Tag Archives: #sepsis

Cell Reprogramming in Sepsis Patients Helps Explain Deterioration of Health Even After hospital Discharge (Medicine)

An article published in Frontiers in Immunology suggests that sepsis can cause alterations in the functioning of defense cells that persist even after the patient is discharged from hospital. This cellular reprogramming creates a disorder the authors call post-sepsis syndrome, whose symptoms include frequent reinfections, cardiovascular alterations, cognitive disabilities, declining physical functions, and poor quality of life. The phenomenon explains why so many patients who survive sepsis die sooner after hospital discharge than patients with other diseases or suffer from post-sepsis syndrome, immunosuppression and chronic inflammation.

The article presents a review of studies conducted to investigate cases of septic patients who died up to five years after being discharged from hospital.

Considered one of the leading causes of death in intensive care units, sepsis is a life-threatening systemic organ dysfunction triggered by the organism’s dysregulated response to an infectious agent, typically a bacterium or fungus. The defense system injures the body’s own tissues and organs while combating the infectious agent. 

If it is not promptly recognized and treated, the condition can lead to septic shock and multiple organ failure. Patients with severe COVID-19 and other infectious diseases run a heightened risk of developing and dying of sepsis. 

New cases of sepsis are estimated to total some 49 million per year worldwide. Hospital mortality from septic shock exceeds 40% globally, reaching 55% in Brazil, according to the Sepsis Prevalence Assessment Database (SPREAD) study, conducted with support from FAPESP

“The massive infection and the accompanying intense immune response with a cytokine outpouring during sepsis may promote irreversible cell metabolic reprogramming. Cell reprogramming is unlikely to occur in leukocytes or bone marrow only. This might happen in several tissues and cells that prompt systemic organ dysfunctions […] Bacteria can transfer genetic material to host cell DNA as eukaryotic cells develop tools to protect themselves against the microorganism invasion. The latter may induce cell biology and metabolic reprogramming that remains even after the infection’s elimination,” the researchers state in the article. 

According to Raquel Bragante Gritte, joint first author with Talita Souza-Siqueira, one of the hypotheses investigated by the group was that metabolic reprogramming starts in the bone marrow, whose cells acquire a pro-inflammatory profile. “Our analysis of blood samples from patients even three years after ICU discharge showed that monocytes [a type of defense cell] were activated and ready for battle. They should have been neutral. Monocytes are normally activated only when they are ‘recruited’ to the tissue,” Gritte told Agência FAPESP. Both Gritte and Souza-Siqueira are researchers at Cruzeiro do Sul University (UNICSUL) in the state of São Paulo, Brazil.

Research line

The Frontiers in Immunology article is one of the first to be published by the group on this subject. The co-authors include two medical doctors and professors at the University of São Paulo (USP): Marcel Cerqueira Cesar Machado, principal investigator for a project supported by FAPESP; and Francisco Garcia Soriano. Their research line has brought to light recent discoveries in studies of post-discharge sepsis patients available from PubMed, a leading database of references and abstracts on life sciences and biomedical topics. 

According to Gritte, the group conducted a follow-up study of 62 patients for three years after discharge from the ICU at USP’s University Hospital, analyzing alterations in monocytes, neutrophils and lymphocytes, as well as microRNAs, to try to identify prognostic markers and factors associated with post-sepsis syndrome.

“Our hypothesis is that white blood cells conserve a memory of sepsis, which helps explain why patients remain sick after they leave hospital,” said Rui Curi, another co-author of the article. Curi is a professor at UNICSUL and a director of Butantan Institute.

In the article, the researchers suggest sepsis may generate a specific macrophage phenotype that remains active after discharge from hospital. “Cell metabolism reprogramming is also involved in the functions and even generation of the different lymphocyte subsets. Several stimuli and conditions change lymphocyte metabolism, including microenvironment nutrient availability,” they write.

According to Gritte, next steps include studies of bone marrow to understand how cells are reprogrammed by sepsis. “We think the key to this alteration is in bone marrow,” she said. “However, another possibility is that activation occurs in the blood. We’ll need to do more in-depth research to find answers.” 

The knowledge acquired in this study can serve as a basis for the development of strategies to minimize or block post-sepsis alterations.

Last year the World Health Organization (WHO) published its first report on the global epidemiology of sepsis, noting that much of the burden of sepsis incidence and mortality weighs on low- and middle-income countries and that a severe lack of population-based sepsis data, especially in such countries, hinders efforts to address the problem.

The WHO report recommends a concerted global effort to increase funding and research capacity for the generation of epidemiological evidence on sepsis, as well as the development of rapid, affordable, and appropriate diagnostic tools to improve sepsis identification, prevention, and treatment.

The article “Why septic patients remain sick after hospital discharge” by Raquel Bragante Gritte, Talita Souza-Siqueira, Rui Curi, Marcel Cerqueira Cesar Machado and Francisco Garcia Soriano is at: www.frontiersin.org/articles/10.3389/fimmu.2020.605666/full#h6.

Featured image: A review article by Brazilian researchers shows that alterations in the defense cell metabolism may explain why many patients who survive sepsis die within a year or suffer from long-term complications (image: Wikimedia Commons)

Provided by FAPESP

Repurposed Heart and Flu Drugs May Help Body Fight Sepsis (Medicine)

Higher platelet counts linked to better outcomes for patients with staph sepsis; repurposed drugs that protect platelets improve survival of septic mice

Despite continued improvements in antibiotics and hospital intensive care, staph sepsis — a bloodstream infection caused by Staphylococcus aureus bacteria — still causes severe illness or death in 20 to 30 percent of patients who contract it.

Rather than continue to throw more antibiotics at the problem, University of California San Diego researchers want to boost the other side of the equation: the patient’s own immune system.

The team recently discovered a battle that occurs between staph bacteria and platelets — blood cells known better for their role in clotting than in immune defense. In some sepsis cases, they found, the bacteria win out and platelet levels plummet. Patients with fewer platelets were more likely to die of staph sepsis than patients with higher platelet counts.

The researchers also determined that two currently available prescription medications, approved by the U.S. Food and Drug Administration (FDA) for other uses, protect platelets and improve survival in mouse models of staph sepsis. The two repurposed drugs were ticagrelor (Brilinta), a blood thinner commonly prescribed to prevent heart attack recurrence, and oseltamivir (Tamiflu), prescribed to treat the flu.

The study publishes March 24, 2021 in Science Translational Medicine.

“In many cases, the antibiotics we give these patients should be able to kill the bacteria, based on lab tests, yet a significant number of patients are not pulling through,” said senior author Victor Nizet, MD, Distinguished Professor at UC San Diego School of Medicine and Skaggs School of Pharmacy and Pharmaceutical Sciences. “If we can reduce mortality in staph sepsis by 10 or 20 percent by arming or protecting the immune system, we can likely save more lives than discovering an additional new antibiotic that may still not cure the sickest patients.”

The study started with a group of 49 University of Wisconsin patients with staph sepsis. The team collected the patients’ blood, bacteria samples, and demographic and health information. To their surprise, it wasn’t white blood cell counts (immune cells) that correlated with patient outcomes — it was the platelet count. Low platelet counts, defined in this case as fewer than 100,000 per mm3 blood, were associated with increased risk of death from staph sepsis. Approximately 31 percent of patients with low platelet counts died from the infection, compared to less than 6 percent of patients with platelets above the threshold.

In laboratory experiments, the researchers worked out what’s likely happening: Platelets secrete antimicrobial peptides that help the immune system destroy staph bacteria. At the same time, staph release an alpha-toxin that’s detrimental to platelets. In addition to poking holes in platelets, the bacteria’s alpha-toxin convinces the blood cells to produce an enzyme that trims off sugar molecules that decorate their own surfaces. The platelet’s new look is recognized by another molecule in the liver called the Ashwell-Morell receptor, which pulls “bald” platelets out of circulation.

Once Nizet and team had an idea of what might be happening in the patients who are less likely to survive staph sepsis, they turned to mouse models of the disease to find ways to tip the balance of what they call the “toxin-platelet-receptor” axis back in favor of the human patient.

The researchers tested several classes of drugs known to be safe in humans and known to act on platelets. Most drugs they tested had no effect, but two drugs made a big difference. Ticagrelor blocks staph’s alpha-toxin so it can’t injure platelets or stimulate its sugar-removing enzyme. Oseltamivir inhibits the platelet sugar-removing enzyme so the cells don’t go bald and aren’t cleared by the liver, even when staph’s alpha-toxin is around.

Mice with staph sepsis and treated with either ticagrelor or oseltamivir maintained more platelets and had less bacteria in their blood. Ultimately, approximately 60 percent of treated mice survived 10 days following infection, compared to 20 percent of untreated mice.

Side effects of these medications may include nausea, diarrhea and nosebleeds, and ticagrelor may cause uncontrollable bleeding. While new clinical trials specifically designed to test the drugs’ safety and efficacy for patients with staph sepsis would be ideal, Nizet said there’s little financial incentive for pharmaceutical companies to do so with an already profitable drug.

Still, repurposing commercially available drugs has many advantages.

“Discovering a new drug is tremendously expensive and takes many, many years,” said Nizet, who is also faculty lead for the Collaborative to Halt Antibiotic-Resistant Microbes (CHARM) at UC San Diego. “But if we look around at what we already have, what we already know to be safe, we may find many opportunities to improve patient outcomes.”

Sepsis can be caused by several types of bacteria in addition to staph, including Streptococcus pyogenesKlebsiellaE. coli and Pseudomonas aeruginosa. According to the Centers for Disease Control and Prevention, each year at least 1.7 million adults in the U.S. develop sepsis and nearly 270,000 die as a result. One in three patients who die in a hospital has sepsis. And it’s one of the costliest of all diseases — in 2013, for example, the Department of Health and Human Services reported that sepsis management added up to more than $24 billion in hospital expenses, or 13 percent of total U.S. hospital costs.

Co-authors of the study include: Josh Sun, Satoshi Uchiyama, Joshua Olson, Ingrid Cornax, Nao Ando, Yohei Kohno, May M. T. Kyaw, Bernice Aguilar, Nina M. Haste, George Sakoulas, UC San Diego; Yosuke Morodomi, Sachiko Kanaji, Taisuke Kanaji, Scripps Research; Warren E. Rose, University of Wisconsin; and Jamey D. Marth, UC Santa Barbara and Sanford Burnham Prebys Medical Discovery Institute.

Disclosure: Warren Rose has received speaking honoraria from Melinta unrelated to the current study. George Sakoulas has consulted for Allergan, Paratek, and Octapharma unrelated to the current study. Victor Nizet has consulted for Cellics Therapeutics, Staurus, Vaxcyte, Clarametyx Biosciences, SNIPR Biome, Boehringer Ingelheim, and Iogen unrelated to the current study.

Featured image: Left: Human platelets are destroyed by Staphylococcus aureus bacteria (circles). Right: With the addition of blood thinner ticagrelor, human platelets (larger blobs) are protected from injury by Staphylococcus aureus (smaller circles). © UC San Diego Health Sciences

Reference: Josh Sun, Satoshi Uchiyama, Joshua Olson, Yosuke Morodomi, Ingrid Cornax, Nao Ando, Yohei Kohno, May M. T. Kyaw, Bernice Aguilar, Nina M. Haste, Sachiko Kanaji, Taisuke Kanaji, Warren E. Rose, George Sakoulas, Jamey D. Marth and Victor Nizet, “Repurposed drugs block toxin-driven platelet clearance by the hepatic Ashwell-Morell receptor to clear Staphylococcus aureus bacteremia”, Science Translational Medicine  24 Mar 2021:
Vol. 13, Issue 586, eabd6737
DOI: 10.1126/scitranslmed.abd6737

Provided by University of California San Diego

Detecting Multiple Sepsis Biomarkers From Whole Blood – Made Fast, Accurate, And Cheap (Medicine)

The Wyss Institute’s eRapid electrochemical sensor technology now enables sensitive, specific, and multiplexed detection of blood biomarkers at low cost with potential for many clinical applications

Many life-threatening medical conditions, such as sepsis, which is triggered by blood-borne pathogens, cannot be detected accurately and quickly enough to initiate the right course of treatment. In patients who suffer infection by an unknown pathogen that then progresses to overt sepsis, every additional hour that an effective antibiotic cannot be administered significantly increases the mortality rate, so time is of the utmost essence.

The challenge with rapidly diagnosing sepsis stems from the fact that measuring only one biomarker often does not allow a clear-cut diagnosis. Engineers have struggled for decades to simultaneously quantify multiple biomarkers in whole blood with high specificity and sensitivity for point-of-care (POC) diagnostic applications, as this would avoid time-consuming and costly blood processing steps in which informative biomarker molecules could potentially be lost.

Now, a multi-disciplinary team at Harvard’s Wyss Institute for Biologically Inspired Engineering and the University of Bath, UK, led by Wyss Founding Director Donald Ingber, M.D., Ph.D., and Wyss Senior Staff Scientist Pawan Jolly, Ph.D., has further developed the Institute’s eRapid technology as an affinity-based, low-cost electrochemical diagnostic sensor platform for the multiplexed detection of clinically relevant biomarkers in whole blood. The device uses a novel graphene nanocomposite-based surface coating and was demonstrated to accurately detect three different sepsis biomarkers simultaneously. The findings are reported in Advanced Functional Materials.

“In this study, we have taken an important step towards deploying our electrochemical sensor platform in clinical settings for fast and sensitive detection of multiple analytes in human whole blood. As the nanocomposite coating we developed here is inexpensive, it has the potential to revolutionize point-of-care diagnostics not only to test for sepsis biomarkers, but a much broader range of biomarkers that can be multiplexed in sets to report on the states of many diseases and conditions,” said Ingber, who also is a lead of the Wyss Institute’s Bioinspired Therapeutics and Diagnostics Platform, and the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

Ingber, Jolly, and their Wyss team are currently also developing eRapid electrochemical sensors with the newly engineered graphene-based nanocomposite coating as a critical component of a point-of-care diagnostic for COVID-19, traumatic brain injury, myocardial infarction, and many other disorders.

By developing their electrochemical sepsis-sensing technology, Ingber’s team built on earlier work published in Nature Nanotechnology, in which they had solved the problem of “biofouling” of electro-chemical sensing elements with their eRapid technology. In theory, electrochemical biosensors would be preferred for many clinical applications because of their ability to quantify the content of biological samples by directly converting the binding event of a biomarker to an electronic signal, their low power consumption and cost, and easy integration with diagnostic readers. However, especially when using whole blood, many blood components nonspecifically bind to the surface coatings of the sensors’ electrodes and lead to their degradation, as well as electric noise in the form of false signals.

A novel antifouling nanocomposite coating covers the electrodes on eRapid chips, which uses graphene-oxide nanoflakes to conduct electricity and has binding reagents for sepsis biomarkers embedded into it. Coupled with additional surface chemistry this enables the conversion of a biomarker binding event to an electrical signal that correlates in strength with the levels of target biomarkers detected. Credit: Wyss Institute at Harvard University

The team’s eRapid technology uses a novel antifouling nanocomposite coating for electrodes to which binding reagents are attached that capture biomarker molecules from small quantities of blood and other complex biological fluids. Upon chemically detecting any one of these biomarker molecules with high sensitivity and selectivity, the eRapid platform generates an electrical signal at the electrodes that correlates in strength with the levels of target molecules that are detected. The initial nanocomposite coating allowed excellent conversion of chemical to electrical signals, and relied on tiny electrically conductive gold nanowires that were embedded in a matrix of a crosslinked protein known as bovine serum albumin. However, the high costs of the gold materials had been the major barrier to commercializing eRapid for clinical applications.

“In our advanced eRapid version, we replaced the coating’s gold nanowires with graphene oxide nanoflakes that also have anti-fouling and electrochemical properties, but they are much less expensive and allow even more sensitive measurements. In fact, the costs of fabricating the nanocomposite were reduced to a fraction of its original cost, which together with the sensing technology’s speed, efficiency, and versatility should enable the eRapid platform to have immediate commercial impact,” said Jolly.

After optimizing and characterizing their nanocomposite coating in binding assays for the inflammatory cytokine interleukin 6, the team applied it to the diagnosis of sepsis. Essentially, by attaching an antibody molecule to the coating that binds procalcitonin (PCT), and adding a second PCT-specific antibody to the complex that is linked to an enzyme, a precipitate is formed from a chemical substrate and deposited on the coating. This changes the current of electrons reaching the electrode, and helps register the PCT binding event as an electronic signal.

“We demonstrated that this electrochemical sensor element can detect PCT with high accuracy in whole blood, and validated it by quantifying PCT levels in 21 clinical samples, directly comparing it with a conventional ELISA assay – with excellent correlation,” said first-author Uroš Zupančič, who was a visiting scholar in Ingber’s group from the University of Bath. Zupančič is a Ph.D. candidate mentored by the study’s co-authors Despina Moschou, Ph.D., a Lecturer at the University of Bath, and Pedro Estrela, Ph.D., Associate professor and the head of the Centre for Biosensors, Bioelectronics and Biodevices at the University.

The team then extended their approach to simultaneously detecting multiple sepsis biomarkers by also designing sensor elements for C-reactive protein, another sepsis biomarker, and pathogen-associated molecular patterns (PAMPs). The PAMP sensor element in particular leverages the Wyss Institute’s broad-spectrum pathogen capture technology that uses a genetically engineered protein called FcMBL, which binds more than 100 different pathogens of all classes, as well as molecules on their surfaces that are released into blood when pathogens are killed (PAMPs) and act to trigger the sepsis cascade.

Video: This animation features an eRapid technology on-a-chip and shows how engineered and spatially separated binding systems capture specific biomarkers without interfering with each other to allow the multiplexed analysis of sepsis in whole blood. Credit: Wyss Institute at Harvard University

“Assembling three dedicated electrochemical sensor elements for biomarkers that can be present in blood at vastly different concentrations on a single chip posed a significant challenge. However, the three elements in the final sensor exhibited specific responses within the clinically significant range without interfering with each other, and they did so with a turnaround time of 51 minutes, which meets the clinical need of sepsis diagnosis within the first hour,” said Zumpančič.

To make the current eRapid technology even more effective and useful for clinical sample analysis, the team integrated it with a microfluidic system that takes out the human element involved in handling the sensor in the laboratory, and enhances the number of biomarker binding events at its surface. This allows biomarker analysis with the system to be automated, and enabled the researchers to decrease the turnaround time for measuring PCT to seven minutes.

The study was funded by the Defense Advanced Research Projects Agency (DARPA) under contract W911NF-16-C-0050, the Wyss Institute for Biologically Inspired Engineering at Harvard University, the Rosetrees Trust under project M681, and the U.S. Army Research Office.

Featured image: Wyss Institute researchers have developed eRapid technology as an affinity-based, low-cost electrochemical diagnostic sensor platform for the multiplexed detection of clinically relevant sepsis biomarkers in whole blood. Credit: Wyss Institute at Harvard University

Reference: Sabaté del Río, J., Henry, O.Y.F., Jolly, P. et al. An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids. Nat. Nanotechnol. 14, 1143–1149 (2019). https://doi.org/10.1038/s41565-019-0566-z

Provided by Wyss Institute

Noncoding RNA Has Surprising Effects on Immune Response and Sepsis (Medicine)

A long noncoding RNA regulates the expression of inflammatory genes and has a surprising effect on vulnerability to septic shock in mice

When the body’s immune response to an infection gets out of control, the result can be sepsis, a life-threatening condition in which an overwhelming inflammatory response can lead rapidly to failure of multiple organs and death.

In a new study, researchers at UC Santa Cruz have identified a long noncoding RNA (lncRNA) molecule that regulates the expression of pro-inflammatory genes in immune system cells called macrophages and affects the susceptibility of mice to septic shock.

This lncRNA, called GAPLINC, was previously studied for its role in cancer, but it turns out to be the most highly expressed lncRNA in macrophages, which play a central role in inflammation. Susan Carpenter, assistant professor of molecular, cell and developmental biology at UC Santa Cruz, said GAPLINC stood out when her lab performed RNA sequencing of human macrophages and their precursors, white blood cells called monocytes.

Subsequent experiments showed that reducing or eliminating GAPLINC led to enhanced expression of inflammatory genes in both mouse and human cells. Paradoxically, this effect protected mice from endotoxic shock and death in a mouse model of sepsis. The difference was dramatic, with all of the normal (“wildtype”) mice dying within a day, while all of the mice in which GAPLINC was knocked out survived.

“Our hypothesis was that the knockout mice would do worse in a model of endotoxic shock, so we were surprised to find that they did much better,” said Carpenter, the corresponding author of a paper on the new findings published February 1 in Proceedings of the National Academy of Sciences.

Noncoding RNAs are RNA molecules that are transcribed from the genome but are not translated into a protein, and lncRNA is the largest class of noncoding RNA. In recent years, scientists have identified thousands of lncRNAs in mammalian genomes that regulate gene expression in different biological processes. GAPLINC is one of the few examples of a lncRNA found in both humans and mice.

Apple Vollmers, a graduate student in Carpenter’s lab and first author of the paper, said the team’s RNA sequencing results showed that GAPLINC is highly expressed during the differentiation of monocytes into macrophages.

“We thought initially that it was involved in regulating differentiation, but when we depleted it, that turned on inflammatory genes,” Vollmers said. “It’s a low level of expression, so the genes are not turning on at the same level as when a macrophage is activated in response to a pathogen.”

Endotoxic shock

The mouse model of sepsis involves exposing mice to a component of gram-negative bacteria called lipopolysaccharide (LPS, also known as endotoxin), which is known to trigger septic shock in bacterial infections. Carpenter said starting from a low baseline level of inflammatory gene expression may make the inflammatory response to LPS less of a shock to the system. “Instead of going from zero to 100, you might go from 10 to 100, and we think that provides some protection, but we’re not sure why,” she said.

Macrophages are among the first cells involved in the body’s response to any injury or infection. They are patrollers and first responders, circulating in the blood as monocytes and differentiating into macrophages that move to sites where they are needed to help fight infection or heal an injury.

“They help turn on inflammation, but they also play an important role in turning it off,” Carpenter said.

The expression of GAPLINC gets turned on when monocytes differentiate into macrophages, and it gets turned down after the cells are exposed to LPS. “Something interesting is happening between those steps, where somehow modulating the expression of GAPLINC can be protective,” Carpenter said.

In sepsis, the inflammatory response goes into overdrive as cells release a deluge of inflammatory proteins called cytokines. Cytokines are often produced in a cascade, as one cytokine stimulates its target cells to make additional cytokines. Uncontrolled cytokine production is often called a “cytokine storm.” This process also triggers clotting, and blood clots then block the flow of oxygen-carrying blood to critical organs, leading to organ failure and death. Sepsis can also be triggered by viral infections and may be involved in severe cases of COVID-19.

“The biggest problem with sepsis is that it happens so fast, and once it gets going there are no good treatment options,” Carpenter said.

A better understanding of the role of GAPLINC in controlling the inflammatory response and septic shock could lead to new opportunities for drug development to target sepsis.

“We are a long way from understanding how you would target this therapeutically, but at least we have identified a pathway to home in on,” Carpenter said.

In addition to Carpenter and Vollmers, the coauthors of the paper include postdoctoral researcher Sergio Covarrubias; undergraduates Daisy Kuang, Aaron Shulkin, and Justin Iwuagwu; sequencing analyst Sol Katzman; assistant professor of biomolecular engineering Christopher Vollmers; and Ran Song and Edward Wakeland at the University of Texas Southwestern Medical Center. This work was funded by the National Institutes of Health. Apple Vollmers was supported by a Ford Foundation Predoctoral Fellowship and a Howard Hughes Medical Institute Gilliam Fellowship.

Featured image: GAPLINC, a highly conserved long noncoding RNA, modulates the immune response during endotoxic shock. Reducing or eliminating GAPLINC led to enhanced expression of inflammatory genes in both mouse and human cells. In a mouse model of sepsis, knockout mice without GAPLINC were protected from endotoxic shock. © Apple Vollmers

Provided by University of California Santa Cruz

No More Needles? (Engineering)

Nearly pain-free microneedle patch can test for antibodies and more in the fluid between cells.

Blood draws are no fun.

They hurt. Veins can burst, or even roll — like they’re trying to avoid the needle, too.

Oftentimes, doctors use blood samples to check for biomarkers of disease: antibodies that signal a viral or bacterial infection, such as SARS-CoV-2, the virus responsible for COVID-19, or cytokines indicative of inflammation seen in conditions such as rheumatoid arthritis and sepsis.

Engineers at the McKelvey School of Engineering at Washington University in St. Louis have developed a microneedle patch that can be applied to the skin, capture a biomarker of interest from interstitial fluid and, thanks to its unprecedented sensitivity, allow clinicians to detect its presence.(Image: Sisi Cao)

These biomarkers aren’t just in blood, though. They can also be found in the dense liquid medium that surrounds our cells, but in a low abundance that makes it difficult to be detected.

Until now.

Engineers at the McKelvey School of Engineering at Washington University in St. Louis have developed a microneedle patch that can be applied to the skin, capture a biomarker of interest and, thanks to its unprecedented sensitivity, allow clinicians to detect its presence.

The technology is low cost, easy for clinicians or patients themselves to use, and could eliminate the need for a trip to the hospital just for a blood draw.

The research, from the lab of Srikanth Singamaneni, the Lilyan & E. Lisle Hughes Professor in the Department of Mechanical Engineering & Material Sciences, was published online Jan. 22 in the journal Nature Biomedical Engineering.

In addition to the low cost and ease of use, these microneedle patches have another advantage over blood draws, perhaps the most important feature for some: “They are nearly pain-free,” Singamaneni said.

Singamaneni © WUSTL

Finding a biomarker using these microneedle patches is similar to blood testing. But instead of using a solution to find and quantify the biomarker in blood, the microneedles directly capture it from the liquid that surrounds our cells in skin, which is called dermal interstitial fluid (ISF). Once the biomarkers have been captured, they’re detected in the same way — using fluorescence to indicate their presence and quantity.

ISF is a rich source of biomolecules, densely packed with everything from neurotransmitters to cellular waste. However, to analyze biomarkers in ISF, conventional method generally requires extraction of ISF from skin. This method is difficult and usually the amount of ISF that can be obtained is not sufficient for analysis. That has been a major hurdle for developing microneedle-based biosensing technology.

Another method involves direct capture of the biomarker in ISF without having to extract ISF. Like showing up to a packed concert and trying to make your way up front, the biomarker has to maneuver through a crowded, dynamic soup of ISF before reaching the microneedle in the skin tissue. Under such conditions, being able to capture enough of the biomarker to see using the traditional assay isn’t easy.

But the team has a secret weapon of sorts: “plasmonic-fluors,” an ultrabright fluorescence nanolabel. Compared with traditional fluorescent labels, when an assay was done on a microneedle patch using plasmonic-fluor, the signal of target protein biomarkers shined about 1,400 times as bright and became detectable even when present at low concentrations.

“Previously, concentrations of a biomarker had to be on the order of a few micrograms per milliliter of fluid,” said Zheyu (Ryan) Wang, a graduate student in the Singamaneni lab and one of the lead authors of the paper. That’s far beyond the real-world physiological range. But using plasmonic-fluor, the research team was able to detect biomarkers on the order of picograms per milliliter.

“That’s orders of magnitude more sensitive,” Wang said.

These patches have a host of qualities that can make a real impact on medicine, patient care and research.

They would allow providers to monitor biomarkers over time, particularly important when it comes to understanding how immunity plays out in new diseases.

Working together

Srikanth Singamaneni, the Lilyan E. Lisle Hughes Professor in the Department of Mechanical Engineering & Materials Science, and Jai S. Rudra, assistant professor in the Department of Biomedical Engineering, worked together to look at cocaine vaccines, which work by blocking cocaine’s ability to enter the brain.

Current candidates for such a vaccine don’t confer long-lasting results; they require frequent boosting. Singamaneni and Rudra wanted a better way to determine when the effects of the vaccine had waned. “We’ve shown that we can use the patches to understand whether a person is still producing the necessary antibodies,” Singamaneni said. “No blood draw necessary.”

For example, researchers working on COVID-19 vaccines need to know if people are producing the right antibodies and for how long. “Let’s put a patch on,” Singamaneni said, “and let’s see whether the person has antibodies against COVID-19 and at what level.”

Or, in an emergency, “When someone complains of chest pain and they are being taken to the hospital in an ambulance, we’re hoping right then and there, the patch can be applied,” said Jingyi Luan, a student who recently graduated from the Singamaneni lab and one of the lead authors of the paper. Instead of having to get to the hospital and have blood drawn, EMTs could use a microneedle patch to test for troponin, the biomarker that indicates myocardial infarction.

For people with chronic conditions that require regular monitoring, microneedle patches could eliminate unnecessary trips to the hospital, saving money, time and discomfort — a lot of discomfort.

The patches are almost pain-free. “They go about 400 microns deep into the dermal tissue,” Singamaneni said. “They don’t even touch sensory nerves.”

In the lab, using this technology could limit the number of animals needed for research. Sometimes research necessitates several measurements in succession to capture the ebb and flow of biomarkers — for example, to monitor the progression of sepsis. Sometimes, that means a lot of small animals.

“We could significantly lower the number of animals required for such studies,” Singamaneni said.

We don’t have to do all of this ourselves

Singamaneni and Erica L. Scheller, assistant professor of Medicine in the Division of Bone and Mineral Disease at the School of Medicine, worked together to investigate the concentration of biomarkers in local tissues. Current approaches for such evaluation require the isolation of local tissues and do not allow successive and continuous inspection. Singamaneni and Scheller are developing a better platform to achieve long term monitoring of local biomarker concentration.

The implications are vast — and Singamaneni’s lab wants to make sure they are all explored.

There is a lot of work to do, he said: “We’ll have to determine clinical cutoffs,” that is, the range of biomarker in ISF that corresponds to a normal vs. abnormal level. “We’ll have to determine what levels of biomarker are normal, what levels are pathological.” And his research group is working on delivery methods for long distances and harsh conditions, providing options for improving rural healthcare.

“But we don’t have to do all of this ourselves,” Singamaneni said. Instead, the technology will be available to experts in different areas of medicine.

“We have created a platform technology that anyone can use,” he said. “And they can use it to find their own biomarker of interest.”

This research was supported by the National Science Foundation (CBET-1900277), and the National Institutes of Health (R01DE027098, R56DE027924, R01CA141521, R21DA036663, R21CA236652).

Reference: Wang, Z., Luan, J., Seth, A. et al. Microneedle patch for the ultrasensitive quantification of protein biomarkers in interstitial fluid. Nat Biomed Eng 5, 64–76 (2021). https://doi.org/10.1038/s41551-020-00672-y

Provided by Washington University in St Louis

About McKelvey School of Engineering at Washington University in St. Louis

The McKelvey School of Engineering at Washington University in St. Louis promotes independent inquiry and education with an emphasis on scientific excellence, innovation and collaboration without boundaries. McKelvey Engineering has top-ranked research and graduate programs across departments, particularly in biomedical engineering, environmental engineering and computing, and has one of the most selective undergraduate programs in the country. With 140 full-time faculty, 1,387 undergraduate students, 1,448 graduate students and 21,000 living alumni, we are working to solve some of society’s greatest challenges; to prepare students to become leaders and innovate throughout their careers; and to be a catalyst of economic development for the St. Louis region and beyond.

Severe Sepsis Predicted By Common Protein

Sugar-binding protein galectin-1 could be a biomarker for patients at risk of life threatening sepsis.

A sugar-binding protein could fuel terrible inflammation and worsen sepsis, a disease that kills more than 270,000 people every year in the US alone, reports a team of researchers led by UConn Health in the 4 January issue of Nature Immunology.

Sepsis is caused mostly by bacterial infections. The immune system runs out of controls and triggers a cytokine storm, a condition in which inflammation-causing proteins flood the blood. Organs may break down, and death often follows.

Other diseases can also cause cytokine storms; medical historians believe cytokine storms were behind the lethality of the 1918-1919 flu epidemic, as well as the Black Death. Cytokine storms are also observed in patients with severe COVID-19 and believed to be involved in death in COVID-19.

A main trigger for the cytokine storms during sepsis is the overreaction of the body when it detects an infection inside the cells. When a cell detects bacteria or pieces of bacteria inside itself, it immediately activates enzymes that in turn activate a protein that pokes holes on the cell membrane from within, eventually causing the cell to burst open and spill cytokines into the bloodstream. Cytokines are alarm signals, calling in the immune system to fight the bacteria. Cytokines also make other cells more likely to burst open and sound the alarm. Usually, the system damps itself after a while and calms down, but in sepsis it spins out of control, causing more and more cells to burst and die and release even more cytokines into the bloodstream.

When cells burst open, they release not only cytokines, but also other danger molecules called alarmins that alarm the body of an infection or injury and can amplify the ongoing cytokine storm.

UConn Health immunologist Vijay Rathinam wanted to know which alarmins were released when a cell detected a specific kind of bacterial molecule called lipopolysaccharide inside itself. Dr. Ashley Russo, then a graduate student in the Rathinam lab, catalogued–in collaboration with immunologists Tony Vella and Antoine Menoret at UConn Health–proteins released by these cells when they detected lipopolysaccharide.

And they found something exciting. Galectin-1, a protein that binds sugars and sugar-coated proteins, seemed to be emanating from the cells. Interestingly, they found that galectin-1 is small enough to be slipping out of the holes poked in the cells’ membrane, even before the cells burst open.

Once they noticed that, they began to look at the role galectin-1 played in sepsis. They found that galectin-1 seemed to be suppressing a brake on inflammation, causing the cytokine storm to ramp up. They also found that mice lacking galectin-1 had less inflammation, less organ damage, and survived longer than normal mice did during sepsis resulting from a bacterial infection and lipopolysaccharide.

To find out if galectin-1 is released during sepsis in human patients, the team collaborated with the Jena University Hospital’s Drs. Deshmukh, Bauer, and Sponholz and found that sepsis patients had higher levels of galectin-1 than other non-sepsis patients in critical care and healthy people.

The team is considering whether galectin-1 might be a good drug target to help dampen cytokine storms during sepsis, as well as a useful marker doctors could use to identify critical ill patients at risk.

This study was made possible by key additional collaborations with the laboratories of Dr. Gabriel Rabinovich of the Laboratorio de Inmunopatología, Instituto de Biología y Medicina Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas in Buenos Aires, Argentina, Drs. Beiyan Zhou, Sivapriya Kailasan Vanaja, and Jianbin Ruan of UConn Health, and Dr. Greg Hudalla of University of Florida.

This project was funded by grants from the National Institutes of Health to Dr. Rathinam.

Reference: http://dx.doi.org/10.1038/s41590-020-00844-7

Provided by University of Connecticut

New Drug to Combat Global Killer Sepsis (Medicine)

A promising new drug to combat sepsis has been developed by researchers at The Australian National University (ANU), potentially saving millions of lives each year.

ANU Professor Christopher Parish and his team have been working on the drug for more than 10 years, with the drug being developed from compounds originally designed to fight cancer.

Fig. 1: Polyanions inhibit histone-mediated endothelial cell cytotoxicity and RBC fragility with minimal structural requirements for the activity identified.

There are some 11 million sepsis-related deaths worldwide each year, according to The World Health Organization (WHO) – accounting for almost 20 percent of all global deaths.

“There is a huge medical need for a treatment for sepsis. It is surprising how many people die from sepsis and the medical profession hasn’t yet found a treatment,” Professor Parish said.

“We hope that we can now treat the untreatable. This drug is very safe, and we are very excited about its potential use against sepsis.”

Sepsis is a dangerous, often fatal response, to many infections including COVID-19.

“Sepsis occurs when pathogens—usually bacteria but sometimes viruses—get out of control and the immune system tries to control them but overdoes the job and causes massive collateral damage,” Professor Parish said.

“Our drug stops patients from feeling very sick due to this uncontrolled collateral damage, a process that can result in multi-organ failure and death.”

The drug, initially developed on-site at ANU was further developed in collaboration with Director and Principal Research Leader Professor Mark von Itzstein AO and researcher Dr. Chih-Wei Chang from Griffith University’s Institute for Glycomics.

It has now passed phase one clinical trials in healthy volunteers and is undergoing another phase one trial in sepsis patients.

The development of the new drug was made possible after a discovery made by US researchers about a decade ago. They found a prominent family of proteins that interacts with DNA, called histones, are the cause of death by sepsis.

“Normally histones are harmless, packaging DNA within cells, but outside cells they can be very toxic,” Professor Parish said.

“In fact, some white cells extrude their histones in net-like structures that entrap pathogens and, via their histones, are very toxic for pathogens.”

Histones are toxic because they can bind to the surface of cells and puncture holes in them—but they are unable to discriminate between pathogens and their own cells.

Professor Parish’s team has been working on a drug which neutralizes histones.

“The drug binds to histones and stops them from punching holes in cell membranes.”

The researchers say their new drug could help some people with COVID-19—particularly those patients with sepsis-like symptoms in their lungs.

“We have spent 10 years developing this drug to treat sepsis and although not planned, we predict it may have some activity against COVID-19,” Professor Parish said.

“But sepsis will be here long after we control COVID-19—because it can come in all shapes and forms and a lot of pathogens can induce it.

“If this drug works as predicted, it should be a game changer in treating sepsis—one of the biggest challenges in 21st century medicine.”

Research on the compound was published in Nature Communications Thursday 17 December 2020.

References: Meara, C.H.O., Coupland, L.A., Kordbacheh, F. et al. Neutralizing the pathological effects of extracellular histones with small polyanions. Nat Commun 11, 6408 (2020). https://www.nature.com/articles/s41467-020-20231-y https://doi.org/10.1038/s41467-020-20231-y

Provided by Australian National University

How To Stop Infections Caused by Carbapenemase-producing Bacteria (Medicine)

Researchers from the Institute of Biomedicine of Seville have published the results of a preclinical study done in collaboration with the University of Fribourg (Switzerland).

In 2017, the World Health Organization published a list of pathogens for which new drugs are urgently needed. Acinetobacter baumannii was ranked in the critical priority group along with other Gram-negative bacteria such as Pseudomonas aeruginosa and carbapenemase-producing Enterobacteria. Specifically, A. baumannii is responsible for more than 10% of hospital infections, often severe, such as pneumonia linked to mechanical ventilation, and bacteremias, especially in intensive care units.

The ‘Bacterial and Antimicrobial Resistance’ group at the Institute of Biomedicine of Seville, led by Dr. José Miguel Cisneros, has published the results of a collaborative preclinical study focused on this specific pathogen. The study was conducted together with the ‘Emerging Antibiotic Resistances’ group, headed by Prof. Patrice Nordmann from the University of Fribourg, Switzerland.

As part of a line of research looking for effective new antimicrobial treatments against infections by carbapenemase-producing bacteria, and based on the results published in 2019 on the in vitro activity of combinations of two carbapenems against clinical strains of carbapenemase-producing A. baumannii, not clonally related, the group launched a study to evaluate in vivo the efficacy of imipenem plus meropenem in an experimental murine model of sepsis caused by clinical isolates of carbapenemase-producing A. baumannii. The results of this study show that the combination of imipenem plus meropenem could be effective in the treatment of infections caused by strains of carbapenemase-producing A. baumannii (OXA-23 and OXA-58).

References: T Cebrero-Cangueiro, P Nordmann, M Carretero-Ledesma, J Pachón, M E Pachón-Ibáñez, Efficacy of dual carbapenem treatment in a murine sepsis model of infection due to carbapenemase-producing Acinetobacter baumannii, Journal of Antimicrobial Chemotherapy, , dkaa487, https://doi.org/10.1093/jac/dkaa487

Provided by University of Seville

Reusing Face Masks: Are Microwaves The Answer? (Medicine)

Researchers from Cardiff University have been testing the feasibility of using microwave ovens and dry heat to decontaminate crucial PPE being used to combat the coronavirus pandemic.

Credit: Cardiff University

Reporting their findings in the Journal of Hospital Infection, the team have shown that certain types of respirators can be effectively decontaminated in just 90 seconds using an industrial-grade microwave oven and a baby bottle sterilizer containing water.

It has been widely reported that access to respirators and surgical face masks has become restricted in many facilities over the course of the pandemic.

“Being unable to access adequate PPE puts frontline workers and patients at risk of contracting coronavirus. Whilst masks are usually considered to be single use items, we wanted to find out whether they could be safely disinfected and used again,” said co-author of the study Prof Jean-Yves Maillard, from the School of Pharmacy and Pharmaceutical Sciences.

The researchers believe microwave decontamination could be used in emergency situations to address supply issues and dramatically increase the number of respirators available to frontline staff.

In the study, respirators were exposed to three microwave disinfection cycles and were shown to retain their ability to filter bacteria and viral-sized aerosols. However, the researchers reported that microwaving surgical masks led to a complete loss of their aerosol filtering capacity.

Michael Pascoe, co-author of the study from the School of Pharmacy and Pharmaceutical Sciences, said: “Surgical masks are known to lose effectiveness once they become moist—we suspected that microwave disinfection would lead to a similar loss in their ability to filter aerosols and this was confirmed by our lab observations.”

The team, which also includes academics from the School of Engineering, also investigated using dry heat ovens as an alternative approach. Dry heat sterilization does not involve any water and so is compatible with items which are damaged by moisture.

Exposure to 70°C dry heat for 90 minutes was effective at decontaminating both surgical masks and respirators. After three dry heat cycles, both types of mask retained their aerosol filtering properties.

It is essential that PPE is effectively decontaminated between uses. Whilst microwave-generated steam and dry heat have both been shown to effectively kill coronaviruses, the researchers wanted to ensure that this method was also effective against bacteria encountered in healthcare environments.

In the study, respirators and surgical masks were purposely contaminated with Staphylococcus aureus, a bacterial species highly prevalent in human airways which can cause soft tissue infections and sepsis. Staphylococcus aureus is also the accepted biological indicator to test the integrity of a mask.

Both methods effectively reduced the number of bacteria on masks to a safe level.

As a result of the study, the team have developed a protocol to determine which types of PPE would be suitable for different treatments with dry heat incubators or microwave ovens.

“Mask and respirator models vary considerably and so it is important to ensure the method of decontamination does not compromise their function.”

The team warns against members of the public using a similar approach at home. Professor Adrian Porch, from the School of Engineering, said: “Domestic microwave ovens typically have much lower power, around 800 W, and use rotating turntables rather than a rotating antenna. Significantly longer exposure times would be needed to achieve similar results and it is unknown how this would affect the functioning of the mask. Masks which contain thin wires can even catch fire when placed in a microwave.”

Reference: M.J. Pascoe et al. Dry heat and microwave-generated steam protocols for the rapid decontamination of respiratory personal protective equipment in response to COVID-19-related shortages, Journal of Hospital Infection (2020). DOI: 10.1016/j.jhin.2020.07.008

Provided by Cardiff University