Biomarker May Predict Early Alzheimer’s Disease

Researchers at Sanford Burnham Prebys Medical Discovery Institute (SBP) have identified a peptide that could lead to the early detection of Alzheimer’s disease (AD). The discovery, published in Nature Communications, may also provide a means of homing drugs to diseased areas of the brain to treat AD, Parkinson’s disease, as well as glioblastoma, brain injuries and stroke.

“Our goal was to find a new biomarker for AD,” says Aman Mann, Ph.D., research assistant professor at SBP who shares the lead authorship of the study with Pablo Scodeller, Ph.D., a postdoctoral researcher at SBP. “We have identified a peptide (DAG) that recognizes a protein that is elevated in the brain blood vessels of AD mice and human patients. The DAG target, connective tissue growth factor (CTGF) appears in the AD brain before amyloid plaques, the pathological hallmark of AD.”

“CTGF is a protein that is made in the brain in response to inflammation and tissue repair,” explains Mann. “Our finding that connects elevated levels of CTGF with AD is consistent with the growing body of evidence suggesting that inflammation plays an important role in the development of AD.”

The research team identified the DAG peptide using in vivo phage display screening at different stages of AD development in a mouse model. In young AD mice, DAG detected the earliest stage of the disease. If the early appearance of the DAG target holds true in humans, it would mean that DAG could be used as a tool to identify patients at early, pre-symptomatic stages of the disease when treatments already available may still be effective.

“Importantly, we showed that DAG binds to cells and brain from AD human patients in a CTGF-dependent manner” says Mann. “This is consistent with an earlier report of high CTGF expression in the brains of AD patients.”

“Our findings show that endothelial cells, the cells that form the inner lining of blood vessels, bind our DAG peptide in the parts of the mouse brain affected by the disease,” says Erkki Ruoslahti, M.D., Ph.D., distinguished professor at SBP and senior author of the paper. “This is very significant because the endothelial cells are readily accessible for probes injected into the blood stream, whereas other types of cells in the brain are behind a protective wall called the blood-brain barrier. The change in AD blood vessels gives us an opportunity to create a diagnostic method that can detect AD at the earliest stage possible.

“But first we need to develop an imaging platform for the technology, using MRI or PET scans to differentiate live AD mice from normal mice. Once that’s done successfully, we can focus on humans,” adds Ruoslahti.

“As our research progresses we also foresee CTGF as a potential therapeutic target that is unrelated to amyloid beta (Aβ), the toxic protein that creates brain plaques,” says Ruoslahti. “Given the number of failed clinical studies that have sought to treat AD patients by targeting Aβ, it’s clear that treatments will need to be given earlier—before amyloid plaques appear—or have to target entirely different pathways.

“DAG has the potential to fill both roles—identifying at risk individuals prior to overt signs of AD and targeted delivery of drugs to diseased areas of the brain. Perhaps CTGF itself can be a drug target in AD and other brain disorders linked to inflammation. We’ll just have to learn more about its role in these diseases”.

Rare Type of Immune Cell Responsible for Progression of Heart Inflammation to Heart Failure in Mice

A new study in mice reveals that eosinophils, a type of disease-fighting white blood cell, appear to be at least partly responsible for the progression of heart muscle inflammation to heart failure in mice.

In a report on the findings, published in The Journal of Experimental Medicine on March 16, researchers found that while eosinophils are not required for heart inflammation to occur, they are needed for it to progress to a condition known as inflammatory dilated cardiomyopathy (DCMi) in mice. The discovery, they say, advances information about the impact of eosinophils on heart function.

“Other studies have shown that people with high levels of eosinophils develop a number of heart diseases. This new work has provided more details about how these immune system cells may lead to deterioration of heart muscle function in mice in a way that lets us draw some parallels to human disease processes,” says Daniela Cihakova, M.D., Ph.D., associate professor of pathology at the Johns Hopkins University School of Medicine and the paper’s senior author.

Heart inflammation, or myocarditis, is rarely diagnosed because it doesn’t always cause severe symptoms and it requires a biopsy to be taken from the patient’s heart. This makes it difficult to study the outcomes of the disease. “We don’t understand why the hearts of some people will heal while those of others develop chronic disease,” says Cihakova.

Different types of myocarditis are distinguished based on the type of immune cell that predominates the inflammation of the heart. For example, in eosinophilic myocarditis, numerous eosinophils infiltrate the heart. It is not known if some types of myocarditis are more likely to progress to DCMi than others. “Our studies show that the presence of eosinophils in the heart makes mice more likely to get DCMi following myocarditis. And if there are a lot of eosinophils, the mice develop even more severe heart failure,” says Nicola Diny, a Ph.D. student in the Bloomberg School of Public Health and the study’s first author. “It will be important to test if the same is true in patients. That way, we may be able to intervene early and prevent DCMi.”

This study, says Cihakova, is the first to examine the role eosinophils play in the development and severity of heart inflammation, and the subsequent progression of inflammation to DCMi. The study addresses a National Institutes of Health-identified need for preclinical models and a clearer understanding of how eosinophils drive heart damage.

For the study, Cihakova and her team first induced myocarditis in two groups of mice: normal mice and a group of mice genetically modified to be eosinophil-deficient. Myocarditis was induced through a process called experimental autoimmune myocarditis, in which mice are immunized with a peptide from heart muscle cells to initiate an immune response against the heart. After 21 days, the researchers found similar levels of acute inflammation in the hearts of both groups by studying the hearts’ tissue. But when the team checked the mice’s hearts later on for evidence of heart failure, the differences between the eosinophil-deficient and the normal mice were striking. The normal mice developed heart failure, while the eosinophil-deficient mice showed no signs of reduced heart function.

“These surprising results told us that it is not the overall severity of inflammation but rather the types of immune cells in the heart that decide whether myocarditis develops into heart failure,” says Diny.

The researchers also examined the hearts for fibrosis, or scar tissue, which develops when mammalian (including human) heart muscles die. This type of scar tissue is also found in DCMi. Although both groups of mice had similar degrees of scar tissue, the eosinophil-deficient mice’s heart functions weren’t negatively affected, while the normal mice developed DCMi.

“This told us that in the absence of eosinophils, heart function can be preserved despite scar tissue formation,” Cihakova says. “It’s also important to note that although eosinophils accounted for just 1 to 3 percent of all heart-infiltrating cells in normal mice, this small percentage can still drive heart failure.”

In another set of experiments, the research team used genetically modified mice, called IL5Tg mice, which have an excess of the protein IL5 that causes the body to make eosinophils. The IL5Tg mice had more inflammation in the atria, or upper chambers of the heart, compared to normal mice in the acute stage and more atrial scar tissue in the chronic stage. IL5Tg mice also had more heart-infiltrating cells in general. Eosinophils accounted for more than 60 percent of heart-infiltrating cells in the IL5Tg mice’s hearts, compared to only 3 percent in normal mice. When the team examined the heart function some 45 days after the start of the experiment, the IL5Tg mice had developed severe DCMi.

To examine whether humans with eosinophil-driven myocarditis also developed inflammation in the atria, the researchers obtained heart tissue samples and cardiac MRI scans from three patients seen at The Johns Hopkins Hospital, all of whom had confirmed eosinophil-driven inflammation.

The images showed that two patients had either inflammation or scar tissue in the atria, which suggests that atrial inflammation and/or scar tissue may also be a feature in humans with eosinophil-driven inflammation, Cihakova says.

To determine whether the IL5 protein is necessary for DCMi development, the research team next examined IL5-deficient mice. The scientists found that they had both inflammation and DCMi severity similar to that of normal mice, suggesting that the IL5 protein is not necessary for DCMi to develop.

Finally, to confirm the differences between the effects of IL5 and eosinophils, the team bred the eosinophil-deficient mice to have excess IL5. Compared to normal mice, these mice showed no decrease in heart function and appeared completely protected from DCMi, which confirms that it is the eosinophils themselves, not high levels of IL5, that are responsible for DCMi development, the investigators say.

To learn more about how eosinophils might drive DCMi progression, the investigators built on the knowledge that eosinophils harbor granules, some of which can kill cells, while others change the function of cells.

“We didn’t see any differences in cell death between the normal mice and those with or without too many eosinophils, so we became interested in the molecules that can change the function of other cells,” says Diny.

In particular, one protein, called IL4, caught the researchers’ attention. Other studies had shown that IL4 made by eosinophils has diverse functions in liver repair and fat tissue. “We wondered if this protein from eosinophils may also be important in the heart,” Cihakova says.

First, the research team used a mouse in which cells that make IL4 turned fluorescent green, thereby allowing researchers to tell where IL4 is made. The team found that eosinophils accounted for the majority of IL4-producing cells. When they used mice that lacked IL4 in all cells, these mice were completely protected from DCMi, just like the eosinophil-deficient mice.

Finally, to determine whether IL4 specifically from eosinophils is necessary for DCMi development, the team used genetically modified mice with no IL4 in their eosinophils but with IL4 in other heart-infiltrating cells. These mice developed less severe DCMi compared to normal mice, which confirms that eosinophils are responsible for DCMi development through IL4.

“The take-home message is that inflammation severity doesn’t necessarily determine long-term disease progression, but specific infiltrating cell types — eosinophils, in this case — do,” says Cihakova. Because eosinophil-driven inflammation is so clinically rare, the percentage of people who develop DCMi is unknown, she notes.

While no drugs are currently available to stop or delay the development of DCMi, the researchers hope their findings will help establish a novel target for IL4-blocking medicines that might be used to treat people with myocarditis, possibly preventing disease progression and the need for heart transplantation.

Flesh-Eating Infections In Rheumatoid Arthritis Patients Spur New Discovery

Rheumatoid arthritis patients taking medications that inhibit interleukin-1beta (IL-1beta), a molecule that stimulates the immune system, are 300 times more likely to experience invasive Group A Streptococcal infections than patients not on the drug, according to University of California San Diego School of Medicine researchers. Their study, published August 19 in Science Immunology, also uncovers a critical new role for IL-1beta as the body’s independent early warning system for bacterial infections.

“The more we know about each step in the body’s immune response to bacterial infections, the better equipped we are to design more personalized, targeted therapies for autoimmune diseases — therapies that are effective, but minimize risk of infection,” said senior author Victor Nizet, MD, professor of pediatrics and pharmacy at UC San Diego School of Medicine and Skaggs School of Pharmacy and Pharmaceutical Sciences.

IL-1beta is a molecule that stimulates an immune response, calling white blood cells to the site of an infection so they can engulf and clear away invading pathogens. The body first produces the molecule in a longer, inactive form that must be cleaved to be activated. The scientific community long believed that only the body itself could cleave and activate IL-1beta, by employing a cellular structure known as the inflammasome. But in experiments using cell cultures and mouse models of infection, Nizet and team found that SpeB, an enzyme secreted by strep bacteria, also cleaves and activates IL-1beta, triggering a protective immune response.

“This finding may explain why some of the more invasive, flesh-eating strep strains have a genetic mutation that blocks SpeB production — it helps them avoid tripping the alarm and setting off an immune response,” said first author Christopher LaRock, PhD, a postdoctoral researcher in Nizet’s lab.

The researchers hypothesize that for less invasive strains, like those that cause strep throat, producing SpeB and activating IL-1beta might be advantageous — the resulting immune response may wipe out competing bacteria and help strep establish a foothold in the body.

While the human immune system can quickly recognize and respond to bacterial infections, sometimes this reaction can go overboard, leading to autoimmune diseases such as rheumatoid arthritis. In this case, a person’s own immune system attacks “self” proteins instead of just foreign invaders.

In their efforts to investigate IL-1beta function, Nizet, LaRock and team analyzed a U.S. Food and Drug Administration (FDA) database on adverse events in rheumatoid patients who took anakinra, a drug that dampens autoimmunity by inhibiting IL-1beta. They found that patients receiving anakinra were more than 300 times more likely to experience invasive, flesh-eating strep infections than patients not taking the drug.

“A likely explanation for this increased risk is that with IL-1beta out of the picture, as is the case with patients taking anakinra, strep strains can progress to invasive infection even while producing SpeB, which goes unnoticed by the immune system,” LaRock said.

This finding underscores IL-1beta’s importance as an early warning system that’s triggered not only by the host, but also directly by bacterial enzymes, essentially “taking out the middle man,” Nizet said. The UC San Diego researchers believe this capacity for direct pathogen detection represents IL-1beta’s original role in immunity, going all the way back in evolution to simpler animals, such as fish.

“Inhibiting the body’s bacterial sensor can put a person at risk for invasive infection,” Nizet said, “but just the fact that we now know that this patient population is at higher risk and why means we can take simple steps — such as close monitoring and prophylactic antibiotics — to prevent it from happening. ”

Researchers Discover Potential Treatment for Sepsis and Other Uncontrollable Responses to Infection

Researchers at the Icahn School of Medicine at Mount Sinai say that tiny doses of a cancer drug may stop the raging, uncontrollable immune response to infection that leads to sepsis and kills up to 500,000 people a year in the U.S. The new drug treatment may also benefit millions of people worldwide who are affected by infections and pandemics.

Their study reported in Science, demonstrates in both cells and animals that a small dose of topoisomerase I (Top 1) inhibitor can dampen an acute inflammatory reaction to infection while still allowing the body’s protective defense to take place. The title of the study is “Topoisomerase 1 inhibition suppresses the transcriptional activation of innate immune responses and protects against inflammation-induced death.”

The treatment may help control not only sepsis — deadly infections often acquired in hospital by patients with a weak immune system — but also new and brutal assaults on human immunity such as novel influenza strains and pandemics of Ebola and other singular infections, says the study’s senior investigator, Ivan Marazzi, PhD, an Assistant Professor of Microbiology at the Icahn School of Medicine at Mount Sinai.

“Our results suggest that a therapy based on Top 1 inhibition could save millions of people affected by sepsis, pandemics, and many congenital deficiencies associated with acute inflammatory episodes — what is known as a cytokine, or inflammatory, storm,” says Marazzi.

“These storms occur because the body does not know how to adjust the appropriate level of inflammation that is good enough to suppress an infection but doesn’t harm the body itself,” he says. “This drug appears to offer that life-saving correction.”

Sepsis is caused by an excessive host response to infection, which in turn leads to multiple organ failure and death. With an overall mortality rate between 20 and 50%, sepsis is the tenth leading cause of death in the U.S. — it kills more people than do HIV and breast cancer.

“To date there has been no targeted treatment for sepsis, or for other infections that promote this inflammatory storm,” says Dr. Marazzi. “Such treatment is desperately needed.”

For example, sepsis is a leading cause of death in infants and children, he says. “Septic shock and lung destruction can occur when a child is suffering from a pneumonia caused by co-infection with a virus and a bacteria even when antibiotic therapy is being used. The elderly are also especially vulnerable to sepsis.”

Following a challenge from the National Institutes of Health to repurpose existing drugs for new uses, the research team used a simple cellular screen to find candidate drugs that could tamp down rampant inflammation.

They discovered that the Top 1 inhibitor class of cancer drugs — four have been previously approved for a variety of cancers — also blocks a set of genes that are activated immediately by immune cells to combat an infection. “These genes are the ones that have the strongest inflammatory effects,” says Marazzi.

The Mount Sinai team found that use of one to three doses of a Top 1 inhibitor that is 1/50th the strength of normal chemotherapy was enough to rescue 70-90 % of mice from an inflammatory storm death due to either acute bacterial infection, liver failure, or virus-bacteria co-infection. The treatment did not produce overt side effects.

They also tested the inhibitor in cells infected with influenza, Ebola, and other viral and bacterial microbes that over-stimulate the immune system, and found the drug blunted a dangerous immune reaction.

“We observed a striking effect of Top-1 inhibitors on expression of pro-inflammatory molecules induced by Ebola virus infection. This study contributes our understanding of pathogenesis of Ebola virus disease and also suggests a direction to develop treatments,” says Alexander Bukreyev, PhD, Professor in the Department of Pathology and Microbiology & Immunology at the Galveston National Laboratory at the University of Texas Medical Branch.

“Finding remedies for these infection-induced inflammatory storms is a global focus, and we look forward to testing the ability of Top-1 inhibitors to save lives,” adds Marazzi.