What is a good color scheme for representing multiple data series on a scatter plot?


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This answer to ‘What is a good color scheme for representing multiple data sets on a scatter plot?‘ shows examples of

  • Ineffective versus effective scatter plots.
  • How Small multiple – Wikipedia helps represent multiple data sets on a scatter plot.
  • Effective color use (symbolisms, intensity, size) to convey quantitative information when displaying scientific data.

Edward Tufte – Wikipedia espouses and vividly demonstrates in his collector-quality books the precepts for excellent data presentation. Today his influence is so pervasive, in the form of Sparkline – Wikipedia and small multiples for example, that many of the best approaches to visually display data, in the New York Times or in Oliver Uberti’s work for the National Geographic for example, borrow heavily from him, as does this answer.

Ineffective versus Effective Scatter Plot

According to Tufte, effective visual data display focuses on the ratio of data : non-data ink and minimizes non-data ink (aka chart junk) as much as possible. See a compelling example of a scatter plot below from his book, ‘The Visual Display of Quantitative Information‘ (apologies, both figures photographed by arguably the world’s worst photographer, yours truly).

As this example shows, when design transparency is accorded the importance it inherently deserves, information rather than its presentation is maximized.

Small Multiples

In his book, ‘Envisioning Information‘ (3), Tufte defined small multiples as (4),

“Illustrations of postage-stamp size are indexed by category or a label, sequenced over time like the frames of a movie, or ordered by a quantitative variable not used in the single image itself.”

Small multiples, i.e., series of similar graphs, help

  • Make large data sets coherent by breaking down the data into more easily digestible ‘small bites’.
  • The reader/audience quickly shift from figuring how the figure works to digesting the information it conveys.
  • Draw the eyes to compare between data sets and make the trends/patterns pop out.
  • Engender simultaneous engagement with the data at different levels, both as broad overview and in fine grain.


Both aesthetics and need to accurately convey quantitative information should guide the choice of color in data visualization. In practical terms, this means

  • Usage should be consistent, i.e., same color used throughout the data series or paper to convey the same attribute.
  • Universally accepted color symbolisms can be leveraged to one’s advantage. For example, in traffic systems, green, amber/yellow/orange and red have stereotypical meanings. These can be easily extrapolated to convey similar meaning about biological data, for example, green to convey stimulation/response increase and red to convey inhibition/response reduction.
  • Black, white and shades of gray can be used to convey quantitative differences between groups within a data set. For example, rather than use the entire rainbow hue for a dose-response series, choosing white for zero/No Rx and black for maximum dose with shades of increasing gray for doses between zero and maximum would reduce unnecessary noise and communicate information within the data series far more effectively.
  • Keep color-blindness in mind when choosing colors in figures. One suggestion is to replace red with magenta in multi-color plots to accommodate those with red-green colorblindness for example (see other tips in 5).
  • Color combined with size to convey quantitative information embodies the adage, ‘less is more‘. Symbol sizes can thus be used to accurately convey quantitative differences, i.e., effect size (see an example below from 6).
  • Color intensity can be used to convey quantitative information (see a beautiful example below from 7).

See below (8) scatter plots that use both small multiples and color to try to display a substantial amount of data while trying to adhere to Tufte’s precepts about good visual data display. Things I’d do differently today:

  • Use either different colors alone or symbols alone, not together (overkill).
  • Use a less harsh, less obtrusive approach to indicate the average.
  • Make the axes and tick marks thinner (less obtrusive).

IMO, scientists interested in improving their data visualization skills would benefit from attending Tufte’s one-day courses (9), typically held in the DC area and in California. US academic institutions such as the NIH usually sign off on such educational expenses or at least they used to, and course attendance used to come with the bonus of getting all his books for free.


1. Tufte, Edward R. “The visual display of quantitative information.” 1983.

2. Kelley, Stanley, Richard E. Ayres, and William G. Bowen. “Registration and Voting: Putting First Things First1.” American Political Science Review 61.2 (1967): 359-379.

3. Tufte, Edward R. “Envisioning Information.” 1990.

4. Small Multiples – InfoVis:Wiki

5. https://www.nature.com/articles/…

6. How to Make Bubble Charts

7. Yoo, Ha-Na, Jin-Won Lee, and Jeong-Chil Yoo. “Asymmetry of eye color in the common cuckoo.” Scientific Reports 7.1 (2017): 7612. https://www.nature.com/articles/…

8. Kamala, Tirumalai, and Navreet K. Nanda. “Protective response to Leishmania major in BALB/c mice requires antigen processing in the absence of DM.” The Journal of Immunology 182.8 (2009): 4882-4890. http://www.jimmunol.org/content/…

9. Edward Tufte: Courses



What’s great and terrible about this draft paper on top journals publishing the least reliable science?


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This answer focuses on biomedical research. The draft paper (‘Top’ journals publish the least reliable science) referenced in the question, “What’s great and terrible about this draft paper on top journals publishing the least reliable science” is both great and terrible.

  • It is great that it claims using data analysis that top journals publish the least reliable science since bold claims that foster open and vigorous debate are a hallmark of science.
  • If independently verified, top journals publishing the least reliable science is terrible since doing so weakens both science itself and the public’s trust in it.

Obsessive & Relentless Focus on Scientific Novelty: The Perverse Incentive that Animates the Entire Biomedical Research Enterprise

That top journals publish the least reliable science shouldn’t be surprising since novelty rather than reliability is their prerogative. The prestige accruing to top science journals is precisely owing to their self-professed ‘high’ and ‘exclusive’ standards that mandate high degree of novelty in the research published by them. So vociferous and pervasive is this demand for novelty that it percolates down the scientific journal food chain. The degree of the novelty demand is the only difference in that standards for novelty are a lot more stringent with the top journals.

It’s self-evident that obsessive focus on novelty could harm biomedical research enterprise by causing irreproducible data to pile up. Problem is current biomedical research enterprise is locked into a vicious self-fulfilling cycle driven almost entirely by such perverse incentives. To understand how such an unfortunate state of affairs dominates biomedical research requires taking a step back to look at the whole picture and not just the scientific journal part of it.

The foundations of the present day scientific enterprise were laid in the post-WW II era. In the US this was largely the doing of mega-administrators such as Vannevar Bush – Wikipedia. Details may vary from country to country but a largely similar biomedical research enterprise structure emerged the world over. The major stakeholders and decision makers within the modern day biomedical research enterprise are (again, details in this answer focus on the US),

Employers: Universities/research institutions along with their presidents, senior management, department heads, professors.

Funding agencies: NIH, NSF, non-profits, foundations, pharma, etc.

Scientific publications: Scientific output gatekeepers who publish original research findings after they’re peer-reviewed.

Today, a biomedical researcher wishing to become an academic starts by getting a Ph.D. (or M.D. or MD-PhD) followed by several years of post-doctoral training. Hopefully this process ends with a faculty position somewhere.

  • How to be competitive in getting a faculty position? Scientific publications, the more prestigious the journal, the better the chances.
  • How to be competitive in getting the grant funding necessary to become an independent researcher? Scientific publications, the more prestigious the journal, the better the chances.
  • How to be a keynote speaker at a prestigious scientific conference? Scientific publications, the more prestigious the journal, the better the chances.
  • How to become an editor on top scientific journals? Scientific publications, the more prestigious the journal, the better the chances.
  • How to …? Rinse and repeat ad nauseam.
  • And what is the most stringent stipulation of top scientific journals? Novelty, not reproducibility.

This in a nutshell is how the perverse incentive for novelty animates the present day biomedical research enterprise structure, one where each of these stakeholders has contributed to creating and maintaining a scientific culture girded almost entirely by this one perverse incentive.

Unrealistic Demand for Scientific Research Novelty Uncouples It from Reproducibility & Fuels Unethical Data Selection Practices

Is it inevitable for scientific novelty and reproducibility to be separate? Absent an a priori reason to separate them, how the scientific enterprise operates in practice makes them so. At its core, scientific research is a journey into the unknown, even sometimes without a navigator. Research results meeting stakeholder demand for novelty cannot be simply churned out periodically on cue and yet that’s what the current system demands. Predictably, biomedical research scientists respond to such obviously untenable pressure to Publish or perish – Wikipedia by cutting corners.

Cutting corners entails doing all that’s necessary to make a viable paper, a veritable ‘sausage’, that will pass muster in the novelty stakes. This demand in turn drives practices such as

Though falling short of outright fraud, such practices are pervasive throughout the biomedical research enterprise, simply a predictable response to relentless and unrealistic demand for far too frequent output of scientific research novelty.

This is also why efforts of self-proclaimed watchdogs such as Retraction Watch – Wikipedia are so much pablum and fluff. They miss the point almost entirely since outright fraud remains a small part of scientific enterprise and thankfully so. Rather the problem is far more consequential and far more pervasive, namely, that official watchdogs such as the United States Office of Research Integrity – Wikipedia (ORI) define scientific fraud far too narrowly, limiting it to three specific acts,

  • Data Fabrication: Making data up from scratch.
  • Data Falsification: Tampering with and improperly altering data.
  • Plagiarism: Stealing another’s ideas and words without their consent.

Such a narrow purview leaves a titanic-sized hole that can be filled with the slew of pervasive unethical, not illegal, cutting corners practices outlined above that fall under the rubric of data selection or data cherry picking (colloquially referred to as ‘cooking’ or ‘massaging’ the data).


Twelve years since John Ioannidis – Wikipedia and his 2005 shot across the bow, ‘Why Most Published Research Findings Are False’, https://www.ncbi.nlm.nih.gov/pmc…, what is it but absurd to still publicly debate the same issue of lack of reproducibility in biomedical research? Nothing has changed since reproducibility still isn’t a priority for any of the three major stakeholders (employers, funders, scientific journals).

With biomedical research scientists not being held accountable if their novel results turn out irreproducible, another generation of academic wannabes graduates from within the same ecosystem and operates in the same culture that demands fealty to scientific novelty and thus the cycle perpetuates.

The entire issue of irreproducibility at the heart of biomedical research thus naturally and automatically sloughs off of stakeholder and practitioner alike to become and remain someone else’s problem. That begs the absurd question of who that someone else is or could be, absurd since this tautology only exists from science stakeholders and practitioners alike being as yet unable to openly admit that their unwavering demand for unrealistically frequent output of scientific novelty is the main reason this problem exists in the first place, one that’s only exacerbated by official scientific watchdogs turning a blind eye to unethical practices that inevitably crop up to sustain such unrealistic demands for output.

Further reading:

Tirumalai Kamala’s answer to How can we create truly large bio repositories to aid medical research?

Tirumalai Kamala’s answer to Which people have considerable potential to become the great scientists of the future?

Tirumalai Kamala’s answer to How does a successful academic research group operate?


Does mental exertion affect your body’s recovery from a cold?


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Mental exertion arguably falls under the rubric of stress. Problem is stress is inherently ambiguous and varies with individual perception such that it’s seemingly impossible to examine stress separate from our individual perception of it.

As Gailen D. Marshall explains (1),

‘Stress is a term often used to connote an adverse situation. Yet our use of the term stress derives from an engineering term that is used to reflect the impact of a situation (often called a stressor) on host homeostasis. It is best thought of as a psychophysiological process that is a product of both the appraisal of a given situation (either acutely or chronically over time) and the ability to cope with that situation. If the situation threatens harm, loss, or danger and/or the host-coping ability is deemed inadequate, the stress is termed distress. Most common uses of the term stress actually mean distress’

While the Psychoneuroimmunology – Wikipedia field hasn’t made much headway into exactly how neuroendocrine functions impact immune health, it’s not surprising that stressful life events and perceived stress both influence immune function (2). Unsurprising then that this in turn could render an individual more susceptible to common infections such as cold or prolong their symptoms.

Problem with evaluating what appears to be quite a logical surmise is that there are few carefully controlled studies which thoroughly evaluate an individual’s psychological stress and then experimentally expose them to an infectious agent.

Having said that, a couple of such studies have indeed been done in the case of the common cold and the results showed that greater an individual’s recent experience of major stressful life events and higher their levels of perceived stress, greater their susceptibility to common cold and longer the duration of their symptoms.

  • A 1991 study reported psychological stress enhanced susceptibility to the common cold (3).
    • In this study, 394 healthy subjects completed questionnaires assessing their levels of psychological stress and were then given nasal drops containing one of five respiratory viruses (either rhino, respiratory syncytial or corona).
    • 26 control subjects got saline drops. All were then quarantined and monitored for their symptoms.
    • Authors analyzed the results controlling for age, sex, education, allergic status, weight, season, number of subjects housed together and virus-specific circulating antibody levels at baseline.
    • Authors found both respiratory infection and its clinical symptoms correlated with an individual’s report of major stressful life events over the past year and were significantly increased with increasing levels of questionnaire-based psychological stress scores.
  • The same first author reported similar results in a more recent 2015 study on 360 healthy adults (4).
    • In this study, poorer an individual’s self-reported health, greater their susceptibility to developing clinical illness after being exposed to a common cold virus (rhinovirus).
    • Again, these results were found independent of age, sex, race, circulating virus-specific antibody levels at baseline, weight, season, education and income.


1. Marshall, Gailen D. “Neuroendocrine mechanisms of immune dysregulation: applications to allergy and asthma.” Annals of Allergy, Asthma & Immunology 93.2 (2004): S11-S17.

2. Moynihan, Jan A., et al. “Stress and Immune Function in Humans: A Life‐Course Perspective.” The Wiley-Blackwell Handbook of Psychoneuroimmunology (2013): 251-265.

3. Cohen, Sheldon, David AJ Tyrrell, and Andrew P. Smith. “Psychological stress and susceptibility to the common cold.” New England journal of medicine 325.9 (1991): 606-612. Psychological Stress and Susceptibility to the Common Cold — NEJM

4. Cohen, Sheldon, Denise Janicki-Deverts, and William J. Doyle. “Self-rated health in healthy adults and susceptibility to the common cold.” Psychosomatic medicine 77.9 (2015): 959. https://www.ncbi.nlm.nih.gov/pmc…


What are the advantages of delivering antibodies through a subcutaneous jab vs. IV?


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Be it antibody or any other drug, safety and efficacy are the most important considerations for injection route. When they are equivalent between two injection routes, patient preference and pharmacological attributes of the medication come into play, the first an important consideration to ensure patients continue with the Rx (Rx adherence) and the latter a time, resource and cost-saving issue. Clearly, each of these criteria is experimentally assessed for any drug. One 2014 systematic review (1) shows a clear patient preference for SC injections and also points out that patient preference unfortunately remains a back burner research issue and not a priority. Empirically, for many therapeutic Monoclonal antibody – Wikipedia (mAbs), subcutaneous (SC) was found comparable or better than intravenous (IV) (2, 3, 4).

  • ~ similar bioavailability, biological effects and rate of elimination.
  • Easier with fewer side-effects.
  • Cheaper since IV injection usually entails need for hospital or infusion center, which often end up comprising as much as 50% of total Rx cost (5).

These observations echo general advantages of SC injections (see below from 3).

Thus, it is not surprising many widely used therapeutic mAbs are indeed delivered SC (see below from 4).

A detailed and careful evaluation showed that while many of the currently prevalent mAbs are more efficacious when given SC, one exception is Abatacept – Wikipedia, used in Rheumatoid arthritis – Wikipedia, which works better when given IV (see below from 6).

All that being said, SC antibody, specifically mAb, have considerable limitations (7, 8, 9) that need to be overcome in order for the practice to supplant IV injection.

  • Like intramuscular (IM), SC drug reaches circulation slowly, only after traveling through the lymphatics or small skin capillaries. Since it requires this kind of absorption, SC is inherently much slower than IV. Being large, not small, molecules, when injected SC, antibodies and mAbs reach the bloodstream not through small blood capillaries but after traversing through lymphatic vessels, i.e., slowly.
  • Unlike IV, only limited volumes, up to 20ml, can be injected at a time SC. This means products have to be specially formulated to be much more concentrated in order to keep the injection volume that low.
  • Since antibodies and mAbs are proteins, concentrating them runs into issues of protein aggregation, solubility and viscosity. This is exacerbated by the fact that patients prefer narrow-gauge needles.
  • Increased concentration combined with slow absorption and flow into bloodstream can cause injected material to become trapped locally in interstitial fluid and get degraded there, which may limit drug bioavailability.
  • Depending on mAb features, increased local concentration could also cause local side-effects such as irritation, swelling, abscess.
  • Since SC injections can be self-administered, how to safely deal with side-effect issues could end up a bigger deal than is the case with IV injections, which are typically given in hospitals or transfusion centers.

Overcoming such limitations requires specific, case-by-case changes in formulations, case-by-case since how specific modifications could affect a particular mAb’s attributes (mode of action, bioavailability, side-effects, etc.) are very much a distinctive feature of each such mAb. Such modifications include mAb derivatization as poly ethylene glycol conjugates (PEGylation) (7) and crystalline suspensions (7, 8).


1. Stoner, Kelly L., et al. “Intravenous versus subcutaneous drug administration. which do patients prefer? a systematic review.” The Patient-Patient-Centered Outcomes Research 8.2 (2015): 145-153. http://sro.sussex.ac.uk/49501/1/…

2. Leveque, Dominique. “Subcutaneous administration of anticancer agents.” Anticancer research 34.4 (2014): 1579-1586. Subcutaneous Administration of Anticancer Agents

3. Duems-Noriega, Oscar, and Sergio Ariño-Blasco. “Subcutaneous fluid and drug delivery: safe, efficient and inexpensive.” Reviews in Clinical Gerontology 25.2 (2015): 117-146.

4. Tabrizi, Mohammad. “Considerations in establishing affinity design goals for development of antibody-based therapeutics.” Development of Antibody-Based Therapeutics. Springer New York, 2012. 141-151.

5. Tetteh, Ebenezer K., and Stephen Morris. “Evaluating the administration costs of biologic drugs: development of a cost algorithm.” Health economics review 4.1 (2014): 26. https://pdfs.semanticscholar.org…

6. Jin, Jing-fen, et al. “The optimal choice of medication administration route regarding intravenous, intramuscular, and subcutaneous injection.” Patient preference and adherence 9 (2015): 923. https://www.dovepress.com/getfil…

7. Dychter, Samuel S., David A. Gold, and Michael F. Haller. “Subcutaneous drug delivery: a route to increased safety, patient satisfaction, and reduced costs.” Journal of Infusion Nursing 35.3 (2012): 154-160.

8. Collins, D. S., et al. “Optimizing the Bioavailability of Subcutaneously Administered Biotherapeutics Through Mechanochemical Drivers.” Pharmaceutical Research 34.10 (2017): 2000-2011. https://pdfs.semanticscholar.org…

9. Jones, Graham B., et al. “Subcutaneous drug delivery: An evolving enterprise.” Science translational medicine 9.405 (2017).


How does the inflammatory tumor microenvironment affect cancer detection?


Question refers to: http://www.cell.com/cancer-cell/fulltext/S1535-6108(17)30296-9

The paper referenced in the question ‘How does the inflammatory tumor microenvironment affect cancer detection‘ describes a method to detect non-small cell lung cancer (NSCLC) using a blood-based Liquid biopsy – Wikipedia, specifically by identifying a tumor-associated change in platelets which the authors describe as tumor-educated platelets. Rather than tumor microenvironment, systemic (circulatory) inflammatory milieu has the capacity to impede liquid biopsy cancer detection.

There is currently a boom, both in terms of research funding as well as commercial investment, in such liquid biopsy methods that sample either blood, saliva, urine, cerebro-spinal fluid, sputum, lung (pleural) fluid or stool. Reasons are self-evident, such methods being minimally invasive and allowing multiple samplings over time.

Liquid biopsies could thus be used for

  • Diagnostic biomarkers to detect cancer.
  • Surrogate biomarkers to detect reproducible cancer-associated changes as in the case of tumor-educated platelets in the referenced paper.
  • Prognostic biomarkers to identify relative risk for disease progression or recurrence.
  • Predictive biomarkers to stratify patients by likelihood of response to particular Rx.
  • Pharmacodynamic biomarkers to help monitor response to Rx.

Detection targets range from circulating tumor cells (CTC) to circulating tumor cell DNA (ctDNA) or microRNA (miRNA) to reproducible changes in other parameters, such as platelets in this case, that could be used as surrogates for tumor. Notwithstanding this recent boom, depending on type and stage of cancer, substantial technical hurdles need to be overcome. Regardless method details, reliable detection of a biological analyte depends on two cardinal features, precision and accuracy.

  • Precision is the result of testing the same sample repeatedly with the same method. Following repeats of the same steps A-to-Z, more identical the results, higher the precision.
  • Accuracy is how well test sample results match those of a reference standard after both are run through the same method. Depending on target, accuracy of a specific liquid biopsy test could even be difficult to define.

These attributes reflect method reproducibility.

Systemic inflammation could play havoc with liquid biopsy test results for two inter-linked reasons.

  • Target material is typically scarce. For example, CTCs are estimated to be ~1 per billion blood cells in circulation in patients with metastatic cancer (1).
  • Systemic inflammation increases circulating levels of both normal blood cells and DNA, i.e., increase in noise.

Given the scope of the task at hand, wouldn’t be overstating to say that separating signal from noise in liquid biopsy cancer diagnosis is akin to finding the proverbial needle in a haystack. Systemic inflammation adds increase in noise to scarcity of target, which could stretch a given method’s detection capacity to breaking point, something best appreciated by visualizing the liquid biopsy process for detecting CTC and ctDNA (see below from 1).

Molecular approaches being highly sensitive, how increase in noise affects a given method’s precision and accuracy depends on its specificity. Higher the specificity, lower the scope for increased noise to impede ctDNA detection.

However, important to keep in mind that most of the current boom in liquid biopsy cancer detection is still at the early validation stage, where the focus is on patients with advanced, usually metastatic, cancer. Such individuals would have theoretically maximal numbers and amounts of CTCs and ctDNA, respectively. Real test would be capability to reproducibly detect even in those with much earlier stages of cancer or with minimal residual disease, where the target material would be much scarcer. Doing so in conditions of systemic inflammation would be the most challenging of all hurdles for such tests.


  1. Heitzer, Ellen, et al. “Circulating tumor cells and DNA as liquid biopsies.” Genome medicine 5.8 (2013): 73. https://genomemedicine.biomedcen…


Why are vaccines not given intravenously?


To understand why vaccines aren’t given intravenously (IV) requires delving into not just science but sociology as well. A combination of convenience, expediency, empirical observations and immunological dogma is why most vaccines are intramuscular (IM) jabs, rest subcutaneous (SC), oral/nasal or intradermal (ID) and why the IV route isn’t used at all.

This answer covers

  • A brief history of vaccine routes.
  • Depot‘ effect: How this immunological dogma got started and brief assessment of its impact.
  • Role practical field conditions play in choice of vaccination routes.

Brief History of Vaccine Routes

Historically, the oldest vaccines such as for smallpox were given by scarification of the skin. When a more systematic approach to vaccines took off in the late 19th century, developers simply stuck to the road already traveled by trying to deposit vaccines in the skin, only needles had arrived on the scene by then which made SC injections possible, the only exception being BCG, the tuberculosis vaccine, which empirically seemed to work better as an ID injection.

Switch away from SC started with research by Alexander Glenny – Wikipedia who discovered purified toxins such as from diphtheria and tetanus were more immunogenic when adsorbed on aluminum salts, inducing stronger immune responses (1). This discovery became the basis for routinely adding aluminum salts to sub-unit vaccines as an Immunologic adjuvant – Wikipedia.

However, giving such adjuvanted vaccines SC tended to give strong injection site reactions, which predictably led to complaints and drop-offs in vaccination rates. This in turn spurred the search for an alternative route that would be as easy as SC but without its drawback. Empirically, IM jabs of the same vaccine formulations seemed to induce just as strong immune responses without the inconvenient injection site reaction. This gave the impetus for newer sub-unit vaccines to be formulated and tested typically for IM delivery. Thus, starting in Glenny’s time in the 1920s, IM injections began to supplant SC and dominate vaccinology, helped along by empiricism and cosmetic considerations (2, see below from 3).

‘Deep intramuscular injection generally is recommended for adjuvant-containing vaccines because subcutaneous or intradermal administration can cause marked local irritation, induration, skin discoloration, inflammation, and granuloma formation.[2,][5] However, subcutaneous injection can lessen the risk of local neurovascular injury and is recommended for vaccines that are less reactogenic but immunogenic when administered by this route, such as live virus vaccines. Intradermal administration is preferred for live bacille Calmette-Guérin (BCG) vaccine.[10]’

Local responses to IM injections of vaccines aren’t readily visible and haven’t been systematically studied. Few animal model studies bothered to examine muscle tissue after an IM vaccine jab and the few that did found signs of intense, even long-term inflammation (4, 5, 6). However, this issue remained both under-researched and unaddressed for decades, in hindsight seeming to await the revitalization of research into the Innate immune system – Wikipedia, something that only got galvanized in the late 1990s. Meantime, IM injections took firm root in vaccinology while modern medicine also took shape over the same time period and again, from convenience, IM became established as a predominant injection route (7) and thus we reach present-day when most vaccines are IM jabs (see below from 8).

‘Depot’ effect: How this immunological dogma got started and brief assessment of its impact

Though practical and cosmetic considerations doubtless helped IM injections dominate vaccinology, another element absolutely crucial in doing so was a dogma that also explains why the IV route became a non-starter for vaccines.

Apart from discovering the adjuvant effects of aluminum salts, currently the most widely used adjuvant in human vaccines, Glenny’s legacy looms over vaccinology and even immunology itself in the form of the dogma called the ‘depot‘ effect.

No question, immunology was in its infancy in Glenny’s time. Key players such as T and B cells were still decades away from being discovered. Question still had to be answered though. How to explain why adding aluminum salts to purified toxins such as diphtheria and tetanus vastly increased the immune responses (measured as antisera) they elicited?

Given the limited understanding of the immune system in the early 20th century, consensus soon coalesced around a physical explanation, namely, that the crystalline aluminum salt and its adsorbed antigen(s) remain at the site of injection as a depot, which would allow the antigen(s) to be released slowly over time to serve as continuing stimulus for sustained antibody production. Clearly, IV injections couldn’t sustain a ‘depot‘ effect so they were never seriously considered for vaccines.

Mechanistic link between antigen-adjuvant ‘depot‘ and robust immunity was challenged almost from the very beginning when animal model studies summarized in a 1950 book (9) showed that strength of the immune response remained unaffected even if the injection site nodules containing antigen-adjuvant were surgically excised a mere 14 days after immunization. However, with no other satisfactory answer forthcoming, the ‘depot‘ effect took root and vaccinology more or less slumbered for decades, at least as far as trying to figure out how aluminum salts and other adjuvants boosted immune responses to the antigens conjugated to them.

The decades-long Rip Van Winkle-like slumber over how adjuvants augment immune responses got jolted in 1989 when Charles Janeway – Wikipedia wrote a hugely influential article (10) about, among other things, the immunologist’s ‘dirty’ little secret, a play of words alluding to the fact that immune responses to purified antigens typically tend to be muted or non-existent unless accompanied by ‘dirt’, by which he meant adjuvants such as CFA (Freund’s adjuvant – Wikipedia) in animal models or aluminum salts in human vaccines.

Janeway’s hypothesis was invigorated and validated by the discovery of the first mammalian Toll-like receptor – Wikipedia (TLR) by his lab in 1997 followed in short order by discoveries of CLRs (C-type lectin – Wikipedia) and NLRs (NOD-like receptor – Wikipedia) and other Pattern recognition receptor – Wikipedia (PRRs), discoveries that offered a biological explanation for the adjuvant effect and opened the door to a molecular basis for understanding how the innate immune system operates and how it co-ordinates with and/or controls the Adaptive immune system – Wikipedia.

Is the ‘depot‘ effect important or even necessary now? Though considerably weakened by the discovery of PRRs, it still retains some of its hold as an immunological dogma to explain the role of even PRRs (11) since we don’t yet fully understand how strong and long-lasting immunological memory forms nor how adjuvants work. However, regardless the need for antigen ‘depot‘, today IM vaccine injections remain a mainstay.

Role practical field conditions play in choice of vaccination routes

Vaccines are typically prophylactic, public health measures given to millions of healthy people. This difference in kind from other medical interventions influences all aspects of vaccines, from how they’re funded to pricing and delivery.

Given their public health nature, meaning intended to be given to as many as individuals as possible, often in field conditions in remote areas, obviously vaccines are designed for ease of administration. Volume, how many can be given vaccine per unit of time, is obviously a function of how easy it is for the person giving the shot. Most often, especially in field conditions, such a person may not even be a doctor or nurse but rather a public health worker. Keeping this frame of reference in mind, IM and SC injections are much easier to give compared to other routes such as ID or IV.

Simplest route would obviously be oral, both from the standpoint of ease of delivery as well as from manufacturing cost since, compared to oral, an injectable is much more expensive, its manufacturing having to pass extremely rigorous quality control measures, making its approval process more cumbersome, lengthy and expensive. Indeed, historically, one of the most used vaccines in the world was oral, the oral polio vaccine, OPV. However, when formulated orally, not all vaccines drive the robust immunity necessary whereas over time, largely empirically, the IM route proved quite effective.

In many parts of the world, birth and infancy still remain the only time period mother and child even come into contact with any type of public health infrastructure, be it a primary health clinic or small local hospital, a logistical reality for why childhood vaccinations are such a crucial fulcrum of public health measures in poorer countries. IV injections are all the more difficult in babies and infants, especially in field conditions, while IM and SC are relatively easy.


1. Tirumalai Kamala’s answer to Why is there aluminum in vaccines?

2. Kroger, A., W. Atkinson, and L. Pickering. “General immunization practices.” Vaccines 6 (2012): 88-111.

3. Kroger, A., Sumaya, C., Pickering, L., Atkinson, W. General Recommendations on Immunization. 2011. https://www.cdc.gov/mmwr/pdf/rr/…

4. RG, WHITE, COONS AH, and CONNOLLY JM. “Studies on antibody production. III. The alum granuloma.” The Journal of experimental medicine 102.1 (1955): 73-82. http://europepmc.org/backend/ptp…

5. Goto, Norihisa, and Kiyoto Akama. “Histopathological Studies of Reactions in Mice Injected with Aluminum‐Adsorbed Tetanus Toxoid.” Microbiology and immunology 26.12 (1982): 1121-1132. Histopathological Studies of Reactions in Mice Injected with Aluminum-Adsorbed Tetanus Toxoid

6. Goto, Norihisa, et al. “Local tissue irritating effects and adjuvant activities of calcium phosphate and aluminium hydroxide with different physical properties.” Vaccine 15.12-13 (1997): 1364-1371.

7. Tirumalai Kamala’s answer to Immunology: Which could induce the best immune response? Same antigen, same dose: Injected intradermally; hypodermally; intramuscularly; IV; and why?

8. http://www.immunize.org/catg.d/p…

9. Holt, Lewis Burnell. “Developments in diphtheria prophylaxis.” Developments in Diphtheria Prophylaxis. (1950).

10. Janeway, Charles A. “Approaching the asymptote? Evolution and revolution in immunology.” Cold Spring Harbor symposia on quantitative biology. Vol. 54. Cold Spring Harbor Laboratory Press, 1989.

11. Lingnau, Karen, Karin Riedl, and Alexander Von Gabain. “IC31® and IC30, novel types of vaccine adjuvant based on peptide delivery systems.” Expert review of vaccines 6.5 (2007): 741-746. https://www.researchgate.net/pro…


How do fevers work?



This answer briefly covers

  • Mechanisms that maintain core body temperature.
  • Mechanisms that raise core body temperature during fever.
  • Benefits to raised core body temperature: Typically Immune Function Enhancement & Harm to Pathogens.

Mechanisms that maintain core body temperature

For many years a single thermoregulatory center was supposed to regulate human body temperature. Now, evidence (1) suggests there are several somewhat independent loops, part of a thermoregulatory circuit, that regulate core body temperature.

Located in various parts of the body such as the hypothalamus, spinal cord, skin, and abdominal organs such as the urinary bladder, Thermoreceptor – Wikipedia monitor the body for temperature changes. These thermoreceptors work through negative feedback loops to autonomously conserve or lose body heat, the former via shivering and vasoconstriction, the latter via sweating and vasodilation. However, one site, the preoptic region of the anterior hypothalamus is still considered the major CNS thermoregulatory center that receives and integrates temperature signals from various parts of the body.

Thermoregulatory neurons present in the median preoptic nucleus of the hypothalamus are either warm or cold sensitive (2). These neurons reduce and increase their firing, respectively, in cold environments. This leads to activation of mechanisms to conserve heat, skin vasoconstriction, piloerection, reduced sweating, increased muscle contraction, non-shivering thermogenesis, and warmth seeking. Reverse occurs in hot environments leading to activation of mechanisms to dissipate heat such as vasodilation, sweating and cold seeking.

For example, the anterior hypothalamus detects the core body temperature rise during exercise (3) through the temperature of the blood passing through it and when the temperature rises past an internal set point, it triggers vasodilation of peripheral blood capillaries and sweat, both of which promote heat loss.

Thus, current thinking holds that the hypothalamus controls core body temperature the way a thermostat regulates room temperature in a house (4), responding by conserving or dissipating heat depending on external conditions (see below from 5).

Mechanisms that raise core body temperature during fever

Fever arose millions of years back in evolution (see below from 6, 7), giving rise to the notion that it is an ancient defense strategy deployed by most animals.

Fever can be both behavioral as well as physiological. An Ectotherm – Wikipedia depends on the environmental temperature to maintain its thermoregulation. Its moving to a warmer place in response to an infection is an example of behavioral fever (8, 9, 10). However, a human who chooses to swaddle in warm clothes in response to sudden drop in environmental temperature is also an example of behavioral fever.

OTOH, in human physiologic fever, the internal set point of the hypothalamic thermoregulatory center shifts upwards, apparently in response to increased local levels of Prostaglandin E2 – Wikipedia (PGE2), a prominent endogenous pyrogen (11, 12, 13, see below from 14). Such changes in turn activate neurons in the vasomotor center that start the process of vasoconstriction.

Benefits to raised core body temperature: Typically Immune Function Enhancement & Harm to Pathogens

Adaptive benefits of fever remain disputed simply because experimental studies and indeed clinical experience of fever in conjunction with serious debilities such as sepsis clearly demonstrate circumstances where outcome of fever (and associated physiological changes) can be unambiguously harmful (15, 16).

Clearly there are costs to fevers (17), costs such as anemia due to iron sequestering, anorexia due to fever-driven loss of appetite and attendant malnutrition as well as higher calorie expenditure necessary to maintain higher body temperature (18). Clearly, fever must have mitigating benefits to make it such a widespread feature across animals. Broadly speaking, two main benefits postulated for fever are

  • Immune function enhancement.
  • Harm to pathogens.

Multiple studies have demonstrated fever enhances immune function (see summations below from 15, 16).

Damage to pathogens is another obvious benefit (15, 19, 20).

Substantial scientific and clinical evidence suggests fever improves survival and reduces the duration of infections (7, 14, 21).

Fever can be Harmful to Pathogens: In Vitro Studies

From malaria parasites to Salmonella to viruses, sustained high temperature hinders their growth (22, 23).

  • Malaria parasites are found not to survive 16 hours at 41oC, with majority already dead at 8 hours (24, 25). This is why malaria parasite lab culture is typically 37oC, same as human body temperature.
  • Iron is critical for normal cellular function, especially for eukaryotic cells. Sequestering it is obviously a cost whose benefit is revealed by examining its effect on pathogens. For example, Salmonella typhimurium is unable to synthesize iron transport compounds it needs at temperatures >40oC and thus stops proliferating at such temperatures. Though poultry are often i carriers, birds are by and large less susceptible to salmonellosis, with their higher body temperature suspected to play a role in such resistance (26, 27, 28).
  • Streptococcus pneumoniae – Wikipedia can be and often is a serious respiratory pathogen in humans. While it replicates easily at 37oC, at 41oC, it cannot and dies (29).

Important to note here that tests of pathogen heat sensitivity in culture are inherently limited in scope and in fact, effect of core body temperature rise on pathogens may be even more profound for the following reasons,

  • Local heat experienced by pathogens at an infected site is largely a black box.
  • Heat exposure in culture is a blunt contrivance that cannot and indeed does not recapitulate the many other ‘inflammatory stressors’ (17) a pathogen would experience during an infection, stressors that are typically set in motion simultaneously with core body temperature rise in response to an infection and that tend to work synergistically. Consider the observation that neither heat nor iron restriction was found effective in vitro to kill Pasteurella multocida, a pathogenic bacterium, while together they could do so (30).

Fever can be Harmful to Pathogens: In Vivo Veritas

  • A mouse model study (see below left from 31) housed them at ambient temperatures ranging from 23 to 35.5oC. Mice were then intraperitoneally injected with Klebsiella pneumoniae. Though the bacteria were growing at identical rates in culture at 37oC and 39.5oC, in vivo, only the febrile temperature yielded better bacterial clearance and survival rate.
  • A seminal 1975 study (see below right from 32) established just how critical behavioral fever could be in ensuring an ectotherm’s survival from an infection. In this study the lizard Dipsosaurus dorsalis was observed to develop a fever of ~2oC when injected with the bacterium Aeromonas hydrophila. Since this bacterial infection is usually lethal, the study explored whether this body temperature increase was related to infection resistance by placing lizards infected with live bacteria either at neutral (38oC), low (34 or 36oC) or high (40 or 42oC) ambient temperature. Elevated body temperature clearly influenced survival from infection. Infected lizards placed at 42oC had maximal survival of ~80% after 7 days of infection while all of those placed at 34oC died within 4 days of infection.
  • There is even a Nobel Prize-winning experiment associated with demonstrating the benefits of fever. Julius Wagner-Jauregg – Wikipedia, the first of only two psychiatrists to have won the Nobel Prize for Medicine or Physiology, induced fever in neurosyphilis patients, Pyrotherapy – Wikipedia (20, 33), by infecting them with malaria using the least aggressive malaria parasite, Plasmodium vivax – Wikipedia, and then treated them later with quinidine. At that time, 1917, neurosyphilis was a terminal diagnosis but this form of pyrotherapy succeeded in curing patients, albeit with a malaria fatality rate of 15% (16, 34, 35).

Microbiota – Wikipedia deserve the final word or more accurately, questions. What is the effect of fever on an individual’s microbiota? Do some stay while others go? Could stable residence during and after physiological fever be an indicator of stable association? Could such stable residence be used to differentiate true symbionts or mutualists from pretenders (Janus-faced pathobionts)? Fever as purifying forest fire in other words. Or do fevers determine otherwise, embolden the more combative or even the downright nasty? Intriguing as-yet unanswered questions.


1. Romanovsky, Andrej A. “Thermoregulation: some concepts have changed. Functional architecture of the thermoregulatory system.” American journal of Physiology-Regulatory, integrative and comparative Physiology 292.1 (2007): R37-R46. http://ajpregu.physiology.org/co…

2. Porat, Reuven, and Charles A. Dinarello. “Pathophysiology and treatment of fever in adults.” UpToDate, Waltham, MA: Reward House (2004).

3. Bradford, Carl D., et al. “Exercise can be pyrogenic in humans.” American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 292.1 (2007): R143-R149. http://ajpregu.physiology.org/co…

4. Kushimoto, Shigeki, et al. “Body temperature abnormalities in non-neurological critically ill patients: a review of the literature.” Journal of Intensive Care 2.1 (2014): 14. https://jintensivecare.biomedcen…

5. Leon, Lisa R., and Robert Kenefick. Pathophysiology of heat-related illnesses. No. USARIEM-MISC-10-37. ARMY RESEARCH INST OF ENVIRONMENTAL MEDICINE NATICK MA THERMAL AND MOUNTAIN MEDICINE DIVISION, 2012. http://www.dtic.mil/get-tr-doc/p…

6. Hasday, Jeffrey D., Christopher Thompson, and Ishwar S. Singh. “Fever, immunity, and molecular adaptations.” Comprehensive Physiology (2014).

7. Mackowiak, PHILIP A. “Temperature regulation and the pathogenesis of fever.” Principles and practice of infectious diseases 6 (2000): 703-718. https://xa.yimg.com/kq/groups/23…

8. Huntingford, Frederick William Goetz, et al. “Behavioural fever is a synergic signal amplifying the innate.” (2013). https://pdfs.semanticscholar.org…

9. Mohammed, Ryan S., et al. “Getting into hot water: sick guppies frequent warmer thermal conditions.” Oecologia 181.3 (2016): 911-917. https://www.researchgate.net/pro…

10. Rakus, Krzysztof, Maygane Ronsmans, and Alain Vanderplasschen. “Behavioral fever in ectothermic vertebrates.” Developmental & Comparative Immunology 66 (2017): 84-91. http://orbi.ulg.ac.be/bitstream/…

11. Engblom, David, et al. “Microsomal prostaglandin E synthase-1 is the central switch during immune-induced pyresis.” Nature neuroscience 6.11 (2003): 1137.

12. Lazarus, Michael, et al. “EP3 prostaglandin receptors in the median preoptic nucleus are critical for fever responses.” Nature neuroscience 10.9 (2007): 1131. https://pdfs.semanticscholar.org…

13. Nakamura, Kazuhiro, and Shaun F. Morrison. “A thermosensory pathway that controls body temperature.” Nature neuroscience 11.1 (2008): 62. https://pdfs.semanticscholar.org…

14. Mackowiak, Philip A. “Concepts of fever.” Archives of Internal Medicine 158.17 (1998): 1870-1881. http://jamanetwork.com/data/Jour…

15. Shephard, Alexander M., et al. “Reverse Engineering the Febrile System.” The Quarterly Review of Biology 91.4 (2016): 419-457.

16. Harden, L. M., et al. “Fever and sickness behavior: friend or foe?.” Brain, behavior, and immunity 50 (2015): 322-333.

17. LeGrand, Edmund K., and Judy D. Day. “Self-harm to preferentially harm the pathogens within: non-specific stressors in innate immunity.” Proc. R. Soc. B. Vol. 283. No. 1828. The Royal Society, 2016. http://rspb.royalsocietypublishi…

18. Anderson, Robert D., Simon Blanford, and Matthew B. Thomas. “House flies delay fungal infection by fevering: at a cost.” Ecological Entomology 38.1 (2013): 1-10. http://www.thethomaslab.net/uplo…

19. Romanovsky, A. A., and M. Szekely. “Fever and hypothermia: two adaptive thermoregulatory responses to systemic inflammation.” Medical hypotheses 50.3 (1998): 219-226.

20. Casadevall, Arturo. “Thermal restriction as an antimicrobial function of fever.” PLoS pathogens 12.5 (2016): e1005577. http://journals.plos.org/plospat…

21. Mackowiak, Philip A., et al. “Concepts of fever: recent advances and lingering dogma.” Clinical Infectious Diseases 25.1 (1997): 119-138. https://pdfs.semanticscholar.org…

22. Bedson, H. S., and K. R. Dumbell. “The effect of temperature on the growth of pox viruses in the chick embryo.” Epidemiology & Infection 59.4 (1961): 457-470. https://www.ncbi.nlm.nih.gov/pmc…

23. Ruiz-Gomez, J., and A. Isaacs. “Optimal temperature for growth and sensitivity to interferon among different viruses.” Virology 19.1 (1963): 1-7.

24. Long, H. Y., et al. “Plasmodium falciparum: in vitro growth inhibition by febrile temperatures.” Parasitology research 87.7 (2001): 553-555. https://www.researchgate.net/pro…

25. Oakley, Miranda SM, et al. “Molecular factors and biochemical pathways induced by febrile temperature in intraerythrocytic Plasmodium falciparum parasites.” Infection and immunity 75.4 (2007): 2012-2025. Molecular Factors and Biochemical Pathways Induced by Febrile Temperature in Intraerythrocytic Plasmodium falciparum Parasites

26. Garibaldi, J. A. “Influence of temperature on the biosynthesis of iron transport compounds by Salmonella typhimurium.” Journal of bacteriology 110.1 (1972): 262-265. http://jb.asm.org/content/110/1/…

27. Cannon, Joseph G. “Perspective on fever: the basic science and conventional medicine.” Complementary therapies in medicine 21 (2013): S54-S60

28. Clint, Edward, and Daniel MT Fessler. “Insurmountable heat: The evolution and persistence of defensive hyperthermia.” The Quarterly review of biology 91.1 (2016): 25-46. http://www.danielmtfessler.com/w…

29. Small, P. M., et al. “Influence of body temperature on bacterial growth rates in experimental pneumococcal meningitis in rabbits.” Infection and immunity 52.2 (1986): 484-487. http://iai.asm.org/content/52/2/…

30. Kluger, Matthew J., and Barbara A. Rothenburg. “Fever and reduced iron: their interaction as a host defense response to bacterial infection.” Science 203.4378 (1979): 374-376.

31. Jiang, Qinqqi, et al. “Febrile core temperature is essential for optimal host defense in bacterial peritonitis.” Infection and immunity 68.3 (2000): 1265-1270. Febrile Core Temperature Is Essential for Optimal Host Defense in Bacterial Peritonitis

32. Kluger, Matthew J., Daniel H. Ringler, and Miriam R. Anver. “Fever and survival.” Science 188.4184 (1975): 166-168.

33. Epstein, Norman N. “Artificial fever as a therapeutic procedure.” California and Western medicine 44.5 (1936): 357. https://www.ncbi.nlm.nih.gov/pmc…

34. Vogel, Gretchen. “Malaria as lifesaving therapy.” Science 342.6159 (2013): 686-686.

35. Whitrow, Magda. “Wagner-Jauregg and fever therapy.” Medical history 34.3 (1990): 294. https://www.ncbi.nlm.nih.gov/pmc…


Why do IgG antibodies cross through the placenta?


, , ,

Maternal IgG transfer across placenta represents transfer of ready-made immunity from mother to fetus, a form of Passive immunity – Wikipedia. After all, it takes one or two years for B cells of human newborns to secrete adult level antibodies.

Antibodies (immunoglobulins) are secreted versions of the B-cell receptor – Wikipedia (BCR) of the B cell – Wikipedia. Through a process called class switch recombination (CSR) or Immunoglobulin class switching – Wikipedia, T cell – Wikipedia ‘help’ enables B cells to switch from native IgM to other antibody Isotype (immunology) – Wikipedia such as IgGs (IgG1, 2, 3, 4), IgA, IgE.

Immunoglobulin G – Wikipedia and IgA are the most abundant and apparently the most consequential since their absence confers serious susceptibility to infections. Given its importance and effectiveness in blocking infections in general, mammals and other classes such as birds and even fishes appear to have evolved a variety of methods to transfer IgG from mother to fetus either antenatally through the placenta or postnatally through colostrum and breastmilk.

Maternal IgG Is Transmitted To Fetus Only In Species With Hemochorial Placenta

Important to recall here the wide variety of placental design in different mammals, with the most invasive form of placenta, the hemochorial, found in higher order primates such as humans, rodents such as mice, rats, guinea pigs, and lagomorphs such as rabbits, where placental Trophoblast – Wikipedia cells are in direct contact with maternal blood (see below from 1).

Wide variety of mother-to-fetus IgG transfer (2, 3) reflects this wide placental design. IgG isn’t transplacentally transported in all mammalian species but rather is a feature of hemochorial placenta. However, even hemochorial placenta does not automatically mean transplacental IgG transfer.

Maternal IgG persists for different lengths of time in different species, as short as 10 days in some fishes to as long as ~9 months in humans. See below from 4 a summation of the variety of ways by which maternal IgGs are transmitted from mother to fetus or newborn, the varying lengths of time such antibodies can persist after transfer and the wide range of species they protect from disease.

  • In humans, rats, mice, maternal antibodies are transferred both pre- (IgG, see below from 4) and post-natally (IgA, little IgG).
  • In rabbits and guinea pigs, maternal antibodies are transferred only pre-natally.
  • In ungulates (ruminants, horses, pigs), there is no pre-natal maternal antibody transmission. Rather, bolus amounts of maternal antibody, largely IgG, pass from mother to newborn through colostrum in the 36 to 48 hours post-birth.

Francis Brambell – Wikipedia, considered the father of the field of transmission of immunity (3),

  • Generated the first modern data to describe transfer of antibodies from mother to fetus via placenta with a 1949 rabbit study (5).
  • Defined the first Fc receptor – Wikipedia system for IgG.
  • Identified the link necessary between mother-to-fetus transfer of IgG and need to protect the IgG molecule from catabolism.

Regardless it is transmitted from mother to fetus through placenta or through milk, in order for maternal IgG or any other type of antibody to be of any immunological usefulness to fetus and neonate, it has to reach the circulation intact after traversing complicated biological barriers such as trophoblast cells and fetal or newborn capillaries. Brambell predicted a unique transport system must exist to transport maternal IgG intact across placenta (human) or newborn GI tract (rodent) and into its bloodstream, a prediction proven years later by the discovery of the Neonatal Fc receptor – Wikipedia (FcRn), also called the Brambell factor (see below from 6 the visualization of how FcRn transports IgG across GI tract and placenta).

History of Maternal Transmission of Immunoglobulins, Specifically of IgG

Starting in the 1870s, data started accumulating to suggest transplacental IgG antibody transfer is important for infant immunity.

  • Studies published in 1877 and 1880 observed that lambs born to ewes recently vaccinated against cowpox (7) and anthrax (8) were also protected against them.
  • Paul Ehrlich – Wikipedia in 1892 (9) and Felix Klemperer – Wikipedia in 1893 (10) showed that transfer of maternal immunity from mother to offspring was important for newborn health.
    • Ehrlich’s observations were crucial and groundbreaking because the experiments were brilliantly conceived to link transfer and outcome.
    • Baby mice born to mothers made immune to toxins were also immune, and though such immunity was limited in time, it could be extended if baby mice suckled immune mothers but not non-immune wet nurses.
    • Something specific clearly needed to pass from mother to fetus in utero and in milk that endowed babies with immune resistance to toxins.
  • In short order followed reports that mothers transferred protective factors, now known to be antibodies, that protected guinea pigs (11, 12) and humans (13, 14) against diphtheria and tetanus.

See below from 4 the wide range of species maternal antibodies, mainly IgGs, protect from disease.


1. Moffett, Ashley, and Charlie Loke. “Immunology of placentation in eutherian mammals.” Nature Reviews Immunology 6.8 (2006): 584-594.

2. Kristoffersen, Einar Klæboe. “Human placental Fcγ‐binding proteins in the maternofetal transfer of IgG.” Apmis 104.S64 (1996): 5-36.

3. Junghans, R. P. “Finally! The Brambell receptor (FcRB).” Immunologic research 16.1 (1997): 29-57.

4. Grindstaff, Jennifer L., Edmund D. Brodie, and Ellen D. Ketterson. “Immune function across generations: integrating mechanism and evolutionary process in maternal antibody transmission.” Proceedings of the Royal Society of London B: Biological Sciences 270.1531 (2003): 2309-2319. https://www.ncbi.nlm.nih.gov/pmc…

5. Brambell, F. W., et al. “The passage into the embryonic yolk‐sac cavity of maternal plasma proteins in rabbits.” The Journal of physiology 108.2 (1949): 177-185. The passage into the embryonic yolk-sac cavity of maternal plasma proteins in rabbits

6. Roopenian, Derry C., and Shreeram Akilesh. “FcRn: the neonatal Fc receptor comes of age.” Nature reviews. Immunology 7.9 (2007): 715. https://www.researchgate.net/pro…

7. Bollinger, Otto. Über Menschen-und Thierpocken, über den Ursprung der Kuhpocken und über intrauterine Vaccination. Breitkopf & Härtel, 1877.

8. Chauveau, A. “Du renforcement de l’immunite des moutons algeriens a l’egard du sang de rate, par les inoculations preventives.” CR Acad. Sci.(Paris) 91 (1880): 148-151.

9. Ehrlich, Paul. “Über immunität durch vererbung und säugung.” Medical Microbiology and Immunology 12.1 (1892): 183-203.; Ehrlich, Paul, and W. Hübener. “Über die Vererbung der Immunität bei Tetanus.” Medical Microbiology and Immunology 18.1 (1894): 51-64.

10. Klemperer, Felix. “Ueber natürliche Immunität und ihre Verwerthung für die Immunisirungstherapie.” Archiv für Experimentelle Pathologie und Pharmakologie 31.4-5 (1893): 356-382.

11. Wernicke, E. “Über die Vererbung der künstlich erzgeuten Diphtherie-Immunität bei Meerschweinen.” Festschrift zur 100 (1895).

12. Smith, Theobald. “Degrees of susceptibility to diphtheria toxin among guinea-pigs. Transmission from parents to offspring.” The Journal of medical research 13.3 (1905): 341. https://www.ncbi.nlm.nih.gov/pmc…

13. Fischl, Rudolf, and Gustav von Wunschheim. Über Schutzkörper im Blute des Neugeborenen: das Verhalten des Blutserums des Neugeborenen gegen Diphtheriebacillen und Diphtheriegift: nebst kritischen Bemerkungen zur humoralen Immunitätstheorie.

14. Polano, O. “Der Antitoxinübergang von der Mutter auf das Kind: Ein Beitrag zur Physiologie der Placenta.” Ztschr. f. Geburtsh. u. Gynäk. 53 (1904): 456.


Given that adults don’t have a functional thymus, how do T cells reconstitute after a bone marrow transplant?



The Thymus – Wikipedia is where T cell – Wikipedia develop. Progenitor cells from the bone marrow enter the thymus to engage in a complicated developmental process and the ones who make it past various bottlenecks leave the thymus as functional, mature CD4 and CD8 T cells.

In the mouse, the thymus dramatically shrinks with age, a process called Involution (medicine) – Wikipedia that severely curtails its output of new, naive, i.e., antigen-inexperienced, T cells, and for long researchers simply extrapolated from mouse that thymic involution must be similarly consequential in humans as well. Important to remember here that the mouse is the most common experimental model for basic immunological research. Hence the assumption ‘Given that adults don’t have a functional thymus‘.

However, over time, the weight of experimental data informs us that the mouse isn’t simply a mini-human, albeit a four-legged, nocturnal, short-lived (maximum lifespan ~2 years) rodent version but rather a species unto itself whose physiological attributes cannot be directly extrapolated to the human (please do imagine my exaggerated eye roll here that it ever even got to the point that something so self-evident even needed saying). Specifically, extrapolating mouse thymus function to human is quite flawed.

This answer describes

  • Key differences between mouse and human thymic function.
  • Effects of thymectomy on T cell numbers and function in humans.
  • Effects of transplant-related thymus impairment in humans.

Key Differences Between Mouse & Human Thymic Function

That a similar process of fewer new T cells coming out of the thymus with age had similar relevance in humans as it does in the mouse was long assumed. However, it turns out naive T cell maintenance is quite a different process in humans, one that makes it less dependent on the thymus over time.

  • Circulating naive T cells in humans self-renew (1), a key difference in kind from naive mouse T cells. Specifically, this groundbreaking study (n=45) showed that the circulating naive T cell pool in elderly humans comprised 10% recent thymic emigrants and 90% self-proliferating (homeostatic proliferation), proportions that were exactly reversed in aged mice. Other studies (2) by other groups including an in silico modeling approach (3) have since corroborated that self-renewal following their emergence from the thymus is a major feature of human naive T cells. Such renewal depends on local levels of IL-7 (Interleukin 7 – Wikipedia) and some other cytokines.
  • While short-lived in mice (1), naive T cells are very long lived in humans (4).
  • Human thymic output may be already less relevant by the 20s since there is little evidence of increasing turnover of circulating naive T cells between 20 and 70 years of age (5). This implies that already by the 20s, the human thymus has pushed out T cell specificities most biologically relevant for an individual to maintain their immunity during their lifetime.
  • Though human thymus begins to involute (reduces its output) early in life, it still remains active until beyond the 60s, only abruptly crashing in the 80s (6). Thus, blunt extrapolation from mouse thymus involution rates to humans is not only inaccurate but also inherently flawed because even the aged human thymus retains measurable capacity to generate new T cells (see below from 7, emphasis mine).

‘As an individual ages, the thymus involutes and the output of new T cells falls significantly [38–40]. In 1985, Steinman et al elegantly demonstrated that thymic function gradually starts decreasing from year one of life [38,39]. The observation of dual components of the human thymus, the true thymic epithelial space, in which thymopoiesis occurs, and the non-epithelial non thymopoietic perivascular space [38,39], was critical to the current understanding of thymic atrophy. The expansion of the perivascular space (adipocytes, peripheral lymphocytes, stroma) with age results in a shift in the ratio of true thymic epithelial space to perivascular space. The thymic epithelial space shrinks to less than 10% of the total thymus tissue by 70 years of age. When extrapolated, Steinman’s data suggest that the thymus would cease to produce new T cells at approximately 105 years of age (Figure 1) [41]…We and others have also demonstrated that while the thymopoietic area of the human thymus decreases with age, the thymopoietic potential per cell, as measured by sjTRECs [47] or by TCR ligation-mediated polymerase chain reaction [3], remains constant at least until approximately 50 years of age [43,45,47–50].’

Such data help understand what had long remained a conundrum, why the naive human T cell population only shrank modestly with age (8, 9, 10, 11), a reduction not in line with the much more rapid pace and extent of thymic involution. Since circulating naive human T cells appear to both hang around longer and be capable of proliferating (homeostatic proliferation) to maintain themselves, in practical terms, this means with age, human T cell repertoire depends less on a functioning thymus, relying instead on maintaining what’s already emerged from the thymus, a difference in kind from the mouse that makes the human T cell system more resilient to withstand damage to the T cell generating capabilities of the thymus. Repertoire refers to the antigen specificities of individual T cell TCRs (T-cell receptor – Wikipedia), with a broader diversity reflecting better anticipatory preparedness. After all, being prepared to specifically engage with antigens not previously encountered is the calling card of the Adaptive immune system – Wikipedia.

  • Homeostatic proliferation is more important for CD4 rather than CD8 T cell maintenance (12), which may be why even the very old have a measurable naive CD4 T cell pool (11) and why impact of aging is greater for human CD8 T cells.
  • None of this negates the importance of the thymus in introducing T cells with new specificities, i.e., T cells with TCRs capable of recognizing new antigens (epitopes). After all homeostatic proliferation can only maintain, not expand, the existing T cell repertoire, a function that’s the sole purview of the thymus. To quote from 1 (emphasis mine),

‘Although these data show that in terms of naive T cell numbers created per day, peripheral T cell proliferation by far exceeds thymic output in human adults, the thymus may still have an essential role – if only because new T cell specificities can only be created by the thymus.’

Effects of Thymectomy on T cell Numbers & Function in Humans

At 1 in 100 newborns, congenital heart disease is among the most common of birth defects (13). Over the past 30 years, having become safer and thus routine, open-heart surgery is increasingly used to correct these defects. However, this also often requires complete or partial thymectomy (thymus removal) since the thymus blocks surgical access to the heart and large vessels, especially in newborns. Researching immune function in such thymectomized individuals in turn provides valuable information on the consequences of such thymectomy.

  • One study (14) on neonatally thymectomized children examined impact in either the short- (within 1 to 5 years, n=17 and 19 healthy controls) or longer-term (at least 10 years later, n=26 and 11 healthy controls) and found it drastically affects T cell diversity (measured as range of TCRs) in the short-term but that the thymus at such a young age is capable of some degree of regeneration since diversity is restored in later life.
  • Another study (15) found rates of autoimmunity and allergy among the neonatally thymectomized (n=7) to be similar to healthy controls (n=7).
  • Though both degree (partial versus total) (16) and age (17) at thymectomy influence outcome on T cell number and function, they don’t affect general health on the whole apart from a tendency for exaggerated responses to cytomegalovirus (CMV) (18). How to explain this? One plausible explanation could be that thymectomy provides impetus for increased plasma IL-7 levels (19), which in turn could support T cell homeostatic proliferation (20, 21).
  • >20 years post-thymectomy, patients infected with CMV had fewer circulating naive T cells and reduced TCR diversity (18) and delayed primary antibody response to tick-borne encephalitis vaccination (22, 23).

Since routine neonatal open-heart surgery and its attendant thymectomy are at 30 years or so of recent vintage, lifelong impact on immunity and health is still work in progress. However, these and other studies suggest impact is individual, hard to predict and highly influenced by chronic infections, especially CMV.

Effects of Transplant-related Thymus Function or Impairment in Humans

Preparing the body for transplant (transplant conditioning regimens) entails the kinds of preparatory treatments (irradiation, chemotherapy, steroids) that severely damage the thymus, impairing its capacity to push out new T cells.

  • Adequate thymic recovery was observed after autologous stem cell transplant
    • Even in those with severe autoimmune diseases (n=10, age range 16 to 49 years old, n=21 controls, age range 20 to 55 years old) (24).
    • In Multiple Sclerosis (MS) patients (n=7, age range 28 to 53) (25).
  • Adequate thymic recovery was observed after heterologous kidney transplant (n=48 patients, 39 controls) (26).
  • One study (27) of 32 adult breast cancer patients treated with autologous peripheral blood stem cell transplant (age range 30 to 69) found thymic recovery to be strictly a function of age, with measurable thymus enlargement (sign of recovery) post-transplant in
    • 4 of 5 of those 30 to 39 years old
    • 6 of 13 in those 40 to 49 years old.
    • Only 2 of 14 in those >50 years old.

Given its capacity for some regeneration as well as its ability to continue to push out new T cells even later in life, albeit at markedly lower rates, no surprise that the human thymus can bounce back after neonatal thymectomy as well as from transplant-related damage. Degree of recovery is also unsurprisingly a function of age and environment. As well, unlike their mouse counterparts, human naive T cell capacity for self-propagation helps maintain them even into old age.


1. den Braber, Ineke, et al. “Maintenance of peripheral naive T cells is sustained by thymus output in mice but not humans.” Immunity 36.2 (2012): 288-297. https://ac.els-cdn.com/S10747613…

2. Thome, Joseph JC, et al. “Longterm maintenance of human naive T cells through in situ homeostasis in lymphoid tissue sites.” Science immunology 1.6 (2016). https://www.ncbi.nlm.nih.gov/pmc…

3. Johnson, Philip LF, et al. “Peripheral selection rather than thymic involution explains sudden contraction in naive CD4 T-cell diversity with age.” Proceedings of the National Academy of Sciences 109.52 (2012): 21432-21437. http://www.pnas.org/content/109/…

4. Vrisekoop, Nienke, et al. “Sparse production but preferential incorporation of recently produced naive T cells in the human peripheral pool.” Proceedings of the National Academy of Sciences 105.16 (2008): 6115-6120. http://www.pnas.org/content/105/…

5. Naylor, Keith, et al. “The influence of age on T cell generation and TCR diversity.” The Journal of Immunology 174.11 (2005): 7446-7452. https://www.researchgate.net/pro…

6. Czesnikiewicz-Guzik, Marta, et al. “T cell subset-specific susceptibility to aging.” Clinical Immunology 127.1 (2008): 107-118. https://www.researchgate.net/pro…

7. Gruver, A. L., L. L. Hudson, and G. D. Sempowski. “Immunosenescence of ageing.” The Journal of pathology 211.2 (2007): 144-156. http://onlinelibrary.wiley.com/d…

8. Bertho, Jean-Marc, et al. “Phenotypic and immunohistological analyses of the human adult thymus: evidence for an active thymus during adult life.” Cellular immunology 179.1 (1997): 30-40.

9. Douek, Daniel C., et al. “Changes in thymic function with age and during the treatment of HIV infection.” Nature 396.6712 (1998): 690.

10. Jamieson, Beth D., et al. “Generation of functional thymocytes in the human adult.” Immunity 10.5 (1999): 569-575. http://www.cell.com/immunity/pdf…

11. Wertheimer, Anne M., et al. “Aging and cytomegalovirus infection differentially and jointly affect distinct circulating T cell subsets in humans.” The Journal of Immunology 192.5 (2014): 2143-2155. http://www.jimmunol.org/content/…

12. Goronzy, Jörg J., et al. “Naive T cell maintenance and function in human aging.” The Journal of Immunology 194.9 (2015): 4073-4080. http://www.jimmunol.org/content/…

13. Hoffman, Julien IE, and Samuel Kaplan. “The incidence of congenital heart disease.” Journal of the American college of cardiology 39.12 (2002): 1890-1900. https://ac.els-cdn.com/S07351097…

14. Van Den Broek, Theo, et al. “Neonatal thymectomy reveals differentiation and plasticity within human naive T cells.” The Journal of clinical investigation 126.3 (2016): 1126. https://www.ncbi.nlm.nih.gov/pmc…

15. Silva, Susana L., et al. “Autoimmunity and allergy control in adults submitted to complete thymectomy early in infancy.” PloS one 12.7 (2017): e0180385. http://journals.plos.org/plosone…

16. Halnon, Nancy J., et al. “Thymic function and impaired maintenance of peripheral T cell populations in children with congenital heart disease and surgical thymectomy.” Pediatric research 57.1 (2005): 42-48. https://www.researchgate.net/pro…

17. Prelog, Martina, et al. “Thymectomy in early childhood: significant alterations of the CD4+ CD45RA+ CD62L+ T cell compartment in later life.” Clinical immunology 130.2 (2009): 123-132. http://www.musiklexikon.ac.at:80…

18. Sauce, Delphine, et al. “Evidence of premature immune aging in patients thymectomized during early childhood.” The Journal of clinical investigation 119.10 (2009): 3070. Evidence of premature immune aging in patients thymectomized during early childhood

19. Mancebo, E., et al. “Longitudinal analysis of immune function in the first 3 years of life in thymectomized neonates during cardiac surgery.” Clinical & Experimental Immunology 154.3 (2008): 375-383. http://onlinelibrary.wiley.com/d…

20. Fry, Terry J., and Crystal L. Mackall. “The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance.” The Journal of Immunology 174.11 (2005): 6571-6576. http://www.jimmunol.org/content/…

21. Silva, Susana L., et al. “Human naive regulatory T-cells feature high steady-state turnover and are maintained by IL-7.” Oncotarget 7.11 (2016): 12163. https://pdfs.semanticscholar.org…

22. Prelog, Martina, et al. “Diminished response to tick-borne encephalitis vaccination in thymectomized children.” Vaccine 26.5 (2008): 595-600.

23. Zlamy, Manuela, et al. “Antibody dynamics after tick-borne encephalitis and measles–mumps–rubella vaccination in children post early thymectomy.” Vaccine 28.51 (2010): 8053-8060.

24. Thiel, Andreas, et al. “Direct assessment of thymic reactivation after autologous stem cell transplantation.” Acta haematologica 119.1 (2008): 22-27.

25. Muraro, Paolo A., et al. “Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients.” Journal of Experimental Medicine 201.5 (2005): 805-816. http://jem.rupress.org/content/j…

26. Nickel, Peter, et al. “CD31+ Naïve Th Cells Are Stable during Six Months Following Kidney Transplantation: Implications for Post‐transplant Thymic Function.” American journal of transplantation 5.7 (2005): 1764-1771. http://onlinelibrary.wiley.com/d…

27. Hakim, Frances T., et al. “Age-dependent incidence, time course, and consequences of thymic renewal in adults.” Journal of Clinical Investigation 115.4 (2005): 930. http://content-assets.jci.org/ma…


Are there any Chimeric Antigen Receptors (CAR-T) that can demonstrate a therapeutic response against solid tumors?


A few case reports here and there show Chimeric Antigen Receptor-T (CAR-T) therapeutic response against solid tumors.

  • Currently one CAR-T case report shows it successfully targeting and eliminating a patient’s solid tumors. One of the earliest CAR-T approaches against Glioblastoma – Wikipedia (GBM) targeted IL13RA2 – Wikipedia which is often over-expressed by such tumors.
    • A small pilot first-in-human clinical trial used such CAR-Ts in 3 GBM patients and reported the Rx generated measurable anti-tumor immune responses with manageable side-effects.
    • In one patient (1), tumors had not only proven resistant to surgery, radiation and chemotherapy but also spread to the spine. For this reason, the researchers decided to infuse anti-IL-13Ra2 CAR-Ts not just in one but in two locations. This approach apparently eliminated their tumors, with the patient reported to be tumor-free as of February 2017.
  • In two case reports (2), CAR-Ts that target Mesothelin – Wikipedia positive tumor MPM (malignant pleural Mesothelioma – Wikipedia) were found to infiltrate tumors with minimal on-target, off-tumor toxicity. However, though completed, no final trial results have yet been posted (3).
  • CAR-Ts targeting the GD2 antigen were effective against pediatric neuroblastoma in 3 of 11 patients (4). No other results have been reported from this trial which is still listed as ongoing (5).

Most CAR-T clinical trials target blood cancer, specifically B cell cancers expressing the cell-surface molecule CD19 (see below from 6, 7).

Practical Obstacles to CAR-Ts Targeting Solid Tumors

The process of making CAR-Ts capable of targeting solid tumors entails

  • Identifying antigens specifically expressed by solid cancer cells on their cell surface and validating them.
    • Targeting tumor-associated antigens (TAAs) rather than tumor-specific antigens (TSAs) increases risk for on-target, off-tumor toxicity which can even be life-threatening. On-target, off-tumor toxicity is a serious problem even with anti-CD19 CAR-Ts thought to only target B cells. Potential for toxicity is theoretically even higher when targeting solid tumors.
    • Germline cancer antigens are a third type of cancer antigen. Such antigens are usually expressed by germ (sperm, eggs) cells but not by somatic cells. Germ cells typically don’t express MHC molecules. This reduces the likelihood they could directly present such antigens to CAR-T cells that could make them a target of their immune responses. This inherent safety feature makes germline cancer antigens an attractive immunotherapeutic target.
  • Generating antibodies against such antigens and then deriving mAbs out of such antibodies.
  • Validating such mAbs to ensure their safety and using their single-chain variable fragment (scFv) to genetically engineer CARs.
    • Apart from identifying and validating tumor-specific antigens, immunologically targeting a tumor using a patient’s own cells requires identifying and isolating tumor-specific T cells from cancer patients. In the case of CAR-T cells, the need to isolate and identify tumor-specific T cells is rendered moot by taking the patient’s T cells out and genetically engineering them in vitro to express tumor-specific CAR.
    • Such CARs are hybrid molecules whose extracellular piece consist of a monoclonal antibody’s (mAb) scFv. Thus, regardless an individual T cell’s antigenic specificity, once it’s genetically engineered to be a CAR-T, it can target cells that express that CAR’s antigen.
  • Successfully targeting solid tumors also requires many more time-consuming genetic engineering steps.
    • For one, CAR-Ts need to be able to efficiently penetrate solid tumors. Ability to kill tumor cells is of little use if CAR-Ts can’t enter the tumor. This requires genetically engineering additional pieces to enable CAR-Ts to do so.
    • Safeguards may also need to be engineered to minimize toxicity, for example, by engineering CAR-Ts to target two TAAs at a time rather than one to increase the chance that the CAR-T in question only got activated by a tumor that expressed both of them rather than by normal tissue that would more likely express one or the other but not both simultaneously.

Each of these steps constitutes several years’ worth of research.

The CARs in CAR-Ts currently being tested started not from scratch but rather by engineering into T cells scFvs from mAbs already approved for clinical anti-cancer use by the FDA or other regulatory agencies. Problem with such an approach is these old-generation mAbs target TAAs rather than TSAs.

For example, skin (keratinocytes) as well as heart muscle cells (cardiac myocytes) express Epidermal growth factor receptor – Wikipedia (EGFR) and HER2/neu – Wikipedia (ERBB2), which is why mAbs targeting these receptors can trigger skin- and heart-related toxicities (8). GD2 is expressed not just by neuroblastoma and sarcoma but also by peripheral sensory nerve fibers and neurons. This is why anti-GD2 mAbs can trigger neuropathic pain (9), which could presumably occur with CAR-Ts that expressed scFv derived from such mAbs.

CAR-T therapies targeting TAAs or cancer germline antigens expressed by solid tumors are at various stages of development and testing (see below from 7). Not many results have yet been published, even though some of these trials are now completed.


1. Brown, Christine E., et al. “Regression of glioblastoma after chimeric antigen receptor T-cell therapy.” New England Journal of Medicine 375.26 (2016): 2561-2569. http://www.nejm.org/doi/pdf/10.1…

2. Beatty, Gregory L., et al. “Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce antitumor activity in solid malignancies.” Cancer immunology research 2.2 (2014): 112-120. http://cancerimmunolres.aacrjour…

3. Autologous Redirected RNA Meso-CIR T Cells – No Study Results Posted – ClinicalTrials.gov

4. Louis, Chrystal U., et al. “Antitumor activity and long-term fate of chimeric antigen receptor–positive T cells in patients with neuroblastoma.” Blood 118.23 (2011): 6050-6056. http://www.bloodjournal.org/cont…

5. Blood T-Cells and EBV Specific CTLs Expressing GD-2 Specific Chimeric T Cell Receptors to Neuroblastoma Patients – Full Text View – ClinicalTrials.gov

6. Klebanoff, Christopher A., Steven A. Rosenberg, and Nicholas P. Restifo. “Prospects for gene-engineered T cell immunotherapy for solid cancers.” Nature medicine 22.1 (2016): 26.

7. Yu, Shengnan, et al. “Chimeric antigen receptor T cells: a novel therapy for solid tumors.” Journal of hematology & oncology 10.1 (2017): 78. https://pdfs.semanticscholar.org…

8. Crone, Steven A., et al. “ErbB2 is essential in the prevention of dilated cardiomyopathy.” Nature medicine 8.5 (2002): 459-465. http://www.salk.edu/pdf/otmd/Art…

9. Yu, Alice L., et al. “Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma.” New England Journal of Medicine 363.14 (2010): 1324-1334. http://www.nejm.org/doi/pdf/10.1…