New Rhabdovirus that causes Hemorrhagic Fever

A novel Rhabdovirus (the same family as rabies) has been discovered in the Democratic Republic of Congo. This new virus is associated with acute hemorrhagic fever. Three cases were identified, but only one has been confirmed. The first two cases died within 2-3 days of presenting with symptoms. The third case was a nurse that had cared for the other two. The nurse survived, and the novel virus was identified in their blood. No other potential cases were found at the time. The source (hemorrhagic fever is generally a zoonotic infection) and mode of transmission have not been determined.

http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002924

Should people be paid for research tissue donations?

A piece in Science on July 6 discussed the case of Henrietta Lacks (the patient HeLa cells are derived from) and whether people who donate tissue for research purposes should receive compensation.

http://www.sciencemag.org/content/337/6090/37

This week, several letters were also published in Science that argue for or against a compensation scheme. The main issues are should compensation be given? how much? would royalties be included? would type of tissue matter? would the number of donors decrease? would compensation induce more people to consent?

Human tissue is an invaluable part of research. Those tissues that are collected are "waste" or dangerous (ex tumours). As someone that will soon rely on human tissue samples, I am in favour of whatever will send tissue my way. However, I don't feel comfortable if people aren't really ok with their tissue being used in research but are donating because they are in need of the money.

Fewer women enrol in HIV vaccine trials

More than half of the new HIV infections occur in women in developing countries, but only about 20% of the participants in phase I and II HIV vaccine trials are women. This makes it difficult for researchers to determine if the virus is actually effective in women. It's thought that this may be due to a couple factors - the first that women can not become pregnant during the trial, but most eligible women are usually within the reproductive age; second, women may need to seek consent from their parents or partners to participate in the trial and this may influence their willingness to participate.

http://www.nature.com/news/hiv-vaccine-trials-struggle-to-enrol-women-1.11415

Same article also on SciDev.Net (I'm not sure if access to the articles is different)

http://www.scidev.net/en/health/hiv-aids/news/hiv-vaccine-trials-struggle-to-enrol-women.html

Vaccine trail identifies targets for immune response that could improve future vaccines

Saw this on Twitter today and thought it was really interesting. Will post the link to the full article later.

http://www.nature.com/news/vaccine-trial-reveals-chinks-in-hiv-s-armour-1.11380

New Male Contraceptive?

There was an article published in Cell today about a potential new male contraceptive option, that would be equivalent to the pill. Matzuk et al describe a small molecule inhibitor that can inhibit the production of spermatozoa to the point of infertility in mice. When they stopped giving the mice the compound, fertility returned and the mice and their offspring appeared normal. The offspring were able to breed, and litter size, pup weight and behaviour were similar to pups sired by mice that hadn't been given the compound. The compound had no effect on hormone levels, which means the mice could engage in normal mating behaviours.

This small molecule inhibitor is very promising, and it may provide a more effective means of contraception for couples that can not use some of the current options.

A preview of the article:
http://www.cell.com/abstract/S0092-8674%2812%2900934-8?utm_source=ECE001&utm_campaign=&utm_content=&utm_medium=email&bid=38A4R3F:SKPGM2F

The article:
http://www.cell.com/abstract/S0092-8674%2812%2900929-4?utm_source=ECE001&utm_campaign=&utm_content=&utm_medium=email&bid=38A4R3F:SKPGM2F

Today's Scrabble word: Jeon for 22 points

Zucchini Brownies

I finally had some time to do some baking, and I decided to try a recipe for Zucchini Brownies that I found on Pinterest. I was looking for a healthier option for a dessert, and zucchini is in season now, so this looked promising. The recipe is available here: TheYummyLife.com/Zucchini_Brownies.

I used 3 normal sized zucchinis to get 2 cups. I don't have a blender or food processor, so I cut the zucchini into fairly small pieces then added it little by little with the yogurt into my Magic Bullet. I used vegetable oil and a low sugar regular yogurt. I had regular whole wheat flour, and it is ok, but he texture of the brownies isn't as smooth as using white flour - I guess saying they were a tiny bit gritty would be accurate. Whole wheat pastry flour (as used in the recipe) would solve this problem. You can see or taste the zucchini in the brownies. If I hadn't made them myself, I would have no idea that there was a vegetable hiding in this treat!. The brownies were very moist. I didn't try the frosting recipe from the website - I had some Betty Crocker chocolate frosting leftover from my holiday Cake Pops, so I iced the brownies with them, then added some crushed pecans on top. These brownies were very tasty, and I will definitely make them again.

Today's Scrabble word: CIG for 18 points

Immunology and Obesity

http://www.nature.com/ni/journal/v13/n8/full/ni.2343.html?WT.ec_id=NI-201208

Article from Nature Immunology discussing the effects of obesity on the immune system. Some facts from the article: Obesity induced inflammation contributes to the development of type 2 diabetes, atherosclerosis, liver disease and some forms of cancer. Diet induced obesity in mice impairs T cell response and causes greater severity of disease and death after infection with influenza. Obesity is associated with aberrant population expansion of cells of the immune system in adipose (fat) tissue. This produces chronic inflammation which can lead to neurodegeneration and type 2 diabetes. Obesity can compromise the efficacy of vaccination against influenza, hepatitis B and tetanus.

Arsenic Life

There's been several articles about this recently, and I've been remiss in not actually writing about them. Last year an article (http://www.sciencemag.org/content/332/6034/1163.long) was published that described a bacterium that broke the basic rules of biology and was able to grow in the presence of arsenic, with no phosphorous present. Phosphorous is one of the basic building blocks of life and is required for DNA synthesis (among other things). The authors reported that the bacteria GFAJ-1 substituted arsenic for phosphorous to sustain growth. This article sparked a lot of controversy and several labs tried to independently verify the original study. None were able to do so.

http://www.jbc.org/content/early/2012/07/13/jbc.C112.394403.full.pdf+html
This article describes the arsenic causing ribosome breakdown, that released phosphorous that the bacteria was able to use to survive. They show the same phenomena occurs with E. coli.

http://www.sciencemag.org/content/337/6093/470.abstract
In this paper they state that they were unable to detect arsenic from the DNA of cells grown in the presence of arsenic, refuting the original paper.

http://www.sciencemag.org/content/337/6093/467.abstract
A paper published at the same time as the above also refutes the original paper. The authors found that the bacteria was able to grow in low phosphorous, high arsenic environments, but that phosphorous was necessary for growth. They conclude that GFAJ-1 is an arsenic resistant, phosphorous dependent bacteria.


I've started playing Scrabble online, so I will now include a Scrabble Word of the Day and points it earned with each post. Today's Scrabble Word of the Day "zeroth" for 76 points.

Christina

The Science of Garbology

Science has a special feature this week called "Working With Waste". There are many articles that discuss ways to reduce the production of waste, or to use the waste in new ways (so it isn't wasted!). There are articles on garbology (it is a real thing), new toilets, waste water, and 0 waste challenges. All the articles can be linked to from here: http://www.sciencemag.org/site/special/waste/index.xhtml

History repeats itself?

I was away for 2 days and am just catching up on all the science reading today . . . hence a bunch of posts about cool articles. This article is particularly awesome. The scientists use "cliodynamics" or scientific methods to illuminate the past (and predict future events). They found that the USA has 50 cycles of violence. The motivating issues vary, but the violent political upheaval is surprisingly regular. The next one is expected to peak in 2020. THey also found that "empires" have cycles that extends over 2-3 centuries, which fits the patterns of instability that occurred across Europe and Asai from the fifth century BC onwards. Last year's Egyptian uprising fits right in with this.

Check out the article at Nature:

http://www.nature.com/news/human-cycles-history-as-science-1.11078

What should we learn in genetics class?

Dr. Rosemary Redfield (UBC) just published a perspective in PLoS Biology discussing how to adapt genetics teaching to be more relevant to the "real world", and to help aid student understanding and retention of the concepts. An interesting read, and (for educators) brings up some questions about if basic science courses need overhauls to better represent the scientific knowledge and applications of today.

http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001356

HIV Treatment as Prevention Feature

PLoS Medicine Volume 9(7) July 2012 has a feature on the use of antiretrovirals as HIV prevention. There are a bunch of articles and different perspectives and aspects are given. I would highly recommend checking it out.

www.plosmedicine.org

Info on the MMR scare

I'm not sure why there were some tweets about this today, since the article is over a year old, but this article explains how Dr. Andrew Wakefield "found" the "link" between MMR vaccine and autism, sparking the vaccination scare, and how Brian Deer, a journalist, exposed the fraudulent data behind the claims. Very good and interesting article. Apparently part of a series that I now must read!

bmj.com/content/342/bmj.c5347

Carrot (cup)Cake

I finally had some time to do some baking this weekend. I made my boyfriends' favourite . . .  carrot cake! Frosted with a cream cheese frosting and topped with crushed pecans and little candy carrots. The recipe I used is from Canadian Living (http://www.canadianliving.com/food/baking_and_desserts/canadas_best_carrot_cake_with_cream_cheese_icing.php/). I followed the recipe as is. Here are some tips: Definitely make sure you check the cake about 5 minutes early - mine was done then. You can omit the pecans if you want. For the pineapple, just drain the juice from the can - don't go overboard and try to squeeze all the juice out of the diced pineapple, it gets messy and takes some of the moisture away from the cake.
For the candy carrots, I use orange Starbursts and a green gel icing pen. Unwrap the Starbursts and place on a plate. Microwave them for about 3 seconds at a time until you can mould them, but they aren't melting. Cut each candy in half along the diagonal and mould them into a cone or carrot shape. Place on the top of the frosted cake and use the icing pen to add a stem to the carrot.
This recipe also works as cupcakes. It makes about 2 dozen (24) cupcakes.

GB Virus C – potential as an HIV therapeutic?

Today I read a paper and an opion piece from advance access for the journal Clinical Infectious Disease discussing GB virus C (GBVC) and its’ role in HIV infection. The opinion piece argued that GBVC should be used as a biovaccine in patients with HIV, because clinical data has demonstrated that those people coinfected with GBVC and HIV have a better prognosis than those with HIV alone.

GBVC infection is common and occurs worldwide. It is estimated that 1-5% of healthly blood donors in developed countries, and up to 20% in developing countries have replicating virus at the time of donation. GBVC has not been associated with any disease. In fact, blood donations are not screened for presence of GBVC because no known diseases occur in those that are infected.

GBVC infection has been associated with prolonged survival in HIV positive people, causing a 2.5 fold reduction in mortality during later (>5 years) stages of HIV infection. In addition, those people coinfected have higher CD4+ T cell counts, lower HIV viral load, delayed progression to AIDS, improved response to antiretroviral therapy, and reduced mother-to-child transmission.

The mechanism(s) used by GBVC to modulate HIV infection haven’t been fully elucidated, but appear to be numerous. GBVC infection alters the expression of HIV entry receptors, and causes the secretion of the natural proteins that bind to the receptors, making it very difficult for the virus to spread to uninfected cells. GBVC infects the same cells that HIV infects, and some of the proteins of GBVC are able to inhibit replication of HIV. Without replication, the virus is unable to make copies of itself, and spread throughout the body. Another protein from GBVC appears to directly interact with HIV, preventing the virus from entering new cells. Most of this should occur when GBVC is actively replicating. Once GBVC virus has been “cleared”, antibodies against the viral protein E2 appear. These antibodies can bind to either a cellular or viral target (it’s not known which) and prevent HIV entry into cells. GBVC is able to alter innate and adaptive immunity, which can help aid the anti-viral immune response to HIV infection. Combined, all of these effects of GBVC lead to improved prognosis for people with HIV infection.

The opinion piece makes a lot of valid points – if GBVC is harmless (and it certainly appears that way) then studies should be undertaken to determine if infecting HIV positive people with GBVC will be a successful therapeutic option. All the data so far points to yes.

For more information see:

Vahidnia et al. “Acquisition of GB Virus Type C and Lower Mortality in Patients With Advanced HIV Disease” Clinical Infectious Diseases

David Gretch “Advocating the Concept of GB Virus C Biotherapy Against AIDS” Clinical Infectious Disease

For lots of research and reviews, look up Jack T Stapleton 

An HIV cure is feasible?

Today, in Nature Reviews Immunology, The International AIDS Society (IAS) Scientific Working Group on HIV Cure published an opinion piece that summarizes their recommended key goals for the international community to establish a cure for HIV (http://www.nature.com/nri/journal/v12/n8/full/nri3262.html).

Combination, compliant use of antiretroviral drugs can improve the health and prolong life in HIV-infected individuals, and can reduce the rates of transmission. However, antiretroviral drugs do not fully restore health or normal immune status in patients – or eliminate the virus from their cells. In addition, only a minority of HIV+ people worldwide have access to antiretroviral therapy. Although the price of antiretroviral therapy has dropped, the cost of providing drugs to all 33+ million people infected with HIV for the rest of their lives is astronomical. But without treatment, the virus will be spread by those that remain untreated.

The IAS identified seven key priorities that will allow us to achieve a sterilizing (elimination of all HIV infected cells) or functional (lifelong control by immune system in absence of therapy, virus not completely eradicated) cure.

The key priorities are:

-       “determine the cellular and viral mechanisms that maintain HIV persistence during prolonged antiretroviral therapy and in rare natural controllers” – focus on establishment of latency, ongoing viral replication, homeostatic proliferations

-       “determine the tissue and cellular sources of persistent simian immunodeficiency virus or HIV in animal models and in individuals on long-term antiretroviral therapy”

-       “determine origins of immune activation and inflammation in the presence of antiretroviral therapy and their consequences for HIV or SIV persistence”

-       “determine host mechanisms that control HIV replication in the absence of therapy”

-       “study, compare and validate assays to measure persistent HIV infection and to detect latently infected cells”

-       “develop and test therapeutic agents or immunological strategies to safely eliminate latent infection in animal models and in individuals on antiretroviral therapy” – reverse latency, clear latently infected cells

-       “develop and test strategies to enhance the capacity of the host immune response to control active viral replication”

While these points are important, likely necessary to develop a cure, I wonder if we are anywhere close to answering these questions. Although a cure is sure to have the greatest impact on eradicating HIV, other issues also need to be addressed. Despite effective prevention options (condoms), 2.7 million people are infected with HIV each year. Alternative prevention strategies (Truvada, microbicides, vaccine) could lower transmission rates.  If you stop (or significantly lower) the spread, new infections can be prevented, which lowers the number of people that need treatment (or the cure, when developed). Preventing the spread of the virus should also be a focus, because there is no guarantee that everyone infected with HIV will receive the cure. This brings me to my second point, which is that many people (both those at “high risk” and those at “low risk”) do not know their HIV status. If a cure is developed, how do you ensure that everyone that is HIV+ receives it? Unless mandatory and regular testing of every single person is undertaken people that don’t want to get tested, or people who think they are “low risk” and have no need for testing, may continue to spread the virus. If the goal is an “AIDS free generation”, more than the science needs to be taken into consideration. 

Human Food Project

I recently started following PaleoDiet4Two on Twitter. They post about something called the Human Food Project, which aims to get a picture of the human microbiome. Today they have a post that is really interesting about the relationship between diet and cognitive function in the elderly. They tie this into the issue of school lunches. This is all based on a study that was published in Nature recently.

Check out the article (and learn more about the Human Food Project) here:

http://humanfoodproject.com/what-can-a-100-year-old-irish-grandmother-teach-us-about-school-lunches/

And go here for the original article from Nature:

http://www.nature.com/nature/journal/vnfv/ncurrent/full/nature11319.html

HIV prevention drug approved by FDA

The FDA has approved the drug Truvada to prevent  HIV infection in adults who do not have HIV. The drug would be used (along with safe sex practices) by people whose partners are HIV positive to prevent transmission.

For more information . . .
http://blogs.nature.com/news/2012/07/fda-approves-landmark-hiv-prevention-drug.html

http://thechart.blogs.cnn.com/2012/07/16/fda-approves-truvada-for-prevention-of-hivaids/

and lots of news stories on Google News

Fat and the Gut Microbiota

Our intestines contain millions of bacteria that help keep us healthy. They do a diverse range of things for us, and the gut microbiota (the total bacteria present) differs not only from person to person, but also differs based on the region that you live in . The gut microbiota of people that live here in North America will be drastically different than that of people from Asia, largely because our diets are very different. The North American diet tends to be high in fat. Little research has been done on the effects of a high fat diet on gut microbiota.

A study published last week in Nature found that dietary fat can alter the gut microbiota of mice, by changing the pool of bile acids. They compared diets high in milk fat and high in plant oils (unsaturated fat). Those mice that were fed a diet high in milk fat were still healthy, but had a change in their gut microbiota. If the mice were lacking an inflammatory signalling molecule called IL-10 they had an increased risk of developing colitis.

An introduction to the article:
http://www.nature.com/nature/journal/v487/n7405/full/487047a.html

The article:
http://www.nature.com/nature/journal/v487/n7405/full/nature11225.html

Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10-/- mice

Paleoanthropology - What did our ancestors eat?

Pretty cool article in Nature that is investigating what australopithecines ate by using proportion of stable isotopes of carbon contained in fossilized teeth.

An overview of the article:
http://www.nature.com/nature/journal/v487/n7405/full/487042a.html

And the article:
http://www.nature.com/nature/journal/v487/n7405/full/nature11185.html

What defines cured for HIV?

http://news.sciencemag.org/scienceinsider/2012/06/evidence-that-man-cured-of-hiv.html?ref=hp

This is a follow-up to my post on the man that was cured of HIV with a bone marrow transplant. A presentation made at a scientific meeting in early June provides data that indicates he still harbours some HIV, which brings into question what constitutes “cured” when you’re talking about HIV? This patient stopped taking antiretroviral drugs after he had the transplant, the virus never returned and his doctors pronounced him cured from HIV.

Steven Yuki (UCSF), who works in Joseph Wong’s lab made the presentation at the meeting. They found some signals of HIV in the man’s body, but are unsure if these are real or from contamination. Yuki used a technique called polymerase chain reaction (PCR) to look for any signs of HIV. PCR allows you to amplify small quantities of (specified sequences) nucleic acid to determine if what you are looking for is present. When they performed PCR on the man’s cells they detected bits of viral nucleic acid, but a collaborator in a different lab didn’t detect any. This is highly suggestive that contamination of Yuki’s sample occurred, but the possibility can’t be ruled out that the man still harbours some HIV. Even more puzzling, another collaborator found signs of the virus, but it was unable to make copies of itself, suggesting it is harmless or defective genetic pieces of HIV. What makes this even more complicated is that the bits of virus don’t match each other, or the virus that he was infected with before the transplant. This provides stronger evidence that contamination of the samples may have occurred. Alain Lafeuillade (General Hospital of Toulon, France) wasn’t involved with the new study, but has issued a press release and a blog post with his interpretation of the results. He questions whether the man was reinfected with HIV and is still infectious to others. This could be possible because the virus detected doesn’t match the original virus the man had.

This new information calls into question what defines being “cured” when you’re discussing HIV. If you can’t detect the virus using p24 assays (the most common detection method), does that mean you’re cured? What if the p24 assay is negative but HIV nucleic acid is present – are you considered infected? Despite the question about whether he has been cured or not, the man has been off antiretrovirals for 5 years and is healthy. I think he, and others with HIV, would consider that cured . . . no matter what scientists decide.

Follow me on Twitter @christinamfarr

A Scientists' Worst Nightmare

http://blogs.nature.com/news/2012/06/brains-thaw-at-harvard-repository.html

There are several things that can make up a scientists worst nightmare, ranging from lack of funding for your research, being “scooped” (someone else publishes the same thing you’ve been working on before you can, so your research is no longer novel), and a myriad of factors within your experiments. So far, the worst that has happened to me is an undergraduate student in the lab accidently contaminating the cancer cells I was working on – I didn’t know the contamination had happened and the cells don’t show any signs that there is a problem for several weeks, so I put the cells in some mice for a 12-20 week long experiment, then found out about the problem. There was no way to tell if the cells that went into the mice were contaminated, so I had to wait out the whole course of the experiment before finding out that it didn’t work. I then had to repeat the experiment, which took an additional 24 weeks (about 6 months!), and this was the last experiment I needed for my MSc thesis, so I finished 6 months later than I wanted, 1 week before my PhD program started across the country, and I had to give up the month-long trip to Europe I had been planning and saving up for over the previous year.

All of this pales in comparison to what happened recently at Harvard’s brain bank – 147 brains were lost when a freezer failed, and a third of those brains were donated from deceased people that had autism. This was one of only a few repositories in the US that distributes autism brain tissue to researchers around the world. A significant source of brain tissue is no longer available, which can hinder all sorts of future research into autism. A bunch of things just seemed to happen all at once to cause this loss to occur. Normally the brains are spread among a bunch of different freezers, but they had been consolidated into one freezer for a visit from the Autism Tissue Program. The freezer went down and the two sensors that monitor temperature and send out an alarm also went down. From the sounds of it, no one knew anything was wrong until the freezer (normal temperature -80C) was opened and it didn’t feel cold inside. The odds of all these things failing during the time so many brains were in one freezer seems highly unlikely, and an investigation is being undertaken.

Something like this is always a fear for a scientist, particularly when you have “precious” samples (especially from human donors, more so when the samples can only be obtained after death) because it can take a lot of time to replace what was lost. If your research relies on these samples, months of time could be lost before enough samples are obtained to resume experiments.

When I was working on my MSc thesis I was always worried about my computer crashing, or losing my USB key, or something, so I had my thesis and all my data backed-up in different locations, and I practically slept with my laptop so I could easily grab it and run if there was a fire. I don’t worry too much about incubators, fridges and freezers in the lab right now, mostly because I don’t have anything that can’t be replaced if something happened. My biggest worry in the lab is that someone will use my sterile water and that will contaminate my experiment, which isn’t a big deal now, but will be a huge issue if I’m working with mice. Soon I will constantly worry about the BLT mice – they are so costly and one small thing could completely destroy months of work.

HIV Prevention Using Microbicides

HIV prevention is a “hot” research area. A lot of time and money is being spent to try to prevent HIV spread. There are several avenues of research for HIV prevention, but 2 of the most popular are vaccines and microbicides. A vaccine (with low production and distribution cost) for HIV would be ideal, particularly if it offers long term protection with no need for a booster. However, it has been very difficult to develop an efficacious vaccine. HIV mutates rapidly, which means that immunity to one version of HIV might not protect you from a different version of the virus (sort of like getting the flu even though you’ve had the flu vaccine). Due to the lack of success with vaccines, several research groups (mine included) are pursuing alternative prevention strategies, such as microbicides.

Now, you may wonder why we are focussing on coming up with new ways to prevent HIV infection when condoms can efficiently prevent HIV infection. There are lots of reasons why people don’t use condoms. In the developing world, where HIV burden is the highest, many women are not able to insist on condom use, due to cultural or societal norms. These women represent more than 50% of the new HIV infections that occur each year. Since the standard prevention options are not feasible for them, an alternative needs to be designed. This is why we are focusing on microbicides – ideally, women could use them prior to intercourse and their partner doesn’t need to know.

Microbicides are drug products that are can be topically applied to the vaginal or rectal tract to prevent infection with HIV. Many microbicides have been under development for HIV, but none have been brought to market yet. Several are undergoing clinical trials, and several have already failed in clinical trials. Tenofovir is one microbicide that I have been hearing a lot about recently. Tenofovir is an anti-retroviral that has been formulated into a microbicide gel. Tenofovir was successful in its’ first clinical trial, showing protection rates of 39%. However, the second trial was recently stopped because no difference was observed between the drug group and the placebo.

An emerging area of microbicide development is the use of engineered bacteria. Several research groups have focussed on Lactobacillus which is a normal component of the vaginal microflora. They hope that by engineering Lactobacillus to express or secrete an anti-HIV protein they can, with one application, provide long-term protection from HIV infection. Ideally, there would be no need to reapply the Lactobacillus microbicide, because the bacteria would be able to live in the vaginal tract, and should (in theory) continually express the protein. However, what do you do if some sort of side effect occurs? How do you kill the engineered bacteria without destroying the natural vaginal microflora? How do you know the bacteria is still expressing the protein?

There are a lot of issues that can arise from the use of Lactobacillus as a microbicide. My research is investigating an alternative engineered bacteria based microbicide system, using a bacteria called Caulobacter crescentus. My lab has developed a system to express a wide variety of different anti-HIV proteins on the surface of C. crescentus. These proteins are expressed at very high levels (20% of total cell protein would contain the anti-HIV protein) and we have had no issue with maintaining long term expression. My research is finding new anti-HIV proteins to put on the C. crescentus, and testing two of the most important factors of a microbicide, safety and efficacy. I have to ensure that topical application of the C. crescentus to the vaginal tract will have no adverse effects. I also have to determine how high protection from HIV infection is. My first round of testing indicates that I can decrease HIV infection rates by 85%! This is all done in a test tube, and I will eventually have to move on to testing this in an animal system (see BLT mice blog post). All of this testing (and some side projects that come up along the way) will compose my PhD thesis.

So far, the only downfall of the C. crescentus system is that the microbicide would need to be applied regularly (I haven’t figured out how often yet). The bacteria can’t survive in humans, so protection from infection will be transient. C. crescentus is cheap to grow, so this shouldn’t be cost prohibitive, but the need for reapplication may be a deterrent for clinical use. Indeed, it is believed that adherence to the dosing regime is the reason why the second clinical trial of tenofovir showed no effect.

The development of a microbicide for HIV has the potential to revolutionize the field of HIV prevention. However, there are still a lot of problems in translating laboratory success to clinical success. In the meantime, prevention strategies should focus on testing for infection, condoms, and pre-exposure prophylaxis (when applicable).

Rubber Ducky Cupcakes

There’s been a lot of cool science news recently, so I’ve fallen behind on posting new cupcake recipes. I thought I’d post instructions on how to make your cupcakes look like rubber ducks. (This is based on the design from the book Hello Cupcake, What’s New, which I love!) You get a huge sugar high from eating one of these, and an even bigger one if you snack on the starbursts and M&Ms that are the wrong colour, lick the frosting off your fingers, and eat the extra Timbits, marshmallows, or starburst molding failures J

Decorating Supplies:

Vanilla frosting (I use Betty Croker, do NOT use whipped)
Yellow food colouring
Mini M&M’s (brown, blue and green)
Marshmallows
Orange starbursts         
Plain Timbits
Vanilla cupcakes in yellow or white liners (start with more than you want because it takes some practice to get them looking the way you want)

 Prep Stuff:

Bake the cupcakes as described and let them cool completely.

Cut the marshmallows in half diagonally.

Cut the starbursts in half and microwave for a few seconds at a time until you can mold them into beaks (or feet). I found it helpful to make a pointy end that could be stuck into the Timbit.

Assembling the Duckies

1.      Once they have cooled, ice the cupcakes and place the marshmallow at one end and the Timbit at the other to make the head and tail of the duck. Use the frosting like glue to help hold them in place.

2.      Put them in the freezer for about 15 minutes.

3.      Microwave the frosting until it is runny and use the yellow food colouring to dye the frosting.

4.      Carefully dip the cupcake into the frosting and let excess frosting drip off.

5.      Carefully add the starburst beak and M&M eyes now OR wait until the frosting has hardened then add them at this point, whatever works out better for you. You may need to use a dab of frosting to hold the M&Ms in place. I stick the pointy end of the beak into the Timbit to help it stay in place

To make upside down duckies:

Place the marshmallow in the opposite orientation from the original ducky. Once it has been dipped, carefully place the starburst feet on either side of the marshmallow tail. You may need a small dab of icing to help them stick in place.

The placement of everything is really important, and it took several tries to get it right. The cupcakes look really good when they are done, but there were a lot of casualties in the process!

BLT Mice

As I’ve alluded to in my recent posts, HIV is kind of difficult to work with. Working with the virus in Petri dishes and cell culture flasks isn’t too bad . . . except for the fact that it’s a level 3 pathogen in Canada, which means I have to work with it in a special facility with restricted access and tons of safety features. Not all research institutions have this type of facility available, so I’m lucky to be able to do all the experiments at my research institute.

Once I complete all the studies I can do with cells, I will have to do my next group of studies using animals. This is done to test the safety in a living organism (cells can only tell me so much) and provides a more real world study system. With no idea on the safety of my compounds, I can’t do any work using humans, which is kind of a problem when studying HIV. As stated in its’ name, HIV is the HUMAN immunodeficiency virus. This means that HIV only infects humans. There are other types of immunodeficiency viruses (like simian immunodeficiency virus), but using SIV in primates, or a hybrid SIV/HIV in primates has not had good translation to work with HIV in humans, because there are a lot of differences between SIV and HIV. This has been a challenge many researchers have tried to solve, and recently success has been observed with humanized mouse models of HIV.

Before I go further I should warn you that these mice involve some very controversial stuff. First off, working with mice, which is always a touchy area. To humanize a mouse you need to perform a minor surgery on them and carefully monitor the mice post-surgery to make sure they don’t develop infections or are in pain. And, to humanize the mouse you use human fetal tissue, which can only be obtained from aborted fetuses that are donated to research. If you have any issues with any of these things, I would suggest you stop reading now.

Each humanized mouse has to be created individually. This is a very lengthy process. You begin with a mouse that, through a combination of genetic defects, does not have an immune system. There are several different types of immunodeficient mice you can use for humanization. I will be using the NOD/SCID/IL2rgamma-/- mouse. That is a fancy name for a mouse that has a severe immune deficiency and is unable to make any functional immune cells. You have to be very careful with these mice, because if they get any sort of infection their body can’t fight it so they will likely die. Their water and food is specially sterilized, their lungs are monitored for infection every other month, and sentinel mice are used to monitor for any other infections.

When the mice are 8 weeks old they have a surgery to implant human fetal liver and thymus under the kidney capsule. They also receive an IV injection of human fetal stem cells (which can be derived from the liver). The human stem cells will allow the development of all the human immune cells, and the liver/thymus allows the T cells of the immune system to mature properly. Because you use bone marrow (the stem cells), liver and thymus, these mice are called BLT mice. Twelve weeks after the surgery you take blood samples from the mice and check that all the immune cells are present. If so, the mice can be used for infection with HIV.

The BLT mice have been shown to be susceptible to vaginal infection with HIV, and the infection course is very similar to what is observed in humans. Whenever I’ve presented my research I’ve been asked how exactly you give a mouse HIV vaginally. The mouse is put under anesthesia, then a small instrument is inserted into the vagina and HIV in saline is deposited. The mouse is held in an inverted position so the saline doesn’t drip out. After a few minutes, the mouse can be woken up from anesthesia. Blood samples can be taken each week to determine HIV infection.

Today I am heading to a collaborators’ lab for 2 weeks to receive the surgical training I need to be able to make BLT mice. These mice will provide me with a lot of information about the “real world” uses of my compounds. I have to do everything perfectly for this mouse experiment, because it is really expensive. With the costs of my training trip, buying the mice, housing the mice, buying reagents and materials, and equipment rental costs, the 45-60 mouse cohort I can make from one set of donor organs will cost $20,000 or more.

Christina

PrEP for HIV Prevention

Another blog post on HIV . . . it’s sort of my thing :p (and the focus of my thesis!)

On May 10^th the Antiviral Drugs Advisory Committee of the US FDA debated whether an anti-HIV drug currently used as an antiretroviral treatment should be approved as a prevention method for uninfected people. It was recommended to the FDA that the drug, Truvada (made by Gilead Sciences), be approved for pre-exposure prophylaxis (PrEP).

PrEP means that the drug is taken routinely by uninfected people at high risk of becoming infected with HIV. It has been shown that taking the drug daily can lower risk of infection by more than 90% in two target groups – men who have sex with men; and uninfected heterosexual (men) with a long-term, HIV positive partner. However, clinical trials of the drug in heterosexual women have failed. (Also, in reading another article about this, it was claimed risk was lowered only by 42% and 73% in separate trials...)

PrEP (like birth control) would need to be taken daily to be effective. And, if adherence is low this could have disastrous consequences, particularly if a backup method of prevention (condoms) is not used. As I mentioned in my last HIV post, the virus replicates really quickly, and doesn’t make perfect copies of itself, leading to mutated forms of the virus. Some of these mutants could be resistant to Truvada. So, if you are not using PrEP as prescribed and have a partner that is HIV positive, it is likely that you will become infected with the virus. If you continue to take the PrEP irregularly, you are putting yourself at risk of being infected with drug-resistant HIV, which would likely make treatment more difficult. Of particular note is that HIV treatment with Truvada must be undertaken in combination with other antiretroviral drugs to prevent resistance.

During the clinical trials participants were monitored monthly for HIV infection, using the most sensitive test to detect HIV, so that PrEP could be stopped upon infection to prevent resistance. A few cases of infection were seen in the trial participants, and just over half of them developed resistance. This testing should be continued for those that are taking Truvada for PrEP, but this testing was not included in the recommendation to the FDA. Without regular testing, it is possible that HIV could be transmitted to someone taking PrEP (remember, it isn’t 100% effective) and they wouldn’t know about it, so they would keep taking the antiretroviral. This will lead to development of a resistant virus, which will create a lot of problems for further treatment once the virus is detected.

I did a PubMed search and wasn’t able to find any clinical trial papers about Truvada, which I would really like to be able to read before forming an opinion on this, particularly since Science and Nature report two different statistics for protection levels. I think this approval of Truvada could drastically lower HIV prevention rates, if used properly (and if affordable!), however, it could also be disastrous if used improperly (particularly without regular HIV screening). Condoms are more effective at preventing HIV transmission and I highly recommend them because they also protect against other STI’s and pregnancy. But, for those who are unable to use condoms for various reasons (particularly women in developing countries), Truvada represents a new hope for HIV prevention.

Christina

A cure for HIV?

According to the World Health Organization and UNAIDS, over 33 million people worldwide are infected with HIV, the virus that causes AIDS. Over 2 million people die AIDS-related deaths each year. HIV/AIDS has enormous social and economic impact, particularly in developing countries. Despite over 30 years of research into HIV, an effective method of prevention (besides condoms) and a cure have not been found. Currently, once someone become infected with HIV they can take a combination of anti-retroviral drugs on a strict regimen, which should suppress the virus for several years. Eventually, the patient develops AIDS which typically leads to death within 1 year. It is thought that antiretroviral therapy can increase survival time by 4-12 years, but the patient would still eventually develop AIDS.

It is really difficult to treat HIV for several reasons. The virus replicates very fast (in less than 2 days) and doesn’t always make a perfect copy when it replicates. This leads to mutations that can allow the virus to become resistant to the antiretrovirals, or to escape the immune system. All of these mutated virus also replicate quickly, so someone infected with HIV will have millions of copies of different versions of HIV. In addition, HIV infects (and kills) CD4+ T cells, which are a very important part of the immune system. So, while your immune system is trying to fight a constantly changing invader, the invader is also killing those cells that have a key role in fighting it. What is really cool is that some genetic combinations provide protection from infection or suppress HIV replication. One that is called Δ32 CCR5 deletion is present in a small percentage of Caucasians descended from Western Europeans, and also prevents infection with bubonic plague.

I have only found 1 case where someone was cured of HIV. An HIV-positive patient with acute myeloid leukemia was given a hematopoietic stem cell transplant (basically a bone marrow transplant). Since this patient had both AML and HIV there wouldn’t be very many (or maybe even any) treatment options available, so this situation provided the opportunity to try something new. The doctors decided to give them a hematopoietic stem cell transplant using cells from a donor that had the Δ32 CCR5 deletion. The transplanted cells would not be infected with HIV, and should be able to destroy any remaining HIV-infected cells in the patient. So far, HIV suppression has been observed in this patient, and they have stopped taking antiretrovirals!

Walker et al (http://jvi.asm.org/content/86/10/5719.abstract) were interested in creating a gene therapy vector that could provide protection from HIV infection. They created a gene therapy vector that would induce the Δ32 CCR5 deletion, in addition to expressing TRIM5α (shown to prevent HIV replication) and a TAR decoy (which should prevent HIV replication).

To determine if this gene therapy vector could be clinically relevant, it needs to be tested in an animal model, which is difficult for HIV. HIV is the HUMAN immunodeficiency virus, which means it can only infect humans. This has posed a huge problem for HIV researchers, but a pretty cool solution has been found – you can create mice that have a human immune system (there will be a blog about how to do this soon, because I’m going to learn how to make my own franken-mice). These mice can be infected with HIV and the infection is similar to what is observed in people.

Walker et al isolated CD34+ hematopoietic stem cells and infected them with the gene therapy vector, then injected the cells into mice. These stem cells developed into immune system cells, including the CD4+ T cells that HIV can infect. The CD4+ T cells appeared functionally normal and contained the gene therapy vector.

Once they verified everything was working as expected, they infected the mice with HIV and monitored the mice. They found that the mice that received the gene therapy vector had significantly enhanced survival of CD4+ T cells after HIV infection. However, the level of HIV virus in the blood was not changed. This suggests that the gene therapy vector was able to keep the CD4+ T cells alive, but didn’t stop HIV from replicating.

This study is a good start for using gene therapy as HIV prevention, but follow-up work on why the HIV levels in blood were unchanged should be undertaken. It is also important to realize that in this study they were sort of putting the cart before the horse – they gave the mice the gene therapy vector before HIV infection. To build on this study they should infect mice with HIV then give them a bone marrow transplant with hematopoietic stem cells that contain the gene therapy vector to see if the HIV infection is cured – it would be more clinically relevant that way. This sort of treatment would be a long way from the clinic, particularly since you’re dealing with a treatment option that could be highly controversial, since it involves stem cells and gene therapy. I am definitely going to keep my eyes on this field!

Christina

Let them eat dirt!

Many epidemiological studies have led to the hygiene hypothesis. The hygiene hypothesis proposes that exposure to microbes in early childhood can decrease susceptibility to allergies and diseases such as asthma, irritable bowel disease (IBD) and autoimmune diseases. It’s believed that this early exposure to microbes helps the immune system function properly, which prevents the “overactivation” of an immune response that occurs in the case of allergies, asthma, etc. When it “overactivates” the immune system sees something that is not dangerous (ex. dust, pollen, peanuts) as harmful, and “freaks out”, releasing all sorts of inflammatory mediators that cause symptoms from a runny nose to full-blown anaphylactic shock. In most cases, this would be annoying or inconvenient, but in some cases it can be deadly. Although a lot is known about WHAT happens when the allergen or disease is triggered, it is not known WHY. Several studies have produced observations that suggest this is all related to hygiene, specifically that exposure to microbes in early childhood lowers the chance someone will develop allergies, asthma, IBD, etc. It has been suggested that this could be related to how “clean” we are now, particularly with the use of antibacterial soaps. It has been observed that more people in developed countries have allergies/asthma/IBD/autoimmune diseases. And, within developed countries, children that are from rural areas have lower rates of allergies and asthma compared to their counterparts in cities. It wasn’t really known why this was the case, but a recent paper in Science (http://www.sciencemag.org/content/336/6080/489.full) has provided some evidence about the immune system cells that are involved.

It has been hypothesized that invariant natural killer T (iNKT) cells play an important role in the pathogenesis of ulcerative colitis and asthma. These cells recognize lipid antigens and release large amounts of proinflammatory factors (called cytokines) when they are activated. Olszak et al investigated the age-dependent regulation of iNKT cells by use of microbes in mouse models of IBD and asthma.

They worked with germ-free (GF) and specific-pathogen-free (SPF) mice. GF mice are born without the bacteria that are normally present, and are protected from any bacterial exposure over their life. SPF mice are free of certain bacteria that would cause disease, but the normal intestinal bacteria are present. They found that the numbers of iNKT cells were increased in GF mice and appeared to be stable for life. When they induced colitis in the mice they found that the GF mice were more sensitive to colitis, with more severe weight loss, pathology, and higher mortality rates compared to SPF mice. If they re-established the microbiota (all the bacteria that should be present) in the GF mice, they found that the iNKT levels and severity of colitis was not reduced. If they took pups that had a GF mother and raised them in SPF conditions, they found a complete normalization of iNKT cells and reduced susceptibility to colitis. This indicated that the microbiota present at birth has life-long effects on health. They repeated these studies with a mouse model of asthma and had very similar observations.

This study fits with the epidemiological studies that led to the hygiene hypothesis, showing that early life exposure to microbes decreases susceptibility to diseases such as IBD and asthma, where as absence of microbial exposure may have the opposite effect. This is one of the first studies that provides a mechanism to explain the epidemiological observations that compose the hygiene hypothesis.

So, I’m not saying that you should feed your kids dirt, or stop them from washing their hands before eating, but let them play in the dirt and don’t freak out if they lick their shovel – it just might lower their risk of inflammatory diseases ;p

Christina