solidified and enhanced his reputation among parents desperately seeking definitive answers to their children’s Autism/ASD diagnosis, and who also felt Wakefield took them seriously while feeling the medical establishment didn’t (9).

How Autism’s Causation Vacuum was Fertile Soil for Wakefield’s Vaccine-Autism Supposition to take Root

On the face of it, it seems astounding that one small study on 12 patients should have had such an outsize impact. And yet, maybe not so surprising from a sociological perspective. At the time the Wakefield et al paper came out, Autism/ASD rates had already been spiking for several years with no satisfactory explanation from the medical establishment. Perhaps unwittingly, this state of affairs helped stoke and sustain this particular controversy.

  • Autism diagnosis remains the purview of behavioral scientists who base the diagnosis on a highly subjective checklist, not an impartial, objective, quantitative diagnostic test.
  • Even as they tweaked and improved their diagnostic toolkit, which in turn led to increasing rates of diagnosis, doctors had no clear answer for why steadily increasing numbers of children were being diagnosed with Autism from the 1980s, especially in the US and UK.
  • Still little understood, neither reliable objective diagnosis nor specific treatment, let alone cure, yet existed for Autism/ASD, a situation little changed in the years since.
  • With increasing numbers of parents desperately seeking answers to their children’s predicament, a causation vacuum concerning Autism was precisely calamitous and in hindsight, the Wakefield paper appears to have arrived at just the right moment to fill it with something that no one had proposed thus far, a ‘medical explanation for the autism epidemic‘ (see below from 9, emphasis mine).

‘However, the fact that there was no other reported or known reason for the ‘epidemic’ did not exactly help matters. Whatever their overall validity, vaccine hypotheses did plug a gaping hole in scientific knowledge about this condition that everyone thought had been measured so precisely and accurately with a wealth of new measurement tools and scales. How could it be that no one actually knew why autism was increasing?…Wakefield’s work was so popular because it promised so much. It promised to fully explain the autism epidemic, thus it was particularly ironic that epidemiological sciences never supported his claims.’

  • Autism/ASD having historically been and tending to remain the purview of behavioral scientists may, in the grand scheme of things, turn out to have been a major stumbling block that stymied accelerated understanding of these conditions.
  • Ironically, by highlighting gastrointestinal issues in autistic children, Wakefield may have done Autism/ASD research a huge service. After all, ~20 years on, the gut microbiota-brain link is so much better appreciated now and indeed gut Dysbiosis – Wikipedia is today well-recognized as a cardinal feature in substantial numbers of Autism/ASD patients (10, 11).
  • There was and is an urgent need for a more multi-disciplinary approach for both research and diagnosis in the Autism/ASD field. Gastroenterologists, immunologists, microbiologists, geneticists and other specialists would only help not impede better understanding of these conditions by helping develop more scientifically robust diagnostic approaches and helping tailor more targeted therapies.
  • Even in 2017, such cross-disciplinary research on Autism/ASD is sorely lacking. A simple literature search is a clear indication of this. My search for ‘Autism’ in both Nature Reviews Immunology and Nature Reviews Microbiology together turned up a total of only 24 articles, 2001-2017 (12), only 19 in Nature Reviews Gastroenterology and Hepatology, though through 2006-2017, which suggests the gut-microbiota-brain axis is becoming a bigger focus of research (13), while the same search in Nature Reviews Neuroscience turned up almost 10X higher articles (219), 2001-2017 (14). For context, the Nature Reviews series are typically considered among the most influential science review journals for various subjects.
  • History also suggests the Wakefield idea fills the Autism/ASD causation vacuum rather like a square peg in a round hole. After all, it is inherently scientifically weak since there are so many Autism/ASD risk factors that effects of vaccines, adverse or otherwise, simply cannot explain.

So many Autism/ASD Risk Factors that Vaccines can’t explain

How could vaccines possibly explain

  • Why Autism/ASD is more common in boys than girls, ranging from ~4:1 in the 1990s (15) to ~9:1 by the 2010s (16, 17, 18)? If vaccines ’cause’ autism, a person’s gender shouldn’t matter.
  • Why Autism/ASD rates are so much higher in monozygotic (identical) (70-90% concordance) compared to dizygotic (fraternal) (0-30% concordance) twins (19, 20, 21)?
    • Found in disparate populations such as in the UK (22) as well as in Scandinavia (23).
    • Monozygotic twin concordance for autism is a long-standing feature, being observed right from the 1970s in pioneering studies by Michael Rutter – Wikipedia (24).
    • Autism thus has an unmistakably strong genetic component (22), something that could not be explained by environmental factors alone such as effects of vaccine(s), adverse or otherwise.
    • If vaccines ’cause’ autism, a person’s genetic background shouldn’t matter.
  • Autism/ASD connection with maternal and child antibiotic use reported in several studies (25, 26, 27)? This alludes to a different environmental trigger, namely, changes in gut microbiota composition.
  • Consistently identified Autism risk factors such as exposure to traffic-related air pollutants, increased parental age, maternal obesity, diabetes and folic acid deficiency, prenatal viral infection, C-section, preterm birth, low birth weight, limited or absent breastfeeding, abnormal melatonin synthesis, hyperbilirubinemia, zinc deficiency, and maternal immigrant status (28, 29, 30, 31, 32, 33)? These factors and vaccines are simply unconnected.

Autism/ASD are clearly multi-factorial, with both genetic and environmental factors intersecting in as-yet undeciphered ways, and since rates started to increase dramatically since the 1980s, clearly some environmental factor(s) are key. However, those factors still remain stubbornly unclear. Rather than vaccines, however, multiple studies since at least 2004 have consistently reported altered gut microbiota composition in ASD subjects (10, 11). Whether that’s cause or effect still remains to be determined.


Basing anti-vaccine sentiment on a purported vaccines-autism link is reckless and dangerous since it inflicts real cost in the form of needless deaths from vaccine preventable diseases. Consider measles where the vaccine is historically one of the safest on record. In June 2017, a six year old Italian leukemia patient died from measles complications after reportedly catching it from his older brother, whom his parents had decided not to vaccinate (34), the latest in a measles ‘tragedy’ that has so far taken 35 lives across Europe (35).


1. Wakefield, Andrew J., et al. “RETRACTED: Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children.” (1998): 637-641. http://www.thelancet.com/pdfs/jo…

2. da Silva, Jaime A. Teixeira, and Judit Dobránszki. “Highly cited retracted papers.” Scientometrics 110.3 (2017): 1653-1661.

3. Collins, Harry M., Luis Reyes‐Galindo, and Paul Ginsparg. “A note concerning primary source knowledge.” Journal of the Association for Information Science and Technology 68.5 (2017): 1105-1110. https://arxiv.org/ftp/arxiv/pape…

4. Taylor, Brent, et al. “Autism and measles, mumps, and rubella vaccine: no epidemiological evidence for a causal association.” The Lancet 353.9169 (1999): 2026-2029. https://www.researchgate.net/pro…

5. Kaye, James A., Maria del Mar Melero-Montes, and Hershel Jick. “Mumps, measles, and rubella vaccine and the incidence of autism recorded by general practitioners: a time trend analysis.” Bmj 322.7284 (2001): 460-463. https://pdfs.semanticscholar.org…

6. Halsey, Neal A., and Susan L. Hyman. “Measles-mumps-rubella vaccine and autistic spectrum disorder: report from the New Challenges in Childhood Immunizations Conference convened in Oak Brook, Illinois, June 12–13, 2000.” Pediatrics 107.5 (2001): e84-e84. http://pediatrics.aappublication…

7. Demicheli, Vittorio, et al. “Vaccines for measles, mumps and rubella in children.” Cochrane Database Syst Rev 4.4 (2005). https://www.researchgate.net/pro…

8. Stratton, Kathleen, et al. “Immunization safety review: measles-mumps-rubella vaccine and autism.” (2001). https://www.ncbi.nlm.nih.gov/boo…

9. Evans, Bonnie. The metamorphosis of autism. Manchester University Press, 2017. https://www.ncbi.nlm.nih.gov/boo…

10. Mayer, Emeran A., David Padua, and Kirsten Tillisch. “Altered brain‐gut axis in autism: Comorbidity or causative mechanisms?.” Bioessays 36.10 (2014): 933-939.

11. Hsiao, Elaine Y. “Gastrointestinal issues in autism spectrum disorder.” Harvard review of psychiatry 22.2 (2014): 104-111. https://pdfs.semanticscholar.org…

12. nature.com search

13. nature.com search

14. nature.com search

15. Baron-Cohen, Simon, and Jessica Hammer. “Is autism an extreme form of the” male brain”?.” Advances in Infancy research 11 (1997): 193-218. https://pdfs.semanticscholar.org…

16. Whiteley, Paul, et al. “Gender ratios in autism, Asperger syndrome and autism spectrum disorder.” Autism Insights 2 (2010): 17. https://www.researchgate.net/pro…

17. Ruzich, Emily, et al. “Sex and STEM occupation predict autism-spectrum quotient (AQ) scores in half a million people.” PloS one 10.10 (2015): e0141229. Sex and STEM Occupation Predict Autism-Spectrum Quotient (AQ) Scores in Half a Million People

18. Baron-Cohen, Simon, et al. “Elevated fetal steroidogenic activity in autism.” Molecular psychiatry 20.3 (2015): 369. https://www.nature.com/mp/journa…

19. Muhle, Rebecca, Stephanie V. Trentacoste, and Isabelle Rapin. “The genetics of autism.” Pediatrics 113.5 (2004): e472-e486. https://www.researchgate.net/pro…

20. Rosenberg, Rebecca E., et al. “Characteristics and concordance of autism spectrum disorders among 277 twin pairs.” Archives of pediatrics & adolescent medicine 163.10 (2009): 907-914. https://www.researchgate.net/pro…

21. Hallmayer, Joachim, et al. “Genetic heritability and shared environmental factors among twin pairs with autism.” Archives of general psychiatry 68.11 (2011): 1095-1102. https://pdfs.semanticscholar.org…

22. Bailey, Anthony, et al. “Autism as a strongly genetic disorder: evidence from a British twin study.” Psychological medicine 25.1 (1995): 63-77. https://www.researchgate.net/pro…

23. Steffenburg, Suzanne, et al. “A twin study of autism in Denmark, Finland, Iceland, Norway and Sweden.” Journal of Child Psychology and Psychiatry 30.3 (1989): 405-416.

24. Folstein, Susan, and Michael Rutter. “A Twin Study of Individuals with Infantile Autism.” Autism. Springer US, 1978. 219-241.

25. Konstantareas, M. Mary, and Soula Homatidis. “Brief report: Ear infections in autistic and normal children.” Journal of autism and developmental disorders 17.4 (1987): 585-594.

26. Niehus, Rebecca, and Catherine Lord. “Early medical history of children with autism spectrum disorders.” Journal of Developmental & Behavioral Pediatrics 27.2 (2006): S120-S127.

27. Adams, James B., et al. “Mercury, lead, and zinc in baby teeth of children with autism versus controls.” Journal of Toxicology and Environmental Health, Part A 70.12 (2007): 1046-1051.

28. Landrigan, Philip J. “What causes autism? Exploring the environmental contribution.” Current opinion in pediatrics 22.2 (2010): 219-225. http://www.autism-society.org/wp…

29. Rossignol, Daniel A., and Richard E. Frye. “A review of research trends in physiological abnormalities in autism spectrum disorders: immune dysregulation, inflammation, oxidative stress, mitochondrial dysfunction and environmental toxicant exposures.” Molecular psychiatry 17.4 (2012): 389. https://pdfs.semanticscholar.org…

30. Grabrucker, Andreas M. “Environmental factors in autism.” Frontiers in Psychiatry 3 (2012). https://www.researchgate.net/pro…

31. Rossignol, D. A., S. J. Genuis, and R. E. Frye. “Environmental toxicants and autism spectrum disorders: a systematic review.” Translational psychiatry 4.2 (2014): e360. https://www.ncbi.nlm.nih.gov/pmc…

32. Ornoy, A., L. Weinstein-Fudim, and Z. Ergaz. “Prenatal factors associated with autism spectrum disorder (ASD).” Reproductive Toxicology 56 (2015): 155-169. https://www.researchgate.net/pro…

33. Ng, Michelle, et al. “Environmental factors associated with autism spectrum disorder: a scoping review for the years 2003-2013.” Chronic Diseases and Injuries in Canada 37.1 (2017). http://www.phac-aspc.gc.ca/publi…

34. Child’s death from measles caught from unvaccinated brother reignites debate in Italy

35. Measles ‘tragedy’ kills 35 across Europe – BBC News


What influences the choice of antibodies in ICC staining?



Immunocytochemistry (ICC) and Immunohistochemistry (IHC) are interchangeably used terms to describe methods that try to identify molecules within cells and tissues using specific antibodies that (could) bind them using stains to visualize such binding (1).

Antibody choice criteria in ICC/IHC staining are the same as with any other antibody-based assay, say ELISA, flow cytometry, Western blot, you name it, i.e., making sure the antibody’s performance (specificity and reproducibility) has been appropriately validated.

Critical issues are false negatives and false positives. As their application in biological techniques has exploded since the 1980s, unfortunately, antibodies have become a major source of problems fueling the ongoing science reproducibility crisis (2, 3, 4, 5, 6, 7, 8), simply because many commonly used antibodies haven’t been properly validated. Problem of antibody quality is of gargantuan proportions considering there are an estimated >180 companies producing and selling >350000 antibodies for research and clinical purposes (9).

Surveys suggest inadequate, even non-existent, antibody validation may be a chronic even epidemic issue (see below from 10, emphasis mine, also read 11).

Responses from more than 500 scientists within the life science research community who responded to the survey showed that, although 70% of respondents validate or have commercial antibodies validated, just 43% of researchers with fewer than five years’ experience reported validating purchased antibodies-and only 22% reported validating antibodies produced in-house. Still more troubling, the survey found that a third of these future lab heads do not validate at all.

Significantly, less than half (44%) of the respondents reported receiving specific training on validation and how to validate research antibodies for specific applications. When assessing barriers to validating antibodies in their research, a clear trend has emerged based on years of experience-more than 25% of researchers with fewer than five years of antibody use indicated that they “do not see the necessity” of performing antibody validation. Other perceived barriers included laboratorians feeling that it “takes too much time” (71%) and “delays my research” (51%).

The research antibody survey clearly identified a need to improve the skills and proficiency of junior scientists in application-specific antibody validation and to develop validation standards that are widely accepted. The biomedical research community must commit sufficient time, resources and expertise to educating and training scientists in best practices for antibody-based experiments and reporting, as well as application-specific validation.3The GBSI survey also indicated that journals should require authors to identify and describe all antibodies used in a study as an explicit condition of publication.4 Essential information included the name of the vendor or individual that supplied the antibody, the immunogen used to generate the antibody, the nature of the antibody preparation (e.g., polyclonal or monoclonal), catalog and lot number and validation status-including primary validation data if not previously published.

Because vendor catalog numbers are not stable over time as products are discontinued or sold,5 GBSI also recommended linking each antibody to one or more online registries or databases and a dedicated bioresource portal like the eagle-i Network6 or the Resource Identification Portal.7 In addition, recent NIH initiatives to improve the rigor and reproducibility of funded research clarified and revised grant application instructions and reviewed criteria as of January 25, 2016. They included expectations, but do not explicitly require that key biological resources such as antibodies will be “regularly authenticated to ensure their identity and validity for use in the proposed studies.”

As stated in the GBSI survey summary, “Despite their prolific use in the laboratory, however, there are no standardized and widely accepted guidelines for how to validate antibodies prior to use in preclinical research, although some approaches have been proposed.”3

Since ICC/IHC are less sensitive compared to other antibody-based assays such as ELISA, false negatives and false positives can be bigger problems with them. Antibody validation is thus essential to ensure ICC/IHC staining data are specific and reproducible. The FDA (12) defines validation as

the process of demonstrating, through the use of specific laboratory investigations, that the performance characteristics of an analytical method are suitable for its intended analytical use

Obviously commercial antibody suppliers for ICC/IHC are a dime a dozen. When looking at company catalogs,

  • Supplied Antibody Data Sheet is the most important piece of information.
  • Methods used to prepare the tissue as well as to stain it for IHC/ICC are key.
  • Antibody Validation data: Most stringent validation would include testing for specificity and reproducibility across a wide variety of antibody-based assays, viz. ELISA, Flow cytometry, Immunofluorescence (IF), IHC/ICC, Immunoprecipitation (IP), Western Blot (WB).
  • Vendor-supplied references that used a particular antibody for ICC/IHC are invaluable resources for type of tissue/cell, tissue preparation especially fixation method (13, 14), type of detection method, etc. that worked. It may well be that the antibody would work as advertised when used exactly according to published parameters but not otherwise.
  • Whenever they use antibodies in any assay, users should also carefully document vendor/supplier name, catalog and lot (or batch) numbers, antibody clone names. This information is necessary for future trouble-shooting when a previously reliably working assay starts becoming scattershot or stops working altogether.
  • Antibodypedia, a web-site that lists validated antibodies and details about the validation process, is a must-use resource for any scientist who cares about the provenance of the antibodies they use in their work.
  • Many antibodies are raised by immunizing research animals not with the entire purified protein but rather with synthetic peptides. However, synthetic peptides may not recapitulate the native protein’s 3-D structure nor its post-translational modifications (15).
  • Even monoclonal antibodies (mAbs) could prove non-specific. This happens because the B cell clones from which they’re derived are grown in research animals and antibodies they secrete collected and purified from Ascites – Wikipedia. Thing is such ascites often turn out to contain antibodies other than the mAb of interest (16).
  • One study found 7 of 20 mAbs (35%) had staining patterns that didn’t match their purported target antigen and further that five even failed to stain them (17).
  • Polyclonals often perform better than mAbs in IHC/ICC.For example, the industry standard for HER2 testing in breast cancer is the FDA-approved Herceptest antibody from Dako (18). Since polyclonal antibodies consist of a pool of different clones against the target antigen, several of whom may be of high affinity, their superiority over mAbs for ICC/IHC may be due to higher likelihood that some or all of them can bind under a range of conditions (16).

Two overarching approaches for antibody validation entail testing its binding in samples that either express or don’t express its target antigen.

  • Positive control: Insert gene for the target in question into cell lines by transient transfection, culture them, harvest and spin them down then perform ICC with the antibody in question. Antibody should specifically bind in cells transfected with the construct in question but not in those transfected with empty plasmid or plasmid with some other construct.
  • Negative control: Perform ICC with the antibody in question on cells and/or tissues where target gene has been knocked out (deleted). There should be no or minimal staining. These days with techniques such as siRNA having become mainstream, this is easier to do than ever so no excuses for not using them to validate antibody targets.

Gold standard of course is to validate the antibody in-house. Currently, consensus recommendation is a suite of five best practices approach the authors call the five pillars for antibody validation (see below from 19).

David Rimm‘s lab at Yale University has published a detailed antibody validation approach specifically applicable for IHC/ICC (see below from 16).


1. Haines, Deborah M., and Keith H. West. “Immunohistochemistry: forging the links between immunology and pathology.” Veterinary immunology and immunopathology 108.1 (2005): 151-156.

2. Rhodes, Kenneth J., and James S. Trimmer. “Antibodies as valuable neuroscience research tools versus reagents of mass distraction.” Journal of Neuroscience 26.31 (2006): 8017-8020. https://pdfs.semanticscholar.org…

3. Pradidarcheep, Wisuit, et al. “Lack of specificity of commercially available antisera: better specifications needed.” Journal of Histochemistry & Cytochemistry 56.12 (2008): 1099-1111. http://journals.sagepub.com/doi/…

4. Couchman, John R. “Commercial antibodies: the good, bad, and really ugly.” Journal of Histochemistry & Cytochemistry 57.1 (2009): 7-8. http://journals.sagepub.com/doi/…

5. Michel, Martin C., Thomas Wieland, and Gozoh Tsujimoto. “How reliable are G-protein-coupled receptor antibodies?.” (2009): 385-388. https://www.researchgate.net/pro…

6. Parseghian, Missag Hagop. “Hitchhiker antigens: inconsistent ChIP results, questionable immunohistology data, and poor antibody performance may have a common factor.” Biochemistry and Cell Biology 91.6 (2013): 378-394. http://www.nrcresearchpress.com/…

7. Schonbrunn, Agnes. “antibody can get it right: confronting problems of antibody specificity and irreproducibility.” (2014): 1403-1407. https://www.researchgate.net/pro…

8. Baker, Monya. “Blame it on the antibodies.” Nature 521.7552 (2015): 274. http://www.abgent.com.cn/assets/…

9. Antibody Resource Page | Antibody Suppliers (A-H)

10. Freedman, Leonard P. “The Case for Validation of Antibodies in Clinical Settings.” Vaccine (2017): 0. The Case for Validation of Antibodies in Clinical Settings

11. Freedman, Leonard P., et al. “The need for improved education and training in research antibody usage and validation practices.” BioTechniques 61.1 (2016): 16-18. http://www.biotechniques.com/mul…

12. https://www.fda.gov/downloads/Dr…

13. Willingham, Mark C. “Conditional epitopes: is your antibody always specific?.” Journal of Histochemistry & Cytochemistry 47.10 (1999): 1233-1235. http://journals.sagepub.com/doi/…

14. Saper, Clifford B., and Paul E. Sawchenko. “Magic peptides, magic antibodies: guidelines for appropriate controls for immunohistochemistry.” Journal of Comparative Neurology 465.2 (2003): 161-163.

15. Jensen, Brian C., Philip M. Swigart, and Paul C. Simpson. “Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific.” Naunyn-Schmiedeberg’s archives of pharmacology 379.4 (2009): 409. https://www.researchgate.net/pro…

16. Bordeaux, Jennifer, et al. “Antibody validation.” Biotechniques 48.3 (2010): 197. https://www.ncbi.nlm.nih.gov/pmc…

17. Spicer, Samuel S., et al. “Some ascites monoclonal antibody preparations contain contaminants that bind to selected Golgi zones or mast cells.” Journal of Histochemistry & Cytochemistry 42.2 (1994): 213-221. http://journals.sagepub.com/doi/…

18. Jacobs, Timothy W., et al. “Specificity of HercepTest in determining HER-2/neu status of breast cancers using the United States Food and Drug Administration–approved scoring system.” Journal of Clinical Oncology 17.7 (1999): 1983-1983. http://ascopubs.org/doi/pdfdirec…

19. Uhlen, Mathias, et al. “A proposal for validation of antibodies.” Nature methods (2016). http://www.novopro.cn/ueditor/ph…


Will a change in diet help to repel mosquitoes?

Change in diet → Change in Skin Microflora (composition and/or metabolism) → Change in Skin Volatilome – Wikipedia (odor profile) → Better repel mosquitoes sounds like a beguilingly intuitive idea. Only problem is there is little experimental data supporting it, at least thus far.

One double-blind trial of 49 American adults found eating garlic to be ineffective in repelling Aedes aegypti (1), another of 40 American adults found Vitamin B to be ineffective in repelling Anopheles stephensi (2).

However, at least one study found higher biting incidence of Anopheles gambiae among beer-drinkers in Burkina Faso compared to those who didn’t (3). Problem is this too was a small study of 25 volunteer beer-drinkers compared to 18 volunteer non-beer-drinkers. Thus, these results are inconclusive unless replicated using larger numbers. Study also didn’t examine whether beer drinking changed body odor and/or skin microflora so it doesn’t provide us with actionable information that could be used to counteract mosquito-attracting effects of beer drinking, if that is indeed the case.


1. Rajan, T. V., et al. “A double‐blinded, placebo‐controlled trial of garlic as a mosquito repellant: a preliminary study.” Medical and veterinary entomology 19.1 (2005): 84-89.

2. Ives, Anthony R., et al. “Testing vitamin B as a home remedy against mosquitoes.” Journal of the American Mosquito Control Association 21.2 (2005): 213-217. https://www.researchgate.net/pro…

3. Lefèvre, Thierry, et al. “Beer consumption increases human attractiveness to malaria mosquitoes.” PloS one 5.3 (2010): e9546. http://journals.plos.org/plosone…


Why are not antibodies formed against self antigens?


Antibodies to self antigens are a common feature of normal B cell function. And of course, antibodies to self antigens are a cardinal feature of some autoimmune diseases such as Graves Disease, Lupus, Myasthenia Gravis. What’s the difference? Plenty,

  • Source of antibodies: Usually the B cell subset is innate in the case of normal anti-self antibodies and adaptive (B2 or Follicular B cells) in the case of pathogenic anti-self antibodies.
  • Antigenic targets of the anti-self antibodies: Breakdown products of normal cell and tissue metabolism in the case of normal anti-self antibodies and critical self-components whose function is impaired when targeted in the case of pathogenic anti-self antibodies.
  • Antibody affinity: Low and high in the case of normal and pathogenic anti-self antibodies, respectively.

Each of these differences is a key component that helps shape the difference in outcome, benign when it’s part of normal B cell function and pathogenic when it’s not. Thus, anti-self antibodies made by ‘innate’ B cells are considered normal.

OTOH, Central tolerance – Wikipedia is the process by which self-reactive B cells from among the adaptive B cell repertoire are deleted during their development in the bone marrow. Specific autoimmunities can ensue when specific parts of this process break down.

Normal anti-self Antibodies

Natural antibodies – Wikipedia (NAbs) is a term used to describe circulating antibodies found in the absence of overt infections. In particular NAbs are antibodies found (1) in germ-free mice as well as human Cord blood, meaning they’re made even in the absence of microbial exposure.

Today we know Nabs

  • Bind with low affinity to various self-antigens. Low affinity means they bind weakly to their target antigens.
  • Are polyreactive, i.e., each NAb can bind multiple different antigens.
  • Mostly belong to the IgM Isotype (immunology) – Wikipedia (isotype = antibody class).

Major source of NAbs are B1a B cells (B-1 cell – Wikipedia), a subset of B cells that, unlike conventional B cells (aka B2 or Follicular), are considered part not of the adaptive but of the innate immune system.

Marginal zone B-cell – Wikipedia (MZ B cell) are another subset of innate B cells that secrete antibodies that can bind self antigens with low affinity (2).

B1a-derived NAbs are nowadays considered general purpose cleaners/janitors that help clear out varieties of molecules produced as a result of cellular metabolism that may be harmful if they persisted or accumulated (3, 4).

One compelling example is a study that reported ~30% of such NAbs In both mice and humans bind oxidation-specific Epitope – Wikipedia (antigenic determinant) (1). Oxidative stress is an inherent feature of aging, cell death, inflammation and tissue injury. It seems NAbs help maintain tissue homeostasis by clearing expected toxic by-products of these metabolic processes (5, 6). NAbs have also been found against other typical products of cell damage and death such as phosphatidylcholine and specific carbohydrate epitopes present on dying and damaged red blood cells (7).

One of the most interesting properties of some NAbs is they function as both general purpose cleaners as well as a first line of defense against specific pathogens. One well-known example in mice is a mouse NAb called the T15 Idiotype – Wikipedia.

  • T15 NAbs bind the phosphorylcholine (PC) antigen present in the cell wall of Streptococcus pneumoniae – Wikipedia (8, 9, 10). In fact, T15 NAbs constitute ~60 to 80% of all the natural anti-PC response in mice (11), where this particular antibody idiotype is conserved across different mouse strains (12), being associated with the use of a specific heavy and light chain (13), experimentally shown to be critical for protection against S. pneumoniae (14).
  • Yet, T15 NAbs seem to also be generated in response to, and to help clear, low-density lipoproteins (15).


1. Chou, Meng-Yun, et al. “Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans.” The Journal of clinical investigation 119.5 (2009): 1335. http://content-assets.jci.org/ma…

2. Lopes-Carvalho, Thiago, Jeremy Foote, and John F. Kearney. “Marginal zone B cells in lymphocyte activation and regulation.” Current opinion in immunology 17.3 (2005): 244-250.

3. Baumgarth, Nicole. “The double life of a B-1 cell: self-reactivity selects for protective effector functions.” Nature Reviews Immunology 11.1 (2011): 34-46.

4. Kaveri, Srini V., Gregg J. Silverman, and Jagadeesh Bayry. “Natural IgM in immune equilibrium and harnessing their therapeutic potential.” The Journal of Immunology 188.3 (2012): 939-945. http://www.jimmunol.org/content/…

5. Silverman, Gregg J., Caroline Grönwall, and Jaya Vas. “Natural autoantibodies to apoptotic cell membranes regulate fundamental innate immune functions and suppress inflammation.” Discovery medicine 8.42 (2009): 151-156.

6. Vas, Jaya, Caroline Grönwall, and Gregg J. Silverman. “Fundamental roles of the innate-like repertoire of natural antibodies in immune homeostasis.” Frontiers in immunology 4 (2013). Fundamental roles of the innate-like repertoire of natural antibodies in immune homeostasis

7. Hardy, Richard R., and Kyoko Hayakawa. “Development of B cells producing natural autoantibodies to thymocytes and senescent erythrocytes.” Seminars in Immunopathology. Vol. 26. No. 4. Springer Science & Business Media, 2005.

8. Briles, David E., et al. “Antiphosphocholine antibodies found in normal mouse serum are protective against intravenous infection with type 3 streptococcus pneumoniae.” Journal of Experimental Medicine 153.3 (1981): 694-705. http://jem.rupress.org/content/j…

9. Briles, DAVID E., et al. “Anti-phosphorylcholine antibodies of the T15 idiotype are optimally protective against Streptococcus pneumoniae.” Journal of Experimental Medicine 156.4 (1982): 1177-1185. http://jem.rupress.org/content/j…

10. Yother, Janet, et al. “Protection of mice from infection with Streptococcus pneumoniae by anti-phosphocholine antibody.” Infection and immunity 36.1 (1982): 184-188. http://iai.asm.org/content/36/1/…

11. Lévy, Martine. “Frequencies of phosphorylcholine‐specific and T15‐associated 10/13 idiotope‐positive B cells within lipopolysaccharide‐reactive B cells of adult BALB/c mice.” European journal of immunology 14.9 (1984): 864-868.

12. Claflin, J. L., and M. Cubberley. “Clonal nature of the immune response to phosphocholine. VII. Evidence throughout inbred mice for molecular similarities among antibodies bearing the T15 idiotype.” The Journal of Immunology 125.2 (1980): 551-558.

13. Crews, Stephen, et al. “A single VH gene segment encodes the immune response to phosphorylcholine: somatic mutation is correlated with the class of the antibody.” Cell 25.1 (1981): 59-66.

14. Mi, Qing-Sheng, et al. “Highly reduced protection against Streptococcus pneumoniae after deletion of a single heavy chain gene in mouse.” Proceedings of the National Academy of Sciences 97.11 (2000): 6031-6036. http://www.pnas.org/content/97/1…

15. Shaw, Peter X., et al. “Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity.” Journal of Clinical Investigation 105.12 (2000): 1731. Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity


Are DNA vaccines used today?

Yes, so far several veterinary but no human DNA vaccines have been approved for use (see below from 1).

Approved Prophylactic Veterinary DNA Vaccines

West Nile Innovator® against West Nile fever – Wikipedia virus in Horses. The US USDA approved this vaccine in 2005. Contains two genes encoding West Nile virus proteins (2).

Apex-IHN® against Infectious hematopoietic necrosis virus – Wikipedia (Rhabdoviridae – Wikipedia) disease in farm-raised Atlantic Salmon. Consists of a plasmid containing a virus glycoprotein gene (3).

Approved Therapeutic Veterinary DNA Vaccine

Oncept™ against Oral Malignant Melanoma in Dogs. This vaccine encodes human tyrosinase. Rationale is it would drive cross-reactive cytotoxic T cell responses against dog tyrosinase highly expressed by melanomas (4). Cross-reactive in this context means once activated by this DNA vaccine, dog T cells specific for peptides derived from human tyrosinase would then respond to similar peptides derived from dog tyrosinase. Keep in mind, this isn’t a stand-alone DNA vaccine effective against a tumor all by itself. It’s only been shown to be effective as an adjunct to surgery for oral malignant melanoma in dogs, i.e., when done after primary tumor’s been surgically removed.

Approved Gene Therapy Veterinary DNA Vaccine

DNA plasmid that expresses the natural form of the Growth hormone–releasing hormone – Wikipedia (GHRH). When given to reproductive age sows, it reduces fetal ill-health and deaths, and thus increases number of surviving babies per litter (5, 6).


1. Pereira, Vanessa Bastos, et al. “DNA vaccines approach: from concepts to applications.” World Journal of Vaccines 4.02 (2014): 50. https://pdfs.semanticscholar.org…

2. Davis, Brent S., et al. “West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays.” Journal of virology 75.9 (2001): 4040-4047. West Nile Virus Recombinant DNA Vaccine Protects Mouse and Horse from Virus Challenge and Expresses In Vitro a Noninfectious Recombinant Antigen That Can Be Used in Enzyme-Linked Immunosorbent Assays

3. Garver, Kyle A., Scott E. LaPatra, and Gael Kurath. “Efficacy of an infectious hematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhynchus tshawytscha and sockeye O. nerka salmon.” Diseases of aquatic organisms 64.1 (2005): 13-22. http://www.int-res.com/articles/…

4. Grosenbaugh, Deborah A., et al. “Safety and efficacy of a xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor.” American journal of veterinary research 72.12 (2011): 1631-1638. https://www.researchgate.net/pro…

5. Khan, Amir S., et al. “Effects of maternal plasmid GHRH treatment on offspring growth.” Vaccine 28.8 (2010): 1905-1910.

6. Khan, Amir S., et al. “A comparison of the growth responses following intramuscular GHRH plasmid administration versus daily growth hormone injections in young pigs.” Molecular Therapy 18.2 (2010): 327-333. http://www.cell.com/molecular-th…


In what ways can antibodies serve as immunogens in the body?

Question appears to ask whether a person’s own antibodies could serve as immunogens to their own immune system so that’s what this answer addresses. An Immunogen – Wikipedia is any substance capable of eliciting an immune response, i.e., target of an immune response. Though immunogen and Antigen – Wikipedia are often interchangeably used terms, key difference between the two is the final outcome. While both antigen and immunogen can bind an immune receptor, typically immunogen is used for something known to trigger an immune response.

Essential building blocks of life, proteins are far and away the singular focus of the human immune system, especially of the adaptive immune system, which consists of T and B cells. Antibodies are protein molecules so of course, they could be targets of immune responses, except there are inbuilt processes that serve as safeguards to minimize, not eliminate, such likelihood. Why minimize but not eliminate the likelihood antibodies could themselves be immunogens?

T cells are the master architects of human adaptive immune responses. B cells typically need T cell help to make antibodies, even antibodies against other antibodies. T cell help for B cells is usually called cognate, meaning the T cell ‘sees’ a portion of the same antigen as the B cell.

Antibodies, just like other protein molecules can be internalized by antigen presenting cells such as dendritic cells, macrophages and monocytes, get digested and their peptides presented to T cells within MHC molecules. However, normally, T cells with receptors specific for most of the peptide pieces generated from digesting antibody molecules would be already deleted during their development in the thymus by a process called Central tolerance – Wikipedia, the key mechanism that ensures our immune system doesn’t constantly attack our own cells, tissues and organs.

Central tolerance though is and never could be complete, no, not even in the healthiest of humans, for the simple reason that not everything that can be expressed by the body (called the periphery by immunologists, as opposed to the thymus where Central Tolerance occurs) is or could be expressed and/or presented by the thymus. This is especially the case for one particular portion of antibodies namely a part within its variable portion.

Antibodies have essentially two parts that help them perform their various functions (which immunologists call effector functions).

Peptides derived from an antibody’s hypermutated region, its CDR3, could not only be antigens but could theoretically also serve as immunogens for T cells. However, this normally rarely happens even in autoimmunity because T cell generation is a Stochastic – Wikipedia process and frequency of T cells with receptors capable of binding peptides derived from any one antibody’s CDR3 are too few to get a full-fledged immune response going.

OTOH, ordinarily an antibody’s constant portion, its FcR, is far less likely to be an immunogen. T cells whose receptors could bind FcR-derived peptides would be deleted during their development in the thymus by the process of Central Tolerance. Thus, antibody’s Fc portion could usually become targets of body’s own immune response only when normal tolerance mechanisms break down, as happens in autoimmunity. This is because as long as they belong to the same isotype (antibody class), a whole bunch of antibodies specific for different antigens would still have the same Fc portion. Thus, provided there is a problem with Central Tolerance, this increases the likelihood of sufficient T cells whose receptors could ‘see’ Fc-derived peptides to initiate and sustain an anti-antibody response.