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Tag: survival curve

The Sickle Cell Salve

I might have been crazy late with April, but for May, my on-timeliness when it comes to the paper a month posts is returning with a vengeance.

This month’s paper from Cell (a journal I usually avoid because their papers are ridiculously long :-)) dives beneath the surface of one of the classic examples of genetics used in almost every intro-to-genetics seminar/class/textbook. As you probably know, living things typically receive two sets of genes: one from the mother and one from the father. If those two sets of genes result in the same protein, then the organism is said to be homozygous for that particular trait. Otherwise, the proper term is heterozygous. In classical genetics (i.e. what was painstakingly discovered by Gregor Mendel, the “father of genetics”), being heterozygous, to a casual observer, was usually something that could only be seen after multiple generations (or with a DNA test). This is because even though the individual has two different versions of the same gene, one of them is “dominant”, expressing itself more loudly than the other.

en93587For the mutation which causes the disease sickle cell anemia (see image to the right as to why the disease is called “sickle cell”), however, the truth was a little different. While heterozygous individuals did not suffer from the problems associated with sickle cell anemia, unlike individuals homozygous for the “normal” gene, they showed a remarkable advantage when it came to surviving infection with malaria. It is one reason scientists feel that sickle cell anemia continues to be endemic in parts of the world where malaria is still a major issue.

But, how the sickle cell disease mutation did this in heterozygotes was not well-understood. The authors for this month’s paper tried to probe one possible explanation for this using mice as an experimental system. The interesting thing that they found was that mice that were heterozygous for the sickle cell trait (HbSAD), despite having better survival against malaria than those which were homozygous for the “normal” gene (HbWT) (see the Kaplan-Meier survival curve in Figure 1A below, showing the proportion of surviving mice over time), did not have a significantly different amount of infected red blood cells (see Figure 1F below).

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So if the sickle cell gene wasn’t reducing the number of infected cells, what was causing the improvement in survival? The researchers knew that red blood cells which are heterozygous for the sickle cell trait will often “leak” an iron-containing chemical called heme into the blood stream. Because heme just floating around is toxic, the body responds to this with an enzyme called heme oxygenase-1 (HO-1) which turns toxic heme into the less toxic biliverdin and carbon monoxide (CO). The researchers considered whether or not HO-1 was responsible for the improved ability of the mice to avoid cerebral malaria. In a creative experiment, they were able to show that the sickle cell mice needed HO-1 to get their better survival – mice which were genetically engineered to be missing one copy of HO-1 (Hmox1+/-), even if they were heterozygous for the sickle cell disease, did not survive particularly well when infected (see Figure 2B below, left for the survival data).

In fact, they were even able to show that if you took mice which did not have any sickle cell trait gene (the HbWT group), and replaced their blood system using irradiation and a bone marrow transplant from a heterozygous sickle cell mouse (HbSAD), you only improve survival if the cells come from a mouse with its HO-1 genes intact (Hmox1+/+) (see Figure 4A below, right).

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So, we know HO-1 is somehow the source of the heterozygous mice’s magical ability to survive malaria. But, how? As I stated earlier, the researchers knew HO-1 produced carbon monoxide (CO) and, they were able to show that heterozygous mice with a defective HO-1 response were able to survive when given carbon monoxide (see Figure 6E below). Interestingly, exposure to carbon monoxide reduces the amount of heme floating around in the bloodstream, something which gets kicked into overdrive when malaria starts killing red blood cells left and right (see Figure 6G below), something the researchers validated when they were able to neutralize the protecting power of carbon monoxide by adding more heme back into the mouse (see Figure 6H)

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So, overall, what did I think? First the positive: these are extremely clearly designed and well-controlled experiments. I could only show a fraction of the figures in the paper, but rest assured, they were very methodical about creating positive and negative controls for all their figures and experiments which is fantastic. In particular, the use of bone marrow transplantation and genetically engineered mice to prove that HO-1 plays a key role in improving survival were creative and well-done.

What leaves me unsettled with the paper is the conclusion. The problem is that the trigger for HO-1, what the authors have shown is the reason mice which are heterozygous for sickle cell anemia survive malaria better, is heme, which happens to also be what the authors say is the cause for many of the survival complications. It’s like claiming that the best way to cure a patient of poison (heme) is to give the patient more poison (heme) because the poison somehow triggers the antidote (HO-1).

In my mind, there are two possible ways to explain the results. The first is that the authors are right and the reason for this is around the levels and timing of heme in the bloodstream. Maybe the amount of heme that the sickle cell heterozygotes have is not high enough to cause some of the malaria complications, but high enough so that HO-1 is always around. That way, if a malaria infection does happen, the HO-1 stays around and keeps the final level of heme just low enough so that problems don’t happen. The second explanation is that the authors are wrong and that the carbon monoxide that HO-1 is producing is not reducing the amount of heme directly, but indirectly by reducing the ability of the malaria parasites to kill red blood cells (the source of the extra heme). In this case, sickle cell heterozygotes have chronically higher levels of HO-1.

Both are testable hypotheses – the first can be tested by playing around with different levels of heme/HO-1 and observing how the amount of free-floating heme changes over time when mice are infected with malaria. The second can be tested by observing test tubes full of red blood cells and malaria parasites under different amounts of carbon monoxide.

In any event, I hope to see further studies in this area, especially ones which lead to more effective treatments for the many millions who are affected by malaria.

(Image Credit – Sickle Cell) (Figures from paper)

Paper: Ferreira et al., “Sickle Hemoglobin Confers Tolerance to Plasmodium Infection.” Cell 145 (Apr 2011) – doi: 10.1016/j.cell.2011.03.049

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Of Ticks and Bacteria

Another month, another paper to blog.

imageOne of the most fascinating things about studying biology is finding out the numerous techniques living things use to survive through adversity. This month’s paper digs into an alliance between a tick species Ixodes scapularis and a bacterium Anaplasma phagocytophilum to help the pair survive through long winter months.

In places where winters can get extremely cold, people will oftentimes use antifreeze to help protect their car engines. Many cold-blooded animals (ectotherms) survive harsh winters in the same way. They produce antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) which are believed to bind to ice crystals and limit their growth and ability to damage the organism.

As Ixodes ticks are fairly active during winter months and are a known carrier of Anaplasma phagocytophilum which is a cause of human granulocytic anaplasmosis, a team of researchers from Yale Medical decided to investigate whether or not Anaplasma had any impact on the ability of Ixodes to survive cold weather.

Panel A of Figure 1 (below) is a survival curve. It shows what % of ticks which have Anaplasma (in dark black circles) and which don’t (in white circles) survived being placed in –20 degrees (Celsius, of course, not Farenheit: this is science after all!) for a given amount of time. While all the ticks died after 45 minutes, at any given timepoint more ticks with Anaplasma survived than the ticks without. While only ~50% of ticks without Anaplasma survived after ~25 minutes in the cold, over 80% of ticks with the bacterium survived!

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What could explain this difference? The researchers suspected some sort of antifreeze protein, and, after combing through the tick’s genome, they were able to locate a protein which they called IAFGP which bore a striking resemblance to other antifreeze glycoproteins.  But, was IAFGP the actual antifreeze mechanism which kept Ixodes alive? And did Anaplasma somehow increase its effectiveness?

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Panel C of Figure 4 (above) shows the key findings of the experiments designed to answer those two questions. Along the vertical axis, the researchers measured the amount of IAFGP gene expression (relative to the gene expression of a control, actin [a structural protein which shouldn’t vary]). Along the horizontal, the researchers tested four different temperature states (23, 10, 4, and 0 Celsius) with Ixodes ticks that were carrying Anaplasma (dark circles) and those that were not (white circles). Each individual circle is an individual tick and the line is the average value of all the ticks in the experimental group (the reason its not in the middle is because the vertical axis is a log scale). This sort of chart is one of my favorites, as it packs in a lot of information in one small area but without generating too much noise:

    • The lines get higher the further to the right we get: Translation: when temperatures go down, IAFGP levels go up – as you would expect if IAFGP was an antifreeze coping mechanism for Ixodes. (And if you could see Panel B of Figure 4, you’ll notice that IAFGP levels at 4 Celsius and 0 Celsius are statistically significantly higher than at 23 and 10)
    • The black dots on average are higher than the white dots: Translation: just carrying Anaplasma seems to push Ixodes’s “natural” levels of antifreeze protein up. And, judging from the P value comparisons, the differences we are seeing are statistically significant.

So, it would seem that IAFGP is somehow related to the affect of Anaplasma on Ixodes, but, is that the only link? To test that, the researchers used an experimental technique called RNA interference (RNAi) which allows a researcher to shut down the expression of a particular protein. In this case, the researchers shut down IAFGP to see what would happen.

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These results are interesting. Although, sadly, the charts (Panels B and F of Figure 5) are not on the same scale and are for different experiments, the numbers are striking. In Panel B, the researchers tested for the survival of ticks which were given a control RNAi (simulates the RNAi process except without what it takes to actually silence IAFGP, white circles) versus those which had IAFGP shut down via RNAi (white triangles). As you can see from the chart, after 25 min at –20 degrees Celsius, the control group hit 50% survival whereas the RNAi group’s survival rate plummeted to only 20%.

The researchers then repeated the experiment with Ixodes ticks which were given control RNAi (black circles) vs. the real thing (black triangles) and then allowed to feed on Anaplasma-infected mice for 48-hours. These ticks were then tested for survival after 50 min at –20 degrees Celsius. As you can see in Panel F, a 75% survival level amongst Anaplasma carrying ticks became less than 50% when IAFGP was shut down with RNAi.

All in all, a very simple positive-control, negative-control experiment showing a pretty clear linkage between Anaplasma, IAFGP gene expression levels, and the ability of Ixodes ticks to survive the cold. However, a few things still bug me about the study and stand out as clear next steps:

    • Panels B and F of Figure 5 are fundamentally different experiments, but presented as comparable. At face value, its hard to tell if IAFGP is the primary mechanism for how Anaplasma alters tick response to the cold. The survival levels of Anaplasma-carrying ticks when IAFGP is shut down is still higher than the survival levels of Anaplasma-free ticks which also undergo the RNAi – but this could be a result of the different experimental conditions (feeding conditions and time). The paper text also reveals that the –20 degrees for 50 min condition was selected because it was supposed to be the point at which there was 50% survival for that particular experimental condition – but clearly, the control group was experiencing 75% survival (and the group with IAFGP shut off was at 50%). Something is off here… but I’m not sure what.
    • Most of this research was conducted on a very abstract level – showing the impact of IAFGP expression levels on cold survival. While the RNAi experiments are very compelling, the lack of clear functional studies is problematic in my mind as I cannot tell from this data if IAFGP is directly responsible for cold survival or linked to other, potentially more important responses to cold.
    • No mechanism was proposed for how Anaplasma increases IAFPG levels in Ixodes. Understanding that would be very powerful and could unveil a whole world of cross-species gene regulation which we were previously unaware of (and could reveal new potential targets for medical treatments of diseases borne by insects).

Regardless of my criticisms, though, this was an interesting study with a very cool result. However, its probably of no comfort to people who have to deal with ticks which can survive cold winter months…

(Image credit – tick) (Figures 1, 4, and 5 from paper)

Paper: Neelakanta, Grisih et al. “Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold.” Journal of Clinical Investigations 120:9, 3179-3190 (Sep 2010) — doi:10.1172/JCI42868

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