Identification of gum disease genes may speed quest for compounds to treat severe periodontitis

Identification of the genes represents a vital step toward developing compounds that can be used in targeted, individualized treatment of severe periodontitis, before loss of teeth and supportive bone occurs.

Findings of the study were published recently in the Journal of Dental Research.

In gene expression studies, investigators find those genes that are most commonly expressed in either healthy or diseased tissue. But such studies cannot identify a causal link between these genes and the disease, and often miss genes that affect a larger number of genetic pathways, which may have a large impact on the disease process.

In this study, a team led by Panos N. Papapanou, DDS, PhD, professor and chair of oral, diagnostic and rehabilitation sciences at the College of Dental Medicine at CUMC, “reverse-engineered” the gene expression data to build a map of the genetic interactions that lead to periodontitis and identify individual genes that appear to have the most influence on the disease. “Our approach narrows down the list of potentially interesting regulatory genes involved in periodontitis,” says Dr. Papapanou. “This allows us to focus on the handful of genes that represent the most important players in the process rather than the whole transcriptome.”

To identify the genes, Dr. Papapanou partnered with CUMC investigators including Ryan Demmer, PhD, assistant professor of epidemiology, at the Mailman School of Public Health, and researchers in Systems Biology who had previously developed algorithms to identify regulatory genes that fuel cancer growth. The researchers examined RNA from healthy and diseased gum tissues of 120 patients with periodontitis. They applied one algorithm to study the interactions among the genes and used another algorithm to identify genes that disrupt healthy tissue and drive the disease process.

Many of the genes identified by Dr. Papapanou and his team are implicated in immune and inflammatory pathways, confirming laboratory and clinical observations of the development of periodontal disease.

Identification of the master regulator genes will allow investigators to test compounds that interrupt their action, creating treatments that stop periodontal disease at its source. “Now it’s important to do the downstream work of validating these master regulators in the lab before we can test these genes in experimental models,” says Dr. Papapanou.

Researchers find byproducts from gum disease incite deadly oral cancer growth

Researchers from Case Western Reserve University have discovered how byproducts in the form of small fatty acids from two bacteria prevalent in gum disease incite deadly oral cancer growth (the growth of deadly Kaposi’s sarcoma-related (KS) lesions and tumors in the mouth).

The discovery could lead to early saliva testing for the bacteria, which, if found, could be treated and monitored for signs of cancer before it develops into a malignancy, researchers say.

“These new findings provide one of the first looks at how the periodontal bacteria create a unique microenvironment in the oral cavity that contributes to the replication the Kaposi’s sarcoma Herpesvirus (KSHV) and development of KS,” said Fengchun Ye, the study’s lead investigator from Case Western Reserve School of Dental Medicine’s Department of Biological Sciences.

The discovery is described in The Journal of Virology article, “Short Chain Fatty Acids from Periodontal Pathogens Suppress HDACs, EZH2, and SUV39H1 to Promote Kaposi’s Sarcoma-Associated Herpesvirus Replication.”

The research focuses on how the bacteria, Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn), which are associated with gum disease, contribute to cancer formation.

Ye said high levels of these bacteria are found in the saliva of people with periodontal disease, and at lower levels in those with good oral health—further evidence of the link between oral and overall physical health.

The deadly oral cancer growth KS impacts a significant number of people with HIV, whose immune systems lack the ability to fight off the herpesvirus and other infections, he said.

“These individuals are susceptible to the cancer,” Ye said.

Deadly oral cancer growth KS first appears as lesions on the surface of the mouth that, if not removed, can grow into malignant tumors. Survival rates are higher when detected and treated early in the lesion state than when a malignancy develops.

Also at risk are people with compromised immune systems: those on medications to suppress rejection of transplants, cancer patients on chemotherapies and the elderly population whose immune systems naturally weaken with age.

The researchers wanted to learn why most people never develop this form of cancer and what it is that protects them.

The researchers recruited 21 patients, dividing them into two groups. All participants were given standard gum-disease tests.

The first group of 11 participants had an average age of 50 and had severe chronic gum disease. The second group of 10 participants, whose average age was about 26, had healthy gums, practiced good oral health and showed no signs of bleeding or tooth loss from periodontal disease.

The researchers also studied a saliva sample from each. Part of the saliva sample was separated into its components using a spinning centrifuge. The remaining saliva was used for DNA testing to track and identify bacteria present, and at what levels.

The researchers were interested in Pg’s and Fn’s byproducts of lipopolysaccharide, fimbriae, proteinases and at least five different short-chain fatty acids (SCFA): butyric acid, isobutryic acid, isovaleric acid, propionic acid and acetic acid.

After initially testing the byproducts, the researchers suspected that the fatty acids were involved in replicating KSHV. The researchers cleansed the fatty acids and then introduced them to cells with quiescent KSHV virus in a petri dish for monitoring the virus’s reaction.

After introducing SCFA, the virus began to replicate. But the researchers saw that, while the fatty acids allowed the virus to multiple, the process also set in motion a cascade of actions that also inhibited molecules in the body’s immune system from stopping the growth of KSHV.

“The most important thing to come out of this study is that we believe periodontal disease is a risk factor for Kaposi sarcoma tumor in HIV patients,” Ye said.

With that knowledge, Ye said those with HIV must be informed about the importance of good oral health and the possible consequences of overlooking that area.

The research was supported by a career development grant at Center for AIDS Research at Case Western Reserve University, and a National Institute of Dental and Craniofacial Research grant.

Contributing to the study were Case Western Reserve University researchers Abdel-Malek Shahir and Nabil Bissada, from the Department of Periodontics; Xiaolan Yu, Jingfeng Sha, Zhimin Feng, Betty Eapen, Stanley Nithianantham, and Aaron Weinberg, from the dental school’s Department of Biological Sciences; and Biswajit Das and Jonathan Karn, from the Department of Molecular Biology & Microbiology at the School of Medicine.

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Katherine Unger Baillie has found that protein drugs, which derive from biological sources, represent some of the most important and effective biopharmaceuticals on the market. Some, like insulin, have been used for decades, while many more based on cloned genes are coming to market and are valued for their precise and powerful functions. Plant-made Antimicrobial Peptide Targets Dental Plaque and Gum Tissues.

Yet the field of dental medicine has very few such drugs due to their high costs, and the ones that are used are delivered invasively, often through surgical procedures, to gum tissues.

Now, a report by University of Pennsylvania School of Dental Medicine scientists in the journal Biomaterials suggests a new approach for delivering a protein drug to treat and prevent oral diseases, including dental caries, commonly known as cavities. Using plants to produce antimicrobial peptides, the researchers were able to rapidly kill tooth-decay-causing bacteria and thwart their ability to form biofilms on a tooth-like surface with a single topical treatment. The peptides were even more effective when combined with an enzyme that degrades the matrix, which surrounds and protects bacteria residing inside biofilms.

In addition, the researchers demonstrated that these peptides, produced in a cost-effective manner in plants, could be taken up by periodontal and gingival cells, indicating that this novel delivery method could be useful in treating diseases that affect the gum tissues, perhaps by promoting wound healing or bone regeneration.

The platform is low-cost compared to the current means of producing biopharmaceuticals and presents a unique opportunity to develop an affordable therapeutic approach that simultaneously attacks disease-causing plaque and promotes gum health, the researchers said.

“As scientists we have many opportunities to develop breakthrough treatments but cost is a huge obstacle,” said Hyun (Michel) Koo, co-corresponding author on the study and professor in the Department of Orthodontics and divisions of Pediatric Dentistry and Community Oral Health in Penn Dental Medicine. “What makes this approach so exciting is not only the science but, because the production costs are low, the feasibility of getting the therapy to the population who truly needs yet can’t afford it.”

The work arose from a partnership between Koo and co-corresponding author Henry Daniell, director of translational research and professor in Penn Dental Medicine’s Department of Biochemistry. Koo was aware of Daniell’s groundbreaking plant-produced therapeutics for a number of important human infectious and inherited diseases. And Daniell learned that Koo had done extensive work on caries-causing biofilms, including searching for alternative approaches to degrade them or prevent them altogether.

“It was a synergism,” Daniell said. “Bringing our research together led to this new concept of a topical protein drug made in plants that can both kill bacteria and break down the oral biofilm.”

Dental caries predominantly affect children and adults of lower socioeconomic status and are responsible for more than $40 billion in health-care spending annually.

In the past, researchers have identified antimicrobial peptides that are potent killers of caries-causing bacteria. But these agents are expensive to make and have had limited success at killing bacteria protected by the extracellular matrix, as is found in dental plaque.

Meanwhile, other groups have investigated enzymes that can break down the biofilm matrix, and these, too, have had limited success at preventing dental caries by themselves.

In the new study, Koo, Daniell and colleagues tried a new approach, combining the antimicrobial peptides with the matrix-degrading enzyme.

To address the prohibitive cost of antimicrobial peptide production, the researchers turned to Daniell’s plant-based protein drug production platform. The process entails bombarding a plant leaf with gold particles coated in a cloned gene in order to reprogram the chloroplasts to synthesize the associated protein. In this case, the researchers coaxed plants to produce two different antimicrobial peptides, retrocyclin and protegrin. Both peptides have complex secondary structures, making them expensive to produce in the lab by traditional means. But the researchers found they could literally grow them in Daniell’s greenhouse and faithfully replicate their unique secondary structures in the plant’s leaves.

They then tested whether the plant-made agents could prevent creation of a biofilm. They exposed a saliva-coated tooth-like surface to the plant-made protegrin for 30 minutes, then exposed the surface to S. mutans cells along with sugar and found that it significantly impaired the ability of the bacterium to form a biofilm compared to an untreated surface.

To see whether the antimicrobials could act not just preventively but therapeutically, the researchers next exposed a pre-formed biofilm on the tooth-mimicking surface to either protegrin alone or a combination of protegrin and a matrix-degrading enzyme. The enzyme alone had no effect on the biofilm, and while the antimicrobial alone was able to kill some bacteria, the combination was powerful, able to degrade 60 percent of the matrix and killing even more bacteria.

“A single topical treatment was capable of disrupting the biofilm,” Koo said. “It’s effectiveness was comparable to chlorhexidine, which is considered the ‘gold standard’ for oral antimicrobial therapy.”

Beyond topical-drug delivery, Daniell’s lab has been investigating molecular “tags” to route protein drugs to human cells to treat several diseases. In this context, delivering growth hormones or other such drugs to gum tissues for wound healing or bone regeneration is of paramount importance to enhance oral health. Their study found that the plant-made antimicrobial peptides could be taken up by human cells in the oral cavity.

“This was unexpected,” Daniell said. “The antimicrobials didn’t harm any of the human cells in gum tissues but had an unusual ability to go across the cell membranes of periodontal and gingival cells. This opens up a completely new field for drug delivery with a topical agent.”

A collaboration with Johnson & Johnson Consumer Inc. will enable Koo and Daniell to continue optimizing their antimicrobial-enzyme production system. One possibility, they note, is to create a chewing gum laced with antimicrobial peptides that could be slowly released as one chews. Alternatively, for Asian cultures where betel leaf chewing is common, the researchers may investigate the possibility of growing these peptides in that plant to promote oral health.

Additional coauthors on the study included co-first authors Yuan Liu and Aditya C. Kamesh and Yuhong Xiao, Victor Sun and Michael Hayes, all of Penn Dental Medicine.

The work was supported by the National Institutes of Health and the Bill & Melinda Gates Foundation.