Assignment in R: slings and arrows

Having recently shared my post about defensive programming in R on the r/rstats subreddit, I was blown away by the sheer number of comments as much as I was blown away by the insight many displayed. One particular comment by u/guepier struck my attention. In my previous post, I came out quite vehemently against using the = operator to effect assignment in R. u/guepier‘s made a great point, however:

But point 9 is where you’re quite simply wrong, sorry:

never, ever, ever use = to assign. Every time you do it, a kitten dies of sadness.

This is FUD, please don’t spread it. There’s nothing wrong with =. It’s purely a question of personal preference. In fact, if anything <- is more error-prone (granted, this is a very slight chance but it’s still higher than the chance of making an error when using =).

Now, assignment is no doubt a hot topic – a related issue, assignment expressions, has recently led to Python’s BDFL to be forced to resign –, so I’ll have to tread carefully. A surprising number of people have surprisingly strong feelings about assignment and assignment expressions. In R, this is complicated by its unusual assignment structure, involving two assignment operators that are just different enough to be trouble.

A brief history of <-

IBM Model M SSK keyboard with APL keys
This is the IBM Model M SSK keyboard. The APL symbols are printed on it in somewhat faint yellow.

There are many ways in which <- in R is anomalous. For starters, it is rare to find a binary operator that consists of two characters – which is an interesting window on the R <- operator’s history.

The <- operator, apparently, stems from a day long gone by, when keyboards existed for the programming language eldritch horror that is APL. When R’s precursor, S, was conceived, APL keyboards and printing heads existed, and these could print a single ← character. It was only after most standard keyboard assignments ended up eschewing this all-important symbol that R and S accepted the digraphic <- as a substitute.

OK, but what does it do?

In the Brown Book, the underscore was actually an alias for the arrow assignment operator.
In the Brown Book (Richard A. Becker and John M. Chambers (1984). S: An Interactive Environment for Data Analysis and Graphics), the underscore was actually an alias for the arrow assignment operator! Thankfully, this did not make it into R.
<- is one of the first operators anyone encounters when familiarising themselves with the R language. The general idea is quite simple: it is a directionally unambiguous assignment, i.e. it indicates quite clearly that the right-hand side value (rhs, in the following) will replace the left-hand side variable (lhs), or be assigned to the newly created lhs if it has not yet been initialised. Or that, at the very least, is the short story.

Because quite peculiarly, there is another way to accomplish a simple assignment in R: the equality sign (=). And because on the top level, a <- b and a = b are equivalent, people have sometimes treated the two as being quintessentially identical. Which is not the case. Or maybe it is. It’s all very confusing. Let’s see if we can unconfuse it.

The Holy Writ

The Holy Writ, known to uninitiated as the R Manual, has this to say about assignment operators and their differences:

The operators <- and = assign into the environment in which they are evaluated. The operator <- can be used anywhere, whereas the operator = is only allowed at the top level (e.g., in the complete expression typed at the command prompt) or as one of the subexpressions in a braced list of expressions.

If this sounds like absolute gibberish, or you cannot think of what would qualify as not being on the top level or a subexpression in a braced list of expressions, welcome to the squad – I’ve had R experts scratch their head about this for an embarrassingly long time until they realised what the R documentation, in its neutron starlike denseness, actually meant.

If it’s in (parentheses) rather than {braces}, = and <- are going to behave weird

To translate the scriptural words above quoted to human speak, this means = cannot be used in the conditional part (the part enclosed by (parentheses) as opposed to {curly braces}) of control structures, among others. This is less an issue between <- and =, and rather an issue between = and ==. Consider the following example:

x = 3

if(x = 3) 1 else 0
# Error: unexpected '=' in "if(x ="

So far so good: you should not use a single equality sign as an equality test operator. The right way to do it is:

> if(x == 3) 1 else 0
[1] 1

But what about arrow assignment?

if(x <- 3) 1 else 0
# [1] 1

Oh, look, it works! Or does it?

if(x <- 4) 1 else 0
# [1] 1

The problem is that an assignment will always yield true if successful. So instead of comparing x to 4, it assigned 4 to x, then happily informed us that it is indeed true.

The bottom line is not to use = as comparison operator, and <- as anything at all in a control flow expression’s conditional part. Or as John Chambers notes,

Disallowing the new assignment form in control expressions avoids programming errors (such as the example above) that are more likely with the equal operator than with other S assignments.

Chain assignments

One example of where  <- and = behave differently (or rather, one behaves and the other throws an error) is a chain assignment. In a chain assignment, we exploit the fact that R assigns from right to left. The sole criterion is that all except the rightmost members of the chain must be capable of being assigned to.

# Chain assignment using <-
a <- b <- c <- 3

# Chain assignment using =
a = b = c = 3

# Chain assignment that will, unsurprisingly, fail
a = b = 3 = 4
# Error in 3 = 4 : invalid (do_set) left-hand side to assignment

So we’ve seen that as long as the chain assignment is logically valid, it’ll work fine, whether it’s using <- or =. But what if we mix them up?

a = b = c <- 1
# Works fine...

a = b <- c <- 1
# We're still great...

a <- b = c = 1
# Error in a <- b = c = 1 : could not find function "<-<-"
# Oh.

The bottom line from the example above is that where <- and = are mixed, the leftmost assignment has to be carried out using =, and cannot be by <-. In that one particular context, = and <- are not interchangeable.

A small note on chain assignments: many people dislike chain assignments because they’re ‘invisible’ – they literally return nothing at all. If that is an issue, you can surround your chain assignment with parentheses – regardless of whether it uses <-, = or a (valid) mixture thereof:

a = b = c <- 3
# ...
# ... still nothing...
# ... ... more silence...

(a = b = c <- 3)
# [1] 3

Assignment and initialisation in functions

This is the big whammy – one of the most important differences between <- and =, and a great way to break your code. If you have paid attention until now, you’ll be rewarded by, hopefully, some interesting knowledge.

= is a pure assignment operator. It does not necessary initialise a variable in the global namespace. <-, on the other hand, always creates a variable, with the lhs value as its name, in the global namespace. This becomes quite prominent when using it in functions.

Traditionally, when invoking a function, we are supposed to bind its arguments in the format parameter = argument.1 And as we know from what we know about functions, the keyword’s scope is restricted to the function block. To demonstrate this:

add_up_numbers <- function(a, b) {
    return(a + b)
}

add_up_numbers(a = 3, b = 5)
# [1] 8

a + b
# Error: object 'a' not found

This is expected: a (as well as b, but that didn’t even make it far enough to get checked!) doesn’t exist in the global scope, it exists only in the local scope of the function add_up_numbers. But what happens if we use <- assignment?

add_up_numbers(a <- 3, b <- 5)
# [1] 8

a + b
# [1] 8

Now, a and b still only exist in the local scope of the function add_up_numbers. However, using the assignment operator, we have also created new variables called a and b in the global scope. It’s important not to confuse it with accessing the local scope, as the following example demonstrates:

add_up_numbers(c <- 5, d <- 6)
# [1] 11

a + b
# [1] 8

c + d
# [1] 11

In other words, a + b gave us the sum of the values a and b had in the global scope. When we invoked add_up_numbers(c <- 5, d <- 6), the following happened, in order:

  1. A variable called c was initialised in the global scope. The value 5 was assigned to it.
  2. A variable called d was initialised in the global scope. The value 6 was assigned to it.
  3. The function add_up_numbers() was called on positional arguments c and d.
  4. c was assigned to the variable a in the function’s local scope.
  5. d was assigned to the variable b in the function’s local scope.
  6. The function returned the sum of the variables a and b in the local scope.

It may sound more than a little tedious to think about this function in this way, but it highlights three important things about <- assignment:

  1. In a function call, <- assignment to a keyword name is not the same as using =, which simply binds a value to the keyword.
  2. <- assignment in a function call affects the global scope, using = to provide an argument does not.
  3. Outside this context, <- and = have the same effect, i.e. they assign, or initialise and assign, in the current scope.

Phew. If that sounds like absolute confusing gibberish, give it another read and try playing around with it a little. I promise, it makes some sense. Eventually.

So… should you or shouldn’t you?

Which raises the question that launched this whole post: should you use = for assignment at all? Quite a few style guides, such as Google’s R style guide, have outright banned the use of = as assignment operator, while others have encouraged the use of ->. Personally, I’m inclined to agree with them, for three reasons.

  1. Because of the existence of ->, assignment by definition is best when it’s structured in a way that shows what is assigned to which side. a -> b and b <- a have a formal clarity that a = b does not have.
  2. Good code is unambiguous even if the language isn’t. This way, -> and <- always mean assignment, = always means argument binding and == always means comparison.
  3. Many argue that <- is ambiguous, as x<-3 may be mistyped as x<3 or x-3, or alternatively may be (visually) parsed as x < -3, i.e. compare x to -3. In reality, this is a non-issue. RStudio has a built-in shortcut (Alt/⎇ + ) for <-, and automatically inserts a space before and after it. And if one adheres to sound coding principles and surrounds operators with white spaces, this is not an issue that arises.

Like with all coding standards, consistency is key. Consistently used suboptimal solutions are superior, from a coding perspective, to an inconsistent mixture of right and wrong solutions.

References   [ + ]

1.A parameter is an abstract ‘slot’ where you can put in values that configure a function’s execution. Arguments are the actual values you put in. So add_up_numbers(a,b) has the parameters a and b, and add_up_numbers(a = 3, b = 5) has the arguments 3 and 5.

Is Senomyx putting baby parts into your fruit juice? The answer may (not) surprise you.

I know a great number of people who oppose abortion, and who are therefore opposed to the biomedical use of tissue or cells that have been derived from foetuses aborted for that very purpose, although most of them do not oppose the use of foetal tissue from foetuses aborted for some other reason.1 Over the last week or so, many have sent me a story involving a company named Senomyx, wondering if it’s true. In particular, the story claimed that Senomyx uses foetal cells, or substances derived from foetal cell lines, to create artificial flavourings. One article I have been referred to is straight from the immediately credible-sounding Natural News:2 3

Every time you purchase mass-produced processed “food” from the likes of Kraft, PepsiCo, or Nestle, you’re choosing, whether you realize it or not, to feed your family not only genetically engineered poisons and chemical additives, but also various flavoring agents manufactured using the tissue of aborted human babies.

It’s true: A company based out of California, known as Senomyx, is in the business of using aborted embryonic cells to test fake flavoring chemicals, both savory and sweet, which are then added to things like soft drinks, candy and cookies. And Senomyx has admittedly partnered with a number of major food manufacturers to lace its cannibalistic additives into all sorts of factory foods scarfed down by millions of American consumers every single day.

Needless to say, this is total bullcrap, and in the following, we’re going on a ride in the Magic Schoolbus through this fetid swamp of lies, half-truths and quarter-wits.

In the beginning was the cell culture…

About ten years before I was born, a healthy foetus was (legally) aborted in Leiden, the Netherlands, and samples were taken from the foetus’s kidney. They were much like the cells in your kidney. Like the cells in your kidney (hopefully!), they had a beginning and an end. That end is known as the Hayflick limit or ‘replicative senescence limit’. Cells contain small ‘caps’ at the end of their chromosomes, known as telomeres.4 At every mitosis, these shorten a little, like a countdown clock, showing how many divisions the cell has left. And once the telomeres are all gone, the cell enters a stage called cellular senescence, and is permanently stuck in the G1 phase of the cell cycle, unable to move on to phase S. This is a self-preservative mechanism: with every mitosis, the cell line ages, and becomes more likely to start suffering from errors it passes on to its descendants. Experimentally, we know that the Hayflick limit is around 40-60 divisions.

Kadir Nelson, The Mother of Modern Medicine. Oil on linen, 59 1/2 x 49 1/2.
Kadir Nelson, The Mother of Modern Medicine. Oil on linen, 59 1/2 x 49 1/2. Image courtesy of the National Museum of African American History & Culture, Washington, DC.

But every rule has an exception. Most obviously, there are the cells that just won’t die – this is the case in cancers. And so, some cancer cells have this senescence mechanism disabled, and can divide an unlimited number of times. They have become practically immortal. This is, of course, a nuisance if they are inside a human, because proliferation and division of those cells causes unpleasant things like tumours. For researchers, however, they offer something precious: a cell line that will, as long as it’s fed and watered, live and divide indefinitely. This is called an immortal cell line. The most famous of them, HeLa, began life as cervical epithelial cells of a woman named Henrietta Lacks. Then something dreadful happened, and the cells’ cell cycle regulation was disrupted – Ms Lacks developed aggressive cervical cancer, and died on October 4, 1951. Before that, without her permission or consent, samples were taken from her cervix, and passed onto George Otto Gey, a cell biologist who realised what would be Ms Lacks’s most lasting heritage: her cells would divide and divide, well beyond the Hayflick limit, with no end in sight. Finally, cell biologists had the Holy Grail: a single cell line that produced near-exact copies of itself, all descendants of a single cell from the cervix of a destitute African-American woman, who died without a penny to her name, but whose cells would for decades continue to save lives – among others, Salk’s polio vaccine was cultured in HeLa cells.5

HeLa cells were immortal ‘by nature’ – they underwent changes that have rendered them cancerous/immortal, depending on perspective. But it’s also possible to take regular mortal cells and interfere with their cell cycle to render them immortal. And this brings us back to the kidney cells of the foetus aborted in the 1970s, a life that was never born yet will never die, but live on as HEK 293. A cell biologist, Frank Graham, working in Alex van Eb’s lab at the University of Leiden,6 used the E1 gene from adenovirus 5 to ‘immortalise’ the cell line, effectively rewriting the cell’s internal regulation mechanism to allow it to continue to divide indefinitely rather than enter cellular senescence.7 This became the cell culture HEK 293,8 one of my favourite cell cultures altogether, with an almost transcendental beauty in symmetry.

HEK293 cells
HEK 293 cells, immunofluorescence photomicrograph.

HEK 293 is today a stock cell line, used for a range of experimental and production processes. You can buy it from Sigma-Aldrich for little north of £350 a vial. The cells you’re getting will be exact genetic copies of the initial cell sample, and its direct genetic descendants. That’s the point of an immortalised cell line: you can alter a single cell to effectively divide indefinitely as long as the requisite nutrients, space and temperature are present. They are immensely useful for two reasons:

  • You don’t need to take new cell samples every time you need a few million cells to tinker with.
  • All cells in a cell line are perfectly identical.9 It goes without saying just how important for reproducible science it is to have widely available, standardised, reference cell lines.

Admittedly, this was a whistle-stop tour through cell cultures and immortal(ised) cell lines, but the basics should be clear. Right? I mean… right?

Unless you’re Sayer Ji.

Until about 1900 today, I had absolutely no idea of who Sayer Ji is, and I would lie if I asserted my life was drastically impoverished by that particular ignorance. Sayer Ji runs a website (which I am not going to link to, but I am going to link to RationalWiki’s entry on it, as well as Orac’s collected works on the man, the myth and the bullshit), and he describes himself as a ‘thought leader’. That alone sets off big alarms. Mr Ji, to the best of my knowledge, has no medical credentials whatsoever, nor any accredited credentials that would allow him to make the grandiose statements that he seems to indulge in with the moderation of Tony Montana when it comes to cocaine:

There is not a single disease ever identified cause by a lack of a drug, but there are diseases caused by a lack of vitamins, minerals and nutrients. Why, then, do we consider the former – chemical medicine – the standard of care and food-as-medicine as quackery? Money and power is the obvious answer.

[su_pullquote align=”right”]Mr Ji’s ‘article’ rests on the fundamental assertion that ‘abortion tainted vaccines’ constitute cannibalism. He is, of course, entirely misguided, and does not understand how cell line derived vaccines work.[/su_pullquote]You may question why I even engage with someone whose cognition is operating on this level, and you might be justified in doing so. Please ascribe it to my masochistic tendencies, or consider it a sacrifice for the common good. Either way, I pushed through a particular article of his which is cited quite extensively in the context of Senomyx, titled Biotech’s Dark Promise: Involuntary Cannibalism for All.10 In short, Mr Ji’s ‘article’ rests on the fundamental assertion that ‘abortion tainted vaccines’, among which he ranks anything derived from HEK 293 (which he just refers to as 293),11 constitute cannibalism. He is, of course, entirely misguided, and does not understand how cell line derived vaccines work:

Whereas cannibalism is considered by most modern societies to be the ultimate expression of uncivilized or barbaric behavior, it is intrinsic to many of the Western world’s most prized biotechnological and medical innovations. Probably the most ‘taken for granted’ example of this is the use of live, aborted fetus cell lines from induced abortions to produce vaccines. Known as diploid cell vaccines (diploid cells have two (di-) sets of chromosomes inherited from human mother and father), they are non-continuous (unlike cancer cells), and therefore must be continually replaced, i.e. new aborted, live fetal tissue must be harvested periodically.

For the time being, the VRBPAC has mooted but not approved tumour cell line derived vaccines, and is unlikely to do so anytime soon. However, the idea that diploid cell vaccines need a constant influx of cells is completely idiotic, and reveals Mr Ji’s profound ignorance.12 WI-38, for instance, is a diploid human cell line, and perfectly ‘continuous’ (by which he means immortal). There is no new “aborted, live fetal tissue” that “must be harvested periodically”.

[su_pullquote]Vaccines do not typically contain cells from the culture, but only the proteins, VLPs or virions expressed by the cells. There’s no cannibalism if no cells are consumed.[/su_pullquote]Equally, Mr Ji is unaware of the idea that vaccines do not typically contain cells from the culture, but only the proteins, VLPs or virions expressed by the cells. There’s no cannibalism if no cells are consumed, and the denaturation process (the attenuation part of attenuated vaccines) is already breaking whatever cellular parts there are to hell and back. On the infinitesimal chance that a whole cell has made it through, it will be blasted into a million little pieces by the body’s immune system – being a foreign cell around a bunch of adjuvant is like breaking into a bank vault right across the local police station while expressly alerting the police you’re about to crack the safe and, just to be sure, providing them with a handy link for a live stream.13

From cell lines to Diet Coke: Senomyx and high throughput receptor ligand screening

If you have soldiered on until now, good job. This is where the fun part comes – debunking the fear propaganda against Senomyx by a collection of staggering ignorami.

Senomyx is a company with a pretty clever business model. Instead of using human probands to develop taste enhancers (aka ‘flavourings’) and scents, Senomyx uses a foetal cell line, specifically HEK 293, to express certain receptors that mimic the taste receptors in the human body. [su_pullquote]To reiterate the obvious: no foetal cells ever get anywhere near your food.[/su_pullquote]Then, it tests a vast number of candidate compounds on them, and sees which elicit a particular reaction. That product (which typically is a complex organic chemical but has nothing to do with the foetal cell!) is then patented and goes into your food. To reiterate the obvious: no foetal cells ever get anywhere near your food.

The kerfuffle around this is remarkably stupid because this is basically the same as High Throughput Screening (HTS), a core component of drug development today.14 Let’s go through how a drug is developed, with a fairly simple and entirely unrealistic example.15 We know that low postsynaptic levels of monoamines, especially of serotonin (but also dopamine and norepinephrine) correlate with low mood. One way to try to increase postsynaptic levels is by inhibiting the breakdown of monoamines, which happens by an enzyme called monoamine oxydase (MAO). But how do we find out which of the several thousand promising MAO inhibitors that our computer model spit out will actually work best? We can’t run a clinical trial for each. Not even an animal test. But we can run a high throughput screen. Here’s a much simplified example of how that could be done (it’s not how it’s actually done anymore, but it gets the idea across).

  1. We take a microtitre plate (a flat plate with up to hundreds of little holes called ‘wells’ that each take about half a millilitre of fluid), and fill it with our favourite monoamine neurotransmitters. Mmm, yummy serotonin! But because we’re tricky, we label them with a fluorescent tag or fluorophore, a substance that gives off light if excited by light of a particular wavelength, but only as long as they’re not oxidated by MAO.
  2. We add a tiny amounts of each of our putative drugs to a different well each of the microtitre plate.
  3. Then, we add some monoamine oxidase to each well.
  4. When illuminated by the particular wavelength of light that excites the fluorophores, some wells will light up pretty well, others will be fairly dark. The bright wells indicate that the candidate drug in that well has largely inhibited monoamine oxidase, and thus the monoamine neurotransmitter remained intact. A dark well indicates that most or all of the monoamines were oxidised and as such no longer give off light. This helps us whittle down thousands of candidate molecules to hundreds or even dozens.

What Senomyx does is largely similar. While their process is proprietary, it is evident it’s largely analogous to high throughput screening. The human cell lines are modified to express receptors analogous to those in taste buds. These are exposed to potential flavourings, and the degree to which they stimulate the receptor can be quantified and even compared, so that e.g you can gauge what quantity of this new flavouring would offset 1 grams of sugar. The candidate substances are then tested on real people, too. Some of the substances are not sweeteners itself but taste intensifiers, which interact to intensify the sweet taste sensation of sugar. It’s a fascinating technology, and a great idea – and has a huge potential to reduce the amount of sugars, salts and other harmful dietary flavourings in many meals.

S2222
S2227, a menthol-like flavouring manufactured by Senomyx. Can you spot the aborted cells? Nope, neither can I.

The end product is a flavouring – a molecule that has nothing to do with aborted cells (an example, the potent cooling sensate S2227, is depicted to the left – I dare anyone to show me where the aborted foetal cells are!). Soylent green, it turns out, is not people babies after all.

Conclusion

Now, two matters are outstanding. One is the safety of these substances. That, however, is irrelevant to how they were isolated. The product of a high-throughput screen is no more or less likely to be toxic than something derived from nature. The second point is a little more subtle.

A number of critics of Senomyx point out that this is, in a sense, deceiving the customer, and with pearl-clutching that would have won them awards in the 1940s, point out that companies want one thing, and it’s absolutely filthy:16

Companies like PepsiCo and Nestle S.A. seek to gain over competitors. To do so, they boast products like “reduced sugar”, “reduced sodium”, “no msg”, etc. Sales profits and stocks increase when consumers believe they eat or drink a healthier product.

The Weston A Price Foundation carries skepticism. They believe that the bottom line is what’s important. Companies only want to decrease the cost of goods for increased profits. Shareholders only care about stock prices and investment potential.

Err… and you expected what? There is absolutely no doubt that something that gives your body the taste of salt without the adverse effects of a high-sodium diet is A Good Thing – so consumers do not merely think they’re getting a healthier product, they’re getting a product with the same taste they prefer, but without the adverse dietary consequences (in other words: a healthier product).[su_pullquote align=”right”]To people who have to adhere to a strict diet, flavour enhancers can give back a craved-for flavour experience, improve quality of life and increase diet adherence.[/su_pullquote] To people who have to adhere to a strict low-sodium diet due to kidney disease, heart disease/hypertension or other health issues, this could well give back a craved-for flavour and improve quality of life. To people struggling with obesity, losing weight without having to say no to their favourite drink can result in better health outcomes. People with peanut allergies can enjoy a Reese’s Cup with a synthetic and chemically different protein that creates the same taste sensation, without risking anaphylaxis. In the end, these are things that matter, and should matter more than the fact that – shock horror! – someone is making money out of this.

All Senomyx does is what drug companies have been doing for decades, and an increasing number of companies will. But yet again, the cynics who see lizardoid conspiracies and corporate deceit behind every wall know the price of everything, but haven’t thought about the value of it for a second, have seized upon another talking point. They did so exploiting a genuine pro-life sentiment so many hold dear, intentionally misrepresenting or recklessly misunderstanding the fact that no aborted tissue will ever make its way into your Coke, nor will there be a need for a stream of abortions to feed a burgeoning industry for artificial taste bud cell lines. If anybody here is exploitative, it’s not Senomyx – it’s those who seize upon the universal human injunction against cannibalism and infanticide to push a scientifically incorrect, debunked agenda against something they themselves barely understand.

References   [ + ]

1.This is not a post about the politics of abortion, Roe v Wade, pro-life vs pro-choice, religion vs science or any of the rest. It is about laying a pernicious lie to rest. My position regarding abortion is quite irrelevant to this, as is theirs, but it deserves mention that all of the people who got in touch hold very genuine and consistent views about the sanctity of life. Please let’s not make it about something it isn’t about.
2.Needless to say, this is not my friends’ usual fare, they too have seen it on social media and were quite dubious.
3.In line with our linking policy, we do not link to pages that endorse violence, hate or discrimination.
4.From Greek τέλος, ‘end’.
5.Finally, Rebecca Skloot’s amazing book, The Immortal Life of Henrietta Lacks, paid a long overdue tribute to the mother of modern medicine in 2010. Her book is a must-read to anyone who wants to understand the ethical complexities of immortal cell lines, as well as the touching story of a woman whom we for so long have known by initials only, yet owe such a debt to.
6.Disclosure: the University of Leiden is my alma mater, I have spent a wonderful year there, and received great treatment at LUMC, the university hospital. I am not, and have never been, in receipt of funds from the university.
7.As such, these cells represent an immortalised cell line as opposed to an immortal cell line like HeLa, where the change to the cell cycle regulation has already occurred
8.Human, Embryonic, Kidney, from experiment #293.
9.Sort of. Like all human processes, cell culturing is not perfect. One risk is a so-called ‘contaminated cell line’, and the classical case study for that is the Chang Liver cell line, which turned out to be all HeLa, all the way. This is not only a significant problem, it is also responsible for huge monetary loss and wasted research money. You can read more about this, and what scientists are doing to combat the problem, here.
10.For ethical reasons, this blog refuses to link to, and thus create revenue for, quacks, extremists and pseudoscientists. However, where the source material is indispensable, an archival service is used to obtain a snapshot of the website, so that you, too, can safely peruse Mr Ji’s nonsense without making him any money. GreenMedInfo has a whole tedious page on how to cite their nonsense, which I am going to roundly ignore because a) it looks and reads like it was written by someone who flunked out of pre-law in his sophomore year, b) 17 U.S.C. §107 explicitly guarantees fair use rights for scholarship, research and criticism.
11.Curiously, he does not mention WI-38 and MRC-5, both cell lines derived from the lung epithelial cells of aborted foetuses, which are widely used in vaccine production…
12.The word ‘profound’ is especially meet in this context.
13.Which sounds like something someone MUST have done already. Come on. It’s 2018.
14.This article is a fantastic illustration of just how powerful this technique is!
15.Unrealistic, because we know all there is to know about monoamine oxidase inhibitors, and there’s no point in researching them much more – but it illustrates the point well!
16.Yes, I brought THAT meme in here!

On dealing with failed trials

To survive this moment, you need to be able to look everyone in the eye and remind them that the trial was designed to rigorously answer an important question. If this is true, then you have likely just learned something useful, regardless of whether the intervention worked or not. Further, if the study was well designed, other people will be more likely to trust your result and act on it. In other words, there is no such thing as a “negative” trial result – the answer given by a well-designed trial is always useful, whether the intervention worked or not. So you simply remind everyone of this. People will still be disappointed of course – we’d be fools to test treatments if we didn’t think they worked, and it’s natural to hope. But at least we know we ran a good trial and added knowledge to the world – that’s not nothing.

I rarely post quotes, but this is important enough to have it here. Go read the whole thing, now.

I tend to say that for scientists, there are no failed trials. Failed trials are investor-speak: and that’s not to say anything bad about investors or their perspective. They’re in the business to make money, and to them, a trial that means the drug candidate they just blew a billion dollars to develop will never make it to the market is, in a profound sense, a failure. But to us as scientists, it’s just another step on the journey of understanding more and more about this world (and as any scientist knows, failure is largely the bread and butter of experimental work). We’ve found another way that doesn’t work. Maybe we’ve saved lives, even – failed experiments have often stemmed the tide of predatory therapies or found toxicities that explain certain reactions with other drugs as well. Maybe we’ve understood something that may be useful somewhere else. And at worst, we’ve learned which road not to go down.

And to a scientist, that should not ever feel like a failure.

Herd immunity: how it really works

There are few concepts as trivial yet as widely misunderstood as herd immunity. In a sense, it’s not all that surprising, because frankly, there’s something almost magical about it – herd immunity means that in a population, some people who are not or cannot be immunized continue to reap the benefit of immunization. On its own, this may even be counter-intuitive. And so, unsurprisingly, like many evidently true concepts, herd immunity has its malcontents – going so far as to condemn the very idea as a ‘CDC lie’ – never mind that the concept was first used in 1923, well before the CDC was established.1

Now, let’s ignore for a moment what Dr Humphries, a nephrologist-turned-homeopath with a penchant for being economical with the truth when not outright lying, has to say – not because she’s a quack but because she has not the most basic idea of epidemiology. Instead, let’s look at this alleged ‘myth’ to begin with.

Herd immunity: the cold, hard maths

Our current understanding of herd immunity is actually a result of increasing understanding of population dynamics in epidemiology, towards the second half of the 20th century. There are, on the whole, two ways to explain it. Both are actually the same thing, and one can be derived from the other.

The simple explanation: effective R_0 depletion

The simple explanation rests on a simplification that makes it possible to describe herd immunity in terms that are intelligible at the level of high school maths. In epidemiology, R_0 (pron. ‘arr-nought‘, like a pirate), describes the basic reproduction rate of an infectious disease.2 To put it at its most simplistic: R_0 is the number of cases produced by each case. The illustration on the side shows the index case (IDX) and the first two generations of an infection with R_0 = 3.

Now, R_0 is a theoretical variable. It is usually observationally estimated, and don’t take measures intended to reduce it into account. And that’s where it gets interesting.

Consider the following scenario, where a third of the population is vaccinated, denoted by dark black circles around the nodes representing them. One would expect that of the 13 persons, a third, i.e. about. 4 , would remain disease-free. But in fact, over half of the people will remain disease-free, including three who are not vaccinated. This is because the person in the previous generation did not pass on the pathogen to them. In other words, preventing spread, e.g. by vaccination or by quarantine, can affect and reduce R_0. Thus in this case, the effective R_0 was closer to 1.66 than 3 – almost halving the R_0 by vaccinating only a third of the population.

We also know that for infections where the patient either dies or recovers, the infection has a simple ecology: every case must be ‘replaced’. In other words, if the effective R_0 falls below 1, the infection will eventually peter out. This happens quite often when everyone in a population is dead or immune after an infection has burned through it (more about that later).

Thus, the infection will be sustainable if and only if

R_{0} \geq 1

Under the assumption of a 100% efficient vaccine, the threshold value \bar{p_V} after which the infection will no longer be able to sustain itself is calculated as

\bar{p_V} = 1 - \frac{1}{R_0}

Adjusting for vaccine efficacy, E_V, which is usually less than 100%, we get

\bar{p_V} = \frac{1-\frac{1}{R_0}}{E_V} = \frac{R_0 - 1}{R_0 E_V}

For a worked example, let’s consider measles. Measles has an R_0 around 15 (although a much higher value has been observed in the past, up to 30, in some communities), and the measles vaccine is about 96% effective. What percentage of the population needs to be vaccinated? Let’s consider \bar{p_V}, the minimum or threshold value above which herd immunity is effective:

\bar{p_V} = \frac{R_0 - 1}{R_0 E_V} = \frac{15-1}{15 \cdot 0.96} = \frac{14}{14.4} \approx 97.22\%

The more complex explanation: \mathcal{SIR} models

Note: this is somewhat complex maths and is generally not recommended unless you’re a masochist and/or comfortable with calculus and differential equations. It does give you a more nuanced picture of matters, but is not necessary to understand the whole of the argumentation. So feel free to skip it.

The slightly more complex explanation relies on a three-compartment model, in which the population is allotted into one of three compartments: \mathcal{S}usceptible, \mathcal{I}nfectious and \mathcal{R}ecovered. This model makes certain assumptions, such as that persons are infectious from the moment they’re exposed and that once they recover, they’re immune. There are various twists on the idea of a multicompartment model that takes into account the fact that this is not true for every disease, but the overall idea is the same.3 In general, multicompartment models begin with everybody susceptible, and a seed population of infectious subjects. Vaccination in such models is usually accounted for by treating them as ‘recovered’, and thus immune, from t = 0 onwards.

Given an invariant population (i.e. it is assumed that no births, deaths or migration occurs), the population can be described as consisting of the sum of the mutually exclusive compartments: P = \mathcal{S}(t) + \mathcal{I}(t) + \mathcal{R}(t). For the same reason, the total change is invariant over time, i.e.

\frac{d \mathcal{S}}{d t} + \frac{d \mathcal{I}}{d t} + \frac{d \mathcal{R}}{d t} = 0

Under this assumption of a closed system, we can relate the volumes of each of the compartment to the transition probabilities \beta (from \mathcal{S} to \mathcal{I}) and \gamma (from \mathcal{I} to \mathcal{R}), so that:

\frac{d \mathcal{S}}{d t} = - \frac{\beta \mathcal{I} \mathcal{S}}{P}

\frac{d \mathcal{I}}{d t} = \frac{\beta \mathcal{I} \mathcal{S}}{P} - \gamma \mathcal{I}

\frac{d \mathcal{R}}{d t} = \gamma \mathcal{I}

Incidentally, in case you were wondering how this connects to the previous explanation: R_0 = \frac{\beta}{\gamma}.

Now, let us consider the end of the infection. If \mathcal{S} is reduced sufficiently, the disease will cease to be viable. This does not need every individual to be recovered or immune, however, as is evident from dividing the first by the third differential equation and integrating and substituting R_0, which yields

\displaystyle \mathcal{S}(t) = \mathcal{S}(0) e^{\frac{-R_0 (\mathcal{R}(t)-\mathcal{R}(0))}{P}}

Substituting this in, the limit of \mathcal{R}, as t approaches infinity, is

\displaystyle \lim_{t\to\infty}\mathcal{R}(t) = P - \lim_{t\to\infty}\mathcal{S}(t) = P - \mathcal{S}(0) e^{\frac{-R_0 (\mathcal{R}(t)-\mathcal{R}(0))}{P}}

From the latter, it is evident that

\displaystyle \lim_{t\to\infty}\mathcal{S}(t) \neq 0 \mid \mathcal{S}(0) \neq 0

In other words, once the infection has burned out, there will still be some individuals who are not immune, not immunised and not vaccinated. These are the individuals protected by herd immunity. This is a pretty elegant explanation for why herd immunity happens and how it works. There are three points to take away from this.

First, herd immunity is not unique to vaccination. The above finding in relation to the nonzero limit of \lim_{t\to\infty}\mathcal{S}(t) holds as long as \mathcal{S}(0) \neq 0, but regardless of what \mathcal{R}(0) is. In other words, herd immunity is not something artificial.

Two, for any i \in \mathcal{S} (that is, any susceptible person) at time t, the probability of which compartment he will be in at t+1 depends on whom he encounters. That, statistically, depends on the relative sizes of the compartments. In this model, the assumption is that the sample i will encounter will reflect the relative proportions of the individual compartments’ sizes. Thus if i meets n people at time t, each compartment will be proportionally represented, i.e. for any compartment \mathcal{C}, the proportion will be \frac{\mathcal{C}(t)}{P-1} for all \mathcal{C} \neq \mathcal{S}, for which the proportion will be \frac{\mathcal{S}(t) - 1}{P - 1}, since one cannot meet oneself. Given that the transition probability \beta_{i}(t) is assumed to equal the probability of meeting at least one element of \mathcal{I}, the following can be said. i‘s risk of infection depends on the relationship of n and \mathcal{I}(t), so that i is likely to get infected if

\displaystyle n \frac{\mathcal{I}(t)}{P-1} \geq 1

This elucidates two risk factors clearly, and the way to reduce them: reduce interactions (quarantine/self-quarantine), thereby reducing n, and reduce the proportion of infectious cases (\frac{\mathcal{I}(t)}{P-1}). The latter is where herd immunity from immunisation comes in. Recall that for a constant n, i‘s risk of infection at t rises as \mathcal{I}(t) rises.4 Recall also that while susceptible cases can turn into infectious cases, recovered (or vaccinated) cases cannot. And so, as \mathcal{R}(0) converges to P-1,5 i‘s risk of infection at any time t, denoted by \beta_{i}(t), falls. In other words,

\displaystyle \lim_{\mathcal{R}(0) \to P-1} \beta_{i}(t) = 0

Or to put it simply: the more are vaccinated at the start, the lower the probability, all things being equal, to meet someone who can pass on the infection.6

A final point to note is that this is primarily a model of statistical dynamics, and deals with average probabilities. It does not – it cannot – take account of facts like that some some susceptible people are just darn unlucky, and bump into a flock of unvaccinated, shiny-eyed snowflakes. Equally, in some places, susceptible people and infected people congregate, creating a viral breeding ground, also known as a Waldorf school. There are agent based models, which are basically attempts at brute force hacking reality, that can take account of such disparities. The takeaway is that herd immunity does not mean no susceptible individual will get infected. What it does mean is that their probability of getting infected is going to be significantly lower, for two reasons. First given a constant number of encounters (n), the likelihood of one of them being with an infectious individual is going to be much lower. More importantly, however, because of herd immunity, the disease is going to be able to persist in the population for a far shorter time – eventually it will burn through the small number of ‘accessible’ susceptible persons. Since the cumulative risk \beta_{i}^T for i \in \mathcal{S} for an infection that dies out after time T is defined as

\beta_i^T = \displaystyle \int\limits_0^T \beta_{i}(t) \, \mathrm{d}t

– the sooner the infection dies out, the smaller the likelihood that i will be infected. With that mathematical basis, let’s tackle a few of the myths about herd immunity.

Myth #1: herd immunity only works with naturally acquired immunity

This argument goes roughly along the following lines: herd immunity does exist, but it only exists if and where the immunity is acquired the ‘natural’ way, i.e. by surviving the disease. Case in point:

The $64,000 question, of course, is what the difference is between the residual immunity from a vaccine and the residual immunity from having survived the illness. A vaccine effectively ‘simulates’ the illness without actually causing the pathological symptoms. From the perspective of the immune system, it is largely irrelevant whether it has been exposed to an actual virus that can damage the body, or merely a capsid protein that is entirely harmless but will nonetheless elicit the same immune reaction. That should suffice to bust this myth, but it’s worth considering immunity quantitatively for a moment. As we have seen above, the source of immunity doesn’t matter. In fact, it doesn’t even have to be immunity: culling every animal except one in a herd is an entirely good way to reduce disease transmission. So is sealing oneself away from the rest of society and spending the evenings telling sexually explicit stories, as the heroes and heroines of Boccaccio’s Decameron have done, since we know that

\displaystyle n \frac{\mathcal{I}(t)}{P-1} \geq 1

Boccaccio’s crowd of assorted perverts knew nothing of all this, of course, but they did know that if they reduced n, the number of contacts with possibly infected persons, their chances of surviving the plague would increase. As it indeed did. Score one for medieval perverts. The bottom line is that it is entirely immaterial how immunity was obtained.

Myth #2: Herd immunity is a concept deriving from animals. It doesn’t work on humans.

This is one of the more outlandish claims, but shockingly, it actually has a tiny kernel of truth.

Now, much of the above is a veritable storehouse of insanity, but the point it makes in the beginning has some truth to it. In human populations, herd immunity sometimes behaves anomalously, because humans are not homogenously distributed. This is true a fortiori for humans who decide not to vaccinate, who – for better or worse – tend to flock in small groups. The term of venery for a bunch of anti-vaxxers is, in case you were wondering, a ‘plague’.7

Herd immunity was, in fact, observed in a range of species. Humans are different as we can knowingly and consciously decide to create herd immunity in our population and protect our fellow men, women and children, the last of whom are particularly susceptible to infectious diseases, from some of the worst killers.

Myth #3: If herd immunity can be obtained through natural immunity, surely we don’t need vaccines.

This argument has recently been peddled by the illustrious Kelly Brogan MD, who bills herself as a ‘holistic psychiatrist’ who threw away her script pad, which means she tends exclusively to the worried well and those with mild mental health issues where medication does not play as decisive a role as it does in, say, schizophrenia, severe PTSD, crippling anxiety disorders or complex neuropsychiatric post-insult phenomena.8 Here’s her foray into epidemiology, something she vaguely remembers studying in her first year of med school.

In this, Dr Brogan has successfully found almost a century old evidence for what everybody knew, namely that herd immunity can be naturally obtained. To anyone who has read the maths part above, this should evoke a sensation of ‘DUH!’. The problem is twofold. One, the ‘actual virus’ has an unsavoury fatality rate of 0.1%, not including the horribly tragic, heartbreaking late consequence of measles known as SSPE.9 Two, and perhaps more important: you don’t get lifelong, natural immunity if you die. This may have somehow escaped Dr Brogan’s attention, but part of the point of herd immunity is to protect those who would not survive, or would suffer serious sequelae, if they contracted the infection. What we don’t know, of course, how many of that 68% suffered permanent injuries, and how many are not included because they died. What we do know is that all 68% probably had a miserable time. Anyone who thinks measles is so fantastic should start by contracting it themselves.

Myth #4: Herd immunity means 95% need to be vaccinated to prevent a disease.

This one comes courtesy of Sarah aka the Healthy Home Economist,10, who, to what I presume must be the chagrin of her alma mater, states she has a Master’s from UPenn. Suspiciously enough, she does not state what in. I am somehow pretty sure it’s not public health.

The tedious conspiracy theory aside, it is quite evident just how little she understands of herd immunity. No – herd immunity is not based upon11 the idea that 95% must be vaccinated, and it is most definitely not based on the idea that 100% must be vaccinated. Indeed, the whole bloody point of herd immunity is that you do not need to vaccinate 100% to protect 100%. In fact, given the R_0 and vaccine efficacy E_V, we can predict the threshold vaccination rate for herd immunity quite simply, as demonstrated earlier: the threshold value, \bar{p_V}, can be calculated as

\bar{p_V} = \frac{R_0 - 1}{R_0 E_V}

As an illustration, the herd immunity threshold \bar{p_V} for mumps, with an efficacy of 88%12 and an R_0 of around 5.5, is \approx 92.98\%, while for Ebola, which has a very low R_0 around 2.0, herd immunity sets in once about 50% are immune.13

And those ‘conventional health authorities’? That’s what we call health authorities whose ideas work.

Myth #5: If vaccines work, why do we need herd immunity?

This argument is delightfully idiotic, because it, too, ignores the fundamental underlying facts of herd immunity. Quite apart from the fact that some people cannot receive some or all vaccines and other people can receive vaccines but may not generate sufficient antibody titres to have effective immunity, sometimes vaccines simply fail. Few things are 100%, and while vaccines are designed to be resilient, they can degrade due to inappropriate storage or fail to elicit a sufficient response for some other reason. Unlike wearing deodorant (or ‘deoderant’, as spelling-challenged anti-vaxxers would say), infections can sometimes be imagined as a chain of transmission. This is a frequently used model to explain the consequences of not vaccinating on others.

In this illustration, an index patient (IDX) is infected and comes in contact with G1, who in turn comes into contact with G2 who in turn comes into contact with G3. In the first case, G1, G2 and G3 are all vaccinated. The vaccine may have a small failure rate – 2% in this case – but by the time we get to G3, his chances of contracting the infection are 1:125,000 or 0.0008%. In the second case, G2 is unvaccinated – if G1’s vaccine fails, G2 is almost guaranteed to also fall ill. By not vaccinating, his own risk has increased 50-fold, from 0.04% to 2%. But that’s not all – due to G2’s failure to vaccinate, G3 will also be affected – instead of the lottery odds of 1:125,000, his risk has also risen 50-fold, to 1:2,500. And this 50-fold increase of risk will carry down the chain of potential transmission due to G2’s failure to vaccinate. No matter how well vaccines work, there’s always a small residual risk of failure, just as there is a residual risk of failure with everything. But it takes not vaccinating to make that risk hike up 50-fold. Makes that deodorant (‘deoderant’?) analogy sound rather silly, right?

Conclusion

Admittedly, the mathematical basis of herd immunity is complex. And the idea itself is somewhat counterintuitive. None of these are fit excuses for spreading lies and misinformation about herd immunity.

I have not engaged with the blatantly insane arguments (NWO, Zionists, Masonic conspiracies, Georgia Guidestones), nor with the blatantly untrue ones (doctors and public health officers are evil and guided just by money as they cash in on the suffering of innocent children). I was too busy booking my next flight paid for by Big Pharma.14 Envy is a powerful force, and it’s a good way to motivate people to suspect and hate people who sacrificed their 20s and 30s to work healing others and are eventually finally getting paid in their 40s. But it’s the myths that sway the well-meaning and uncommitted, and I firmly believe it’s part of our job as public health experts to counter them with truth.15

In every social structure, part of co-existence is assuming responsibility not just for oneself but for those who are affected by our decisions. Herd immunity is one of those instances where it’s no longer about just ourselves. Many have taken the language of herd immunity to suggest that it is some sort of favour or sacrifice done for the communal good, when it is in in fact the very opposite – it is preventing (inadvertent but often unavoidable) harm to others from ourselves.

And when the stakes are this high, when it’s about life and death of millions who for whatever reason cannot be vaccinated or cannot form an immune response, getting the facts right is paramount. I hope this has helped you, and if you know someone who would benefit from it, please do pass it on to them.

References   [ + ]

1.Topley, W. W. C. and Wilson, G. S. (1923). The spread of bacterial infection; the problem of herd immunity. J Hyg 21:243-249. The CDC was founded 23 years later, in 1946.
2.Why R_0? Because it is unrelated to \mathcal{R}, the quantity denoting recovered cases in \mathcal{S(E)IR} models – which is entirely unrelated. To emphasize the distinction, I will use mathcal fonts for the compartments in compartment models.
3.I hope to write about SIS, SEIR and vital dynamic models in the near future, but for this argument, it really doesn’t matter.
4.Technically, as \frac{\mathcal{I}(t)}{P - 1} rises, but since the model presupposes that P is constant, it doesn’t matter.
5.Since otherwise \mathcal{R} = P and \mathcal{S} = 0, and the whole model is moot, as noted above.
6.Note that this does not, unlike the R_0 explanation, presuppose any degree of vaccine efficacy. An inefficiently vaccinated person is simply in \mathcal{S} rather than \mathcal{R}.
7.Initially, ‘a culture’ was proposed, but looking at the average opponent of vaccination, it was clear this could not possibly work.
8.In other words, if you have actual mental health issues, try an actual psychiatrist who follows evidence-based protocols.
9.Subacute sclerosing panencephalitis is a long-term delayed consequence of measles infection, manifesting as a disseminated encephalitis that is invariably fatal. There are no adjectives that do the horror that is SSPE justice, so here’s a good summary paper on it.
10.As a rule, I don’t link to blogs and websites disseminating harmful information that endangers public health.
11.Correct term: ‘on’
12.As per the CDC.
13.Efficacy E_V is presumed to be 100% where immunity is not acquired via vaccination but by survival.
14.Anyone in public health is happy to tell you those things don’t merely no longer exist, they never even existed in our field.
15.And, if need be, maths.

Epidemiologists, here’s your chance to make your parents proud.

I found this t-shirt as a stocking stuffer for my dad, who still doesn’t really know what I do for a living, other than that it has ‘to do with viruses’ (yeah… almost, Dad.). Epidemiology geeks, there’s hope: here’s your chance to make your parents proud this Christmas!

Taken on Dec 06, 2017 @ 23:26 near Budapest II. kerület, this photo was originally posted on my Instagram. You can see the original on Instagram by clicking here..