Exercising could help to resolve inflammation quickly!

Written by: Abby Stahl

Original Article: Zheng et al. Journal of Immunology 2019
The Gist of It
Exercise has long been known to improve health, and not getting enough of it increases risks of cancer, diabetes, heart disease, depression, and dementia. Part of the benefit of exercise comes from reduced blood pressure and body weight, but chemical signals are also involved. Catecholamines are chemicals released from long-duration or high-intensity workouts that are believed to help reduce inflammation, but how they actually work is still unknown. In this exciting new research, scientists found that a catecholamine called epinephrine is released in mice after they exercise. The epinephrine caused the production of a specialized fat molecule known to be important for resolving inflammation called resolvin D1 (RvD1). The increased amount of RvD1 in the mice that exercised helped to clear inflammation much more quickly than in the mice that were sedentary by acting on immune cells called macrophages. One of the key jobs of a macrophage is to phagocytose, or to engulf (or, to think about it more simply, to eat), things around it to clear dead/dying cells and debris from the body. In the mice that exercised and had increased RvD1, the macrophages could phagocytose more, allowing them to get rid of molecules that were driving the inflammation. These findings show just how important exercise is to enhance the ability of the cells in our body to function at the highest level possible and keep us safe from inflammation.

Epinephrine is released after exercise and can act on macrophages through the alpha1-adrenergic receptor (a1-AR) and Alox5 to produce resolvin D1, which increases phagocytic uptake of dying neutrophils and quickly resolves inflammation.

The Nitty Gritty
In this work, mice were divided into two groups, one that was exercised for four weeks on a treadmill and one that was not. Then all mice received an injection of zymosan into the peritoneal cavity to stimulate acute inflammation. Following the injection, leukocytes were recruited in both groups, including neutrophils and macrophages. Inflammation peaked at 12–13 hours after zymosan treatment in both exercised and non-exercised mice. There were no differences in the peak number of leukocytes or the amounts of cell death or macrophage recruitment occurring between the groups. Interestingly, however, the number of neutrophils was reduced at 48 hours in exercised animals versus sedentary animals, signifying that the “resolution phase” of inflammation was hastened in the exercised animals.
Although macrophage numbers increased similarly in the exercised and sedentary mice, the researchers hypothesized that phagocytic activity of the macrophages might be enhanced in the exercised group and thus explain the greater decrease in the number of neutrophils at 48 hours post-zymosan injection. Excitingly, macrophages isolated from the peritoneal cavity of exercised mice did have increased phagocytosis of opsonized zymosan particles in vitro. Resolvin D1 (RvD1) is known to enhance the phagocytosis of cellular debris through interactions with surface receptors. Researchers found that exercised animals had larger amounts of RvD1 in peritoneal exudates as well as more cells expressing the mRNA coding for RvD1 and its biosynthetic enzymes Alox15 and Alox5 compared with their sedentary counterparts. Intriguingly the increases in both RvD1 expression and macrophage phagocytosis were observed even after just 1 week of exercise training!
Since catecholamines are known to act directly on macrophages and to be produced following exercise, the authors checked for levels of these chemicals in the urine of the mice and found that exercised mice had increased levels of the catecholamines epinephrine and metanephrine compared with sedentary mice. In addition, the expression levels of catecholamine receptors, including alpha1a, alpha1b, alpha2a, and beta2, as well as enzymes for catecholamine metabolism were higher in cells from exercised mice. When macrophages derived from bone marrow were treated with epinephrine, there was an increase in RvD1 production, showing that RvD1 can both be produced by and act on macrophages.
To elucidate how catecholamines cause the production of RvD1 in macrophages, the authors added inhibitors of various receptors. Treatment with prazosin, which blocks alpha1 adrenergic receptors (a1-AR), prevented the production of RvD1 after epinephrine administration. In addition, an activator of a1-AR called phenylephrine was able to activate RvD1 production. The enzyme responsible for RvD1 production, 15-LOX-1 encoded by Alox15, was required for phenylephrine to elicit this effect.
Finally, the scientists administered the α1-AR antagonist prazosin to exercised mice and found the production of RvD1 was blocked and macrophage phagocytosis was no longer enhanced, leading to a four-hour delay in the resolution of inflammation after zymosan treatment. This work demonstrated that exercise leads to the release of epinephrine that acts on macrophages through the a1-AR, leading to production of RvD1 and enhanced phagocytosis, thereby resolving inflammation more quickly.
Original Research Article: Zheng, J.-J. et al. “Exercise promotes resolution of acute inflammation by catecholamine-mediated stimulation of resolvin D1 biosynthesis.” J Immunol 203 (2019): 3013–3022. doi: 10.4049/jimmunol.1900144

Preventing Hype and Promoting Innovation

Written By: Rebecca Tweedell & Kaitlyn Sadtler

“Scientists Discover Cure for Ebola Virus”; “Scientists Discover ‘Cure’ for Cervical Cancer…” These headlines are meant to be eye-catching, and they certainly do a good job of capturing people’s attention. But in an age of hyper-competitive science, wherein the ivory towers are ever present, and nepotism heavily affects career trajectory, from smaller things like awards to tenure-track positions at universities, it is often tempting to inflate results or make broader claims to make a splash and garner the public’s attention. So, the negative headlines tend to accompany the positive ones: “Wakefield Study Linking MMR Vaccine, Autism Uncovered as Complete Fraud”; “Scientist Falsified Data for Cancer Research Once Described as ‘Holy Grail’, Feds Say”. But why does this happen, and what can we do about it to prevent the hype and promote innovation?
Disconnect between science and journalism
The first major reason you may often read news articles in which scientific findings are exaggerated is that there is a disconnect between scientists themselves and the journalists writing the article. Scientists are often guilty of over-simplifying and even exaggerating their findings in conversation or interviews because they are incredibly excited about the potential for what they have found to help people. This excitement tends to manifest as researchers speaking very broadly about what all their work COULD mean for the future of science and medicine. Unfortunately, when scientists talk this way to people who are not other scientists, like journalists for example, it is hard for everyone to realize what the limitations of the data and true scientific findings are. For example, a scientist might say “this could potentially lead to targets for the development of new therapeutics,” but it is instead construed as “these researchers have developed new therapeutics.”  For researchers who are used to speaking to other researchers, who would be familiar with all the intricacies of the data and how exactly the study was done and what those details mean for the applicability of the findings, it can be challenging to change the way you talk about your results to make sure others do not walk away thinking you have discovered something that you haven’t. This ties into the difficulties with the game of telephone – where a scientist describes their research to a journalist, and maybe a press release is put out by their institution, then another press group decides to report on it, but mainly uses another press release as their source, further exaggerating the claims.
Sometimes the reason for the overstatement is not so innocently motivated, though. As we alluded to above, science has become a highly competitive industry that values things like “impact factors” as a measure of success. An impact factor is a pseudo-scientific way to measure the importance of publications in a particular journal by measuring how many times on average articles published in that journal in the past have been cited by other researchers. Getting your research published in a journal with a higher impact factor looks better to hiring managers and promotions committees than publishing in a “lower-tier” journal. This makes it very tempting for researchers to “bend the truth” to make their story “sexier” to get accepted by these higher journals. But the truth is that, while great papers do end up in these journals, impact factors are not necessarily a true measure of how important the research is, and the competition for having the highest impact factor can cause good, important science to get lost in the noise.
Pressure to deliver products
Another reason you may see stories that exaggerate a scientific finding is because scientists feel pressure from the public to deliver products and therapies for general use. This often stems from the fact that most individuals outside of research do not realize the amount of time and work required for proper translation of mechanistic discoveries to products for the clinic. It makes sense that once we have a concept for a technology, we hope and even expect to see it in the clinic as soon as possible. However, the development pipeline is much more complicated. Say I have a material that could help regenerate skin. Well, the first step in the path from my research on this material to the use of it in the clinic is to develop the technology enough to file for intellectual property – aka submit a patent application. Initial research is years and years of scientific analysis, and the patent application has to be filed before any of the details of the work can be published, as publishing would be considered “public disclosure” and therefore nullify an argument for a patent. After these years and years of initial research and the issuing of the patent, then the information about the work with this material is ready to be submitted for publication in a scientific journal. Once submitted, there is a peer review process that can take months, and even in some cases a year (or more). When the article is finally published, the press will be able to access and share the information about this research for the first time. From this point, the technology will have to be spun off into a company or licensed by an existing company in order to provide a nexus for the clinical trials. Clinical trials are expensive, and all drugs/products that go into the body must pass through three phases.
Phase I is focused on safety – where usually less than 100 healthy patients are enrolled to determine if the product is safe for use in humans. This phase normally lasts about a year, depending upon the product being tested; devices that are meant to stay in the body long-term will take longer to assess than a drug that clears from the body relatively quickly.
Phase II is focused on efficacy – does the product actually work? And this can take a very long time. Many, many more patients must be recruited to the trial, including multiple control patients, and more outcomes are studied than just safety. The majority of products that have gone into trials have failed by this point.
Phase III is focused on large-scale safety and efficacy – involving hundreds of patients. Just over half that get into Phase III do ultimately pass and are given the FDA seal of approval.
In total, a product that has gone through these three phases of trials will have taken 10 – 15 years from when the first Phase I trial started until it is given FDA approval and can be used in the clinic. Ten to fifteen years. And that does not include the development time prior to that initial publication of results after the patent was received and the time from that publication to the start of clinical trials.

The Gartner Hype Cycle is a graphical representation developed by the Gartner consulting firm to describe the hype generated by the broader community in the context of product development. Most technologies go through a period of massive hype, which is not necessarily where the technology will end up after thorough vetting and testing.

What can we do about it?
One critical step to reducing the hype and emphasizing innovation is to ensure accurate communication between scientists and the public. This includes both communication coming directly from scientists (often seen on social media and in conversations) and from journalists reporting on scientific discoveries. Scientists need to learn how to appropriately communicate their science, and there are an increasing number of science communication courses and seminars being hosted to help with this. Additionally, the media needs to be held responsible for their portrayal of research, and for the love of puppies and all that is holy – link to the primary research articles so that people can assess the merits of the study for themselves. Finally, it’s important to keep in mind that just because a scientist’s discovery wasn’t picked up by the New York Times doesn’t mean their work isn’t important. Scientists need to be supported for their research even if it isn’t picked up by mainstream media. Furthermore, more scientists need to be given direct connection to the general public – if you ask someone to name a scientist you will probably hear Bill Nye or Neil DeGrasse Tyson, but there are a lot more scientists out there.
Another important step to reduce overstatements is to take some of the competition out of science. This can be achieved partly by reducing the reliance on the “traditional” metrics, such as number of publications and impact factor of those publication, when assessing a scientist’s career in the job market. While academic institutions are beginning to realize that this is an issue, there is still much work to be done in this arena. Emphasizing number of collaborations over impact factor of publications could be a positive step in the right direction. This will lead to more researchers sharing their data in early stages of projects with collaborators, allowing more minds to focus on a particular topic and greatly increasing the innovative power. Additionally, funding agencies could play a key role in reducing the amount of competition in science, since scientists are competing for funding as much as they are competing for jobs. Adopting improved guidelines for the assessment of grant applications that place added emphasis on aspects other than a researcher’s publication history could help with this.
Finally, to emphasize innovation, it is important for EVERYONE to remember that good science takes time. This applies to both scientists and the rest of society. When scientists try to short circuit this and wind up overstating their results, it creates more confusion and animosity than innovation. These overstatements can take many forms, from claiming that you have developed a novel technique, which is simply using a reagent purchased online as per manufacturer’s instructions, to exaggerating the effects and applications of a finding. Now, it is important to note that some science does move quickly, but this should not be expected in all cases, especially in biomedical research. However, new ways of getting science to other researchers quickly, such as preprint servers wherein a scientist can post their work prior to formal publication in a journal, are resources to promote the rapid sharing of scientific findings and allow for feedback from a larger scientific community beyond the 3-4 reviewers that are normally used in the standard peer review process. This can increase the speed with which good science can get out there for others to build on, accelerating the discovery process.
Overall, there are lots of reasons for the broad and almost wild scientific claims we have become accustomed to seeing in the news, but it’s important to be able to separate the hype from the actual scientific advances. This is critical to promote good science that will actually be able to move our world forward and improve the lives of all.

Can we deliver drugs into cartilage – with sound?

Written by: Ken Estrellas

Original Article: Nieminen et al. Scientific Reports 2019
The Gist of It
The advent of modern medicine, combined with lessons learned from engineering and material science, has led to the development of numerous ways to deliver medicine to the body. Pills, syrups, injections, infusions – the list goes on. None of these methods are perfect, though; not all of the molecules of the drug administered will ultimately end up at the site or site(s) of interest, whether that is the tiniest immune cell or the largest achy joint. One approach being studied by several scientists uses sound – more specifically, ultrasound waves – to manipulate different bodily tissues in a non-destructive fashion. Ultrasound has been tested to improve the delivery of medicines at various sites, ranging from the skin to even the brain. In this study, Nieminen and colleagues looked at the use of high-intensity ultrasound to deliver small molecules to cartilage. Thirty samples of articular cartilage from cows were exposed to a small molecule (the dye methylene blue) either before ultrasound treatment (group C1), during ultrasound treatment (group T1), or without ultrasound treatment (group C2) over the course of 15 minutes. The cartilage exposed to the dye at the same time as ultrasound treatment showed higher levels of dye absorption compared with the other two groups at several different depths of tissue. Importantly, the ultrasound treatment did not appear to negatively affect the cartilage tissue itself. Molecules related to the metabolism and inflammation associated with osteoarthritis were present in similar amounts in the cartilage treated with ultrasound compared to unexposed cartilage, and the number of cell death markers in the fluid surrounding ultrasound-exposed cartilage was not increased. Taken together, these results demonstrate that ultrasound treatment has the potential to promote drug delivery to cartilage, which could potentially improve treatment outcomes in diseases such as osteoarthritis where cartilage is significantly affected.
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High-frequency ultrasound was used to more efficiently deliver a small molecule compound into cartilage

The Nitty Gritty
Diseases such as osteoarthritis are characterized by focal lesions of articular cartilage, but localized delivery of active pharmaceutical ingredients specifically to these sites to counteract these lesions has been characterized by low bioavailability. Nieminen et al. conducted significant previous work to increase cartilage-specific bioavailability, ranging from the delivery of larger molecules (2.8 kDa) to the use of laser-induced ultrasound. In the current study, the authors used high-frequency ultrasound focused through a calibrated 0.2 mm needle hydrophone to facilitate the delivery of a small molecule (methylene blue [MB], 320 daltons) to samples of bovine articular cartilage. Osteochondral plugs (13 mm in size) were exposed to 0.005% w/v MB in phosphate-buffered saline (PBS) under three conditions: simultaneous sonication with exposure to MB (group T1), sonication in PBS followed by exposure to MB (control group C1), and sample immersion in MB only with no sonication (control group C2). All samples were exposed to MB for a total of 20 minutes, and sonicated samples were sonicated (either in PBS or MB itself) for 15 minutes. Characterization of Napierian light absorbance at multiple tissue depths was used to quantify the efficiency of MB absorption, and samples from group T1 showed significantly increased absorption up to a tissue depth of 600 µm from the articular cartilage surface (P < 0.05). Expression of several genes related to metabolism and inflammation in osteoarthritis (TGFB, TNF, NOS2, TIMP3, MMP1/3/9/13, COL2A1, ACAN) was not shown to be significantly different between sonicated and non-sonicated cartilage plugs, and levels of lactate dehydrogenase did not differ in the supernatant 24 hours after sonication / non-sonication, suggesting no negative effects on chondrocyte viability. Taken together, these results build upon previous work to further support the use of ultrasound techniques to increase the bioavailability of delivered compounds to articular cartilage.
Original Research Article: Nieminen, H.J., et al. “Localized delivery of compounds into articular cartilage by using high-intensity focused ultrasound.” Sci Rep 9.15937 (2019).

Viral competition: How insect-specific viruses may help prevent the spread of human diseases

Written By: Rebecca Tweedell

Original Article: Baidaliuk et al. Journal of Virology 2019
The Gist of It:
We all know that we can get infected with viruses that make us sick, but did you know that mosquitoes can get infected with insect-specific viruses, too? One such virus that has been infecting mosquitoes for hundreds of years is called cell-fusing agent virus (CFAV). No one really knows exactly what this virus does in mosquitoes (like if it makes them sick or causes some kind of damage). But some people have wondered whether being infected with this virus would have any impact on the abilities of other viruses to infect the mosquito, including viruses that can be passed along to humans like dengue virus and Zika virus. To answer this question, researchers at the Institut Pasteur in Paris and the Institut Louis Malardé In Tahiti infected mosquitoes with CFAV and either dengue or Zika virus. They paid particular attention to the amount of time it took for dengue or Zika virus to become present in the mosquito’s head because this is the point in time when the mosquito could infect a person (before there is virus in the head, even if the mosquito bites a person, it really can’t transmit the virus successfully). The researchers tried infecting the mosquitoes with CFAV 2 days before they infected them with dengue or Zika virus. Being infected with CFAV didn’t keep the mosquitoes from being subsequently infected with dengue or Zika virus, but it did slow down the speed at which the mosquito had detectable virus in its head, and it reduced the amount of dengue or Zika virus present in the head. This is an exciting finding because slowing down the speed at which a mosquito becomes infectious to humans and reducing the amount of virus present in the mosquito are both key strategies that can reduce the transmission of these deadly viruses to people. Building on this work and understanding more about how CFAV can impair the ability of both dengue and Zika virus to multiply and spread in the mosquito can have significant global impacts to reduce the spread of these and other deadly mosquito-borne pathogens.
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Mosquitoes infected with CFAV do not develop dengue or Zika virus in their heads as quickly or at as high a level as mosquitoes not infected with CFAV, slowing the mosquito’s ability to transmit these viruses to people.

The Nitty Gritty:
Baidaliuk et al. began to address the question of whether cell-fusing agent virus (CFAV) had an impact on the replication and dissemination of dengue virus (DENV-1) and Zika virus (ZIKV) in mosquitoes by performing in vitro assays in the Aedes albopictus cell line C6/36. They infected C6/36 cells with CFAV 3, 2, or 0 days before DENV-1 infection or 3 days before ZIKV infection. They found that no matter when CFAV infection was given, DENV-1 and ZIKV replication rates were significantly reduced over a 7-day time course. Based on these findings, they moved into an in vivo model using female A. aegypti mosquitoes that were bred and raised CFAV-free. They infected the mosquitoes with CFAV by intrathoracic injection and then provided an infectious bloodmeal containing DENV-1 or ZIKV 2 days later. They monitored the dissemination rate (the proportion of DENV-1– or ZIKV–positive mosquitoes with a DENV-1– or ZIKV–positive head) and dissemination titer (the infectious titer in the DENV-1– or ZIKV–positive head). At 5, 7, and 13 days post-infectious bloodmeal, they found that CFAV-infected mosquitoes had significantly lower dissemination rates and dissemination titers compared with CFAV-uninfected mosquitoes. UV-treatment of the CFAV stock to inactivate the virus particles eliminated this reduction in dissemination rate and titer. In CFAV-uninfected mosquitoes, there was a linear relationship between DENV-1 titer in the body and the head tissue; however, CFAV infection abolished this linear relationship, suggesting that CFAV interferes with the systemic dissemination of DENV-1. This study suggests that insect-specific viruses like CFAV could play an important role in a strategy to reduce DENV-1 or ZIKV transmission and reduce the impact of these diseases on the human population.
Original Research Article: Baidaliuk, A., et al. “Cell-fusing agent virus reduces Arbovirus dissemination in Aedes aegypti mosquitoes in vivo.J Virol 93.18 (2019): e00705-19.