As this therapeutic approach gains regulatory approval, clinical laboratory tests to determine condition of patient’s gut microbiota and monitor therapy will be needed
Some developments in the clinical laboratory industry are less about diagnostic tests and more about novel approaches to therapy. Such is the case with a new carbon bead technology developed by researchers from University College London (UCL) and the Royal Free Hospital intended to remove harmful bacteria toxins from the gut before they leak to the liver. The macroporous beads, which come in small pouches, are delivered orally and could be utilized in the future to treat a number of diseases.
Why is this relevant? Once a new treatment is accepted for clinical use, demand increases for a clinical laboratory test that confirms the therapy will likely work and to monitor its progress.
In collaboration with Yaqrit, a UK-based life sciences company that develops treatments for chronic liver disease, the UCL and Royal Free Hospital scientists engineered the carbon beads—known as CARBALIVE—to help restore gut health. They measured the technology’s impact on liver, kidney, and brain function in both rats and mice.
“The influence of the gut microbiome on health is only just beginning to be fully appreciated,” said Rajiv Jalan, PhD, Professor of Hepatology at UCL in a press release. “When the balance of the microbiome is upset, ‘bad’ bacteria can proliferate and out-compete the ‘good’ bacteria that keeps the gut healthy.
“One of the ways [the ‘bad’ bacteria] do this is by excreting endotoxin, toxic metabolites, and cytokines that transform the gut environment to make it more favorable to them and hostile to good bacteria,” he continued. “These substances, particularly endotoxin, can trigger gut inflammation and increase the leakiness of the gut wall, resulting in damage to other organs such as the liver, kidneys, and brain.”
“I have high hopes that the positive impact of these carbon beads in animal models will be seen in humans, which is exciting not just for the treatment of liver disease but potentially any health condition that is caused or exacerbated by a gut microbiome that doesn’t work as it should,” said Rajiv Jalan, PhD (above), Professor of Hepatology, University College London, in a press release. “This might include conditions such as irritable bowel syndrome (IBS), for example, which is on the rise in many countries.” Though not a clinical laboratory diagnostic test, new therapies like CARBALIVE could be a boon to physicians treating patients with IBS and other gastrointestinal conditions.
Developing the Carbon Beads
The team discovered CARBALIVE is effective in the prevention of liver scarring and injury in animals with cirrhosis when ingested daily for several weeks. They also found a reduced mortality rate in test animals with acute-on-chronic-liver-failure (ACLF).
After achieving success with CARBALIVE in animals, the researchers tested the technology on 28 cirrhosis patients. The carbon beads proved to be safe for humans and had inconsequential side effects.
“In cirrhosis, a condition characterized by scarring of the liver, it is known that inflammation caused by endotoxins can exacerbate liver damage,” Jalan explained. “Part of the standard treatment for cirrhosis is antibiotics aimed at controlling bad bacteria, but this comes with the risk of antibiotic resistance and is only used in late-stage disease.”
The beads, which are smaller than a grain of salt, contain an exclusive physical structure that absorbs large and small molecules in the gut. They are intended to be taken with water at bedtime as harmful bacteria is more likely to circulate through the body at night which could result in damage. The carbon beads do not kill bacteria, which decreases the risk of antibiotic resistance. They eventually pass through the body as waste.
“They work by absorbing the endotoxins and other metabolites produced by ‘bad’ bacteria in the gut, creating a better environment for the good bacteria to flourish and helping to restore microbiome health,” said Michal Kowalski, M.Sc.Eng, Director and VP of Operations at Yaqrit, in the UCL news release.
“This prevents these toxins from leaching into other areas of the body and causing damage, as they do in cirrhosis,” he added. “The results in animal models are very positive, with reduction in gut permeability, liver injury, as well as brain and kidney dysfunction.”
Additional Research
The researchers plan to perform further clinical trials in humans to determine if the carbon beads are effective at slowing the progression of liver disease. If the benefits that were observed in lab animals prove to be compelling in humans, the technology may become an invaluable tool for the treatment of liver disease and other diseases associated with poor microbiome health in the future.
According to the American Liver Foundation, 4.5 million adults in the US have been diagnosed with liver disease. However, it is estimated that 80 to 100 million adults have some form of fatty liver disease and are unaware of it. Liver disease was the 12th leading cause of death in the US in 2020 with 51,642 adults perishing from the disease that year.
According to BMC Public Health, globally there were 2.05 million new cases of liver cirrhosis diagnosed in 2019. In that year, 1.47 million people around the world died from the disease.
More research and clinical studies are needed before this novel technology can be used clinically. When and if that happens, the demand for clinical laboratory tests that measure microbiome deficiencies and monitor patient progress during therapy will likely be high.
Pathologists may be interested to learn that though a substitute for horseshoe crab blood has been available for nearly 20 years, it has not been widely adopted in the US
Since the 1970s, the blue blood of the horseshoe crab has saved countless human lives by detecting deadly toxins in medical products, as well as its use in a number of medical laboratory tests. Now, that unique blood is playing a vital role in the fight against COVID-19 as well, by enabling pharma companies to ensure the coronavirus vaccines they are giving are free of bacterial contaminants—such as endotoxins—that can harm or kill patients.
Microbiologists and clinical laboratory scientists are familiar with the Endotoxin Activity Assay (EAA), which uses LAL as a way to detect if a critically-ill patient may have whole blood endotoxemia. Published studies show that the detection of elevated endotoxin activity levels is associated with an increased disease severity in patients with sepsis and septic shock.
Horseshoe crab blood is an opaque blue color due to its high copper content. The blood contains limulus amebocyte lysate or LAL (pronounced “el-ay-el”), which either clots or changes color in the presence of bacterial endotoxins. LAL has become the standard to test the safety of many medical devices and pharmaceuticals—including coronavirus vaccines. There are currently no other tests that can analyze the purity of medications and cleanliness of medical devices with the same accuracy as the LAL test.
“The crabs live only on the east coasts of Asia and North America,” Yahoo News reported. “The world’s largest population of American horseshoes winters off Maryland shores and spawns each spring in Delaware Bay. The latest trawl survey puts this population at more than 14 million mature crabs.”
Futuristic Clean Rooms for Extracting Blood
After the crabs are dredged from the ocean floor, or captured as they come ashore for breeding, they are transported to one of a handful of facilities authorized to perform the bleeding process. Once there, the horseshoe crabs are cleaned and sterilized, suspended upside down, and inserted with a needle which extracts approximately one third of their blood.
“These things are being produced in ‘clean rooms’ that look like the stuff they make microchips in,” Glenn Gauvry, Founder and President of the Ecological Research and Development Group Inc. (ERDG) told Yahoo News. ERDG is a 501(c)3 non-profit wildlife conservation organization. Its primary focus is the conservation of the world’s four horseshoe crab species.
In futuristic-looking labs (above), technicians withdraw the prized blood cells from the tubular heart of the crab and then spin the blood in a centrifuge to isolate the LAL. The resulting product is then sold as endotoxin test kits to pharmaceutical companies that test medical and clinical laboratory supplies, including COVID-19 vaccines. (Photo copyright: Popular Mechanics.)
Certain laboratory tests developed from horseshoe crab blood are used to detect bacterial contamination in everything from heart stents, saline drips, and flu shots, to surgical implants such as pacemakers and prosthetic devices.
Federal law mandates that any medical device that is inserted or injected into a human body must pass the LAL test for contamination. This makes horseshoe crab blood vital for ensuring the new COVID-19 vaccinations are free of potentially-deadly toxins.
“Without LAL, you’re not going to be able to produce the billions of inoculations that are going to have to come about to handle this pandemic,” John Tanacredi, PhD, Professor of Earth and Environmental Sciences, Department of Biology, Chemistry and Environmental Studies at Molloy College, told CBS News.
A $60 Million/Year Industry
Once the LAL has been withdrawn, the crabs are returned to the wild. The horseshoe crabs typically spend one to three days in the lab while the LAL is extracted. They can survive out of water for up to four days, as long as their gills stay moist.
The crabs are then returned to the fishermen who originally caught them, to be gently placed back into the sea. “We treat these crabs like babies,” Yahoo News reported one fisherman saying. “Blue bloods save lives,” said another fisherman.
The Atlantic States Marine Fisheries Commission estimates that in 2019 labs extracted blood from approximately 640,000 horseshoe crabs in the US. Researchers estimate the industry makes about $60 million per year and that the coveted blue blood is worth about $60,000 per gallon, Yahoo News reported.
There are only four labs in the US authorized to extract blood from the horseshoe crabs. They are located in Maryland, Virginia, South Carolina, and Cape Cod. In Maryland, only three trawlers hold permits to catch the horseshoe crabs from the waters surrounding that state.
“We have a 450 million-year-old creature that for the last 45 years has improved the safety of medicines for humans and animals,” John Dubczak, Director of Operations at Charles River Laboratories, told CBS News.
Allen Burgenson, Global Subject Matter Expert-Testing Solutions, Lonza Walkersville, a division of Lonza Bioscience, told Yahoo News “the [pharmaceutical] industry produces enough tests in one day to screen five billion doses of coronavirus vaccine.”
Synthetic Substitute for Horseshoe Crab Blood Available, But Not Widely Used in US
In response to pressure from conservation groups, researchers in Singapore successfully cloned the blood of horseshoe crabs and developed a synthetic test for endotoxins. The synthetic test, known as Recombinant Factor C (rFC), has been commercially available since 2003 and in some countries in Europe is accepted as equal to tests using horseshoe crab blood. However, rFC has not yet been approved for widespread use in the US.
“You’ve got a very large, biomedical bleeding industry with a vested interest in keeping those horseshoes crabs coming in and basically protecting this monopoly,” Ryan Phelan, co-founder and Executive Director of Revive and Restore, a wildlife conservation group based in California that lobbied for the synthetic, told Yahoo News.
In 2017, Dark Daily reported on the potential threat to horseshoe crabs due to blood harvesting. In “Medical Laboratories Could Be Threatening the Survival of Horseshoe Crabs,” we noted growing concerns that the biomedical industry was having a negative impact on the horseshoe crab population. Since then, not much movement has been made toward establishing the synthetic as the standard test in the US.
Recombinant Factor C might eventually eliminate the need to harvest blood from living creatures. But for now, horseshoe crabs continue to provide a critical component for safeguarding humans against contaminants in medical supplies and pharmaceuticals, including COVID-19 vaccinations.
According to Dubczak, the LAL test “has unequivocally elevated the quality and safety of injectable pharmaceutical drugs and medical devices, and that includes all of the vaccines that protect us,” he told USA Today.
