Human Immunodeficiency Virus – the sneaky sleep thief

Kirsten Redman

 

Stepping into the field of physiology was not something I took lightly. It was largely intimidating; hugely intimidating, in fact, especially given that I came from a microbiology background! When the option came for me to exercise my knowledge of virology in my Honours year, I jumped at the opportunity.

 

Research had already been widely conducted on HIV and sleep in other parts of the world. Indeed, with the success of highly active antiretroviral therapy (ART), HIV has increasingly taken the position as a chronic illness. And in fact, quality of life aspects have become more prominent as HIV-related life expectancy has increased. As more was discovered elsewhere, it was a shame for me to realise that not much research on sleep and HIV was being done here in South Africa. So that is where I fitted myself in – well, my supervisors created this niche, but for the purposes of this story I’ll say that I inserted myself.

 

During my Honours year, I had run a cross-sectional study in a treated HIV cohort of South African patients, which unexpectedly revealed a relationship between higher CD4 counts and poorer sleep quality. Just to give some background, having a low CD4 count is one of the markers of AIDS, and the aim of ART is, by various means, to increase CD4 counts.
This relationship was seemingly counterintuitive, because one would expect there to be better sleep quality with better immune status (higher CD4 counts), as has been shown in studies in other countries, which found that lower sleep quality was associated with lower CD4 counts. Our cohort at the time differed from other studies’ cohorts as our patients had started ART later in their disease and their baseline CD4 counts were very low. We then hypothesized that during immune reconstitution, there may have been an increased immune activation in these patients, which may explain why the higher CD4 counts were associated with worse sleep quality. So we designed a longitudinal study to see how sleep quality progressed from an ART naïve state, up until a few months on treatment. In this longitudinal study, surprisingly, we found that patients did not complain about their sleep per se but seemed to start off with high daytime sleepiness which reduced (got better) across the study. In this new study, we found no relationship with CD4 counts and sleep quality, but instead found that people who had low viral loads at the time of initiation onto ART had worse sleep than those who had high viral loads.

 

So what happened between my honours and my masters research studies?

 

First of all, my cross-sectional study patients had been diagnosed for seven years and treated on average for four years, very different from the longitudinal cohort who had just been put on treatment and for whom we had followed up ‘only’ until the 18th month, though it is possible that sleep disruption occurs after our cut-off time period. Second, as mentioned before, patients in my cross-sectional study had started their treatment late in the disease when CD4 counts had dropped below 100 cells/μl (AIDS is defined by <250 CD4 T cells/μl and below 100 cells/μl, there are higher chances of developing immune reconstitution inflammatory disease when put on ARV treatment). In our longitudinal cohort, the guidelines had changed and patients started ARVs on average around 250 CD4 counts/μl. So it is possible that what we observed in the first study was a unique effect of starting treatment so late.  In fact, current ‘reservoir’ studies (which follow HIV infection in CD4 memory T-cells) show that immune reconstitution is associated with a higher HIV reservoir load in CD4 memory cells. It is also associated with CD4 activation when treatment was started at lower CD4 counts, while there is nearly no HIV reservoir and very low CD4 activation when treatment is started when CD4 counts are still above 500 cells/μl.

 

And what about this new finding on the relationship between viral load and daytime sleepiness?

 

Currently we postulate that there is an underlying immune-mechanism affecting sleep quality. Indeed cytokines such as TNFa, Il-6, and Il1 have been shown to affect sleep quality. It may be that those who showed lower viral loads may have had a higher immune response against HIV, with concomitant higher production of cytokines, which may have led the patients to experience higher daytime sleepiness. Conversely, those with high viral loads may be those who are not able to build such a strong immune response and would have lower cytokine secretion, leading to lower daytime sleepiness. So my next step is to perform some cutting-edge (not-really) totally awesome (yes, really) cytokine analyses (actually I just ran it last week!)  and flow cytometry, assessing the different activation of the CD4 T-cells as people start treatment up until over a year on treatment and investigate its association with sleep measurements. As you can see from the picture below, I’m already on the task of telling the T-cell’s story.

 

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Kirsten hard at work in the lab.

 

Believe me, nobody is more excited than me to find out what the answer is… but we’ll have to wait and see. On to PhD!!

 

 

How to do an ELISA (or A day in the Fever Lab)

Arista Botha

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An example of a 96-well ELISA plate. Photo by Arista Botha

 

When you are writing up your PhD protocol, your experiments all seem simple. Then, when it gets to actually carrying out the experiment, things are often a lot more complicated than what they appeared like on paper. For example, my methods description was strewn with all of the blood chemistry tests one could possibly think of. Once my protocol and my ethics were approved, I had to start making it happen. How hard could it be?

 

As it turns out, finding an ELISA kit for any animal other than humans, rats or mice is tricky, so I ended up sending about twenty emails back and forth to various companies. Finally I found a company that could send me the right ELISA kit. The second surprise came when I saw what ELISA kits cost! I never knew that such a small thing could be so expensive. Luckily, my supervisor had a grant that was just about the right amount to cover my ELISA kits. So I had a product, a supplier, a quote and a grant. Satisfied, I placed my order. I was so excited when, a few days later, my kits were delivered. Like a little kid opening a Christmas present, I unpacked my kits, marked them clearly with my name, and stored them neatly into the fridge.

 

I stood back and thought: Now what!

 

Panic started to set in. During my Master’s degree I had spent most of my time in the field, tracking my study animals and doing behavioural observations. The result being that I have not spent any time in a laboratory since my undergraduate degree! How did I get myself into this?

 

Luckily Tanya and Lois from the Fever Lab were doing ELISA’s for their research, and they agreed that I could tag along and they would teach me how to do an ELISA. So I joined the Fever Lab for a day.

 

First off, I have to say that the Physiology Department have the most amazing equipment in Monica’s assay lab! To start off with, Tanya used an ultrasonic homogenizer (also called a sonicator) to prepare rat brain tissue before performing an ELISA for detecting rat IL-1β protein. A sonicator is similar in purpose to a homogeniser (or the blender in your kitchen) except that a sonicator uses ultrasonic sound waves to disintegrate the tissue. ULTRA-SONIC SOUND WAVES! That sounds like something from a science fiction movie…

 

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An ultrasonic liquid processor (sonicator) is used in our lab to sonicate the samples. Photo by Arista Botha

 

And then, they have this “super-pipette”, which we call the “Pipette-man”! The first part of doing an ELISA is pipetting your sample and other solutions provided by your ELISA kit into the sample wells. This little piece of equipment makes a big difference in speeding up the whole process.

 

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This pipette allows you to pipette simultaneously into eight wells, saving a lot of time during the ELISA process. Photo by Arista Botha

 

After incubating your samples the wells have to be washed out properly. You can either manually empty each well and then wash it out with wash buffer using an ordinary pipette (repeat times five!), or (the better option) you can use an automatic well washer, such as the one shown below:

 

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The automatic well washer saves you hours of repeatedly pipetting wash buffer into each well. Photo by Arista Botha.

 

After washing your samples, you add a substrate solution (consisting of colour reagents), which makes your samples turn blue, if for example, IL-1β is present in your sample. After leaving it to incubate for a set time period you add the stop solution, which changes the colour from blue to yellow.

 

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Adding the stop solution changes the sample colour from blue to yellow. Arista Botha

 

The final step in the ELISA process is, of course, getting the results! The concept is that the darker yellow your samples, the higher the concentration of the substance you want to measure in your sample (in this case, IL-1β). You put your 96-well plate into a microplate reader, which is set at a certain wavelength, and which will give you optical density (OD) readings. Further calculation of these OD readings ultimately will give you your sample concentration.

 

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The ELISA microplate reader measures the optical density of each well, which you then use to calculate the concentration of your sample. Photo by Arista Botha

 

After spending the day with the Fever Lab, I feel a lot more confident (and excited) about doing my own ELISAs. I am so glad that we have these equipment and skilled people in our research group and that we can learn from one another. This is why the BFRG is a team and why we do research TOGETHER!