USF research teams led by Dr. Yu Chen and Dr. Xingmin Sun describe ways to control the No. 1 hospital-acquired bacterial infection in a paper published in the journal Nature Communications.

The ironic joke goes that if you want to get sick, stay in a hospital. That’s because hospitals can harbor germs that take advantage of a patient’s weakened state, complicating the illness that brought them there in the first place.

But health officials have an arsenal to keep people safe, including cephalosporins, strong antibiotics that fight bacteria such as staphylococcus and streptococcus. Cephalosporins are often used against skin, soft tissue and surgery related infections.

Dr. Yu Chen

However, treatment with β-lactam antibiotics – particularly cephalosporins – is a major risk factor for the virulent Clostridioides difficile infection (CDI), which attacks the large intestine and can cause diarrhea and life-threatening colitis.

These complications are explained in a recent paper published in the journal Nature Communications by teams that includes senior author Dr. Yu Chen, professor in the Department of Molecular Medicine in the USF Health Morsani College of Medicine, and co-corresponding author Dr. Xingmin Sun, associate professor in the Department of Molecular Medicine in the Morsani College of Medicine. Several other USF research teams, led by Rays Jiang, PhD, Prahathees Eswara, PhD, and Ioannis Gelis, PhD, also contributed to the study.

“When you give a person an antibiotic to treat a disease, one of the consequences is the antibiotic can wipe out a lot of the good bacteria in the gut,’’ Dr. Chen said. “But in this case, C. difficile is resistant to cephalosporins, so it creates a high-risk factor. And if people are under prolonged antibiotic treatment, they are at an even higher risk for CDI.’’

Cephalosporin resistance in CDI is well documented, but the underlying mechanism has, until this point, remained unclear. The USF Health team used a combination of experimental techniques to characterize the molecular basis of cephalosporin resistance in CDI, which is the No. 1 hospital-acquired bacterial infection in the United States, according to the Centers for Disease Control and Prevention.

Initially, antibiotics are administered for an unrelated infection or prophylaxis, causing the gut flora diversity to diminish. Without competition from the good bacteria in the large intestine, CDI can easily proliferate, secreting toxins that cause cell death.

“The primary risk factor for CDI are broad-spectrum antibiotics, specifically those with weak activity against C. difficile and strong activity against other gut bacteria,’’ the authors state.

These broad-spectrum antibiotics irreversibly inhibit a bacterium’s penicillin-binding proteins (PBPs), which are enzymes that assemble in the bacterial cell wall. These proteins are critical not only for the growth of C. difficile, but also to produce its spores, which are resistant to harsh environmental conditions and contribute to the high recurrent rates of CDI. The challenge for researchers is that, prior to the Nature Communications report, there was little information about the PBPs of C. difficile.

As a common hospital-acquired infection, the pathogenesis of CDI is well-understood. It causes about 500,000 infections each year in the United States, and one in about 10 people over 65 with the infection die within a month, according to the CDC.

“We want to know more about C. difficile resistance so it (data) can be used to create new therapies for the future,’’ Chen said. “This research will help us understand more about certain drugs that are risks factors for infection.’’

The researchers emphasized two key findings in the journal report. First, by elucidating the three-dimensional structures of key PBPs from C. difficile and how they interact with beta-lactam antibiotics, the USF Health teams showed that cephalosporins do not have strong inhibitory activity against the PBPs essential for C. difficile growth and are thus unable to kill the bacterium.

Second, they also found that many of these proteins require zinc to be functional, partly explaining why dietary zinc is also a risk factor for CDI. Furthermore, the results can be used to develop new inhibitors of these PBPs to kill C. difficile and eliminate its spores. Such compounds can be developed into new antibiotics to treat CDI.

CDI can affect anyone, and symptoms often are painful and life threatening. Risk factors include:

• Being 65 or older
• Recent stay at a hospital or nursing home
• A weakened immune system, such as people with HIV/AIDS, cancer, or organ transplant patients taking immunosuppressive drugs
• Previous infection with CDI or known exposure to the germs

For more information, visit https://www.cdc.gov/cdiff/risk.html

The journal Nature Communications is an open access, multidisciplinary journal dedicated to publishing high-quality research in all areas of the biological, health, physical, chemical and Earth sciences. Papers published by the journal aim to represent important advances of significance to specialists within each field.

Story by Kurt Loft

USF Health Heart Institute scientists developed nanoparticles deployed to suppress inflammation in the bowels of anemic infant mice, preventing transfusion-associated intestinal injury

Babies born prematurely who are severely anemic can develop necrotizing enterocolitis, or NEC, within 48 hours after receiving a red blood cell transfusion.

Physicians have long suspected that red blood cell transfusions given to premature infants with anemia may put them in danger of developing necrotizing enterocolitis, or NEC, a potentially lethal inflammatory disease of the intestines. However, solid evidence for the connection has been difficult to obtain in part because of the lack of a practical animal model able to accurately represent what physically occurs when a baby gets NEC.

Now, a research team led by Johns Hopkins Medicine, including USF Health Morsani College of Medicine collaborators, describes the development of a new model — using infant mice, or pups, that are first made anemic and then given blood transfusions from neonates of a different mouse strain. The method, the researchers say, mimics what happens when blood transfusions are given to human babies from a non-related donor.

“We needed a working live mouse model in order to learn if a blood transfusion alone leads to NEC or does it only happen if the transfusion is given when anemia is present,” says Akhil Maheshwari, MD, professor of pediatrics at the Johns Hopkins University School of Medicine, director of neonatology at Johns Hopkins Children’s Center, and senior author of the research paper.

A description of the mouse model, along with significant findings and potential benefits from its first uses, is published in a new paper in the journal Nature Communications.

Maheshwari, a professor of pediatrics at USF Health before joining Johns Hopkins Medicine (Baltimore) in 2018, conducted much of the research published in the paper while he was at the USF Health and Tampa General Hospital. Nanoparticles designed by USF Health coauthors Samuel Wickline, MD, and Hua Pan, PhD, were employed to test whether immune cells (macrophages) in the intestines of the anemic infant mice must undergo inflammatory activation to cause bowel injury.

More common in severely anemic preemies after transfusion

Seen in approximately 10% to 12% of infants weighing less than 3.5 pounds at birth, NEC is a rapidly progressing gastrointestinal emergency in which bacteria invade the wall of the colon, causing inflammation that can ultimately destroy healthy tissue at the site. If enough cells are necrotized (killed) that a hole results in the intestinal wall, fecal material can enter the bloodstream and cause life-threatening sepsis.

Since 2004, Maheshwari says, research studies have repeatedly shown that babies born prematurely who are severely anemic — those with a proportion of red blood cells to total blood volume between 20% and 24% at birth — can develop NEC within 48 hours after receiving a red blood cell transfusion. By comparison, the American Academy of Pediatrics says that babies delivered at term normally have red blood cell volumes between 42% and 65%, dropping to between 31% and 41% at age 1.

In search of a useful and practical mouse model, Maheshwari and his colleagues had to overcome a size problem. “Newborn mouse pups are about the size of a quarter and weigh less than an ounce, so it’s extremely difficult to remove enough blood from them for laboratories to analyze,” Maheshwari says.

A new working mouse model was used to investigate whether blood transfusions alone leads to NEC, or if the potentially deadly inflammatory disease only occurs only when tranfusions are administered in the presence of anemia.

To get past that obstacle, a private medical diagnostic equipment company donated the use of its advanced blood analysis system that only requires a 5 microliter (5 millionths of a liter) sample instead of the 50 microliters — 60% of a mouse pup’s total blood supply — that most testing labs require.

Next, the researchers designed a procedure to induce severe anemia in the pups by removing about half of their blood volume every other day for 10 days after birth. This dropped their red blood cell counts to levels approximating those in severely anemic newborn babies.

Seven days after birth, the researchers introduced bacteria that had been isolated and cultured from a premature infant with NEC. Finally, red blood cell transfusions were given on the 11th day after birth.

Over the next 48 hours, the researchers looked for development of NEC-like symptoms in their experimental group and three other sets of mouse pups: (1) a control group without any intervention, (2) a group without anemia that received transfusions and (3) a group with anemia but not transfused.

“Only the severely anemic pups who received blood transfusions showed intestinal damage that resembled human NEC with necrosis, inflammation and separation of the tissues supporting the lining of the colon,” Maheshwari says. “The next step was to see if we could find a mechanism for why this occurred.”

A “double whammy” on the intestinal wall

Examining the blood of the pups with NEC-like conditions after they were transfused, the researchers discovered that it contained three components not seen in the blood of the other test mice: (1) a large number of macrophages, the immune cells that engulf and digest cellular debris, bacteria and viruses, (2) freely circulating hemoglobin, the iron-based molecules that normally carry oxygen throughout the body when attached to red blood cells, and (3) elevated levels of inflammation-inducing proteins, indicating that the macrophages had been activated even without a biological threat to the intestine.

The researchers also observed that levels of haptoglobin, a protein that removes free hemoglobin from the blood, were extremely low.

“These findings suggest that anemia reduces the amount of haptoglobin in the neonate, preventing the free hemoglobin that comes in via transfusion from being properly removed as it normally would,” Maheshwari says.

What apparently happens, he says, is that free hemoglobin attaches to a protein receptor on the intestinal wall that is the same site where bacterial poisons bind. As a result, the immune system mistakenly believes the intestine is being attacked and activates the macrophages.

Once those immune cells go to work, Maheshwari explains, they trigger release of the inflammatory proteins seen in the blood of the anemic, transfused mice. “That event starts a double whammy on the intestinal wall,” he says. “First, the macrophage proteins inflame and weaken the tissues, making them vulnerable, and then, bacteria move in and produce endotoxins that kill the individual cells.”

Johns Hopkins Medicine senior author Akil Maheshwari, MD, says he hopes the findings from the current study can be used to develop blood biomarkers that could indicate which human newborns are most at risk of developing NEC.

With evidence for a probable mechanism to explain the connection between anemia and transfusion in the development of NEC, the researchers next sought to confirm it by seeing if they could block two of its stages, and perhaps, advance the search for potential therapies.

“In one trial, we gave haptoglobin to our model anemic mice before transfusing them and blocked macrophage activation, so they did not develop NEC-like symptoms,” Maheshwari says.

Controlling a master regulator of inflammation in NEC

In another test, nanoparticles that Samuel Wickline, MD, and his colleagues at the University of South Florida  Health developed were used to deliver a genetic interruption — an RNA molecule known as a small interfering RNA, or siRNA — that blocks the chemical signal telling macrophages to start producing inflammatory proteins. The nanoparticles were tagged with a fluorescent dye to track their movement and included a non-toxic compound derived from honeybee venom.

Maheshwari says the macrophages in the blood of anemic mouse pups engulfed the nanoparticles and enclosed them within vacuoles. The bee venom derivative, he explains, broke open the vacuoles so that the siRNA could be released inside the macrophages.

The genetic signal blocker worked well, Wickline says, protecting the anemic mouse pups from intestinal inflammation after red blood cell transfusions.

“Because we showed that the inhibitory nanoparticles with siRNAs were able to control a master regulator of inflammation in NEC, perhaps this technology can one day be applied to not only treat or prevent NEC, but other diseases where inflammation plays a key role such as arthritis and atherosclerosis,” he says. “We had previous shown in preclinical studies that while these peptide siRNA nanoparticles can suppress harmful inflammation, they do not affect levels of an inflammatory signaling molecule (NF-kB) important in protecting normal tissues. That sets the stage for clinical testing in the near future.”

Maheshwari says he hopes the new mouse model and the findings from the current study can be used to develop blood biomarkers that could indicate which human newborns are most at risk of developing NEC.

Research paper coauthors Samuel Wickline, MD, and Hua Pan, of the USF Health Morsani College of Medicine, developed the nanoparticles used in the study.

Funding for this investigation came from National Institutes of Health (NIH) awards HL124078 and HL133022, and American Heart Association award 14GRNT20480307, given to Maheshwari; and NIH awards HL073646, DK102691 and AR067491, given to Wickline. Sysmex America donated the use of its automated veterinary hematology analyzer that made possible the mouse model developed in this study.

Collaborating on the study were researchers from the Johns Hopkins University School of Medicine, the Morsani College of Medicine at the University of South Florida, the Yale School of Medicine and the University of Alabama at Birmingham. Along with Maheshwari and Wickline, the team members included lead author Mohan Kumar Krishnan, Kopperuncholan Namachivayam, Tanjing Song, Byeong Jake Cha, Andrea Slate, Jeanne Hendrickson, Hua Pan, Joo-Yeun Oh, Rakesh Patel, Ling He and Benjamin Torres.

-This story is an edited version of a news release by Johns Hopkins Medicine. 

Tampa, Fla. (April 10,  2012) – A research team led by the University of South Florida’s Department of Psychiatry & Behavioral Neurosciences has found that a fragment of the amyloid precursor protein (APP) — known as sAPP-α and associated with Alzheimer’s disease — appears to regulate its own production.  The finding may lead to ways to prevent or treat Alzheimer’s disease by controlling the regulation of APP.

Their preclinical study is published online today in Nature Communications.

“The purpose of this study was to help better understand why, in most cases of Alzheimer’s disease, the processing of APP becomes deregulated, which leads to the formation of protein deposits and neuron loss,” said study senior author Dr. Jun Tan, professor of psychiatry and the Robert A. Silver Chair, Rashid Laboratory for Developmental Neurobiology at the USF Silver Child Development Center.   “The many risk factors for Alzheimer’s disease can change the way APP is processed, and these changes appear to promote plaque formation and neuron loss.”

Microscopic image showing the merging of the amyloid precursor protein fragment,
sAPP-α, and the APP-converting enzyme BACE 1, in neuronal cells.  This co-localization
suggests that sAPP-α may serve as the body’s mechanism to inhibit BACE1  activity and
thus lower production of the toxic amyloid beta characteristic of Alzheimer’s disease.

An estimated 30 million people worldwide and 5 million in the U.S. have Alzheimer’s.  With the aging of the “Baby Boom” generation, the prevalence of the debilitating disease is expected to increase dramatically in the U.S. in the coming years.  Currently, there are no disease-modifying treatments to prevent, reverse or halt the progression of Alzheimer’s disease, only medications that may improve symptoms for a short time.

“For the first time, we have direct evidence that a secreted portion of APP itself, so called ‘ sAPP-α,’ acts as an essential stop-gap mechanism,” said the study’s lead author Dr. Demian Obregon, a resident specializing in research in the Department of Psychiatry & Behavioral Neurosciences at USF Health. “Risk factors associated with Alzheimer’s disease lead to a decline in sAPP-α levels, which results in excessive activity of a key enzyme in Aβ formation.”

Dr. Demian Obregon is one of the study's coauthors.

In initial studies using cells, and in follow-up studies using mice genetically engineered to mimic Alzheimer’s disease, the investigators found that the neutralization of sAPP-α leads to enhanced Aβ formation.  This activity depended on  sAPP-α’s ability to associate with the APP-converting enzyme, BACE1.  When this interaction was blocked,  Aβ formation was restored.

The authors suggest that through monitoring and correcting low sAPP-α levels, or through enhancing its association with BACE, Alzheimer’s disease may be prevented or treated.

Dr. Demian Obregon and Dr. Lucy Hou of the USF Department of Psychiatry and Behavioral Neurosciences, the study’s lead authors, collaborated with colleagues from the Laboratory of Neurosciences at the National Institute on Aging and colleagues at the USF Center for Aging and Brain Repair, the James A. Haley Veterans’ Hospital and Saitama Medical University in Japan.  Other study authors included: Juan Deng, MD, Brian Giunta, MD, Jun Tian, BS, Donna Darlington, MS, Md Shahaduzzaman, MD, Yuyuan Zhu, MD, PhD, Takashi Mori, DVM, PhD, and Mark P. Mattson, PhD.

The research was supported by a grant from the National Institutes of Health, National Institute on Aging, and a Veterans Affairs Merit grant.

– USF Health –

USF Health’s mission is to envision and implement the future of health. It is the partnership of the USF Health Morsani College of Medicine, the College of Nursing, the College of Public Health, the College of Pharmacy, the School of Biomedical Sciences and the School of Physical Therapy and Rehabilitation Sciences; and the USF Physician’s Group. The University of South Florida is a global research university ranked 34th in federal research expenditures for public universities.

Media contact:
Anne DeLotto Baier, USF Health Communications, abaier@health.usf.edu or (813) 974-3300