Florian Hladik, Fred Hutchinson Cancer Research Center, University of Washington
Something that you canít pick up from a handshake is HIV but thousands of people are infected with the virus everyday through sexual contact. One strategy to prevent this is referred to as pre-exposure prophylaxis. The idea is that an at-risk individual can apply a gel laced with an anti-virus agent to potential exposure sites, and this stops the virus gaining a toe-hold, but to make this work, you need to use very high concentrations of the drug and this could have unexpected effects on the tissue as Florian Hladik has discovered.
Florian - If we use a drug topically, it has been shown that the concentrations locally in the mucosa are much higher than if you take the same drug orally. And so thereís some concerns that if you achieve much higher concentrations that you might also encounter side-effects. We were interested to look at the effect of microbicides across the whole human genome. Essentially check whether we can find certain patterns of expression that indicate problems.
Chris - And how did you approach this? What were you actually doing in your study?
Florian - The study had four trial arms, and in each arm there were 20 patients. One arm had no drug used, one arm had a control gel, one arm the patients received a gel containing Nonoxynol-9 a detergent that is an irritant as positive control, and then the fourth arm the patient received Tenofovir 1% gel. When the patients were enrolled, before starting the study drug, biopsies were taken from the rectum in all four study arms. Then after the first dosage, so that would be about 1 hour after receiving the first dosage, another biopsy was taken. After that, the patients received the study drug daily over 7 days and at the end of that period we took biopsies again.
Chris - And how did you then process those biopsies? What did you do to see what the Tenofovir was or wasnít doing to the tissue?
Florian - Biopsies were put in a preservative for RNA isolation and then after that we ran Illumina microarray chips where with certain probes, you look at expression changes of genes across the whole human genome.
Chris - So, youíre able to then say what genes are being turned on or off in each instance to see what effects there were or werenít on the genes in those tissues?
Florian - Yeah, the first readout really is the number of genes that are changing. So, essentially how many genes are upregulated and how many genes are downregulated by the treatment. And that was a real surprise to us because we were expecting all the changes in the Nonoxynol-9 arm which we had used as a positive control arm. There was one arm that showed by far the most changes and we were assuming it must be the N-9 arm but it actually turned out the Tenofovir arm.
Chris - What is the Tenofovir doing then? The fact that itís actually producing these gene changes argues that youíre getting what are called off-target effects because Tenofovir should be affecting only virus function, shouldnít it? So the fact that youíre seeing gene changes argues that it is doing something to those cells. Which genes were being impacted?
Florian - We saw a lot of impact on transcription factors. Tenofovir, overall had relatively strong inhibitory effect on many transcription factors. One of them was a transcription factor called, CREB1 and also another one, CREB-binding protein. And these are transcription factors that are important for transcription of Interleukin-10. The Interleukin-10 is regulating, toning down an immune response that has been set in motion by a pathogen and by inflammatory cytokines that has this kind of action. That would indicate that Tenofovir inhibits the anti-inflammatory arm of immunity.
Chris - So, if Tenofovir in this setting can de-repress inflammation, in other words it can facilitate an inflammatory process. That means long-term exposure to it might have consequences that we hadnít anticipated then.
Florian - That could be. And the hypothesis from our data is that this inflammation prolonging effect of Tenofovir would come into play only if you already have a background level of inflammation due to some other reasons. In particular in people who are in need of using these kinds of strategies to prevent HIV transmission, inflammation is often present.
Chris - Certainly quite a worry.