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Wednesday, July 1, 2020

Review finds major weaknesses in evidence base for Covid-19 antibody tests

Major weaknesses exist in the evidence base for COVID-19 antibody tests, finds a review of the latest research published by The BMJ today.
The evidence is particularly weak for point-of-care tests (performed directly with a patient, outside of a laboratory) and does not support their continued use, say the researchers.
Serological tests to detect antibodies against COVID-19 could improve diagnosis and be useful tools for monitoring levels of infection in a population. The UK Prime Minister Boris Johnson has described antibody tests as “game-changing” in its response to the pandemic, but it is important to formally evaluate whether there is sufficient evidence that they are accurate.
So an international team of researchers set out to determine the diagnostic accuracy of antibody tests for COVID-19.
They searched medical databases and preprint servers from 1 January to 30 April 2020 for studies measuring sensitivity and/or specificity of a COVID-19 antibody test compared with a control test.
Sensitivity measures the percentage of people who are correctly identified as having a disease, while specificity measures the percentage of people who are correctly identified as not having a disease.
Of 40 eligible studies, most (70%) were from China and the rest were from the UK, US, Denmark, Spain, Sweden, Japan and Germany.
Half of the studies were not peer reviewed and most were found to have a high or unclear risk of bias (problems in that can influence results). Only four studies included outpatients and only two evaluated tests at the point of care.
When sensitivity results for each study were pooled together, they ranged from 66% to 97.8% depending on the type of test method used, meaning that between 2.2% and 34% of patients with COVID-19 would be missed.
Pooled specificities ranged from 96.6% to 99.7%, depending on the test method used, meaning that between 3.4% and 0.3% of patients would be wrongly identified as having COVID-19.
Pooled sensitivities were consistently lower for the lateral flow immunoassay (LFIA) test compared with other test methods. The LFIA test is the potential point-of-care method that is being considered for ‘immunity passports.’
Based on these results, the authors explain that, if an LFIA test is applied to a population with a COVID-19 prevalence of 10%, for every 1000 people tested, 31 who never had COVID-19 will be incorrectly told they are immune, and 34 people who had COVID-19 will be incorrectly told that they were never infected.
Pooled sensitivities were also lower with commercial kits (65%) compared with non-commercial kits (88.2%) and in the first and second week after symptom onset compared with after the second week.
The researchers point to some limitations, such as differences in study populations and the potential for missing studies. However, strengths include thorough search strategies and assessment of bias.
“These observations indicate important weaknesses in the evidence on COVID-19 serological tests, particularly those being marketed as point-of-care tests,” they write.
“While the should be lauded for the pace at which novel serological tests have been developed, this review underscores the need for high quality clinical studies to evaluate these tools,” they conclude. “With international collaboration, such studies could be rapidly conducted.”
More information: Diagnostic accuracy of serological tests for COVID-19: systematic review and meta-analysis, BMJ (2020). DOI: 10.1136/bmj.m2516 , www.bmj.com/content/370/bmj.m2516

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New ultrafast insulin developed

Researchers at Stanford University are developing a new insulin formulation that begins to take effect almost immediately upon injection, potentially working four times as fast as current commercial fast-acting insulin formulations.
The researchers focused on so-called monomeric , which has a molecular structure that, according to theory, should allow it to act faster than other forms of insulin. The catch is that monomeric insulin is too unstable for practical use. So, in order to realize the ultrafast potential of this insulin, the researchers relied on some materials science magic.
“The themselves are fine, so we wanted to develop a ‘magic fairy dust’ that you add into a vial that would help to fix the stability problem,” said Eric Appel, assistant professor of materials science and engineering at Stanford. “People often focus on the therapeutic agents in a but, by focusing only on the performance additives—parts that were once referred to as ‘inactive ingredients’ – we can achieve really big advancements in the overall efficacy of the drug.”
After screening and testing a large library of additive polymers, the researchers found one that could stabilize monomeric insulin for more than 24 hours in stressed conditions. (By comparison, commercial fast-acting insulin stays stable for six to ten hours under the same conditions.) The researchers then confirmed the ultrafast action of their formulation in diabetic pigs. Their results were published July 1 in Science Translational Medicine. Now, the researchers are conducting additional tests in hopes of qualifying for clinical trials in humans.
One step back, two steps forward
Current commercial formulations of insulin contain a mix of three forms: monomers, dimers and hexamers. Scientists have assumed monomers would be the most readily useful in the body but, within vials, the insulin molecules are drawn to the surface of the liquid where they aggregate and become inactive. (Hexamers are more stable in the vial but take longer to work in the body because they first have to break down into monomers to become active.) This is where the “magic fairy dust”—a custom polymer that is attracted to the air/water interface—comes in.
“We focused on polymers that would preferentially go to that interface and act as a barrier between any of the insulin molecules trying to gather there,” said Joseph Mann, a graduate student in the Appel lab and co-lead author of the paper. Crucially, the polymer can do this without interacting with the insulin molecules themselves, allowing the drug to take effect unimpeded.
Finding just the right polymer with the desired properties was a long process that involved a three-week trip to Australia, where a fast-moving robot created approximately 1500 preliminary candidates. This was followed by processing and testing individually by hand at Stanford to identify polymers that successfully exhibited the desired barrier behavior. The first 100 candidates didn’t stabilize commercial insulin in tests but the researchers pressed on. They found their magic polymer only weeks before they were scheduled to run experiments with diabetic pigs.
“It felt like there was nothing happening and then all of the sudden there was this bright moment … and a deadline a couple of months away,” said Mann. “The moment we got an encouraging result, we had to hit the ground running.”
In commercial insulin—which typically remains stable for about 10 hours in accelerated aging tests—the polymer drastically increased the duration of stability for upwards of a month. The next step was to see how the polymer affected monomeric insulin, which on its own aggregates in 1-2 hours. It was another welcome victory when the researchers confirmed that their formulation could remain stable for over 24 hours under stress.
“In terms of stability, we took a big step backward by making the insulin monomeric. Then, by adding our polymer, we met more than double the stability of the current commercial standard,” said Caitlin Maikawa, a graduate student in the Appel lab and co-lead author of the paper.
With a seed grant from the Stanford Diabetes Research Center and the Stanford Maternal and Child Health Research Institute, the researchers were able to evaluate their new monomeric insulin formulation in diabetic pigs—the most advanced non-human animal model—and found that their insulin reached 90 percent of its peak activity within five minutes after the insulin injection. For comparison, the commercial fast-acting insulin began showing significant activity only after 10 minutes. Furthermore, the monomeric insulin activity peaked at about 10 minutes while the commercial insulin required 25 minutes. In humans, this difference could translate to a four-fold decrease in the time insulin takes to reach peak activity.
“When I ran the blood tests and started plotting the data, I almost couldn’t believe how good it looked,” said Maikawa.
“It’s really unprecedented,” said Appel, who is senior author of the paper. “This has been a major target for many big pharmaceutical companies for decades.”
The monomeric insulin also finished its action sooner. Both beginning and ending activity sooner makes it easier for people to use insulin in coordination with mealtime glucose levels to appropriately manage their blood sugar levels.
A multifaceted success
The researchers plan to apply to the Food and Drug Administration for approval to test their insulin formulation in with human participants (although no trials are planned yet and they are not seeking participants at this time). They are also considering other uses for their , given how significantly it increased stability in commercial insulin.
Because their insulin formulation activates so quickly—and, therefore, more like insulin in a person without diabetes—the researchers are excited by the possibility that it could aid the development of an artificial pancreas device that functions without the need for patient intervention at mealtimes.
Additional Stanford co-authors include former visiting scholar Anton Smith (from Aarhus University in Denmark); graduate students Abigail Grosskopf, Gillie Roth, Catherine Meis, Emily Gale, Celine Liong, Doreen Chan, Lyndsay Stapleton and Anthony Yu; clinical veterinarian Sam Baker; and postdoctoral fellow Santiago Correa. Researchers from CSIRO Manufacturing in Australia are also co-authors. Appel is also a member of Stanford Bio-X, the Cardiovascular Institute, the Stanford Maternal and Child Research Institute and a faculty fellow at Stanford ChEM-H.
More information: J.L. Mann el al., “An ultrafast insulin formulation enabled by high-throughput screening of engineered polymeric excipients,” Science Translational Medicine (2020). stm.sciencemag.org/lookup/doi/ … scitranslmed.aba6676

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‘Absurdly high’ levels of toxins released by fireworks

Ooh, ahh, uh oh: An explosive new study on the dangers of fireworks has revealed a major health hazard just in time for the 4th of July 2020.
It’s not just the immediate risks of accidental fire and life-threatening injury you might expect. Everyday fireworks — ones that any amateur pyro could acquire — emit “absurdly high” amounts of harmful toxins, including lead and copper, said Dr. Terry Gordon, the senior author of the study.
Exposure to these toxins could leave lasting damage on lungs, previous research has shown.
The findings are especially troubling given the recent uptick in illegal firework activity in New York City and elsewhere, Gordon said.
“These are high levels of … toxic metals that [usually] only occur a few days a year,” he told The Post. “But this has been increased.”
In New York City, fireworks-related noise complaints reached 9,000 cases between June 1 and 21. And some retailers say they’re seeing a surge in sales this year like they’ve never seen before — perhaps resulting in the confiscation of thousands of dollars worth of fireworks during the past few weeks.
Gordon claimed the study — 14 years in the making — is one of the only to analyze consumer fireworks for their toxic, respiratory hazards. He and his team in the Department of Environmental Medicine at NYU Langone Health published their findings this month in the journal Particle and Fibre Toxicology.
“To me, this has always been overlooked,” he said. “There hasn’t been any real studies to look at the toxicity of fireworks.”
As a result of the study, he suggests anyone with asthma or other respiratory issues situate themselves upwind of larger pyrotechnic displays, to prevent emissions from rolling your way. This could exacerbate existing issues.
Their experiment included a dozen popular brands, such as the Black Cuckoo, the Color Changing Wheel and Blue Storm firecrackers. To get a sense of the potential immediate damage these toxins can do, researchers detonated the fireworks in a chamber containing mice subjects as well as human cells, and later assessed the health of each case study.
“Most surprising was that, in this very small sample of consumer fireworks … two out of the 12 had high levels of lead,” he said. In particular, he called the amount of lead found in the Black Cuckoo “absurdly high” at 40,000 parts per million. It also contained 12,000 ppm of copper.
The Blue and Purple Colorful Storms also turned up 53,000 and 44,000 ppm of copper, respectively. Other potentially harmful metals found in their analysis included bromine, zinc, barium and cobalt.
Their chamber experiment revealed that acute, high exposure to the fireworks were enough to cause human cell damage as well as inflammation in the lungs of mice.

“This isn’t [just] contamination level,” Gordon says of the Black Cuckoo. “This is like purposely putting it in there.”
He added, “I don’t know why someone would put lead in,” which, per the American Fireworks Standards Laboratory guidelines, should not be there, he pointed out. He speculates the chemical was “cheaper” to use than an alternative ingredient.
He says there are a “very good set of … guidelines” in place. Unfortunately, he continued, they’re not enforced. Gordon blames an industry that fails to sample its products for the egregious oversight. He estimated that some “90% or more” of fireworks are being imported from China.
He believes the industry should be regularly sampling their fireworks inventory “to make sure that the imports are safe.”
And for those pyros who just can’t keep away, Gordon feels comfortable recommending sparklers. “I’ve always let my kids use sparklers,” he said.

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Asthma drug salbutamol’s potential as Alzheimer’s treatment

A new study reveals that the common asthma drug salbutamol may offer potential as a treatment for Alzheimer’s disease.
Alzheimer’s disease is the most common form of dementia, affecting 47 million people worldwide and its prevalence is expected to triple to more than 130 million cases by 2050.
No effective treatments that cure the disease or slow down its progression have been discovered. However, this new early-stage study has revealed that repurposing an existing drug, salbutamol, offers significant potential as a low cost and rapid response option.
Extensive analytical in-vitro experiments conducted by the research team show that salbutamol is effective at reducing the accumulation of insoluble fibres of the tau protein – which is found in the brains of people with Alzheimer’s disease. These microscopic fibres accumulate into neurofibrillary tangles and can cause neuron destabilisation, brain cell death, and are a key characteristic of the disease’s progression.
Much Alzheimer’s disease research has focused on the build-up of amyloid plaques, caused by misfolding of the amyloid-β protein. However, because of disappointing results from numerous therapies targeting Aβ aggregation, more attention is shifting towards tau.
This study, led by researchers at Lancaster University, used a new automated ‘high throughput’ screening approach to study the structure of the misfolding tau protein with a special analytical technique called ‘Synchrotron Radiation Circular Dichroism’ (SRCD) at Diamond Light Source, the UK national synchrotron light source in Oxfordshire. With this powerful technique they were able to look at a selection of more than 80 existing compounds and drugs simultaneously to determine their effectiveness at preventing the formation of tau fibrils.
This method confirmed the compound epinephrine, more commonly known as adrenaline, was effective at stabilising the tau proteins and preventing the formation of tau tangles. However, our bodies do not easily absorb epinephrine and it rapidly gets metabolised, so the scientists then looked at a range of readily available compounds with similar chemical structures. This search revealed four current drugs as possible candidates – etamivan, fenoterol, dobutamine and salbutamol.
Etaminvan and fenoterol were found to have little effect on the assembly of tau tangles. Dobutamine, which is used for the rapid treatment of heart attacks and heart failure, was found to have some benefit. However, because its effects are very short-lived, and because it needs to be administered intravenously, it is not ideal as a basis for treatment of Alzheimer’s disease.
Further tests using a range of analytical techniques all revealed salbutamol could inhibit tau aggregation in vitro. Tests where salbutamol was added to solutions containing tau resulted in drastically reduced density of fibrous tau structures responsible for the tau neurofibrillary tangles.
The researchers believe that salbutamol interacts with an early stage of tau fibril formation, reducing their ability to form an initial nucleus which drives the aggregation process.
Because it is easily ingested, absorbed into the brain, and remains in the body for several hours, salbutamol has attractive properties as a research avenue for potential new treatment for Alzheimer’s.
Dr David Townsend, of Lancaster university and lead author of the research, said: “Our work highlights the potential impact of repurposing drugs for secondary medical uses, by discovering a novel therapeutic strategy that impedes the molecular pathology of Alzheimer’s disease, and which may have otherwise gone unstudied.
“Salbutamol has already undergone extensive human safety reviews, and if follow up research reveals an ability to impede Alzheimer’s disease progression in cellular and animal models, this drug could offer a step forward, whilst drastically reducing the cost and time associated with typical drug development.”
Professor David Middleton, co-author of the research, said: “This work is in the very early stages and we are some way from knowing whether or not salbutamol will be effective at treating Alzheimer’s disease in human patients. However, our results justify further testing of salbutamol, and similar drugs, in animal models of the disease and eventually, if successful, in clinical trials.”
Dr Rohanah Hussain, of Diamond Light Source, Senior Beamline Scientist and co-author said: “Diamond B23 beamline unique micro-collimated beam has made high throughput CD possible allowing the screening of many compounds through structural activity correlation crucial in drug discovery.”
The researchers say that current asthma inhalers result in only a small amount of salbutamol reaching the brain and so, if further research is successful, a new delivery method would also need to be developed. They add that future research could also focus on other asthma drugs that are chemically similar to salbutamol, but which circulate in the bloodstream for much longer.
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The research is outlined in the paper ‘Circular dichroism spectroscopy identifies the β-adrenoceptor agonist salbutamol as a direct inhibitor of tau filament formation in vitro’, which has been published by the journal ACS Chemical Neuroscience.
The authors are David Townsend, Barbora Mala, Eleri Hughes, Nigel Fulwood and David Middleton of Lancaster University, and Rohanah Hussain and Giuliano Siligardi of Diamond Light Source.
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A simpler way to make sensory hearing cells

Scientists from the USC Stem Cell laboratories of Neil Segil and Justin Ichida are whispering the secrets of a simpler way to generate the sensory cells of the inner ear. Their approach uses direct reprogramming to produce sensory cells known as “hair cells,” due to their hair-like protrusions that sense sound waves. The study was published in the journal eLife.
“We’ve succeeded in directly reprogramming a variety of mouse cell types into what we’re calling ‘induced hair cell-like cells, or iHCs,” said PhD student Louise Menendez, the study’s lead author. “This allows us to efficiently generate large numbers of iHCs to identify causes and treatments for hearing loss.”
The scientists successfully reprogrammed three different types of mouse cells to become iHCs. The first two types were embryonic and adult versions of connective tissue cells, known as fibroblasts. The third was a different type of inner ear cell, known as a supporting cell.
To achieve reprogramming, the scientists exposed fibroblasts and supporting cells to a cocktail of four transcription factors, which are molecules that help convey the instructions encoded in DNA. The scientists identified this cocktail by testing various combinations of 16 transcription factors that were highly active in the hair cells of newborn mice.
“The four key ingredients turned out to be the transcription factors Six1, Atoh1, Pou4f3, and Gfi1,” said Menendez.
The resulting iHCs resembled naturally occurring hair cells in terms of their structure, electrophysiology, and genetic activity. The iHCs also possessed several other distinct characteristics of hair cells, including vulnerability to an antibiotic known to cause hearing loss.
“Hair cells are easy to damage, and currently impossible to repair in humans,” said Segil, a professor in the Department of Stem Cell Biology and Regenerative Medicine, and the USC Tina and Rick Caruso Department of Otolaryngology – Head and Neck Surgery, and one of the corresponding authors of the study. “Aging, loud noises, and certain chemotherapy drugs and antibiotics can all lead to the permanent loss of hair cells, which is the leading contributor to hearing loss worldwide.”
iHCs have the potential to accelerate hearing loss research in at least two important ways, according to Ichida, who is the John Douglas French Alzheimer’s Foundation Associate Professor of Stem Cell Biology and Regenerative Medicine at USC, and the other corresponding author of the study.
“In the near term, researchers can use iHCs to screen large numbers of drug candidates that might prevent or treat hearing loss,” said Ichida, who is also a New York Stem Cell Foundation-Robertson Investigator. “And further in the future, it could become possible to directly reprogram supporting cells in the inner ear of a deafened individual, as a way to restore hearing.”
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This paper is the result of a collaboration between the Segil and Ichida labs, initiated by Menendez and Suhasni Gopalakrishnan. Two additional groups joined the collaboration to characterize the new iHCs: Radha Kalluri, assistant professor in the USC Tina and Rick Caruso Department of Otolaryngology – Head and Neck Surgery, and Alex Markowitz characterized the physiological properties of the new cells; and James Lee and Chichou Huang of DRVision Technologies developed software for robotically imaging and quantifying the growth and death of the iHCs. Other key contributors from the Segil Lab were Talon Trecek and Litao Tao, who collected and analyzed data, as well as Juan Llamas, Xizi Wang, and Haoze Vincent Yu.
The collaboration was catalyzed by a Regenerative Medicine Initiative grant from the Office of the Dean of the Keck School of Medicine of USC. Seventy percent of this work was supported by federal funding from the National Institutes of Health (R01DC015530, R00NS077435, and R01NS097850). Additional sources of funding include the New York Stem Cell Foundation and the Merkin Family Foundation.

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