Bt maize and mouse immune responses - what's wrong with this picture?

I have to admit, I haven't been paying close attention to anti-GMO material lately. I've been very busy writing my PhD thesis* and so have largely left GM related myth detection and busting in other very capable hands, such as the guys at the Genetic Literacy Project and the Plant Science Panel at Sense about Science. I also don't get a whole lot of exposure to unscientific anti-GM arguments during my average work day, what with working in a plant science lab.


However, by night (and morning, and weekend), I'm a fancy rat owner and breeder, which exposes me to all sorts of different people and views, especially where rat nutrition is concerned. I've become quite used to discussing (dare I say debunking) concerns relating to the safety of Roundup Ready (RR) maize in rat feed that followed the widely refuted, retracted and then re-plublished Seralini study on long term exposure to glyphosate and RR maize. Less expected, however, were concerns regarding the below poster shared from the GMO Free USA facebook account, referencing a recently published study on mouse immune responses to the Bacillus thuringiensis (Bt) protein 'Cry1Ab' - a naturally occurring insecticidal protein which has been modified and inserted into maize and other crop species. This makes the crops more resistant to some insect pests, thus making growers less reliant on pesticide sprays.

Post by GMO Free USA.

Dare I say it? I smelled a rat. So I decided to review the evidence behind the poster. (I will confess, for the sake of my sanity I stuck to the referenced publication on inhalation of Bt proteins. I believe studies on gut responses have been reviewed elsewhere but if I fail to find the links I may do it myself.)


It has to be said, the creators of the poster have done a good job of evoking fear without saying anything too outlandish - note the use of the word 'may'. However, the implication was that the referenced study proved that inhalation of GM Bt maize induced an allergic reaction in mice. The creators also cherry-picked the data which could potentially support their cause, without acknowledging the data that didn't...



What the paper says


The researchers exposed the mice to a number of treatments by anaesthetising them and then dripping liquid solutions directly into their nostrils. The treatments were purified Cry1Ab from B. thuringiensis, the transgenic Cry1Ab found in maize, pollen and leaf preparations of GM Bt maize, and pollen and leaf preparations from non-Bt maize as controls. This was accompanied by a group which were just treated with liquid buffer. From what I can tell, no other purified protein was used as a control. They then measured the immune response of the mice (looking at variables such as IgE antbibody production, which is associated with allergic responses, and proliferation of different sorts of white blood cells and proteins important for immune responses).

The main piece of 'positive' data presented in this study, which the poster takes inspiration from, is the finding that the pure Bt protein treatments resulted in Cryp1Ab-specific antibody production. However, these antibodies were not detected from the mice treated with the Bt maize extracts, or the non-GM maize extracts. None of the other immune response variables (with the exception of one type of cytokine in one experiment) differed significantly between mice treated with the Bt protein containing extracts and those treated with extracts from non-Bt plants. However, this is somewhat obfuscated in the paper:

The authors repeatedly, and rather confusingly, refer to the mice treated with liquid buffer as the controls, rather than those treated with the non-GM maize extracts. This allows the authors to highlight the significant differences between the GM plant extract treatments and their 'control group'. However, as they quite rightly point out, there is no significant difference between the GM and non-GM plant treatments, indicating that the observed responses are not due to the Bt protein. Indeed, one type of white blood cell (eosinophils) were elevated in response to plant extracts, but not in response to the purified Bt protein, which rather suggests that this response was caused by another plant component. Indeed, the authors conceded in their discussion that many of the observed responses were likely a generalised response to plant extracts rather than to Bt protein.

So to summarise - purified Bt protein (Cry1Ab) treatment resulted in antibody production, but neither this nor the other investigated variables differed between mice treated with Bt maize extracts compared to non-Bt maize extracts.

Limitations of the study

Aside from the aforementioned lack of a non-allergenic protein control, this study has a few limitations that I've picked up on, many of which the authors themselves highlight in their discussion:

The premise for this research was to analyse the risks of allergic reactions to inhaled Bt protein, either from insecticide sprays (more on that later) on inhaling GM Bt pollen. However, it's not clear how well their experimental design of dripping liquid into the nostrils of unconscious mice placed on their backs reflects natural nasal exposure of humans to the investigated substances. Indeed, the authors state that "the model is optimised to induce allergy", indicating that it may not reflect normal exposure in humans. This contradicts the GMO Free USA statement accompanying the poster that "the protein may actually be MORE [sic] allergenic and immunotoxic than this study suggests"

Furthermore, again acknowledged by the study authors, "the clinical relevance of the detected specific IgE was not evaluated" meaning that no assessment was made of whether the IgE production led to a symptomatic allergic reaction in mice. Further work is therefore required to demonstrate whether, as the poster puts it, "mice that inhale Bt toxin insecticidal proteins from GM corn may develop immune and allergic reactions".

Bt isn't just a 'GM' thing

One major caveat of this study's use by anti-GMO campaigners is that the use of Bt proteins isn't unique to GM crop production. On the contrary, B. thuringiensis is a widely used 'biopesticide', and has been used in organic crop production for over 50 years. Opponents of GM have previously argued that the 'natural' Bt protein and the transgene product are not substantially equivalent, since the naturally occurring protein is activated in insect guts, while the transgenic protein is 'pre-activated', which could cause health issues in humans and animals. However, this particular study does not support that argument, since both forms of the protein resulted in antibody production when applied in purified form. The fact that this was not the case for Bt protein containing maize extracts again rather turns this argument on its head. The authors state in their conclusions that:

"Given the importance of Bt-transgenic maize as food and feed across the world, a considerable number of individuals may be exposed to Cry1Ab by inhalation, in the field as well as along the food/feed chain."

However, since antibody production was induced by purified Bt protein but not by plant extracts, the relevance of transgenic Bt-crops as opposed to Bt treatment of conventional and organic crops in terms of human exposure is arguably overstated.



Conclusions


The referenced study does provide evidence that purified Bt protein Cry1Ab can induce an immunogenic response in mice, when applied in liquid form directly to their nasal passages. However, this does not translate into evidence that Bt maize causes allergic reactions (in mice or humans), especially since no clear immunological response to Bt-maize extracts was observed compared to non-GM plants.

I for one will be leaving my face mask in the growth room, where it protects me from mycotoxin producing Fusarium species. Incidentally, there is evidence that cultivation of Bt-maize might help to lower contamination of maize cobs with fungal mycotoxins, since the fungi normally infect the plants through holes made by certain insects (which are killed by Bt protein). I personally would rather risk consuming Bt protein than deoxinivalenol (a.k.a vomitoxin), but perhaps that's just me.

Don't get me wrong, I'm not saying Cry1Ab isn't a potential human allergen. It might well be, but the findings from this study are a long way from demonstrating it. In addition, this study suggests that all forms of Bt protein might be equally allergenic, rather than just those produced by GM crops.

Finally, and only because I'm being very pedantic, that's a baby rat wearing a mask, not a mouse. Here's a picture of a baby rat without a mask, which is much cuter.

*As a side note, this blog post has taken me over a day to research and write. This included reading the paper a couple of times, brushing up on my knowledge of antigens, allergens and the human immune system (quite different to plants) and reading other relevant documents, and being accused of working for Monsanto when I tried to engage with other commenters on the original post. All this when I arguably should have been writing up papers from my PhD thesis in order to increase the likelihood of securing funding to carry on doing research into how to protect plants from diseases. This was without addressing other questions such as the credibility of the journal, the interests and funding source of the authors, and the validity of the statistical tests used. Small wonder, then, that many anti-GMO statements like this one go unrefuted by scientists. It's not that they're irrefutable, rather that doing all the research required to present a solid, evidence-based critique is likely far more laborious than generating the statements in the first place.

Plant Pathology PhD students - flex your presentation muscles at MBPP!

Once every year, scores of budding plant pathologists emerge from the confines of their greenhouses and laboratories and flock to a single location somewhere in the UK, where they share stories and alcohol….

This event is neither a natural mating phenomenon nor a ceremonial cult gathering. It is, rather, Molecular Biology of Plant Pathogens (MBPP) – an annual meeting held at a different venue in the UK each year. Its tried and tested formula – 1.5 days of student/postdoc oral presentations intermingled with keynotes from senior academics – is a brilliant way for PhD students (and postdocs) to flex their presentation muscles in front of a friendly, supportive audience of global plant pathology researchers. It also provides an excellent networking opportunity, all of which makes it a valuable exercise both in its own right but also in terms of preparing students for larger, international conferences (more on that in a follow-up post).

There is also the (in)famous ‘Pathology and Alcohol’ talk to attend, this year given by Dr. Chris Ridout on improving Barley for craft beer brewing. I can’t make any promises, but I rather envisage that this might involve some form of taste-based ‘sampling’, for scientific purposes of course, which is in itself a wonderful bonding exercise for plant pathologists. This is followed by a poster session and wine reception, and then of course the conference dinner, which provides further opportunities to network and perhaps scope out future collaborators or employers.

While accommodation is not included, the registration fee for MBPP also makes it an attractive and accessible meeting for students, at just £50 for both days, including lunches and the conference dinner. You may even be able to claim this back from your studentship grant. This year MBPP is taking place on 8^th-9^th April at the University of the West of England (UWE, Bristol), so there’s still plenty of time to plan your experiments around it!

Meeting and registration details: http://www.mbpp2015.co.uk/

From the organisers:

“MBPP provides an excellent forum for networking between junior and senior scientists. The primary focus is on providing PhD students and post-doctoral scientists the opportunity to give oral presentations in front of a wide range of national and international researchers.”

'Irresistable' antibiotic isolated from caged soil microbe

I often find myself terrified by the development of antimicrobial resistance (resistance to antibiotics, fungicides, etc.) in harmful bugs. I'm terrified as a plant pathologist, because I understand all too well the threat that plant pathogens and pests pose to global food security, and just how reliant we are on biocides to control them. I'm terrified as a pet owner and breeder, observing the staggering frequency with which companion animals present with antibiotic-resistant bacterial infections. And, of course, I'm terrified as a human being, envisaging a future where death due to bacterial infections becomes the norm, rather than the stuff of nightmares, or indeed where infection risks from surgery are deemed too high for currently treatable conditions to be treated.


Sometimes though, I take time out from being terrified to get on with my own research on making plants more resistant to infectious disease (thus reducing the need for antimicrobials in the first place) or to eavesdrop on my colleagues and find out what they're up to. That's how I know that soil is absolutely chock full of unculturable bacteria, which are hard to study in the lab since they just won't grow on a dish or in a test tube. These bacteria are involved in all sorts of processes and ecosystem functions, such as nitrogen and carbon cycling, degradation of agricultural chemicals, and plant growth promotion. As research published yesterday in Nature shows, they might also be an invaluable source of novel antimicrobials.


The researchers in this study were trying to isolate novel antimicrobials from unculturable soil bacteria. The problem, of course, is how to cultivate and study bacteria that won't grow on in the lab. To get around this, the researchers used an Ichip. A what? Quite. In my mind's eye, the Ichip ('I' for isolation) is like a series of tiny cages. A single bacterial cell is captured in each cage, and the Ichip is then placed back in the soil. The gaps between the 'bars' of the cages are too narrow to let bacteria in or out, but big enough to allow fluid and nutrients to move across. It's a bit like the cages divers use around sharks. The diver can't get out and the shark can't get in, but they're both submerged in the same water. This allowed a single 'unculturable' bacterial cell to multiply in each cage - i.e. be cultured.

'Artist's' interpretation of an Ichip

Using Ichips, the researchers managed to culture about 50% of the 'captured' cells, which is a big step up from the 1% that will grow on a petri dish. They did this for 10,000 bacterial cells, and then took extracts from each culture, and tested whether they were any good at killing Staphylococcus aureus - a bug that can cause all sorts of serious infections in humans and animals. Antibiotic resistant strains of S. aureus already pose a major health threat, so finding new ways to kill it are a top priority.
One extract, taken from the culture of a new bacteria called Eleftheria terrae, seemed particularly good at killing S. aureus. The researchers isolated the 'killer' molecule in this extract, which doesn't seem to have been isolated ever before, which meant they got to name it.


They went with teixobactin, which is probably a more modest name than I might have come up with if I discovered a new bug busting molecule. Especially since it wasn't just really good at killing S. aureus, but also a whole load of other disease causing bacteria including Mycobacterium tuberculosis (which causes TB), Clostridium difficile, and Bacillus anthracis. There were other bacterial types that it wasn't so good at killing, but since antibiotic resistant 'staph' and TB are already a major problem it wasn't off to a bad start.


Of course, finding a new class of antibiotic is only part of the battle. The reason why we need new ones is due to the development of resistance to the existing ones. The likelihood of resistance development can be tested in the laboratory by repeatedly exposing a bacterial colony to a sub-lethal dose of an antibiotic, and monitoring whether this alters the minimum dose needed to kill the bacteria. Encouragingly, the researchers didn't find any change in the dose needed to kill S. aureus when doing this, and didn't manage to isolate resistant strains of S. aureus or M. tuberculosis when using lethal doses. The reason for this 'resistance to resistance' is thought to be that teixobactin (I forgot the name of it then, I said I'd have thought of something more snazzy) prevents cell wall formation by binding to the fatty building blocks. Changes in these building blocks, which could create resistance to the teixobactin, are less likely than changes in the protein building blocks, which are targeted by other antibiotics.


However, many promising antimicrobials get this far and then fall down, either because they prove to be toxic to mammals, or because they don't manage to kill bacterial cells inside a living animal. Sometimes because they can't get to the infection site, or because the animal's own cells break down the antimicrobial compound before it can kill the bacterial cells. However, teixobactin jumped all of these hurdles too. At the petri dish stage, it was found to be non-toxic to human cells. Then it was found to be non-toxic to living mice, and persist inside them at bacteria-killing levels for at least 4 hours. It was also completely successful in treating septicaemia (blood poisoning) in mice, after a single dose, and pretty good at treating lung infection too.


So there we have it. A new compound from a mysterious caged microbe, which kills a number of dangerous disease-causing bacteria on plates and in mice and evades resistance by targeting fatty building blocks. Of course, teixobactin hasn't reached its last hurdles. Just because no resistance occurred under laboratory conditions doesn't mean that resistance to teixobactin will never emerge. Also, it has yet to prove safe and effective for human use, but the researchers are optimistic that human trials could start within the next few years. So it might still be one less reason to be quite so terrified.