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For three of the most common hand conditions, MUSC Health’s highly skilled surgical team offers specialized and minimally invasive solutions.

Milton B. Armstrong, M.D., professor of Surgery and Division Chief of Plastic, Reconstructive and Hand Surgery at MUSC Health, details the less invasive treatment options that exist for carpal tunnel syndrome, Dupuytren's contracture and Raynaud's disease. MUSC’s surgeons have the specialized training to consistently execute successful results.

Carpal Tunnel Syndrome: Endoscopic Treatment Approach

According to Dr. Armstrong, carpal tunnel syndrome is one of the most common conditions that bring patients into his office. This condition is caused by compression of the median nerve at the wrist, causing numbness and tingling in the fingers and weakness in the hand.

For the past century, open surgery has been the primary treatment for carpal tunnel syndrome. In this procedure, surgeons make an incision on the palm, cut the ligament that covers the nerve causing the problem and then close the skin.

Over time, surgeons have attempted this procedure using smaller and smaller incisions at the wrist. Today, Dr. Armstrong says he uses a very small incision about a centimeter in length and uses an endoscope to view the ligament from the underside. A knife attached to the endoscope then cuts the ligament and the surgeon closes the skin with one or two sutures—in a procedure that can be completed in 15 minutes or less.

For patients who are a good fit for this procedure, Dr. Armstrong says the less invasive nature can make for a quicker recovery. Often, patients can return to work 10 days to 2 weeks faster than if they had undergone the open technique.

“That helps people who have jobs that require significant use of their hands for activities as well as others, such as students, who have busy lives to return to. It's a very useful procedure,” he says.

Dupuytren's Contracture: Injection Treatment

A less common hand condition that Dr. Armstrong treats, Dupuytren's contracture is primarily seen in patients of Northern European ancestry. Patients develop contractures in the palm and the fingers, which pull the fingers down toward the palm over time. While not generally painful, the condition can cause stiffness and rigidity, making movement of the fingers difficult.

Dupuytren's contracture has traditionally been treated with an open technique. In the procedure, a hand surgeon opens the skin, then separates and takes out tissue that's fibrotic, which is otherwise normal tissue that becomes problematic in certain groups of people. This can be a lengthy procedure, lasting up to 2 hours in some cases, and requires meticulous dissection of important structures, such as digital nerves and flexor tendons.

Today, Dr. Armstrong and other MUSC Health surgeons can often bypass surgery entirely with an injection treatment. “We have an enzyme, called Xiaflex, that we can inject into the contracted cord to break it up,” he says. “In many cases, we can avoid open surgery.”

Patients receive the injection and then follow up with their doctor, anywhere from 24 hours to 7 days after the procedure. The doctor then breaks up the contracture manually—no operating room necessary.

“That's been a boon for these patients, especially people who may be older or have other medical problems that make them not ideal candidates to go under anesthesia,” explains Dr. Armstrong. “We can now treat this problem without having to put a patient under an anesthetic and open the skin with a scalpel.”

Raynaud's Disease: Botox® Offers Relief

Raynaud's disease, a type of vasospastic disorder, happens when a spasm of the arteries travels to the fingers. This can either be an isolated problem, more common in young women, or an issue related to other disease processes, such as lupus.

In severe cases, patients can develop ulcers and pain in their fingers due to lack of blood flow. “In some patients where it's very severe, to the point where the tissues have died off, we have to do amputations of some parts of the fingers,” says Dr. Armstrong.

Traditionally, doctors have performed an open operation that aims to release tissues around the arteries to allow for better blood flow.

“The smooth muscles within arterial walls are controlled by little nerves, the digital nerves, and so we separate the nerves from the arteries using magnification, sometimes with the microscope,” says Dr. Armstrong. “It’s a technically demanding operation and doesn’t guarantee success. Those patients have significant pain from the surgery, and then we have to wait for days or weeks to the results of the surgery.”

Hand surgeons treating Raynaud's disease now have another treatment option to consider: Botox®, or botulinum toxin A. Commonly used to treat a host of cosmetic problems, such as wrinkles of the face and forehead, Dr. Armstrong says Botox® can be a useful adjunct for this problem.

“What we have found is that the botulinum toxin, which relaxes smooth muscles, can be injected into some patients’ hands,” says Dr. Armstrong. “It breaks up the spasm and can have as good a result for some patients as doing the open surgery.”

Carpal Tunnel and Other Techniques Require Intensive Training

These three procedures illustrate how advanced, minimally invasive approaches can offer a multitude of benefits to patients, possibly limiting the need for surgery, often with a similar or better result. However, Dr. Armstrong emphasizes the need for specialized training in performing these hand procedures.

In the division of plastic surgery alone, MUSC Health has four physicians who are fellowship-trained hand surgeons, three of whom are also board certified in surgery of the hand. All of this additional training helps guide surgeons toward the best treatment results for patients.

“Physicians should ensure they’re referring patients to a surgeon who is trained and who understands the anatomy and pathophysiology of these problems,” says Dr. Armstrong, noting that an open procedure may be the next option if a less invasive technique doesn’t offer patients sufficient relief or isn’t recommended.

When patients discuss their treatment options with an expert in minimally invasive and open hand techniques, they can ensure they receive well-rounded recommendations and effective results.

For more information, contact Dr. Armstrong at armstrom@musc.edu.
 

A new approach to breast reconstruction offers better results for more women. According to MUSC plastic surgeon Kevin O. Delaney, M.D., pre-pectoral breast reconstruction can benefit women who:

  • Are seeking reconstruction for the first time
  • Underwent reconstruction surgery years or decades ago but aren’t happy with the results or suffer side effects

If women aren’t satisfied with how their breasts look or feel after reconstruction—or have lingering pain from the procedure—this new technique may help them.

Pre-Pectoral Breast Reconstruction: What Is It?

MUSC Health provides a range of treatment options for women seeking breast reconstruction after a mastectomy. Whether a mastectomy was performed to treat or prevent breast cancer, Dr. Delaney works closely with MUSC Health’s Hollings Cancer Center team to guide women toward the best available options.

MUSC Health offers 2 main types of breast reconstruction today. Free-flap DIEP (deep inferior epigastric perforator) breast reconstruction relies on a patient’s own skin and fat tissue to rebuild breast tissue, whereas other procedures use breast implants.

As Dr. Delaney explains, pre-pectoral breast reconstruction is a new way of performing implant-based procedures, an approach MUSC Health has been offering for close to 2 years.

“We're one of the first centers in the region to perform this pre-pectoral, or subcutaneous, breast reconstruction,” says Dr. Delaney. “In this procedure, we place a breast implant just beneath the breast skin, which means we don't need to cut a patient’s pec muscles.”

Previously, surgeons would need to cut a patient’s pec (or pectoralis major) muscles in order to set the implant underneath these muscles. That’s how implant-based breast reconstruction has traditionally been performed for the past 20 to 30 years.

Breast Reconstruction Technique Offers Many Benefits

According to Dr. Delaney, the new pre-pectoral approach offers many pros and few cons. Two significant benefits to patients: a better cosmetic outcome and less pain.

“One main benefit to this approach is that the cosmetic outcome of the reconstructed breast looks a lot better,” Dr. Delaney explains. “Pre-pectoral breast reconstruction avoids what is commonly known as an animation deformity, which happens to most women who’ve had implant reconstruction under the muscle, to varying degrees.”

This animation deformity occurs when a woman moves her arms or flexes her muscles. The implant, as well as the overlying breast skin, flattens and moves towards the armpit, which many women consider undesirable, says Dr. Delaney.

Because the pre-pectoral technique eliminates the need for cutting the pec muscle, women experience significantly less pain in the short and long term. This equates to a much faster and easier recovery following the surgery. Women also don't lose any functionality of their pec muscle, which is a possibility with the traditional approach.

Dr. Delaney says this new technique is now the preferred route of implant-based breast reconstruction at MUSC. He says it’s slowly catching on nationwide but that as of now, it’s largely offered only through academic centers.

Pre-Pectoral Breast Reconstruction: The Right Candidates

Dr. Delaney assesses the best reconstruction approach on a case-by-case basis, but he says there are only a few reasons why he would recommend against the pre-pectoral approach for implant-based reconstruction.

The main caveat is if a woman has previously undergone radiation therapy to the breast. “Since the breast skin typically doesn't heal as well after it's been exposed to radiation, it makes the pre-pectoral breast reconstruction more risky from a healing and ultimately infection standpoint,” says Dr. Delaney.

However, the majority of women seeking breast reconstruction can benefit from this approach. In addition to offering current patients a better treatment option, this procedure could also benefit a wide swath of women who received breast reconstruction previously but aren’t happy with the results.

“Many women who have undergone sub-pectoral implant-based breast reconstruction in the past, whether it was 3 years ago or 20 years ago, have significant complaints,” says Dr. Delaney. “Patients come to us with tightness in their chest or up into their arm, chronic pain in that area, as well as complaints about the animation deformity and how they don't like the look of their reconstructed breasts. Those are all real complaints that we hear every day.”

Previously, Dr. Delaney says he couldn’t offer those patients many solutions. But now, with this pre-pectoral technique, he says he can significantly improve many patients’ symptoms or complaints in a straightforward outpatient surgery that takes just a few hours.

“We can remove their old implant, put their muscle back down to where it belongs anatomically, and then put a newer, better implant in below the skin,” he says. “Their pain significantly improves. Their animation deformity goes away. And they're incredibly pleased with how much better their reconstructed breasts look and feel as opposed to when they were below the muscle.”

A Better Breast Reconstruction Option Now Available

Dr. Delaney wants to spread the message that there’s a new option available for women who may fall into this category—and that their health insurance would likely cover it because it’s a part of breast reconstruction care.

“As plastic surgeons, if patients have had breast cancer treated but they're displeased with their reconstruction, we want to help,” he says. “If they’re not local, we can often talk to patients via telehealth and let them know whether this procedure might benefit them.”

For more information, contact Dr. Delaney at delaneyk@musc.edu.
 

The Musculoskeletal Institute at MUSC Health brings orthopaedics, rheumatology and endocrinology specialists under one roof to treat an array of bone and joint disorders as well as metabolic bone diseases and osteoporosis.

This multidisciplinary care model offers care and convenience benefits to patients: The team’s depth of expertise in musculoskeletal care makes the institute uniquely capable of coordinating the care of patients with complex conditions. And patients don’t have to travel to see the specialists they need—they can get all of their care in one place, saving them time and energy.

Treating Hip Pain: What Multidisciplinary Looks Like

The institute’s multi-specialty approach ensures a smooth process for both patients and doctors. Patients can easily get the services they need, and physicians can seamlessly coordinate that care.

“Our advantage as a functional unit for patient care is that the physicians within those three groups interchange and freely move patients among one another based upon the diagnoses and the needs of the patients,” says  Vincent Pellegrini, M.D., chief of the Musculoskeletal Institute and chair of the department of orthopaedics at MUSC.

Consider a patient with hip pain, who makes an appointment with Dr. Pellegrini. If he determines that a patient’s pain originates from the back, he can simply walk around the corner to a spine surgeon colleague.

“The spine surgeon would then see that patient,” explains Dr. Pellegrini. “And he may decide that day that the patient really doesn't need an operation but might benefit from injection therapy.”

The coordination continues from there: “The spine surgeon would then walk around the corner and take that patient to see a physiatrist or a physical medicine physician, who might then do a therapeutic and diagnostic injection.”

Diagnostic tests, evaluation by multiple specialists and treatment: All this could happen in the same space, on the same day, even though the patient came in for hip pain and it turned out to be a back condition. These are not “what-if” scenarios—stories like this happen daily at the Musculoskeletal Institute at MUSC Health.

Orthopaedics, Endocrinology and Rheumatology: A Triad of Care Focus

The Musculoskeletal Institute combines orthopaedics, endocrinology and rheumatology for a reason: These disciplines frequently cross over and require complex care decisions, such as physical rehabilitation.

Patients who benefit from this type of care can include:

  • An orthopaedic patient who thought he or she needed surgery but could benefit from a joint injection and a rehabilitative specialist consult
  • A patient with rheumatoid arthritis who is having severe hand pain, who could benefit from consulting with a hand surgeon and a hand therapist
  • A patient with osteoporosis who has been using Fosamax® for a long time and has unique fractures that require the collaboration of an endocrinologist (to adjust the medication and dosing) and an orthopaedic surgeon (to surgically treat the problem)
  • A patient with hip or knee arthritis who needs an orthopaedic surgeon to address inflammatory arthritis and a rheumatologist to help adjust the medication before surgery

At many centers, all of those appointments would need to be on separate days, with separate specialists. At the Musculoskeletal Institute, we bring together an extensive team to care for a wide swath of people—in a more effective, efficient way.

“With many of our patients, it can take a village to have all of the resources needed to take care of certain problems that are a little more unusual,” says Dr. Pellegrini.

Hip Pain, Osteoporosis and More: Patient and Physician Benefits

The Musculoskeletal Institute has a patient-centric focus, resulting in care that’s improved, coordinated and timely. Dr. Pellegrini says these benefits extend to referring physicians as well—particularly for complicated cases that could use an extra set of eyes from a different specialist.

“I believe most referring physicians are primarily interested in taking care of their patients in a way that’s efficient and expedient,” he says. “Because we put an array of resources under one roof, it allows us to take care of some of the more complicated patient needs that can overwhelm a smaller practice with fewer resources. Our team approach can be very beneficial in certain patient situations.”

For more information, contact Dr. Pellegrini at pellegvd@musc.edu.
 

Co-administering a monoclonal antibody that neutralizes tumor-released soluble MHC I chain-related molecule (sMIC) improves anti-CTLA4 antibody therapy effectiveness and reduces treatment-related colitis, report Medical University of South Carolina (MUSC) investigators in an article published online May 17, 2017 by Science Advances.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) can be thought of as an 'off switch' or 'immune checkpoint' for effector T cells that are activated to fight cancer tumors. Researchers have developed a therapy to block CTLA4, specifically, anti-CTLA4 antibody immunotherapy, to help sustain T-cell activation and improve patient survival.

Unfortunately, while anti-CTLA4 therapy is highly effective in animals, response rates in humans vary widely and serious adverse events such as colitis (gastrointestinal inflammation) are common. For example, Ipilimumab, an FDA-approved anti-CTLA4 antibody therapy for advanced melanoma, is highly effective in controlling tumors in mice but has a response rate of only fifteen percent in humans. Clinical investigators have tried to improve the efficacy and safety of anti-CTLA4 therapy by combining it with other agents but response rates and toxicity remain suboptimal.

MUSC cancer immunologist Dr. Jennifer WuA team of MUSC researchers led by Jennifer Wu, Ph.D., professor of Microbiology and Immunology, suspected that response disparities between humans and animals may be due to differences in immune modulators that human tumors express.

"When we use animals to study therapies for humans we often neglect certain human-specific biological pathways simply because they don't exist in the animal," explains Wu. "MIC is one of those molecules that is expressed in human tumors but is absent in mice. We knew that the soluble form, sMIC, is highly immunosuppressive in humans and we knew it was important, but we had no way to study it. We had to create a new model."

Their work to unravel these molecular-level differences has now paid off, with the discovery of a new combination therapy that dramatically improves CTLA4 therapy effectiveness and avoids therapy-induced colitis.

The team developed a clinically relevant, MIC-transgenic spontaneous mouse tumor model that closely resembled the onco-immune dynamics seen in human cancers. Using this model, they investigated how tumor-derived, human sMIC affected anti-CTLA4 therapy. They found that high blood levels of sMIC not only reduced the antitumor efficacy of anti-CTLA4 therapy but also directly evoked colitis.

"It was a total surprise, and we were a little nervous about the results," says Wu. "So, we repeated the test using multiple models and antibodies and different batches of animals to make sure it was reproducible and that what we were seeing was real. Sometimes knowledge itself gets in the way. Because there are certain accepted beliefs that make you think what you're seeing can't be true-that it's impossible. But when multiple experiments are all coming up with the same results, what can you say? That's when we have to let go and come to a new understanding."

Next, the team co-administered an antibody called B10G5 that neutralizes sMIC, alongside anti-CTLA4 therapy. The new combination therapy not only remarkably improved anti-CTLA4 immunotherapy effectiveness, but also alleviated therapy-induced colitis.

"We've been studying B10G5 for a while and published a paper in Clinical Cancer Research in 2015 demonstrating that using B10G5 to target sMIC can alleviate tumor-induced immune suppression and also has a huge immune-stimulating ability," explains Wu. "So those results led us to think it would be a good strategy to combine B10G5 with antibodies that target immune checkpoint molecules, the 'off-switch' of an ongoing immune response."

Finally, inspired by a case study published in 2006, the team decided to try to validate its current findings in humans. The original case reported a melanoma patient who developed anti-MIC autoantibody during anti-CTLA4 therapy and had a superior therapeutic response. So, they contacted a long-time collaborator at Oregon Health and Science University and Knights Cancer Center to obtain plasma samples collected during a clinical trial they were conducting in metastatic prostate cancer patients.

Wu's team looked for anti-sMIC autoantibodies in samples from ten patients receiving anti-CTLA4 therapy with Ipilimumab. One sample showed high levels of anti-MIC autoantibody. When the team followed up with their colleague, it turned out that this particular cancer patient not only demonstrated a remarkable therapeutic response (his prostate-specific antigen fell from 191 to 4.6 ng/ml after eight treatment cycles) but also did not develop autoimmune colitis.

That case reinforced the team's preclinical in vivo findings that coadministering the sMIC-neutralizing antibody (B10G5; CanCure, LLC) enhances anti-CTLA4 therapy and deters colitis. Overall, these results indicate a new, powerful combination immunotherapy for cancer. They also suggest that pre-screening serum sMIC levels might help clinicians to identify patients who are most likely to have a positive therapeutic response.

"I hope that our findings will inspire investigators to revisit other cancer immunotherapies that were successful in animals but presented no efficacy in humans," says Wu. "Anti-CTLA4 therapy is approved for melanoma and also is still in trials for other cancers. Maybe our study will inspire clinical investigators to think about screening their patients to identify who will be a better responder versus a poor responder to anti-CTLA4 therapy."

MUSC Health Sports Medicine, a specialized group of orthopedic doctors and other medical providers within MUSC’s Musculoskeletal Institute, is passionate about providing comprehensive, personalized care to athletes. 

“We’re focused on the evaluation, diagnosis, treatment (both operative and nonoperative) and rehabilitation of injury or pain conditions in the muscles, bones and joints that impact athletes and active people,” says Shane Woolf, M.D., chief of orthopedic sports medicine at MUSC Health.

At MUSC Health Sports Medicine, patients have access to integrated care—from the time of injury until they’re back at play. Patients also benefit from the group’s concentration on education and research, which promotes best practices and novel techniques that demonstrate an expert level of sports medicine care.

Beyond Orthopedic Treatment for Athletes

What sets MUSC Health Sports Medicine apart from most orthopedic groups? According to Dr. Woolf, the answer is in the scope of what—and who—they treat. “I like to consider orthopedic sports medicine as primarily soft tissue orthopedic trauma, as opposed to general orthopedic trauma, which involves primarily treating fractures,” he says. “We all treat broken bones, but at MUSC Health Sports Medicine, our orthopedic sports medicine specialists are skilled in the repair or reconstruction of cartilage, muscle, ligament and tendon injuries and joint instability.”

MUSC Health’s sports medicine group is affiliated with numerous area sports teams, including local high schools as well as the Charleston River Dogs, a minor league baseball team, and the Charleston Battery, a USL pro soccer team. While the focus is on sports medicine, the team doesn’t discriminate based on athletic ability.

“We have experience in treating active and athletic people of all age ranges, activity levels and skill levels,” says Dr. Woolf.

Many athletes may require medical care outside of orthopedic injury issues. MUSC Health Sports Medicine coordinates that care, too. “When you’re playing a sport, even at a recreational level, many medical problems need to be managed differently compared to people who don’t engage in athletics or an active lifestyle,” explains Dr. Woolf.

Conditions such as exercise-induced asthma, diabetes and heart issues can benefit from a sports medicine focus. “Our primary care sports medicine colleague, Alec DeCastro, M.D., is skilled at helping patients with medical issues remain active,” says Dr. Woolf.

Importance of Coordinated Sports Medicine Care

At MUSC Health Sports Medicine, specialists take a big-picture approach to athlete care, coordinating among many specialties.

“We integrate with other specialists very closely,” says Dr. Woolf. “We work with referring primary care physicians as well as our colleagues in radiology, primary care sports medicine, neurology, cardiology and other divisions within our institution. We get athletes the evaluation they need, even if it’s not necessarily an orthopedic issue. We keep it personal, customizing treatment plans for each patient.

“We want to make sure that every patient is safe to play, and we use every resource within our disposal to find a way to allow them to participate in their chosen activity, safely,” adds Dr. Woolf.

Two examples of how patients benefit from this coordination:

  • Joint injuries: A patient receiving joint injections sees a radiologist for image-guided injections, to confirm treatments are reaching the area of concern. Physical therapy professionals can then assist the same patient with rehab of the joint.
  • Gastrointestinal issues: MUSC Health specialists work with the Women’s Tennis Association when athletes are in town for a tournament. When an athlete has a gastrointestinal issue, for example, she might be connected with an MUSC Health specialist for a quick evaluation. “Even though the condition isn’t a musculoskeletal problem, it still affects athletic performance, and we’re happy to coordinate that care,” says Dr. Woolf.

A Leader in Sports Medicine Research and Education

MUSC Health sports medicine specialists are also educating future sports medicine providers and promoting evidence-based care through evolving research.

“We are very much engaged in research and education for sports medicine. We’re not only evaluating and treating these problems, but we’re studying them as well to learn how to provide better care,” says Dr. Woolf.

Specialists teach a range of skills to sports medicine residents and students, from the intricacies of open and arthroscopic surgery techniques to education on the importance of rehabilitation after an injury.

Current research studies are delving into a range of sports medicine points of interest, such as:

  • Improving rehabilitation after ACL tears
  • How foot pain relates to weakness in an athlete’s core (the muscles and joints in the back, pelvis, abdomen and hips that make up the fundamental foundation of the body)
  • Identifying injuries and best treatment plans for cartilage problems in the shoulder and knee

Whatever an athlete’s medical concern, MUSC Health Sports Medicine can help coordinate care and ensure the best treatment, and the fastest recovery, for each individual.

For more information about the MUSC Health Sports Medicine program, contact 843-876-0111.

Primary colorectal tumors secrete VEGF-A, inducing CXCL1 and CXCR2-positive myeloid-derived suppressor cell (MDSC) recruitment at distant sites and establishing niches for future metastases, report Medical University of South Carolina (MUSC) investigators in an article published online ahead of print on April 28, 2017 by Cancer Research. Liver-infiltrating MDSCs help bypass immune responses and facilitate tumor cell survival in the new location. This research illuminates mechanisms by which primary tumors contribute to premetastatic niche formation and suggests CXCR2 antagonists may reduce metastasis.

Recent cancer research shows that premetastatic 'niches' form at sites far from the original tumor before new tumors occur. In colorectal cancer (CRC), these supportive microenvironments form in preferred secondary organs, such as the liver and lung, and facilitate the colonization, survival, and growth of metastasizing tumor cells. However, the mechanisms responsible for the formation of these premetastatic 'niches,' including what role(s) the primary tumor may play, are not well understood. It is critical to better understand the mechanics of CRC metastasis, as it is the second leading cause of cancer deaths in the US and patients with advanced cases often die because current treatments for widely metastasized disease are not effective.

MUSC investDr. Raymond N. DuBois, dean of the Medical University of South Carolina College of Medicineigators led by Raymond N. DuBois, M.D., Ph.D., dean of the MUSC College of Medicine and professor of Biochemistry and Molecular Biology, have now illuminated how primary CRC tumors contribute to premetastatic 'niche' formation.

"The idea that some sort of 'priming' needs to take place for metastasis to occur in distant organs - that there is some sort of activity in the future tumor location - is not new. But most research has focused on growth factors, chemokines and pro-inflammatory cytokines. There hasn't been much work looking at immune cell activity in distant organs prior to metastasis," explains DuBois. "We knew that the type and density of immune cells in the primary tumor plays a role in progression. For example, when more immature myeloid cells are present in the tumor, it becomes resistant to immune attack. But we didn't know what to expect in a metastatic model."

To explore this area, the team first evaluated whether the presence of a primary tumor affected immune cell profiles in premetastatic liver and lung tissues of mice. They found that the presence of a primary cecal tumor caused MDSCs to begin infiltrating the liver before metastasis began. Working backward from this finding, they used a series of experiments to reveal the chain of events that led up to MDSC infiltration.

Because CXCR2 is essential for drawing MDSCs out of the bloodstream and toward CRC tumors and colonic mucosa, the team began looking for CXCR2 and its ligands (CXCL1, CXCL2, and CXCL5) in mouse liver tissue. The team not only found that the ligand, CXCL1, attracted MDSCs from the bloodstream into premetastatic liver tissue, but also that administering a CXCR2 antagonist inhibited CXCL1 chemotaxis. This demonstrated that CXCR2 is required for CXCL1 to induce MDSC liver infiltration. In other words, the CXCL1-CXCR2 axis is required to recruit MDSCs to the liver. Importantly, they also found that liver- infiltrating MDSCs secrete factors that promote cancer cell survival and metastatic tumor formation without invoking the innate and adaptive immune responses.

Next, because VEGF is known to induce CXCL1 expression in lung cancer, the research team examined whether VEGF secreted by CRC tumors also regulated CXCL1 expression. Their results demonstrated that VEGF-A secretion by primary CRC tumor cells stimulates macrophages to produce CXCL1. Interestingly, although VEGF-A knockdown inhibited liver metastasis, it did not affect the growth of the primary tumor.

"We did not expect to find that a primary tumor could affect a distant organ before any of the cancer cells arrived on site," says DuBois. "We were surprised to see these changes before a single metastatic cell took up residence."

Together, these studies reveal that VEGF-A secreted by the primary CRC tumor stimulates macrophages to produce CXCR1, which recruits CXCR2-expressing MDSCs from the bloodstream into healthy liver tissue. The MDSCs then create a premetastatic 'niche' or micro-environment where cancer cells can grow to form new tumors. These results demonstrate for the first time that cells in the primary tumor contribute to forming distant pre-metastatic 'niches' which facilitate the spread of disease.

"Now that we know the primary tumor puts things in motion remotely prior to metastasis, we should be able to inhibit this process and have a positive impact on survival," explains DuBois. "We now know which molecules and immune cells are involved and that if we disrupt the CXCL1-CXCR2 axis we can possibly reduce the spread of disease. Both antibodies and small molecules can inhibit this pathway, but they have not yet been optimized. I hope these findings will speed up the development of inhibitors of the CXCR2 pathway."

"Juicing" Th17 cells with FDA-approved small molecule beta-catenin and p110 delta inhibitors during in vitro expansion for adoptive T cell therapy (ACT) profoundly improves their therapeutic properties, report investigators at the Medical University of South Carolina (MUSC) in an article published online ahead of print on April 20, 2017 by JCI Insight.

MUSC cancer immunologist Dr. Chrystal M. PaulosACT involves harvesting T cells, rapidly amplifying and/or modifying them in the laboratory to boost their cancer-fighting ability, and then reinfusing them back to the patient to boost anticancer immunity. One challenge for ACT has been that the rapid expansion of T cells in the laboratory can cause them to age and wear out, decreasing their longevity after reinfusion.

"Juicing" Th17 cells with the FDA-approved small molecules enhanced their potency, function and stem-like (less differentiated) quality, suggesting that they would persist better after reinfusion into patients, and also reduced regulatory T cells in the tumor microenvironment, which can blunt the immune response. These findings highlight novel investigative avenues for next-generation immunotherapies, including vaccines, checkpoint modulators, and ACT.

"This is exciting because we might be able to overcome some of the delays and disadvantages of rapid expansion in the laboratory," explains senior author Chrystal M. Paulos, Ph.D., associate professor of immunology and Endowed Peng Chair of Dermatology at MUSC and a member of the MUSC Hollings Cancer Center. "We might be able to use fewer cells (for ACT) because we can pharmaceutically 'juice' these T cells to make them more fit in the oppressive tumor microenvironment."

Building upon their previous findings that ICOS costimulation is critical for generating human Th17 cells and for enhancing their antitumor activity, an MUSC research team led by Paulos and including postdoctoral fellow Kinga Majchrzak report for the first time that repurposing FDA-approved small molecule drugs that inhibit two ICOS-induced pathways greatly enhances the antitumor potency of T cells.

Several biologic properties of the Wnt/ beta-catenin and P13K delta pathways led the team to suspect that they supported the antitumor activities of Th17 cells. For example, these pathways are active in both regulating T cell cytokine production during the immune response and in promoting self-renewal of hematopoietic stem cells (HSCs) and sustaining HSCs in an undifferentiated state. So, they designed a series of experiments to determine whether these two pathways were also active in enhancing Th17 antitumor memory and effectiveness.

To test this idea, they pharmaceutically inhibited PI3K delta and beta-catenin in Th17 cells (using idelalisib [CAL-101] to block the PI3K delta pathway and indomethacin [Indo] to inhibit beta-catenin)-anticipating that this would weaken Th17 cells' antitumor activity. To their surprise, the exact opposite occurred. ICOS-stimulated Th17 cells that were treated in vitro with CAL-101 plus Indo elicited a more potent antitumor response against melanoma in mice.

"My post-doc student came to me and said, 'I think I made a mistake because the data are going in the opposite direction to what we originally predicted!" says Paulos. "So, she repeated the experiment several times but we kept getting the same result. The data showed that using drugs to inhibit these pathways actually made the Th17 cells even better at killing tumors."

The team found that Th17 cells treated with CAL-101 express less FoxP3, suggesting that the drug suppresses Treg conversion while sustaining central memory-like Th17 cells. This finding is highly important because the phenotypic plasticity of Th17 cells in vivo allows their conversion to Tregs or Th1 cells with weak antitumor properties. These data suggest that treatment with CAL-101 can halt the development of these poorly therapeutic phenotypes and, thus, enhance the T cells' antitumor activity.

While the findings were initially counterintuitive and perplexing from a mechanistic perspective, in retrospect Paulos sees that they make sense. "Essentially, the T cells are younger," explains Paulos. "We know that T cells used for ACT age and wear out over time. Somehow these drugs sustain their youth and function. They're able to keep all the properties of their youth-they expand better and they're more functional and handle the oppressive tumor microenvironment better."

The discovery that existing FDA-approved drugs that block p110 delta and beta-catenin can make T cells more efficient tumor killers in vivo is an exciting prospect for Paulos' team. "From a clinical standpoint, this finding indicates that the therapeutic effectiveness of ACT could be improved by simple treatments with readily available drugs. It opens a lot of new investigative avenues for next-generation immunotherapy trials," she says.

"This research offers tremendous promise for the treatment of patients with serious forms of skin cancer," says Dirk M. Elston, M.D., chair of the Department of Dermatology and Dermatologic Surgery at MUSC.

Paulos has a patent on ICOS signaling in adoptive T cell transfer therapy (US 9133436), and Paulos, Majchrzak, and J.S. Bowers have a patent on pharmaceutical drug combinations or genetic strategies that instill durable antitumor T cell memory and activity (patent application P1685).

For many of the millions of patients treated annually in hospitals for upper gastrointestinal (GI) bleeding, there is little value in placing a nasogastric (NG) tube in patients to determine the source of that bleeding or size of a lesion, report investigators in an article published online ahead of print on January 9, 2017 by the Journal of Investigative Medicine.

Study authors, including Don C. Rockey, M.D., Medical of University of South CarDr. Don C. Rockeyolina (MUSC) Department of Medicine chair and professor of gastroenterology, position the research as improving patient care by doing less when possible, in terms of procedures or treatments that don't provide significant benefit to patients and are costly and uncomfortable.

"Placing a tube through the nose and down into the stomach makes sense if we are talking about delivering nutrition to a patient or to get an idea of what is in someone's stomach, but the value of placing this tube for patients who have an upper GI bleed has been unclear," Rockey said. "Our goal was to examine that value, and our results suggest that for millions of patients with an upper GI bleed, placing this tube had little clinical benefit and produces unnecessary cost and discomfort for all involved. If it doesn't help the patient or the clinician trying to diagnose the cause of this kind of bleed, we don't need it as a standard of care when there is no value."

The single-blind, randomized, prospective, non-inferiority study compared NG placement (with aspiration and lavage) to no NG placement (control) and demonstrated that NG tube placement in patients with typical upper GI bleeding had no impact on outcomes. In addition, the placement of NG tubes was often unsuccessful or associated with patient discomfort.

T cell attacking a tumor

T cell attacking a tumor

 

 

Stylized image of a T cell attacking a tumor. Illustration by Emma Vought.


 

 

 

 

 

 

 

 

 

 

 

 

Release Summary: The protein moesin could be a target for cancer immunotherapy, report Medical University of South Carolina (MUSC) investigators in an article in the Journal of Clinical Investigation. Their data suggest that moesin promotes conversion of naive T cells into regulatory T cells that suppress the immune response against cancer. Inhibiting moesin could help restore the anti-tumor T cell response and also improve the survival of cancer-killing CD8+ cells after adoptive T cell transfer.

In an article published online ahead of print on March 13, 2017 by the Journal of Clinical Investigation, Medical University of South Carolina (MUSC) investigators report preclinical research showing that moesin, a membrane-domain organizing protein, controls regulatory T cell (Treg) function as well as the abundance and stability of transforming growth factor-beta (TGF-beta) receptors on the surface of cells, providing a potential therapeutic target for cancer immunotherapy.

Their findings show that TGF-beta acts at the protein level to generate Tregs in the tumor microenvironment. Although the human immune system is capable of eradicating cancer, Tregs dampen the immune response and protect cancer cells against tumor-killing (i.e., cytotoxic) T cells. The MUSC study is the first to show that eliminating moesin reduces TGF-beta receptor expression and subsequent Treg generation to restore anti-tumor immunity.

T cells, a subtype of white blood cells, can effectively attack and kill tumor cells when activated by the protein TGF-beta. However, the immune system has a sophisticated network of checks and balances to ensure that the body does not produce so many of these cytotoxic T cells that it harms its own cells and tissues. When the immune reaction is complete, TGF-beta signals naive T cells to become Tregs that suppress and degrade the activated, inflammatory T cells, ensuring that they do not overproduce the immune factors that can lead to autoimmune disease.

Cancer cells have learned to hijack this system of checks and balances to hide from the tumor-killing T cells. Many cancers produce TGF-beta that binds the receptors on the tumor-killing helper T cells so they can’t be recruited to fight the tumor. The T cells convert instead to Tregs, which suppress the immune response against the cancer.

Inhibiting moesin could help prevent conversion of naive T cells into Tregs, thereby restoring the anti-tumor immune response. 

"Because moesin supports greater Treg production, we could design moesin inhibitors to halt or slow active TGF-beta signaling and slow down Treg conversion so that anti-tumor T cells can have a chance to see the cancer and eradicate it,” explains Zihai Li, M.D., Ph.D., chair of the Department of Microbiology and Immunology at MUSC and senior author on the paper.

Earlier studies by Philip Howe, Ph.D., chair of MUSC's Department of Biochemistry and Molecular Biology and a co-author on the paper, demonstrated that many TGF-beta-mediated epithelial mesenchymal transition genes, including moesin, were repressed by an RNA-binding protein in healthy epithelial cells and that moesin expression could be restored through TGF-beta stimulation.

This ability of TGF-beta to dramatically increase moesin expression led the team to investigate moesin’s role in Treg generation. Jointly with other colleagues at MUSC, the team compared the abilities of helper T cells with and without moesin to become Tregs. They found that moesin promotes Treg generation by interacting with a TGF-beta receptor to make it more available, thereby enhancing TGF-beta signaling. Conversely, TGF-beta signaling was reduced in the absence of moesin, impairing the development and function of Tregs.

Perhaps the most compelling results were provided by studies involving adoptive T cell therapy in a mouse model of melanoma. In adoptive T cell therapy, tumor-killing T cells are “harvested” from a human or animal with cancer and amplified or otherwise “supercharged” before being reinfused into the donor. Although these reinfused cells can be very effective at killing tumors, they do not always survive long-term, setting the stage for recurrence. 

The MUSC research team showed that these reinfused anti-cancer CD8+ T cells not only underwent rapid activation and expansion in mice lacking moesin, but that they also survived longer, reducing the likelihood of recurrence. Indeed, after adoptive T cell transfer, all of the mice having moesin relapsed while most of the mice lacking moesin were cured.

"When the mice lacking moesin had no recurrence, this was really exciting. We were not only deleting moesin but, when we gave T cells to the active tumors, those T cells could control the cancer for a very long time,” explains Ephraim Ansa-Addo, Ph.D., a postdoctoral fellow in the Department of Microbiology and Immunology and lead author on the paper.

These findings suggest that moesin could be a therapeutic target in developing new treatments for cancer and Treg-related immune disorders. Chemical modulators of moesin could control the function of T cells by inhibiting moesin in cancers or inducing it to treat autoimmune diseases. Moesin modulators could also be combined with current immunotherapy regimens.

“These findings are very interesting for the field and provide a lot of directions for further research into alternative therapies," says Li.

Fluorescent labeling of KV7 channel expression in neurons

 

Researchers at the Medical University of South Carolina identified potassium channel genes as novel preclinical pharmacogenetic targets that show early promise for reducing heavy alcohol drinking.

 

 

 

 

 

 

Fluorescent labeling of  KV7 channel expression in neurons. Image courtesy of Dr. Patrick Mulholland.

A handful of FDA-approved drugs exist for treating individuals with alcohol use disorder but they have been largely ineffective at reducing the high rates of relapse. As such, there remains a critical need to identify and develop alternative pharmacological treatment options.

Researchers at the Medical University of South Carolina (MUSC), through collaborative efforts with the NIH-funded INIAstress Consortium, have identified novel potassium (K+) channel genes within addiction brain circuitry that are altered by alcohol dependence and correlate with drinking levels in a mouse model of alcohol drinking. Significant reduction of heavy alcohol drinking after administration of a KV7 channel–positive modulator validated Kcnq, one of the identified genes that encodes KV7 type K+ channels, as a potential pharmacogenetic target. These preclinical findings, published in the February 2017 special issue of Alcohol on mouse genetic models of alcohol-stress interactions, suggest that K+ channels could be promising therapeutic targets that may advance personalized medicine approaches for treating heavy drinking in alcoholics.

Alcohol is known to change how neurons fire, and K+ channels play a crucial role in modulating a neuron’s excitability by returning the cell membrane potential back to baseline after the neuron has fired an action potential. Although there is an old literature that links K+ channels and alcohol use disorder, the alcohol field has not actively pursued this line of research.

Recently, the MUSC research team lead by Patrick J. Mulholland, Ph.D., associate professor of Neuroscience and Psychiatry & Behavioral Sciences and senior author on the article, revisited this research area in a novel way. By applying new genomic database technologies, the team became the first to use an experimental genetic bioinformatics approach to determine the relationship between expression levels of brain K+ channel genes with alcohol consumption.

“We looked at all 79 K+ channel genes in an alcohol drinking model using genetically diverse strains of mice and were trying to find the genes that might be risk genes for drinking and the genes that are changed by alcohol dependence,” said Mulholland. “More critically, we wanted to determine how alcohol changed expression of K+ channel genes and how those changes predicted how the mice drank after they were rendered dependent. In other words, we wanted to know what the mechanisms are that facilitate enhanced drinking in alcohol dependence.”

In this preclinical study, INIAstress researchers exposed strains of mice with diverse genetic backgrounds and varied drinking behaviors (BXD recombinant inbred) to alcohol drinking bottles. Half of the mice remained on this protocol and represented non-dependent mice (i.e., mice that consumed alcohol but were not rendered dependent). Alcohol dependence was induced in the other half of mice using a chronic intermittent ethanol exposure model. After 10 weeks, microarray analyses were completed in the prefrontal cortex and nucleus accumbens. Mulholland and colleagues then performed a targeted analysis of K+ channel genes and alcohol drinking in BXD strains using the GeneNetwork software system.

In non-dependent mice, expression levels of several K+ channel genes significantly correlated with the amount of alcohol consumed. Along with identifying novel genes (e.g., Kcnd2), the findings validated genes that were previously implicated in alcohol use disorder.

In particular, low expression levels of Kcnq genes were significantly correlated with high drinking levels. As these correlations were seen prior to dependence, they may represent risk markers for heavy alcohol consumption.

In dependent mice, the expression levels of Kcnq5 were significantly dysregulated across BXD strains, and as the researchers expected, these gene adaptations correlated with the degree of escalated drinking during dependence.

Mulholland and his team were particularly excited by the findings implicating Kcnq genes and KV7 channels in non-dependent and dependent drinking behavior as these findings replicated their previous study in rats (published November 2016 in Addiction Biology). In this prior study, retigabine, an FDA-approved KV7 channel–positive modulator, significantly reduced alcohol consumption in high-drinking non-dependent rats. This study was the first to identify KV7 channels and Kcnq genes as a potential target to reduce heavy drinking.

To further validate Kcnq as a therapeutic target, the researchers induced chronic alcohol drinking in a strain of mice with high drinking behavior (C57BL/6J). After seven weeks, the mice were treated with retigabine. Consistent with the rat studies, retigabine significantly reduced alcohol consumption in high-drinking non-dependent mice. These findings were also consistent with clinical evidence in humans that mutations in KCNQ genes associate with early-onset alcohol dependence.

Together, these studies provide, both genetically and pharmacologically, strong evidence that KV7 channels and KCNQ genes are promising pharmacogenetic targets for treating alcohol use disorder.

“With all of the preclinical and clinical genetic evidence linking KV7 channels and heavy drinking, it would be great to have a precision medicine follow-up study examining the relationship of KCNQ single-nucleotide polymorphisms (i.e., mutations) with retigabine’s response at reducing heavy alcohol drinking and alcohol relapse,” said Mulholland.

Given that retigabine is an FDA-approved drug, its use in a clinical trial on alcohol use disorder is theoretically feasible. However, there is a roadblock to clinical trial development since retigabine’s manufacturer recently announced they will stop making the drug due to commercial reasons.

Fortunately, the path to translating these promising preclinical findings to humans does not end here.  

“There are better drugs that target KV7 channels that are available on the preclinical side,” said Jennifer A. Rinker, Ph.D., postdoctoral fellow in the Department of Neuroscience and first author on the Alcohol paper. “For example, retigabine hits most of the KV7 channel subtypes. There are selective drugs that target just two of the subunits instead of all of them. That’s where we are headed, to figure out which of the subunits are critical for the effects of retigabine to reduce drinking.”

Dr. Robert Gemmill (front) and Dr. Patrick Nasarre (back) were authors on the article.

Medical University of South Carolina (MUSC) investigators report preclinical research showing that the tumor-promoting properties of neuropilin (NRP)-2 reside predominantly on isoform NRP2b, while NRP2a has the opposite effects in non-small cell lung cancer (NSCLC), in the January 17, 2017 issue of Science Signaling. In mouse models, NRP2a inhibited tumor cell proliferation, while NRP2b promoted metastasis and progression. This new understanding may lead to improved therapies that specifically target NRP2b, while sparing the tumor-inhibiting functions of NRP2a.

Lung cancers are highly invasive, metastatic, and drug resistant—accounting for one fifth of cancer deaths worldwide each year. In part, the malignant properties of lung cancer are driven by the epithelial-mesenchymal transition (EMT), which is primarily induced by transforming growth factor beta (TGF-beta), and results in the proliferation of cancer stem cells.

It is known that the two human NRPs—NRP1 and NRP2—are often up-regulated in tumors and associated with poor patient prognosis. Previous work by MUSC Hollings Cancer Center researchers Robert Gemmill, Ph.D. and Harry Drabkin, M.D., first and senior authors on the Science Signaling article, demonstrated that NRP2, in particular, was up-regulated in cultivated NSCLC lines during TGF beta-mediated EMT. Furthermore, inhibiting NRP2 was found to reduce TGF-beta-mediated responses, including invasive tumor growth.

However, to date, nearly all studies of NRP2 have focused on the 2a isoform. Until now, NRP2b, the alternatively spliced isoform, has been largely uninvestigated and poorly understood.

"Most research has been focused on understanding the major effects of NRP2 overall and wasn't really concerned with breaking down the roles of its component parts—the 2a and 2b isoforms,” explained Gemmill, who holds the Melvyn Berlinsky Endowed Chair for Cancer Research at MUSC. “So, we know that NRP2a and NRP2b are nearly identical – only about the last 100 amino acids at the C terminus are different. There was some speculation that they might have different functions, but most of us assumed those differences were minor."

"It's turning out that there are lots of molecule variants that people just haven't looked at before and that we've only recently been discovering are important,“said Drabkin, who holds the Mary Gilbreth Endowed Chair in Clinical Oncology at MUSC. “We usually find the big stuff first and then work down into the deeper layers and that takes time. As they say, 'the devil is in the details' and there's lots of details." 

The team was drawn into their investigation of NRP2b by the results of experiments they were conducting on a potent tumor suppressor, semaphorin 3F (SEMA3F), which uses NRP2 as its receptor. When analyzing NRP2 expression, they unexpectedly observed a double band and that induction appeared to affect one band more than the other. This led them to question why one band was altered more than the other. In addition, they found that, during the progressive changes that lead to tumor metastasis, SEMA3F was lost.

 "So, we asked, what happens to it? Where does it go? And we found that, during lung cancer progression, SEMA3F is lost and NRP2b is induced,” explained Gemmill. “That led us to investigate the 2b isoform to see what it does because no one knew."

To this end, the team designed a series of experiments. Real-time, reverse transcription polymerase chain reaction assays revealed that TGF-beta stimulation substantially increased NRP2b but not NRP2a. This was the first time that NRP2 up-regulation by TGF-beta had been shown to preferentially involve the uninvestigated 2b isoform. They then looked at tumor cell migration and invasion patterns and found that cancer cell migration across Matrigel-coated membranes was inhibited in NRP2b knockdown models and enhanced by NRP2a knockdown. Repeated experiments confirmed that TGF-beta-mediated cancer cell migration and invasion was dependent on NRP2b.

That's when the team realized that the two isoforms had very different functions in terms of cancer progression, leading them to extend their studies to in vivo animal models.

 "As soon as we saw the migration results, we knew we had to put it in an animal model using cancer cell lines where we could control the isoform expression. We thought, 'this is just too good to be true,' but it was true,” explained Gemmill. ”We got the same results time after time. Whenever we expressed NRP2b the cancer metastasized, and whenever we expressed NRP2a progression and metastasis were suppressed. Clearly, with the 2b isoform, we have found something that promotes metastasis."

"Other people had looked at NRPs as co-receptors but never got into the details of which isoform was playing what role,” said Drabkin. “It's sort of like detective work. You follow leads and ask questions. Sometimes you follow a false lead—but in this case we found something new that turned out to be important."

Additional experiments showed that cancer stem cell tumor-spheres, which are highly tumorigenic and resistant to chemo- and radiation-therapies, were substantially reduced in NRP2b knockdown models.

Specifically, significantly fewer cells developed gefitinib chemotherapy resistance in models that knocked out NRP2b (i.e., tumor environments with very low levels of 2b) and significantly more cells developed resistance in NRP2a knockout (i.e., tumor environments with very low levels of 2a).

Finally, the researchers looked at human tissue samples and found that NRP2b abundance in human lung tumors was correlated with a higher cancer stage and more advanced progression.

"EMT produces a wholesale change in the repertoire of growth factors and receptors. In tumors where EMT is underway, there's more resistance to treatment,” explained Drabkin. “What we found was that, in these epithelial tumors, if we block NRP2—especially the 2b protein—on the cell surface, they just did not respond in any way like control cells in terms of their ability to take on the EMT phenotype and migrate. By inhibiting that receptor we'd made a big dent in their ability to become resistant."

"Honestly, I was very, very surprised at how distinctly different the two isoforms were,” said Gemmill. “When we first realized that there was differential expression, I thought we would have to look really hard to find some very minor differences. The fact that they were opposites—one inhibiting and one promoting cancer cell migration—and that this difference fell out so quickly was astounding because there's such a short list of things that are different about these 2 isoforms."

Although there is still a lot to learn about how NRP2a and NRP2b function in both normal tissue as well as cancers, these discoveries open new avenues for potential therapies. Possibilities range from developing monoclonal antibodies to target the 2b isoform, to immunotherapies, to using NRP2b as a biomarker for predicting patients' responses to particular therapies.

Gemmill anticipates that, in the wake of their findings, researchers will start looking at the role of NRP2b in other disease areas.

"I think a lot of people are going to sit up when they read this article and say, 'I wonder what it does in my system?'” said Gemmill. “For example, fibrosis is often associated with a TGF-beta response and we now know that TGF-beta induces NRP2b. So, maybe, NRP2b plays a role in fibrosis that affects the kidneys, liver, and lungs.”   

Both researchers agree that this work exemplifies the importance of not dismissing small but bothersome findings.

Gemmill notes that many of the biggest breakthroughs in science start with one person sitting in a laboratory, pursuing a single, seemingly minor, phenomenon.

"If you see something that isn't quite right, don't dismiss it,” said Gemmill. “This is an exciting finding that came from not letting a detail slip by.”

"That's how we find out what's really going on in a system,” said Drabkin. “If you work out enough of the details, you start to see how things interact. The more you learn, the more you see how things connect and where the pivot points are that can have biologic consequences. Progress happens little-by-little until we find a weakness where we can direct therapy."

The team recently received a four-year Veteran's Administration Merit Award to further fund their work.

Summary: Boosting the immune system’s cancer-fighting ability is the aim of adoptive T cell immunotherapy, in which the patient’s T cells are harvested, expanded, and then reinfused. Ensuring T cell persistence after reinfusion has been a challenge. In the October 15, 2016 Cancer Research, investigators at the Medical University of South Carolina and Loyola University report that culturing T cells in N-acetyl cysteine (NAC) significantly improves persistence. Adding NAC to melanoma immunotherapy protocols could improve outcomes.

A collaborative team of investigators at the Medical University of South Carolina (MUSC) and Loyola University have demonstrated for the first time that culturing T cells in N-acetyl cysteine (NAC) before they are infused as immunotherapy improves effectiveness and outcomes in a preclinical model of melanoma. These findings were reported in the October 15, 2016 issue of Cancer Research.

Both incidence and mortality rates for metastatic melanoma continue to rise. Only about 15% of Stage IV melanoma patients receiving standard treatment can expect to survive for five years. By contrast, clinical trial data show that up to 40% of Stage IV melanoma patients survive for five years when treated with adoptive cell therapy (ACT), a form of immunotherapy that calls for infusion of autologous, melanoma-specific T cells.

ACT aims to boost a patient’s own immune responses against the cancer. To do this, the patient’s own T cells are harvested, genetically modified with a therapeutic T cell receptor, activated, and then rapidly expanded to generate large numbers of T cells for therapeutic re-infusion. Unfortunately, patient responses vary. Better outcomes are positively correlated with persistence of the transferred cells. The rapid expansion of harvested T cells before reinfusion increases their susceptibility to activation-induced cell death (AICD), prompting the authors to hypothesize that AICD reduces ACT’s overall effectiveness.

Researchers have long known that factors limiting T cell persistence also limit ACT efficacy but, until now, no one knew that something as simple as changing the culture condition by supplementing with NAC could improve survival of the reinfused T cells. The research team showed that adding NAC to the in vitro T cell expansion culture prevents increases in the DNA damage marker ?H2AX and significantly improves T cell persistence and immunotherapy outcomes, including reduced tumor growth and enhanced survival.  

The team found that nearly 40% of NAC-cultured T cells were detectable in tumors after transfer compared to approximately 1.2% of standard-culture T cells. They also found that mice receiving NAC-cultured cells experienced significantly delayed tumor growth compared to mice receiving standard-culture cells (P<.0001).

"We were really surprised by the number of adoptively transferred T cells we saw in the tumor,” said Christina Voelkel-Johnson, Ph.D., Associate Professor of Microbiology and Immunology at MUSC's Hollings Cancer Center and the senior author on the article. “Given the harsh environment T cells encounter within tumors, we did not expect that the number of NAC-cultured T cells would be 33-fold higher than T cells not cultured in NAC.”

Adding NAC to existing protocols should pose little risk to patients, according to Voelkel-Johnson, because NAC is already in clinical use for many indications and the culturing of T cells in NAC would occur outside of the patient. “The only difference would be a change to the cell culture protocol in an effort to generate cells with an improved phenotype," said Voelkel-Johnson.

The road to discovering the protective role of NAC began with the MUSC/Loyola research team hypothesizing that preventing T cells from becoming susceptible to AICD during in vitro expansion might improve their persistence and effectiveness upon reinfusion.

The team started by examining the role of p53, which is crucial for coordinating cellular stress responses and determining the fates of damaged cells (i.e., whether they will be repaired or allowed to die). While it is known that inhibiting p53 protects T cells from AICD, the molecular-level mechanisms that provide this protection were unknown. The investigators created conditions similar to those that occur during in vitro T cell expansion by re-stimulating the T cell receptors (TCR) to assess p53 status and cell death. They found that AICD following TCR re-stimulation is accompanied by phosphorylation of p53 on Ser15 and accumulation of p53 in the cell nucleus.

The next set of experiments clarified that the PI3K-like serine kinase, ataxia telangiectasia mutated (ATM), is necessary for the phosphorylation of p53 on Ser15 after TCR restimulation. They also found that inhibiting ATM almost completely prevents cell death (99%) after TCR restimulation. Thus, ATM appears to be a required upstream factor for AICD onset.

While activation of ATM and p53 are parts of a known DNA damage response pathway, ATM also responds to oxidative stress or hypoxia. So, the team needed to determine whether the changes in ATM and p53 were being caused by DNA damage or oxidative stress/hypoxia. They looked at two established DNA damage markers, gH2AX and p-SMC-1, and found that, within 15 minutes of TCR restimulation, both gH2AX and p-SMC-1 increased three-fold. This suggested that DNA damage and ATM activation occur in parallel, leading the authors to conclude that TCR restimulation causes DNA damage, which then triggers the ATM/p53 DNA damage response pathway.

Because it is known that AICD depends on the generation of reactive oxygen species (ROS), the investigators focused on ROS as a potential cause of DNA damage. They incubated some T cells with the antioxidant NAC prior to restimulation and compared them to T cells that were expanded using a standard protocol. Results showed that ROS levels were significantly lower in the NAC cultures than in the standard cultures. In addition, pretreatment with NAC reduced activation of the DNA damage markers, ?H2AX and p-ATM, by 94% and 69%, respectively. These results confirmed that ROS generated during TCR restimulation play a central role in damaging the DNA.

This is the first study to show that expanding therapeutic T cells in the presence of NAC prior to adoptive transfer improves their ability to resist AICD. Most importantly, using these "AICD-resistant" T cells improves therapeutic outcomes in a preclinical model by enhancing T cell persistence, increasing tumor control, and improving survival.

"Now we are looking at studies to help us understand exactly how NAC changes the phenotype of T cells,” said Voelkel-Johnson. “How does it make these cells survive? How is trafficking to tumors improved? There may be benefits to culturing T cells in NAC aside from generating AICD resistance that we haven't yet recognized.”

MUSC Health Rheumatologist Dr. Jim Oates

 

Summary: Medical University of South Carolina (MUSC) investigators report preclinical research showing that prognostic models for lupus nephritis that include novel biomarkers have significantly improved predictive power over models using only traditional markers, in the August 2016 issue of Arthritis & Rheumatology. Data reveal that chemokines, cytokines, and markers of cellular damage were most predictive of patients' therapeutic response. This is a critical first step to developing clinically meaningful, decision-support tools in lupus nephritis.

 

 

Caption: MUSC Health rheumatologist Jim C. Oates, M.D.

Results of preclinical studies by investigators at the Medical University of South Carolina (MUSC) reported in the August 2016 issue of Arthritis & Rheumatology demonstrate for the first time that including novel biomarkers in lupus nephritis (LN) prognostic models significantly increases their power to predict therapeutic efficacy. Identifying biomarker models with sufficient predictive power is a critical step toward developing clinical decision-making tools that can rapidly identify patients who require a change in therapy and potentially reduce onset of renal fibrosis during induction therapy.

Approximately half of all patients with systemic lupus erythematosus (SLE) develop LN, an immune complex-mediated glomerulonephritis. Lupus nephritis, in turn, leads to renal failure in up to 50% of patients within five years. American College of Rheumatology guidelines recommend changing LN treatment after six months of induction therapy if response to therapy is not achieved. However, 'response to therapy' is not clearly defined and renal damage can occur during the six-month induction period.

Currently, clinicians monitor response to treatment via blood pressure measurements, serum complement levels, anti-double-stranded DNA (anti-dsDNA) antibody levels, urinary sediment, urinary protein-to-creatinine ratios, and surrogates of renal function. Unfortunately, predicting disease progression is difficult using these traditional biomarkers due to their low sensitivity and high LN heterogeneity at presentation. Even when machine learning models are employed, traditional biomarkers are only 69% accurate in predicting a LN diagnosis among SLE patients. There is a need for individualized, decision-support tools that can better define 'therapeutic response' at the start of therapy and allow clinicians to tailor induction therapy to disease severity to prevent renal damage and unnecessary drug toxicity.

"We saw our colleagues' frustration in trying to come up with predictive models,” said Jim C. Oates, M.D., Associate Director of the MUSC Clinical and Translational Research Center, Associate Professor of Rheumatology, and senior author on the article. “The traditional markers we use in clinic today have quite limited predictive capacity. All lupus patients have varying degrees of kidney damage and levels of involvement of the different kidney structures. So, we wanted to account for this heterogeneity and the stages of disease progression. We wanted to include markers for pathways of inflammation as well as for damage."

The research team hypothesized that a targeted panel of urinary biomarkers reflecting initial resident and inflammatory cell activation (cytokines), signals for homing to the kidney (chemokines), activation of inflammatory cells (growth factors), and damage to resident cells, combined with artificial intelligence/machine learning modeling, might provide an early LN decision-support tool that could predict outcomes better than standard biomarkers alone. The team also chose to assess urine biomarkers rather than serum/plasma markers to increase the tool's sensitivity and specificity to signals of renal (rather than systemic) processes.

Urine samples from 140 patients with biopsy-proven LN who had not yet started induction therapy were analyzed for a panel of novel biomarkers using pre-mixed, commercially available kits. Univariate, receiver operating characteristic (ROC) curves were generated for each biomarker and compared to ROC area under the curve (AUC) values from machine learning models developed using random forest algorithms. Outcome models using novel biomarkers plus traditional clinical markers demonstrated greater AUC and significance compared to models developed with traditional markers alone ([AUC 0.79; P<0.001] vs. [AUC 0.61; P=0.05], respectively). The combined models also demonstrated greater power to correctly predict LN therapy outcomes (responder versus non-responder) than models using only traditional markers (76% vs. 27%, respectively [p<0.002]).

The team identified chemokines, cytokines, and markers of cellular damage as most predictive of LN therapy response. Race, anti-double-stranded DNA antibodies, and induction medication did not significantly contribute to the model.

"We were somewhat surprised by some of the analytes that were important in the model,” said Oates. "One traditional marker, protein-to-creatinine ratio, was the third most important, and a standard kidney function measure was the ninth. I was also surprised to see interluekin-8 so high. This is in keeping with recent publications highlighting the importance of neutrophils in the pathogenesis of lupus, however."

Including multiple mechanisms of disease pathogenesis and cellular damage likely provides a more effective diagnostic approach by better reflecting the multi-stage, heterogeneous nature of LN. This is the first study to combine a broad biomarker panel with machine learning techniques to optimize disease outcome models. "This could apply to any model where there is kidney inflammation leading to damage,” said Oates. "It's proof of concept for other kidney diseases that you can take a discovery model and incorporate machine learning to develop and validate predictive models."

The team is now testing other biomarkers and applying the model in a larger patient population to ensure external validity and improve power. They are also exploring other inputs.

"Our next approach is to harness existing data in the medical record to enhance predictions,” said Oates. “This is much more immediately translatable in the clinic than getting through a long FDA validation process and the industry pipeline. Using medical record data is cheaper, and there are patient and system factors in the medical record that you can't measure with an assay, such as economic and societal disparities, which affect outcomes. This approach could also be used to enhance biomarker predictive models”

Induced Pluripotent Stem Cell

Researchers from the Medical University of South Carolina and elsewhere devise new method to enhance genome-wide association studies for liver disease.

Caption: This image shows induced pluripotent stem cells expressing a characteristic cell surface protein called SSEA4 (green).Image courtesy of Stephen A. Duncan of the Medical University of South Carolina.

A research team including developmental biologist Stephen A. Duncan, D. Phil., SmartStateTM Chair of Regenerative Medicine at the Medical University of South Carolina (MUSC), has found a better way to purify liver cells made from induced pluripotent stem cells (iPSCs). Their efforts, published August 25, 2016 in Stem Cell Reports,1 will aid studies of liver disease for the National Heart, Lung, and Blood Institute (NHLBI)’s $80 million Next Generation Genetic Association Studies (Next Gen) Program. The University of Minnesota (Minneapolis) and the Medical College of Wisconsin (Milwaukee) contributed to the study.

 This new methodology could facilitate progress toward an important clinical goal: the treatment of patients with disease-causing mutations in their livers by transplant of unmutated liver cells derived from their own stem cells. Previous attempts to generate liver-like cells from stem cells have yielded heterogeneous cell populations that bear little resemblance to diseased livers in patients.

NHLBI’s Next Gen was created to bank stem cell lines sourced from patients in genome-wide association studies (GWAS). The goal of the NHLBI Next Gen Lipid Conditions sub-section—a collaborative effort between Duncan and Daniel J. Rader, M.D., and Edward E. Morrisey, Ph.D., both at the University of Pennsylvania—is to help determine the genetic sources of heart, lung, or blood conditions that also encompass the liver. These GWAS studies map the genomes in hundreds of people as a way to look for genetic mutation patterns that differ from the genomes of healthy individuals.

A GWAS study becomes more powerful—more likely to find the correct genetic mutations that cause a disease—as more genomes are mapped. Once a panel of suspected mutations is built, stem cells from these individuals can be “pushed” in culture dishes to differentiate into any of the body’s cells, as for example liver-, heart-, or vascular-like cells. The cells can be screened in high-throughput formats (i.e., cells are expanded and cultured in many dishes) to learn more about the mutations and to test panels of drugs that might ultimately help treat patients harboring a disease.

 The problem arises during the “pushing.” For example, iPSCs stubbornly refuse to mature uniformly into liver-like cells when fed growth factors. Traditionally, antibodies have been used to recognize features of maturity on the surfaces of cells and purify cells that are alike. This approach has been crucial to stem cell research, but available antibodies that recognize mature liver cells are few and tend to recognize many different kinds of cells. The many types of cells in mixed populations have diverse characteristics that can obscure underlying disease-causing genetic variations, which tend to be subtle.

“Without having a pure population of liver cells, it was incredibly difficult to pick up these relatively subtle differences caused by the mutations, but differences that are important in the life of an individual,” said Duncan.

Instead of relying on antibodies, Duncan and his crew embraced a new technology called chemoproteomic cell surface capture (CSC) technology. True to its name, CSC technology allowed the group to map the proteins on the surface of liver cells that were most highly produced during the final stages of differentiation of stem cells into liver cells. The most abundant protein was targeted with an antibody labeled with a fluorescent marker and used to sort the mature liver cells from the rest.

The procedure was highly successful: the team had a population of highly pure, homogeneous, and mature liver-like cells. Labeled cells had far more similar traits of mature hepatocytes than unlabeled cells. Pluripotent stem cells that had not differentiated were excluded from the group of labeled cells.

“That’s important,” said Duncan. “If you’re wanting to transplant cells into somebody that has liver disease, you really don’t want to be transplanting pluripotent cells because pluripotent cells form tumors called teratocarcinomas.”

Duncan cautions that transplantation of iPSC-derived liver cells is not yet ready for translation to the clinic.  But the technology for sorting homogeneous liver cells can be used now to successfully and accurately model and study disease in the cell culture dish.

“We think that by being able to generate pure populations, it will get rid of the variability, and therefore really help us combine with GWAS studies to identify allelic variations that are causative of a disease, at least in the liver,” said Duncan.

Reference

Mallanna SK, et al. Mapping the cell-surface N-glycoproteome of human hepatocytes reveals markers for selecting a homogeneous population of iPSC-derived hepatocytes. Stem Cell Reports. ePub August 25, 2016. doi: http://dx.doi.org/10.1016/j.stemcr.2016.07.016

Summary: Medical University of South Carolina (MUSC) investigators report preclinical research showing that Krüppel-like factor 12 (KLF12) promotes colorectal cancer (CRC) cell growth by activating early growth response protein 1 (EGR1), in the July 2016 issue of PLOS One. Data also reveal that levels of KLF12 and EGR1 correlate synergistically with a poor CRC prognosis. Results indicate that KLF12 plays an important role in CRC progression and provides a potential novel prognostic marker and therapeutic target.

Results of preclinical studies by MUSC investigators reported in the July 2016 issue of PLOS One (doi:10.1371/journal.pone.0159899) demonstrate for the first time that the transcription factor Krüppel-like factor 12 (KLF12) promotes poor colorectal cancer (CRC) cell growth, in part, by activating EGR1. Furthermore, data demonstrate that KLF12 and early growth response protein 1 (EGR1) levels synergistically correlate with  CRC prognoses.

CRC is the third most common and third deadliest cancer in the US. Like most cancers, CRC development is spurred by a series of genetic mutations and epigenetic changes that alter gene expression. In turn, this altered gene expression initiates tumors and supports their progression. Thus, transcription factors that regulate gene expression and signaling pathways during carcinogenesis have long been studied as potential therapeutic targets.

Dr. Raymond DuBois, Dean of the MUSC College of Medicine, Professor of Biochemistry and Molecular Biology, and senior author on the article is focused on understanding the role of inflammation in cancer. "We've been studying the connections between inflammation and cancer in my lab for some time now and have determined that some inflammatory mediators stimulate the progression of cancer,” DuBois said. “We found that KLF12 was increased dramatically in the presence of inflammation in certain cancers, so we were trying to determine the specific molecular mechanisms responsible for these effects."

Other researchers who were studying kidney development previously identified transcription factor KLF12 as a transcriptional repressor of the AP-2? gene. It was then discovered that AP-2? expression is also reduced in advanced CRC tumor tissue compared to matched normal tissue and that loss of AP-2? promoted CRC invasion. This connection illuminated a potential link between KLF12 and CRC. In vitro studies show that KLF12 promotes gastric cancer (GC) cell proliferation and invasion, and that KLF12 levels are elevated in about 40% of poorly differentiated GCs and correlate with tumor size. Furthermore, recent genome-wide analyses find high KLF12 levels in approximately 40% of esophageal adenocarcinomas and in 45% of salivary tumors. Until now, however, the role of KLF12 in CRC remained unclear.

The MUSC research team designed a series of in vitro and in vivo experiments to clarify the role of KLF12 in CRC. The first set of studies examined KLF12 expression in seven human CRC cell lines. They found not only that KLF12 was expressed in six of the seven cell lines, but also that its overexpression led to increased cell numbers and KLF12 knockdown led to reduced cell numbers. In addition, they also found that overexpression of KLF12 led to the formation of larger cecal tumors while KLF12 knockdown led to formation of smaller cecal tumors, compared to controls. Thus, this set of experiments indicates that KLF12 promotes CRC growth by enhancing cancer cell proliferation and/or survival.

The next set of experiments focused on clarifying which KLF12 target genes may be involved in regulating CRC growth. Using microarray assays, the researchers found that KLF12 overexpression altered multiple genes including EGR1. It has been previously reported that KLF12 regulates expression of some target genes by binding to the CACCC motif.  They found that the EGR1 promoter contains two possible KLF12 DNA-binding motifs located at -1488bp (motif 1) and -808bp (motif 2) relative to the transcription start site. Using ChIP assay, the MUSC researcher team found that KLF12 does, indeed, bind strongly to the EGR1 promoter motif 2 but not to motif 1. In vitro experiments demonstrated that, at both the mRNA and protein levels, CRC cells with undetectable levels of KLF12 expressed the lowest levels of EGR1 compared to cells expressing high levels of KLF12. In vivo studies using mice implanted with CRC tumor cells that overexpressed KLF12 showed that EGR1 expression was up-regulated compared to mice implanted with control cells. Furthermore, staining of human CRC tissue specimens produced the same pattern. Taken together, these results indicate that KLF12 directly activates EGR1 in CRC.

The third set of experiments looked at whether EGR1 mediated the effects of KLF12 on tumor cell growth. Results showed that EGR1 knockdown reduced KLF12-induced tumor cell growth, whereas EGR1 overexpression promoted CRC cell growth in vitro as well as tumor growth in the mouse model. The results of this set of studies, thus, indicate that KLF12 enhances CRC cell growth by activating EGR1.

The final set of experiments evaluated whether KLF12 and EGR1 levels correlate with CRC patients' prognoses. Using gene expression data from publicly available microarray databases (Moffitt [n = 177]; Vanderbilt Medical Center [n = 55]), CRC patients were stratified by level of KLF12 and/or EGR1 expression. These data showed that patients with high levels of either KLF12 or EGR1 had worse outcomes compared to those with low levels of these genes, and that those with high levels of both KLF12 and EGR1 had the lowest survival rates.

This is the first study to clarify the role of KLF12 in CRC tumor growth and progression, which appears to occur, at least in part, through EGR1 activation. The finding that synergistic contributions of KLF12 and EGR1 produce the worst outcomes among CRC patients illuminates their potential in developing novel therapies. More studies are needed to further clarify the role of KLF12 in CRC progression and its potential as a novel prognostic marker and therapeutic target. 

Vitamin D Illustration

Summary: Investigators at the Medical University of South Carolina and Ralph H. Johnson VA Medical Center report clinical research showing that African-American and European-American men with prostate cancer exhibit significantly different expression of genes associated with immune response and inflammation, in the July 2016 issue of  Pharmacogenomics. Systems-level, RNA analyses support the concept that inflammatory processes may contribute to racial disparities in disease progression and that vitamin D3 supplementation can modulate pro-inflammatory transcripts.

The results of clinical studies by investigators at the Medical University of South Carolina (MUSC) and the Ralph H. Johnson VA Medical Center (VAMC), reported in the July 2016 issue of Pharmacogenomics (doi:10.2217/pgs-2016-0025) , demonstrate transcriptome-level linkages between racial disparities in circulating levels of vitamin D and expression of pro-inflammatory genes in African American (AA) patients with prostate cancer compared to European American (EA) patients.

Racial disparities in prostate cancer are well documented with AA men having significantly higher risk of developing prostate cancer and significantly higher mortality rates than EA men. In addition, among patients presenting at the same disease stage, AA men often have higher prostate-specific antigen (PSA) levels and higher-grade tumors than EA men. However, the biological mechanisms underlying these substantial and persistent disparities are unclear.

Researchers at MUSC and VAMC noticed that racial disparities in prostate cancer mirror differences in circulating levels of vitamin D between AA and EA patients. Vitamin D3 is known to have multiple anti-cancer actions including suppression of cyclo-oxygenase-II (an independent predictor of cancer recurrence) and inhibition of IL-8 (an angiogenic, pro-inflammatory cytokine). Prostate cells express the vitamin D receptor, vitamin D-25-hydroxylase, 25 hydroxyvitamin D-1-alpha-hydroxylase, and 25-hydroxyvitamin D-24-hydroxylase. Thus, normal prostate cells can synthesize 25(OH)D3 (calcidiol) from vitamin D (cholecalciferol), and 1,25(OH)2D3 (calcitriol) from 25(OH)D3. 1,25(OH)2 D3 (calcitriol) is the bioactive, hormonal, and most potent form of vitamin D and facilitates cell-to-cell communication via paracrine/autocrine pathways.

Sebastiano Gattoni-Celli, M.D., Professor of Radiation Oncology at MUSC, and senior author on the article, explains how his team came to explore the therapeutic potential of vitamin D supplementation in prostate cancer, "A lot of previous work shows that D3 levels are much lower in African Americans than in European Americans and it's well established that prostate cells are very sensitive to vitamin D levels. So this raised the possibility that long-term vitamin D deficiency may contribute to the progression of prostate cancer, especially among African American men. We began to wonder whether eliminating racial disparities in circulating levels of vitamin D, through supplementation, could help reduce the disparities we see in prostate cancer outcomes."

The team designed a prospective, placebo-controlled, clinical study to investigate the effects of a daily 4,000 IU vitamin D3 supplementation over a two-month period among 27 men (10=AA, 17=EA) who had elected to treat their prostate cancer via prostatectomy. A trial length of two months was chosen to leverage the recommended, standard-of-care recovery period between their biopsy and surgery procedures. Using high-throughput RNA sequencing, they performed a series of genome-wide expression profiling experiments to generate transcriptional profiles of patients' prostate tissue samples and assessed them using systems-level analyses. Their primary aims were to: (1) illuminate any molecular differences in gene expression that may be related to prostate cancer disparities between AA and EA men; and (2) investigate any effects vitamin D supplementation may have on the prostate transcriptome.

Not only did the team find significant differences in gene expression between AA and EA men but also between AA men receiving vitamin D supplements and AA men receiving placebo. Due to the size of the RNA sequencing dataset, results are reported as adjusted p-values (or q-values) which represent the smallest 'false discovery rate' at which a result can be called significant. A total of 3,107 prostate genes were differentially expressed between the AA and EA groups (q<0.1) with 8,238 differentially expressed transcripts between AA and EA subjects (q<0.4). Analyses of these found that AA study patients had substantially elevated expression of transcripts related to immune response and inflammation.

"The number of genes expressed differently in AA and EA was a really big surprise—we found differences in over 8,000 genes,” said Gattoni-Celli. “I expected something but not this massive difference and it was not a fluke. When we compared our results with previous studies using a less advanced technology, we saw that they, too, found these differences, but not as many.”

“Our findings captured all of the differences observed in previous studies but also many more because newer RNAseq technology and Big Data analytical approaches allowed us to see the transcriptome in greater detail,” noted Gary Hardiman, Ph.D., Professor of Medicine and Public Health Sciences and Bioinformatics Director for the Center for Genomic Medicine at MUSC, and co-senior author on the article. “This analysis was performed using the OnRamp BioInformatics Genomics Research Platform we deployed at MUSC a little over a year ago. Our approach converged advanced genomics analysis, comprehensive data management, big data analytics and hyperscale servers. A ‘Big Data’ analytical pipeline that utilized Hadoop software was implemented. This enabled an automated RNAseq workflow to process the patient data and explore differential prostate gene expression analysis between AA and EA men and sensitively interrogate the effects of vitamin D supplementation with robust statistical power.”

When comparing AA men receiving vitamin D supplementation to AA men receiving placebo,  the team found 817 differentially expressed genes (q<0.4). However, no similar difference in gene expression was observed between EA men receiving vitamin D supplementation versus placebo. Comparing the 8,238 differentially expressed transcripts between AA and EA subjects with the 817 genes that were differentially expressed among AA men receiving vitamin D supplementation and AA men receiving placebo, the team found an overlap of 346 genes. This finding suggests that a substantial number of genes that are differentially expressed across racial groups can be affected by a brief (2-month) course of vitamin D3 supplementation in AA patients.

This research is an important step in understanding the molecular underpinnings of health disparities in prostate cancer. Further clarification of race-based transcriptome differences and the role of vitamin D in prostate tissue may lead to use of vitamin D3 supplementation as an early-stage therapeutic option in prostate cancer. Furthermore, results from studies among AA and EA women with breast cancer could extend these findings because breast cancer, like prostate cancer, is an endocrine cancer, with many similarities including sensitivity to vitamin D.

An accompanying editorial by Batai K and Kittles RA, "Can vitamin D supplementation reduce prostate cancer disparities?" was published in the same issue of Pharmacogenomics (volume 17, number 11, 2016, pages 1117-1120).   

Predicting speech fluency after stroke. Brain images showign features of damage to grey-matter and white-matter regions of brain, reflecting their importance in predicting speech fluency.

Image Caption: Predicting speech fluency after stroke. These are features of gray-matter cortical regions (left) and white-matter tracts (right), reflecting their importance in predicting speech fluency scores. Regions/connections are marked in red when they strongly influence speech fluency, in blue when their influence is moderate, and are left uncolored when the influence is weak or non-existent. Image used courtesy of Dr. Leonardo Bonilha and Dr. Grigori Yourganov of the Medical University of South Carolina, who own the copyright for the image. Published in the June 22 issue of the Journal of Neuroscience (doi:10.1523/JNEUROSCI.4396-15.2016).

Loss or impairment of the ability to speak is one of the most feared complications of stroke—one faced by about 20% of stroke patients. Language, as one of the most complex functions of the brain, is not seated in a single brain region but involves connections between many regions.

In an article published in the June 22, 2016 issue of the Journal of Neuroscience (doi:10.1523/JNEUROSCI.4396-15.2016), investigators at the Medical University of South Carolina (MUSC) and the University of South Carolina (USC) report that mapping all of the brain’s white matter connections after stroke, in addition to imaging the areas of cortical tissue damage, could better predict which patients will have language deficits and how severe those deficits will be. The totality of the brain’s connections is referred to as the connectome.

“Imaging the connectome of patients after stroke enables the identification of individual signatures of brain organization that can be used to predict the nature and severity of language deficits and one day could be used to guide therapy,” said MUSC Health neurologist Leonardo Bonilha M.D., Ph.D., senior author on the Journal of Neuroscience article, whose laboratory focuses on connectome imaging, particularly as it relates to language loss after stroke. Grigori Yourganov, Ph.D., is the first author on the article. Julius Fridriksson, Ph.D., Chris Rorden, Ph.D., and  Ezequiel Gleichgerrcht, Ph.D, aphasia researchers at USC who recently received NIH funding to establish a Center for the Study of Aphasia Recovery and who are long-time collaborators of the Bonilha laboratory, are also authors on the article.

This study is the one of the first to use whole-brain connectome imaging to examine how disruptions to white matter connectivity after stroke affect language abilities. White matter fiber tracts are the insulated wires that connect one area of the brain to others. White matter is named for the myelin sheaths (insulation) that cover the many axons (wires) that make up the fiber tracts.

“If you have two brain areas and both of them have to work together in order to carry out a function and the stroke lesion takes out axons that connect those brain areas—the two areas are intact but the communication between them is disrupted and so there is dysfunction,” said Yourganov.

Currently, structural magnetic resonance imaging (MRI) is used after stroke to assess lesions in the cortical tissue—the brain’s grey matter. However, the extent of cortical damage often does not correlate with the severity of language deficits.

“Stroke patients sometimes have significant impairments beyond the amount of cortical damage,” said Bonilha. “It is also hard to predict how well a patient will recover based on the cortical lesion alone.”

Could connectome-based imaging be a useful complement for assessing damage to the brain’s connections after stroke and for guiding rehabilitative therapy?

The study led by Bonilha took an important first step toward answering these questions. The study, which enrolled 90 patients at MUSC and USC with aphasia due to a single stroke occurring no less than six months prior, assessed four areas related to speech/language using the Western Aphasia Battery—speech fluency, auditory comprehension, speech repetition, and oral naming—as well as a summary score of overall aphasia. Within two days of behavior assessment, each of the patients underwent imaging studies—both T1- and T2- weighted MRI, typically used after stroke to map cortical damage, and diffusion imaging, used for connectome mapping. The team then used a type of machine learning algorithm—support vector regression (SVR)—to analyze the imaging results and make predictions about each patient’s language deficits.  In essence, an algorithm was created that could derive the WAB score from either a feature relevant to imaging of the grey matter damage by structural MRI or a feature relevant to connectome imaging of the brain’s white matter fiber tracts. The team used 89 of the 90 patients as training sets for SVR and then used the algorithm to predict language defect/preservation in the 90th patient. This was done for each of the 90 patients and, in each patient, for both features identified via structural MRI and connectome imaging.

The accuracy of the algorithm’s prediction of WAB score for each patient was then assessed by comparing it to the WAB score determined via behavioral testing. Connectome-based analysis was as accurate as cortical lesion mapping for predicting WAB scores. In fact, it was better at predicting auditory comprehension scores than was lesion-based imaging using structural MRI and only slightly less accurate at predicting speech fluency, speech repetition, and naming scores.

The study demonstrates that damage to the white matter fiber tracts that connect the brain’s regions plays a role beyond cortical damage in language impairment after stroke. Furthermore, this study also discloses that connections in the brain’s parietal region are particularly important for language function, especially fluency. This region is less likely to sustain damage after stroke, even in patients who experience aphasia, suggesting that damage or preservation of the brain’s connections in this region could play a key role in determining who will experience aphasia and who will have the best chances for recovery. The integrity of these connections could not be mapped with conventional structural MRI but can now be assessed through connectome-based analysis.

The study findings also suggest that connectome-based analysis could be used to inform a more individualized approach to stroke care. Because the algorithms developed using these study patients as the training set are generalizable to a broader stroke population, connectome-based analysis could one day be used to identify the distinctive features of each patient’s stroke—which connections have been lost and which preserved—and then the algorithm could be used to predict the type and severity of language impairment and the potential for recovery. This information could then be used to direct rehabilitative therapy to improve outcomes. 

“By mapping much more accurately the individual pattern of brain structural connectivity in a stroke survivor, we can determine the integrity of neuronal networks and better understand what was lesioned and how that relates to language abilities that are lost,” said Bonilha. “This is, broadly stated, a measure of post-stroke brain health. It is the individual signature pattern that could also be used to inform about the personalized potential for recovery with therapy and guide treatments to focus on the deficient components of the network.”

Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days.

 

 

 

 

 

 

 

 

 

Summary: In an article published online in Nature Cell Biology on July 11, 2016, investigators at the Medical University of South Carolina report preclinical findings suggesting that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis. Maintaining Dab2 by inhibiting cathepsin B could prevent tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

The results of preclinical studies by investigators at the Medical University of South Carolina (MUSC) reported in an article published online on July 11, 2016 in Nature Cell Biology (doi: 10.1038/ncb3388) demonstrate that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis.

While expression of Dab2—an endocytic adaptor and tumor suppressor—is known to occur during transforming growth factor-beta (TGF-beta)–mediated epithelial-mesenchymal transition (EMT), the mechanisms by which it regulates apoptosis were, until now, poorly understood.

Exploring the pathways by which Dab2 is degraded and the effects of maintaining Dab2 levels reveals its pivotal role in preventing tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

TGF-beta induces EMT—a process by which cells transform from a polarized epithelial phenotype to a fibroblastic or mesenchymal one. Dab2 is expressed during TGF-beta–mediated EMT. While EMT is essential for normal cellular growth and homeostasis, it is abnormally activated in tumor cells and contributes to their chemo-resistance and metastasis.

TGF-beta has also been reported to regulate autophagy, which, in established tumors, ensures tumor cell survival through times of stress, as for example during chemotherapy. In other words, autophagy supports the chemo-resistance, growth, and metastasis of tumor cells.

Researchers focused on the Dab2 protein after noticing that, in cells treated with TGF-beta, Dab2 levels rose over the initial 24-48 hours as they went through EMT but then fell with continued TGF-beta treatment. By day 7, the tumor cells had transitioned to a morphological state suggestive of either autophagy or apoptosis. Furthermore, the mesenchymal markers N-cadherin and vimentin, which like Dab2 initially rose during EMT, began to decline with longer exposure to TGF-beta.

"This was an unexpected finding that we followed,” explains senior author Philip Howe, Ph.D., Professor and Chair of Biochemistry and Molecular Biology and Hans and Helen Koebig Chair in Clinical Oncology at MUSC. “We knew that if you give cells TGF-beta they go through EMT, and we knew you needed Dab2 for TGF-beta–mediated EMT. But, when we kept adding TGF-beta for more sustained periods (after EMT took place), cells took on a different morphology and we noticed a loss of Dab2. We investigated this loss of Dab2 and discovered that it was being cleaved and that the cells were undergoing autophagy. Upon sustained TGF-beta treatment, the cells had lost their mesenchymal phenotype they'd gained in EMT and entered into an autophagic state.”

The team began to explore how prolonged TGF-beta treatment led to loss of Dab2 and the mesenchymal phenotype.

First, they found that longer TGF-beta exposure significantly increased cathepsin B (CTSB) expression and promoted its co-localization with Dab2. The team then not only demonstrated that CTSB is responsible for cleaving Dab2 but also that it recognizes the cleavage site by the flanking amino acids Val499 and Gly500. Thus, while an unaltered Dab2 sequence (Leu-Val-Gly-Leu) was degraded by CTSB, it did not cleave a mutant Dab2 sequence (Leu-Val-Leu).

Second, findings showed that, after 7 days, continuous TGF-beta treatment induced autophagy and down-regulated markers of apoptosis. This was particularly notable because these conditions promote tumor cell chemo-resistance and metastasis.

Third, they found that CTSB inhibition or expression of a mutant Dab2 without the CTSB cleavage site (i.e., the Leu-Val-Leu mutant) led to time-dependent increases in pro-apoptotic markers. When TGF-beta was withdrawn, cells in which Dab2 had been preserved underwent cell death. This series of experiments show not only how Dab2 is modulated by CTSB but also that it serves as a switch for regulating TGF-b–induced autophagy and apoptosis.

Another series of experiments were undertaken to clarify exactly how Dab2 functions to prevent autophagy and promote apoptosis. These findings show that Dab2 inhibits TGF-beta–induced autophagy by blocking the Vps-Beclin-1 interaction and promotes apoptosis by attenuating ERK-Bim interactions.

Finally, the team used the chemotherapeutic agent doxorubicin (DOXO) to determine whether the role of Dab2 in inhibiting autophagy might affect tumor cell chemo-sensitivity. They found that cells in which CTSB was overexpressed had increased survival in the presence of DOXO. However, cells with high Dab2 levels due to CTSB inhibition or expression of the CTSB-resistant Dab2 mutant were more chemo-sensitive and underwent apoptotic changes. Thus, Dab2 was shown to promote chemotherapeutic drug–induced cell death by attenuating drug-induced autophagy. In vivo tumor studies in mice further found that Dab2 both enhanced DOXO-mediated cell death and attenuated tumor cell metastasis.   

These direct insights into molecular mechanisms supporting tumor cell survival and death are crucial for maximizing the effectiveness of existing chemotherapeutic agents. "This is important because there aren't a whole lot of drugs out there," explains Howe. "Most of what we use today has been around for 20 or 30 years because of a lack of investment in basic science." 

The team's next steps are to investigate in vivo models for combination therapies using DOXO and a CTSB inhibitor to further illuminate the potential for targeting Dab2 as a means of reducing tumor recurrence and metastasis.

Image Caption: In the absence of Disabled-2 (Dab2), Vps34/Beclin-1 Interactions are maintained. Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days. Photos were taken by confocal microscope. Scale bars, 10 mm. The data show that in the absence of Dab2, due to CTSB overexpression, Vsp34/Beclin-1 interactions are maintained and autophagy is initiated. Adapted from a figure originally published in an article by Jiang Y, Woosley AN, Sivalingam N, Natarajan S, and Howe PH in Nature Cell Biology (doi: 10.1038/ncb3388).

 

 

Summary: Investigators at the Medical University of South Carolina report impressive 90-day outcomes in patients with large-vessel ischemic stroke who underwent thrombectomy using a direct-aspiration, first pass technique.

In an article published online April 16, 2016 by the Journal of Neurointerventional Surgery (doi: 10.1136/neurintsurg-2015-012211), investigators at the Medical University of South Carolina (MUSC) report promising 90-day outcomes for stroke patients with large-vessel clots who underwent thrombectomy or clot removal using the direct-aspiration, first pass technique (ADAPT).  Approximately 58% of stroke patients with a large-vessel clot removed using the technique achieved a good outcome at 90 days, defined as a Modified Rankin Score (mRS) of 0 to 2.

ADAPT aims to remove the clot in its entirety with a large-diameter aspiration catheter in a single pass. In contrast, stent retrievers, currently considered standard of care, frequently fragment the clot for removal and can require several passes.

ADAPT was developed by MUSC Health neuroendovascular surgeons M. Imran Chaudry, M.D., Alejandro M. Spiotta, M.D., Aquilla S. Turk, D.O., and Raymond D. Turner, M.D., all co-authors on the April 2016 Journal of Neurointerventional Surgery article. MUSC Health neurosurgery resident Jan Vargas, M.D., is first author on the article.

“The goal in ADAPT is to take the largest-bore catheter available up to the blood clot and put suction where it’s blocked and pull it out of the head to reestablish flow in that blood vessel,” said Turk. If the first-pass attempt is unsuccessful, stent retrievers can still be used to remove the clot.

In the article, the investigators report the results of a retrospective study of 191 consecutive patients with acute ischemic stroke who underwent ADAPT at MUSC Health. In 94.2% of patients, blood vessels were successfully opened—by direct aspiration alone in 145 cases and by the additional use of stent retrievers in another 43 cases. Good outcomes at 90 days (mRS, 0-2) were achieved in 57.7% of patients who were successfully revascularized with aspiration alone and in 43.2% of those who also required a stent retriever. The average time required to reopen the blocked blood vessels was 37.3 minutes—29.6?minutes for direct aspiration alone and 61.4 minutes for cases that also required stent retrievers. Patients presented for thrombectomy on average 7.8 hours after stroke onset.

These results confirm the promise of ADAPT, which was first described by the MUSC Health team in a seminal 2014 article in the Journal of Neurointerventional Surgery. Since the publication of that article, a number of single-center series studies have reported impressive recanalization times (the time it takes to open the blood vessel) and good neurological outcomes with ADAPT using a large-bore catheter, suggesting that it could offer an alternative approach to stent retrievers for mechanical thrombectomy.

Stent retrievers have been considered standard of care for stroke patients since the publication in the October 2015 issue of Stroke of a scientific statement on thrombectomy by the American Heart Association. That statement recommended rapid clot removal in addition to tissue plasminogen activator (tPA), a clot-busting drug that can minimize stroke complications if administered in a tight time window. The recommendation was based on the promising findings of five large clinical trials comparing treatment with tPA alone versus treatment with tPA plus thrombectomy using stent retrievers in large-vessel clots: MR CLEAN, EXTEND-IA,  ESCAPE, SWIFT PRIME, and REVASCAT.

A definitive answer as to whether ADAPT could likewise become standard of care for stroke patients with large-vessel clots will require clinical trials comparing the efficacy of the direct aspiration technique versus stent retrievers in this population of stroke patients.

The MUSC Health neuroendovascular surgery team is currently running the COMPASS trial (COMParison of ASpiration vs Stent retriever as first-line approach; Clinicaltrials.gov identifier NCT02466893) in conjunction with colleagues Dr. J. Mocco of Mount Sinai and Dr. Adnan Siddiqui of the University of Buffalo. The trial is randomizing patients to either ADAPT or a stent retriever as the initial thrombectomy technique. The trial, scheduled to enroll 270 patients, has enrolled 90 patients in the past year at ten sites in the United States.

Image Caption: Left: Frontal view of the skull showing occlusion of the right internal carotid artery (ICA) beginning at the level of the vertical petrous potion (arrow). Right: Frontal view of the skull after thrombectomy shows the revascularization of the ICA and the distal arteries supplying the right side of the brain.

SUMMARY: A genomics approach at the Medical University of South Carolina (MUSC) has unmasked genetic signatures in breast cancer cells that predict their sensitivity to certain drugs. The findings, published in the May 2, 2016 issue of Oncotarget, provide proof of concept for personalized pharmaceutical therapies that target the genes responsible for driving tumor growth.

Drug treatments for breast cancer patients might soon be designed based on the unique genetic autograph of their tumor.

A genomics approach at the Medical University of South Carolina (MUSC) has unmasked genetic signatures in breast cancer cells that predict their sensitivity to certain drugs. The findings, published in the May 2, 2016 issue of Oncotarget, provide proof of concept for personalized pharmaceutical therapies that target the genes responsible for driving tumor growth.

Dr. Stephen EthierCertain oncogenes drive solid tumor growth in some breast cancer patients but are just passenger genes in others—expressed but not essential for growth. As a result, tumors in different breast cancer patients may respond differently to the same treatment depending on which oncogenes are active and which are just along for the ride. Identifying the panel of active genes in a patient’s tumor—called the functional oncogene signature—could help an oncologist select therapies that target its growth, according to Stephen P. Ethier, Ph.D., Interim Director of the Center for Genomic Medicine at MUSC and senior author on the study.  

“In order to move the field forward, we now need to be able to use oncogene signatures to tailor therapies using combinations of targeted drugs so that multiple driving oncogenes can be targeted at once,” said Ethier.  “Doing this successfully requires the identification of the oncogenes to which the cancer cells are addicted, as this allows the use of targeted drugs at very low doses. Low doses are essential if we are to use combinations of different targeted drugs.”

Ethier’s group identified unique functional oncogene signatures in four different human breast cancer cell types. Using next-generation genome sequencing and genome silencing as each cancer cell type grew and multiplied, they assembled a list of genes for each cell type’s functional oncogene signature—those genes that were copy number amplified or point mutated, and most essential to cancer cell survival. Although thousands of candidate oncogenes were screened during experimentation, only a handful made the list—fewer than 20 for each cell type.

The brevity of each list facilitated selection of the best oncogene for pharmaceutical targeting. Because lower doses of targeted drugs can be highly effective, side effects could be reduced. For example, Ethier’s group found that targeting two or more members of a signature with much lower total drug concentrations in combination still killed cancer cells better than one higher-concentration drug alone.

Remarkably, a BCL2L1-targeted drug  that worked in one cell line also then worked in a fifth breast cancer cell line with a similar oncogene signature containing BCL2L1, an oncogene not normally associated with breast cancer. This work demonstrates that one signature-targeting treatment can be extended to more than one cancer cell type. This means that patients with other types of cancer who have a similar functional oncogene signature might benefit from drugs that target BCL2L1, which are already in development.

Ethier thinks that oncogenes identified in a tumor biopsy might one day soon provide a rational and individualized approach to pharmaceutical treatment with targeted drug combinations. Meanwhile, these findings from his laboratory—showing the importance of considering a patient’s functional oncogene signature before testing a new drug— could provide a rationale for redesigning clinical trials for breast cancer.

Stephen T. Guest, Ph.D., of the MUSC Department of Pathology & Laboratory Medicine, was first author on the study.

Stylized DNA replication fork. Illustration by Madelaine Price Ball

Mechanism of genome replication arrest provides pioneering insight about cell life span and aging.

A research collaboration between the Medical University of South Carolina, the Institute of Human Genetics in France, and Howard Hughes Medical Institute at Rockefeller University has revealed the means by which cells accomplish programmed DNA replication arrest. Their results in the June 13, 2016 issue of the Proceedings of the National Academy of Sciences describe the conditions that require a replication fork to stop, and in doing so explain why terminator sites on DNA don’t always successfully stop a replication fork. It is a matter of different proteins working together to calibrate fork movement.

In a process similar to a rail system in which trains follow a coordinated schedule of stops, cells use programmed fork arrest to halt the replication machinery at predetermined places along the DNA strand called terminator sites. Terminator sites minimize collision between replication machinery and transcription machinery traveling along the same track of DNA by blocking both processes at the halted fork. A collision might otherwise cause the DNA strand to break or become unstable. Programmed fork arrest also prevents replication and transcription machinery from running constantly, which helps conserve the amount of energy a cell needs to function.

These measures control cell life span and preserve genome stability, according to Deepak Bastia, Ph.D., Endowed Chair for Biomedical Research in the MUSC Department of Biochemistry and Molecular Biology and co-senior author of the study.

“Programmed fork arrest interconnects DNA replication with aging, transcription and gene differentiation,” says Bastia. “You have to maintain the genome so that genetic integrity and life span is maintained.”

During DNA replication and transcription, DNA polymerases travel along the double helix. During replication, one enzyme, a helicase, unwinds the double-stranded DNA into two single strands that travel behind it as it moves. DNA polymerases serve as templates on each single strand, allowing synthesis of two double-stranded daughter copies from one parent DNA strand. The junction where double-stranded DNA is separated into two single strands is aptly called the fork.

Only large proteins called histones that bind tightly to DNA are guaranteed to stop a replication fork in its tracks. The replication fork machinery easily sweeps other DNA-bound proteins out of the way. In one sense, this process keeps replication moving smoothly along the DNA strand. But in order to fine-tune their life cycle, cells need a more precise measure to stop replication other than the bulky histones. It turns out that a protein called Fob1 resides at terminator sites on DNA and works intermittently to halt fork progression, much like a gate. Its biochemical signal is phosphorylation.

It is this process that Bastia and his colleagues worked out. DDK, one of the two major cell cycle dependent kinases that sense which phase of life a cell is in, is responsible for assembling a replication fork blockage on terminator sites where Fob1 is bound. During active replication, the replication machinery easily pushes Fob1 off the DNA track and continues past the terminator sites. However, when DDK phosphorylates the helicase that unwinds double-stranded DNA at the head of the fork, it initiates the formation of a protein-based landing pad that connects to the helicase. An enigmatic protein named “Timeless” then docks on the landing pad and restrains other helicases that would normally sweep  off of the DNA terminator sites ahead of the moving fork. The Fob1 gate then stops the replication fork as programmed.

Bastia’s group showed this in yeast by genetically inactivating a component of DDK that is responsible for phosphorylating the helicase. In “chromosome combing” microscopy experiments, where single-stranded and double-stranded DNA were labeled with different colored fluorescent molecules and gently extended on coverslips, inactivated DDK failed to stop the replication fork. When active DDK was blocked from phosphorylating the helicase, Timeless protein could not reach the landing pad and the replication fork proceeded uninhibited. This physiologic program, which is similar across many organisms, is also likely to be conserved in humans, according to Bastia.

Bastia states that this new understanding will inform research on aging. Deciphering the means to prolong programmed fork arrest in healthy cells might eventually extend healthy life span in humans. “Aging is a disease,” he says, “not a natural process.”

Image caption: Stylized DNA replication fork with nucleotides matched, 5'->3' synthesis shown, no enzymes in diagram. Illustration by Madeline Price Ball. Obtained via wikimedia (Creative Commons License).

  The Structure of the Myo1c-Neph1 Complex

 

 

 

 

 

 

 

 

 

 Summary: Researchers at the MUSC used small-angle X-ray scattering to determine the full structure of the motor protein Myo1c and it complex with Neph1, a protein crucial for kidney filtration. Their findings suggest that Myo1c uses the actin cytoskeleton as a “track” for Neph1 transport—a finding with translational relevance for glomerular diseases such as diabetic nephropathy, as movement of Neph1 to and from the surface membrane triggers the injury/recovery response.

The motor protein Myo1c binds to Neph1, a protein crucial for ensuring effective filtration by the kidney, and serves as one mode of its cellular transport, according to findings by investigators at the Medical University of South Carolina (MUSC) and their collaborators report in an article in press at Molecular and Cellular Biology.

Neph1 is essential for the maintenance of podocytes—neuron-like cells with long finger-like projections that wrap around the glomerular capillaries, serving as the final barrier between the blood and the urine. When podocyte structure fails, the kidney is no longer able to act as an effective filter, and excessive protein escapes the capillaries into the urine. The resulting proteinuria can lead to kidney failure over time. Faulty podocyte structure and filtration also characterize diabetic nephropathy and glomerular diseases (e.g., focal and segmental glomerular sclerosis), which were once considered orphan diseases but have become more prevalent in recent years as sedentary lifestyles and diets high in processed foods have become commonplace.

The MUSC investigators and their collaborators used small-angle X-ray scattering (SAXS) to determine the full structure of Myo1c and its complex with Neph1. The crystallographic structure of two of Myo1c’s three segments had previously been determined but never that of the entire protein or the complex it forms with another protein.

 “Having a 3D crystal structure is more like a snapshot of the protein in three-dimensional space but of more physiological relevance is understanding how the proteins behave in solution,” says senior author Deepak Nihalani, Ph.D., an Associate Professor in the College of Medicine, Division of Nephrology, at MUSC. “We did a solution-based structure where we could actually get the structure of the entire protein and study that structure in complex with a cargo protein (Neph1).”

Nihalani’s team sent solutions of purified Myo1c to Brookhaven National Laboratory for SAXS analysis, examined the resulting SAXS intensity profiles to determine the structure of Myo1c in solution, and used molecular modeling to fill in the gaps of the 3D crystal structure. They also showed that Neph1 binds to the C tail region of Myo1c.

These findings suggest that Myo1c uses the actin cytoskeleton as a “track” for cellular transport of its Neph1 cargo (bound to its C tail). The movement of Neph1 through cellular space is known to be linked to the injury/recovery response. When injury occurs, Neph1 and other surface proteins are dislodged into the cytoplasm and must find their way back to the surface of the cell membrane to trigger the events needed for recovery.

As a means of transport for Neph1, Myo1c likely plays an important role in the injury/recovery response as well. However, it is unclear whether Myo1c favors renal protection or injury since it is not yet known in which direction Myo1c transports Neph1.

If it carries Neph1 from the surface into the cytoplasm and perinuclear space, it could be associated with more severe glomerular disease. In that case, Myo1c could be an attractive therapeutic target, as inhibiting it would prevent these proteins from leaving the cell membrane, an event that triggers the injury response. Over time, the inflammation associated with the injury response can lead to renal damage. If instead it ferries Neph1 back to the cell membrane, it could be essential in recovery after injury.

“These are early findings, but they show that Myo1c is critical to the transport of Neph1. Understanding whether that transport contributes to protection or injury could have translational importance for the treatment of glomerular diseases,” says Nihalani.

To solve that mystery, Nihalani and his collaborators have developed a mouse model in which Myo1c is knocked out in the podocytes and are conducting experiments to better understand the effects of Myo1c knockout on the injury/recovery response. They also hope to study whether Myo1c forms complexes with other proteins key for the maintenance of healthy renal function.

Image Caption: The Structural Domain of the Myo1c-Neph1 Complex. Image courtesy of  Dr. Deepak Nihalani. Reproduced from Molecular and Cellular Biology (10.1128/MCB.00020-16), with permission of the American Society of Microbiology.

Nonocarriers to help prevent kindney rejection after transplant

 

Congratulations to ToleRaM Nanotech, LLC  for winning a National TechConnect Innovation Award, which is meant to recognize innovative technology that has the potential to make a difference in a special sector, such as medicine. Only 15% of submitted entries receive the prestigious award.

ToleRaM Nanotech is a start-up company that grew out of a collaboration among MUSC Health transplant surgeon Satish Nadig, M.D., Ph.D,  biomedical engineer and nanotechnologist Ann-Marie Broome, Ph.D., MBA, and immunologist Carl Atkinson, Ph.D.

ToleRaM Nanotech, LLC was developed to use novel graft targeting nanoparticles to attenuate both acute and chronic rejection after transplant. Learn more by reading Progressnotes article Special Delivery.

 

Dr. Vincent Pellegrini, Chair of the Department of Orthpaedics, leading a revision hip replacementFigure 1 Logo

Join Dr. Vincent Pellegrini, Chair of the Department of Orthopedics at MUSC Health, on April 26 at 8 pm as he leads a virtual grand rounds on revision hip replacement on the free case-sharing app Figure 1 (http://figure1.com, app available on iOS and Android). Annotated surgical photos highlighting key moments in this complex surgery will drop one by one, with Dr. Pellegrini available live online to answer questions and respond to comments. Follow @MUSChealth on the app to participate in this event and see photographs from other complex and innovative surgeries at MUSC Health.

Anti-fibrotic effects of M10 in a mouse model of interstitial lung disease

Caption: Lung tissue isolated from mice that received bleomycin is characterized by extensive infiltration of inflammatory cells, thickening of the alveolar walls, and multiple fibrotic lesions with excessive amounts of extracellular matrix proteins (left). Lung tissue from mice receiving bleomycin + M10 shows significant reduction of cellular infiltrates, decreased thickness of alveolar septa, and reduced accumulation of extracellular matrix proteins (right). Images courtesy of Galina S. Bogatkevich, M.D., Ph.D. Reproduced from Translational Research (http://www.translationalres.com), Volume 170, April 2016, Pages 99–111, with permission from Elsevier.

Summary: Investigators at the Medical University of South Carolina report preclinical findings showing that the M10 peptide reduces collagen production and reverses fibrotic damage due to systemic sclerosis (SSc)–associated interstitial lung disease (ILD) in the April 2016 issue of Translational Research. ILD is one of the deadliest complications of SSc, a chronic autoimmune disease characterized by vasculopathy, autoimmunity, and excessive collagen production and deposition. Lung fibrosis carries a high risk of morbidity/mortality in SSc patients.

 

The results of preclinical studies by investigators at the Medical University of South Carolina (MUSC) reported in the April 2016 issue of Translational Research suggest that the M10 peptide could help protect against fibrotic damage in patients with systemic sclerosis, particularly in those who develop interstitial lung diseases (ILD), its deadliest complication.

Fibrotic diseases, which are characterized by excessive scarring due to overproduction by fibroblasts of collagen or extracellular matrix, account for more than 45% of U.S. deaths—more than cancer—and are estimated to cost $10 billion annually. Despite the prevalence of fibrotic diseases, only a handful of anti-fibrotic agents have been approved by the U.S. Food and Drug Administration, and none is available for systemic sclerosis.

In many ways, systemic sclerosis is the quintessential fibrotic disease, since its scarring can damage any part of the body. “Systemic sclerosis is often more than skin deep, affecting the gastrointestinal tract, the lungs, the heart, the kidneys, and the blood vessels, so it is a model for many other more prevalent fibrotic diseases,” says Richard M. Silver, M.D., Director of the Division of Rheumatology and Immunology at MUSC and a co-author on the article. “Whereas there may be 300,000 Americans with scleroderma/systemic sclerosis, millions of others suffer from fibrosis of these other organ systems.”

M10 is a ten-amino acid peptide formed from the natural cleavage of the receptor tyrosine kinase MET by the caspase 3. MET, also known as hepatocyte growth factor receptor, is thought to protect against injury and fibrosis, but the mechanisms by which it does so have remained unclear.

The MUSC investigators showed that M10 could protect against fibrotic injury in a bleomycin-induced model of ILD and that its anti-fibrotic effects are likely due to its modulation of the transforming growth factor beta 1 (TGF-?1) pathway. TGF-?1 is a cytokine that has been implicated in inflammation and fibrosis.

“We observed that treatment with M10 by intraperitoneal injection markedly improved bleomycin-induced lung fibrosis in mice, suggesting that the M10 peptide may have potential for use in the treatment of scleroderma-associated interstitial lung disease and other forms of pulmonary fibrosis, for example, idiopathic pulmonary fibrosis,” says Galina S. Bogatkevich, M.D., Ph.D., senior author on the Translational Research article. Lead authors for the article are Ilia Atanelishvili, M.S., of MUSC and Yuichiro Shirai, M.D., Ph.D., who holds a dual appointment at MUSC and the Nippon Medical School.

When instilled intratracheally, bleomycin induces fibrotic changes in the lungs, including peribronchial and interstitial infiltration of inflammatory cells, thickening of alveolar walls, and the development of numerous fibrotic lesions with excess deposition of extracellular matrix protein. Using this bleomycin-induced model of lung fibrosis, the MUSC investigators evaluated the anti-fibrotic effects of M10, using a scrambled peptide as a control. The scrambled peptide had the same ten amino acids as M10 but arranged in a different order. Fibrosis was quantitated using the Ashcroft scale, which ranges from 0 (normal) to 8 (totally fibrotic).

As expected, mice receiving bleomycin plus a scrambled peptide showed greater than eight times more lung fibrosis than controls receiving saline and scrambled peptide (Ashcroft scores, 5.63±1.72 vs. 0.69± 0.35). However, Ashcroft scores dropped to 1.67±1.01 when mice were administered both bleomycin and M10, suggesting the anti-fibrotic potential of this peptide.

Because M10 was given on the same day as bleomycin, its anti-fibrotic effects are considered preventive. To establish the therapeutic anti-fibrotic efficacy of M10, Bogatkevich and her MUSC colleagues are planning experiments in which M10 will be administered a week after the instillation of bleomycin, when fibrotic damage has already occurred. If these additional experiments suggest therapeutic efficacy, Bogatkevich hopes to find an industry partner to help take M10 forward into clinical trials.  

Many researchers speculate that there is a final common pathway to fibrosis in many different organ systems. If an anti-fibrotic agent is demonstrated to be effective in systemic sclerosis, which can affect many different organs, it could potentially hold promise for treating fibrotic disease that is confined to particular organ systems as well.

Bogatkevich and the other MUSC investigators also performed in vitro studies to assess the efficacy of M10 in decreasing abnormal collagen production/deposition and to shed light on the mechanism by which it does so. Skin and lung fibroblasts were obtained from three deceased patients with systemic sclerosis with confirmed lung involvement. As expected, these fibroblasts showed high levels of collagen production. Incubation of these fibroblasts with M10 reduced collagen expression in a dose-dependent manner. M10 likewise reduced collagen production induced in normal cells by administration of TGF-?1 without affecting baseline collagen levels.

These findings suggest that the anti-fibrotic effects of M10 may rely on its suppression of the TGF-?1 pathway. Indeed, protein interaction assays showed that M10 likely achieves such suppression by interacting with the Smad2 protein, thereby preventing it from binding to Smad3, which is necessary for the downstream inflammatory effects of the TGF-?1 pathway.
 

Reb-1Researchers at the Medical University of South Carolina and elsewhere resolve the first protein structure in a family of proteins called transcription terminators that could provide insight into aging and cancer. The work reveals the protein Reb1 to be a traffic signal for coordinating transcription and gene replication, rather than a passive roadblock as previously thought.

Image Caption: Space-filling model of Reb1 bound to DNA. Reused with permission from PNAS.

 

In a study published on 28 March 2016 in the Proceedings of the National Academy of Sciences, researchers at the Medical University of South Carolina (MUSC) and Virginia Commonwealth University have resolved the first protein structure in a family of proteins called transcription terminators. The crystal structure of the protein, called Reb1, provides insight into aging and cancer, according to Deepak Bastia, Ph.D., Endowed Chair for Biomedical Research in the MUSC Department of Biochemistry and Molecular Biology and co-senior author of the study.

During transcription, large molecular machines read genes by traveling along double-stranded DNA. This machinery simultaneously reads out the gene code in continually lengthening chains of single-stranded RNA. The RNA code is then used to assemble proteins that cells use for growth and division. At certain times during the life of a cell, transcription must be stopped–in order to conserve cellular energy or prevent uncontrolled growth, for example. At other times, cells may be preparing to divide, during which period trouble can arise.

Before a cell can divide, the DNA must be exactly replicated for use in the new cell. During part of this process, two types of machinery are now moving along the DNA strand–transcriptional machinery and replication machinery. In regions where the two machines are moving in opposite directions, collisions can occur and DNA broken, causing mutations. Harmful gene mutations can be passed into the new cell. That’s where Reb1 comes in.

One way to prevent genome instability is to prevent replication from colliding with transcription,” says Bastia. That’s what these terminator proteins do.”

Bastia’s group knew that there are specific sites on the DNA strand called terminator regions to which Reb1 binds itself. Reb1 was thought of as a simple physical barrier that sits on DNA and blocks both the transcriptional and replication machinery from moving further along the DNA strand and colliding with each other. Then Bastia’s group did an experiment to cut the transcription terminator region (tail) off of Reb1. Intriguingly, Reb1 was no longer able to halt the transcription machinery without its tail but was still able to bind to DNA. Therefore, the simple roadblock theory couldn’t be correct.

The insight came when they solved the crystal structure–a laborious process during which Carlos R. Escalante, Ph.D., Bastia’s co-senior author from Virginia Commonwealth University, made monthly drives transporting freshly made crystals from MUSC to the X-ray crystallography facility at Brookhaven National Laboratory in New York. The crystal structure showed that, when bound to DNA, the transcription terminator tail of Reb1 can interact with a specific part of the transcriptional machinery, acting as a tether between the two.

The work illuminates Reb1 as a traffic signal for coordinating transcription and gene replication, rather than as a simple roadblock as previously thought.

Though the tether between Reb1 and the transcriptional machine is clear, the team is still not sure exactly how terminator proteins stop transcription, a question which drives their current work. And the connection between terminator proteins and colorectal cancer has been made, but work in other cancers and in aging has yet to be undertaken.

Still, Bastia suspects that this coordination prevents the type of gene errors that lead to many types of cellular aging and tumor growth, both of which are processes that result from uncontrolled transcription and replication. The group is currently researching another type of terminator protein, work which Bastia hopes will lend further knowledge to the diseases of aging.

 

Placement of a single, coated, self-expanding metallic stent achieved similar rates of resolution of benign pancreatobiliary strictures as placement of multiple plastic stents, the current standard of care, and required fewer sessions of endoscopic retrograde cholang­iopancreatography, according to the results of a randomized controlled trial led by an endoscopist at the Medical University of South Carolina and reported in the March 22 JAMA.

Pictured above: MUSC Health endoscopist Gregory A Coté, M.D., performing ERCP

Placement of a single covered, self-expanding metallic stent (cSEMS) via endoscopic retrograde cholang­iopancreatography (ERCP) resolved benign obstructions of the pancreatic and biliary ducts as well as placement of multiple plastic stents, the current standard of care, and required fewer ERCP sessions, according to the results of a randomized controlled trial reported in the March 22, 2016 issue of the Journal of the American Medical Association. These findings will change practice in the opinion of Gregory A. Coté, M.D., an endoscopist at the Digestive Disease Center at the Medical University of South Carolina, lead author of the article, and the national principal investigator for the eight-center study. B. Joseph Elmunzer, M.D., of the MUSC Digestive Disease Center is also a co-author.

“For appropriately selected patients who are presenting the first time with a blockage, many endoscopists will change their strategy and use these newer stents, in an effort to reduce the total procedures that are required,” says Coté.

For now, the use of cSEMS (pictured below right) in benign biliary strictures would be off-label, as they have been approved by the U.S. Food and Drug Administration only for the treatment of malignant pancreatobiliary strictures, such as those which develop in the setting of unresectable pancreatic cancer.

Left untreated, benign biliary strictures can lead to jaundice, cholangitis (i.e., infection of the bile duct), and secondary biliary cirrhosis. Benign strictures most often occur after liver transplantation and gallbladder surgery or as a result of chronic pancreatitis. ERCP is preferred to surgery in these patients because it is less invasive.

ERCP is an endoscopic procedure in which a camera is introduced through the mouth and advanced first to the proximal duodenum and then, using endoscopy and fluoroscopy, into the pancreatic and bile ducts.  Although highly effective, three to four ERCP sessions are typically required to fully stretch the blockage and minimize the chance of recurrence once the stents are removed.  Because ERCP is not without its risks—complications include pancreatitis, infections, bowel perforation, and bleeding—minimizing the number of sessions needed to successfully treat the stricture benefits patients. Coté designed the trial to determine whether using the larger cSEMS could successfully treat benign strictures with fewer ERCP sessions. cSEMS are coated to prevent in-growth of tumor or other tissue and to facilitate removal.

The primary endpoint of the trial, which enrolled 112 patients, was the rate of benign stricture resolution after no more than 12 months of stenting. cSEMS were found to be noninferior to plastic stents for achieving stricture resolution and they did so more quickly (181 vs. 225 days) and with fewer ERCP sessions (2.14 vs. 3.24). It should be noted that the study had careful enrollment criteria; patients with small (<6 mm) bile ducts and those with intact gall bladders were excluded. “We were careful not to cross the gall bladder insertion into the bile duct in patients who still have their gall bladder because you don’t want to block the gall bladder and potentially create a new problem,” says Coté.

The procedure can be performed by anyone proficient in ERCP but best results will likely be seen when it is performed by a high-volume provider (2-3 ERCPs per week) who is comfortable placing and removing cSEMS.

“We can’t universally change practice based on these findings, but, in appropriately selected patients with benign pancreatobiliary strictures, deployment of cSEMS via ERCP should be first-line treatment,” says Coté.

Accumulation of DihydroceramidesSummary: Sphingosine kinase inhibitors are a new category of drugs that act on specific enzymes involved in sphingolipid metabolism to reduce the formation of a pro-cancer, pro-inflammatory lipid signaling molecule known as sphingosine-1 phosphate (S1P). Preclinical studies led by immunologist Christina Voelkel-Johnson, Ph.D., of the Medical University of South Carolina showed that a first-in-class sphingosine kinase 2 inhibitor slowed growth of aggressive prostate cancer cells.

A first-in-class sphingosine kinase 2 inhibitor slowed the growth of castration-resistant prostate cancer cells, in part by inhibiting the enzyme dihydroceramide desaturase (DEGS), but did not kill them, according to the results of preclinical in vitro and in vivo studies published in the December 2015 issue of Molecular Cancer Therapeutics by researchers at the Medical University of South Carolina (MUSC) and others.

Christina Voelkel-Johnson, Ph.D., Associate Professor of Microbiology and Immunology at MUSC, led the study, which was funded by a pilot grant from MUSC Hollings Cancer Center. Co-authors include Charles D. Smith, Ph.D., who developed the compound and led an earlier phase 1 trial at MUSC Hollings Cancer Center; oncologist Michael Lilly, M.D., a prostate cancer specialist; and Richard Drake, Ph.D., director of the Proteomics Core at MUSC, who has developed techniques to use MALDI imaging mass spectrometry to measure sphingolipid levels.

Sphingosine kinase inhibitors are a new category of drugs that reduce the generation of sphingosine-1-phosphate. This lipid signaling molecule promotes cancer cell growth and survival, thereby supporting the development of resistance to chemotherapy and radiation by cancer cells.

The study reported in Molecular Cancer Therapeutics showed that the compound YELIVA™ (ABC294640; RedHill Biopharma Ltd.; Tel Aviv, Israel) slowed prostate cancer cell proliferation by inhibiting sphingosine kinase 2, but also that it did something unexpected. “By inhibiting a second sphingolipid enzyme (DEGS), the compound increases levels of another class of lipids - dihydroceramides - which may contribute to the growth suppressive effects of the drug,” says Voelkel-Johnson. This study is the first to show activity for this compound against DEGS and to potentially link inhibition of DEGS to slowing the growth of castration-resistant prostate cancer cells. Treatment with YELIVA™ (ABC294640) increased dihydroceramide levels even in the absence of sphingosine kinase 2. 

The MUSC team conducted both in vitro and in vivo studies with YELIVA™ (ABC294640) in castration-resistant prostate cancer, relying on the MUSC Lipidomics Shared Resource for measurement of sphingolipid levels and the MUSC Proteomics Center for MALDI imaging mass spectrometry.

 In vitro studies conducted with castration-resistant mouse prostate cancer cells (TRAMP-C2) showed that treatment with YELIVA™ (ABC294640) reduced expression of the androgen receptor and the oncogene c-Myc, both important therapeutic targets for prostate cancer. Although many existing prostate cancer therapies target the androgen receptor, none directly target c-Myc.

To test in vivo response, one million TRAMP-C2 cells were injected under the skin of mice with an intact immune system, which were then treated with YELIVA™ (ABC294640) three days later. MALDI imaging mass spectrometry showed the presence of YELIVA™ (ABC294640) within murine tumors and confirmed in vitro findings of increased dihydroceramide levels.

“The significance of these findings is that this compound might be a novel therapeutic for advanced prostate cancer,” says Voelkel-Johnson, who believes that combination regimens of YELIVA™ (ABC294640) and focal radiation in this difficult-to-treat patient population deserve further study.

See full EurekAlert! release at http://www.eurekalert.org/pub_releases/2016-01/muos-anc012816.php

Image Caption:

The signal for ABC294640 is detected only when the drug but not the vehicle was administered (upper panel). The intensity for two different dihydroceramides is shown in the middle panel (dhC16-cer) and lower panel (dhC18-cer). A color bar indicates the signal intensity. Adapted with permission from the American Association for Cancer Research : Venant H, et al. The Sphingosine kinase 2 inhibitor ABC294640 reduces the growth of prostate cancer cells and results in accumulation of dihydroceramides In vitro and In vivo. Molecular Cancer Therapeutics; 2015 Dec; 14(12):2744-52. doi: 10.1158/1535-7163.MCT-15-0279.

fatty liver disease image 3

In results published on October 19, 2015 in the Journal of Lipid Research (http://dx.doi.org/10.1194/jlr.M063511), a team of translational scientists at the Medical University of South Carolina (MUSC) report a new reason why non-alcoholic steatohepatitis (NASH) worsens in people who are obese. The results may help prevent cirrhosis and liver cancer, according to co-senior authors Kenneth D. Chavin, M.D., Ph.D., a transplant surgeon in the MUSC Health Department of Surgery, and Lauren Ashley Cowart, Ph.D., Associate Professor in the Department of Biochemistry and Molecular Biology and Co-Director of the MUSC Center of Biomedical Research Excellence in Lipidomics and Pathobiology.

 NASH (also called non-alcoholic fatty liver disease) has become a major cause of liver disease requiring transplant. “In my 17 years of doing liver transplants, it’s gone from 4% of patients to around 20% of patients who get transplants because of NASH,” says Chavin. “In 10-15 years, because of advances with Hepatitis C, it will probably become the number one reason why patients get transplants.”

When excess dietary fats are consumed over time, fat deposits form in the liver and NASH can develop. Early-stage NASH is typically not associated with any physical symptoms; nearly 30% of people in the U.S. have it. Though obesity is correlated with the development of NASH, the team wanted to know exactly why NASH worsens to a stage requiring transplant in certain obese people. “Obesity doesn’t cause disease in every obese person and we don’t understand why it does in some but not others,” explains Cowart.

The team suspected that inflammation stemming from a lipid molecule called sphingosine-1-phosphate (S1P) might be responsible. They’d previously discovered in other organs that S1P is increased by excess dietary saturated fat.

Chavin took biopsies from human livers during transplant surgery and supplied them to Cowart, who determined the levels of sphingosine kinase 1, the enzyme that makes S1P. They found double the normal amount of sphingosine kinase 1 in livers of obese people with non-alcoholic steatohepatitis.

The team wanted more understanding of why S1P causes inflammation, but NASH has previously been difficult to mimic in the laboratory setting. They developed a new and highly improved preclinical model of NASH, wherein mice were fed on custom-designed diets of either high saturated fat or high unsaturated fat. Curiously, mice on each type of diet became obese, but only mice on the saturated fat diet developed inflammation and NASH-like pathology stemming from S1P. Taking the human and pre-clinical studies together, it’s likely that saturated fat, but not unsaturated fat, raises S1P levels in obese people, and it’s S1P that unleashes the inflammation that characterizes NASH.

Performing lipid studies in the laboratory is not easy—most biochemistry is water-based, and fat and water don’t easily mix. The group relied on the MUSC Sphingolipidomics Core laboratory, one of only a handful of such facilities in the country capable of developing the new methods needed to examine S1P for their study. Without lipidomics, we never would have understood that saturated fats activate this pathway,” says Cowart. The team is working to identify the S1P receptors responsible for inflammation in NASH, with the ultimate goal of designing treatments to prevent the need for a liver transplant in obese patients with NASH.

Does this work support the idea that it’s the type of fat, but not all fat, that leads to health problems? After all, mice fed a high unsaturated fat diet still became obese but were metabolically healthy. “Because the unsaturated fat diet didn’t cause NASH, it may provide a clue as to what actually links obesity to disease,” says Cowart. “Even if it’s difficult to lose weight, dietary modifications might prevent some disease associated with obesity.”

 MUSC researchers Tuoyu Geng, Ph.D., Alton Sutter, M.D.,Ph.D., Arun Palanisamy Ph.D., and Michael D. Harland also contributed to this study.

 This work was supported by a Veterans Affairs Merit Award, National Institutes of Health Grants 1R01HL117233 and 5P30GM103339-03 (L.A.C.), and National Institutes of Health Grant 1R01DK069369 to K.D.C .

Nursing motherIn the October 2015 issue of Pediatrics, two MUSC Children’s Hospital faculty members—vitamin D researcher Bruce W. Hollis, Ph.D., and neonatologist Carol L. Wagner, M.D.—reported clinical trial findings definitively showing that sufficient vitamin D can be transmitted via breast milk to meet the needs of the exclusively breastfed infant, provided that the mother is adequately supplemented.

Breastfeeding is encouraged by the medical community in part because breast milk meets all nutritional needs of the child, with the glaring exception of vitamin D. Why such an essential vitamin would be missing from breast milk has always been puzzling. Many physicians erroneously believe that vitamin D simply cannot be transmitted via breast milk. To prevent deficiency in exclusively breastfed babies, the American Academy of Pediatrics recommends that they be supplemented with 400 IU/d of vitamin D3, delivered via liquid drops. Unfortunately, the drops can be difficult to administer and not all mothers adhere to this directive, leaving some infants vulnerable to rickets or fractures.

The study results reported by Hollis and Wagner suggest that a more natural and effective way to supplement the child would be to adequately supplement the nursing mother.  At the time the study was designed, the Institute of Medicine (IOM) recommended that adults, even nursing mothers, receive 400 IU/d of vitamin D3; the IOM has since increased the recommended dose to 600 IU/d. The study randomized mother/infant dyads to either 400 IU/d of vitamin D3 each or 6,400 IU/d for the mother and none for the infant. The infants in both arms of the trial achieved vitamin D sufficiency, and no adverse effects were reported for mothers receiving the 6,400/IU day dose.  The results suggest that adequate maternal supplementation—6,400 IU/d of vitamin D3 vs. the current IOM recommendation of 600 IU/d—offers a safe and effective alternative to direct infant supplementation.

Fibrotic heart

The summer of 2015 saw approval of the first new drug for heart failure in almost two decades. The angiotensin receptorneprilysin inhibitor Entresto (formerly LCZ696, Novartis Pharmaceuticals) was approved for heart failure with reduced ejection fraction (HFrEF) on the basis of the results of the PARADIGM-HF trial published in the September 11, 2014 issue of the New England Journal of Medicine.1 MUSC Health cardiologist Michael R. Zile, M.D., who served on the national steering committee of the PARADIGM-HF trial, predicts that the new therapy “will  replace ACEs and ARBs as the cornerstone of standard therapy for patients with HFrEF.”

 However, patients with HFrEF account for only about half of the 3 million heart failure cases in the U.S. each year. No agent has been shown to improve morbidity and mortality in patients with heart failure with preserved ejection fraction (HFpEF), who make up the other half of heart failure cases. In patients with HFrEF, the left ventricle does not fully contract, while in those with HFpEF, the ventricle does not fully relax. The two largest clinical trials to date showed that traditional heart failure therapy with angiotensin receptor blockade (Irbesartan or Candesartan) did not improve heart function in patients with HFpEF. According to Zile, “The biggest unmet need in cardiology today is a treatment for HFpEF.”

That could be changing. Zile is on the national steering committee of the PARAGON-HF trial, which will assess the efficacy of Entresto in patients with HFpEF, and MUSC Health is one of the sites enrolling patients into the trial. Results are expected in 2017. “PARAGON-HF will be largest clinical trial in patients with HFpEF,” says Zile. “We hope that Entresto will form the foundation for novel and effective treatment that reduces symptoms and increases survival in HFpEF.”

For more information about the trials, read "A New Paradigm for the Treatment of Heart Failure."

 Reference

1 McMurray JJ, et al. Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med. 2014 Sep 11;371(11):993-1004. doi: 10.1056/NEJMoa1409077. Epub 2014 Aug 30.

Photo of veteranA study among military veterans needing psychotherapy for depression has demonstrated that such therapy delivered via telemedicine in patients’ homes achieves outcomes that are not significantly inferior to those of traditional in-person clinical encounters.1 In their editorial in the August 2015 The Lancet Psychiatry, Hoge and Rye describe this study as the kind of rigorous clinical trial that is necessary to establish acceptable standards of care, calling it “a vanguard of demonstration of safety and efficacy of in-home telemental health.”  All research team members were affiliated with the Medical University of South Carolina (MUSC) or the Ralph H. Johnson Veterans Affairs Medical Center in Charleston, SC. The Principal Investigator was Leonard E. Egede, M.D., Professor of Medicine at MUSC, Director of the MUSC Center for Health Disparities Research, and the Director of the Charleston VA HSR&D Health Equity and Rural Outreach Innovation Center. In the 2007 - 2011 study (NCT00324701), 204 veterans aged 58 years and older were treated via either telemedicine or same-room treatment. Their response to the treatments did not differ significantly. The researchers’ interpretation of this data, which is in the same issue of The Lancet Psychiatry2, is that evidence-based psychotherapy delivered to patients in their homes is not only not inferior, it is advantageous because it overcomes distance, attendance, and stigma barriers faced by some veterans. "This is the first study in the elderly that shows that in-home telemedicine works as well as face-to-face sessions for the treatment of depression,” says Egede. “It provides strong evidence for using in-home telemedicine to treat depression and possibly other mental health conditions in those with stigma or mobility challenges, the home-bound, or those who are geographically isolated and cannot get to care easily. In addition, there were no adverse events, which have always been a concern for mental health treatment delivery via telemedicine.”

1 Egede LE, Acierno R, Knapp RG, et al. Psychotherapy for depression in older veterans via telemedicine: a randomized, controlled, open-label, non-inferiority trial. The Lancet Psychiatry 2015; 2(8): 693-701.

2 Hoge, CW, Rye, CB. Efficacy and challenges of in-home telepsychotherapy. The Lancet Psychiatry 2015; 2(8): 668-669.

brain optics graphicIn 2015, the National Science Foundation's (NSF) Experimental Program to Stimulate Competitive Research (EPSCoR) focused on three priority areas: the complexity of the brain, clean energy, and food security. After reviewing nearly sixty applications, the NSF awarded eight awards to the tune of $42 million in early August. Only three of these awards were for brain research, and MUSC was a recipient. A $4 million grant to MUSC will be used to develop and implement new optical technologies to image brain function at a very high resolution. The Principal Investigators of the grant (Peter W. Kalivas, Ph.D., and Prakash Kara, Ph.D., both from the Department of Neurosciences at MUSC) felt that partnering with the University of Alabama at Birmingham (UAB) played an important role in securing this grant. UAB and MUSC will bring complementary technologies to develop a parallel pipeline of state-of-the-art brain scanners in both states. Optical technologies previously used to look at the stars in the sky will now be miniaturized to look inside the brain. Additional partners in this grant include Furman University, the University of South Carolina Beaufort, and Clemson University.

The NSF recognizes the importance of funding new research projects that merge science, technology, engineering, and mathematics (STEM). Grant recipients must also develop a STEM workforce in order to grow their research programs. While the NSF funds basic science research, this infrastructure often leads to new discoveries that improve health care. For example, this grant thematically focuses on developing tools to determine the precise mechanisms (on the microcircuit scale) by which neurons normally communicate with blood vessels in the brain. However, this “neurovascular” communication breaks down in many neurological diseases. Thus, this grant will have far-reaching impacts in the fields of neuroimaging, neurology, and neuroscience education.

fingertips touchingSending imperceptible white-noise vibrations through the wrist could increase the dexterity of those recovering from stroke, according to an article published in Physiological Results July 14.

Patients who have experienced a stroke can have lasting side effects, such as a diminished tactile response in their fingers and hands. A decrease in sensory feedback from the fingers can cause insufficient grip force control, decreased dexterity, decreased fine object manipulation, and an unstable grip, often causing patients to drop objects.

Na Jin Seo, Ph.D., Assistant Professor in the Division of Occupational Therapy at MUSC Health, was the senior investigator for this study, which applied perceptible and imperceptible white-noise vibrations at various intensities and in separate locations on patients’ extremities to determine the optimal level of white-noise vibration needed to improve signal detection. V. Ramesh Ramakrishnan, Ph.D., and Abigail W. Lauer, M.S., in the Department of Public Health Sciences at MUSC Health performed the statistical analyses for the study. The patients’ non-dominant hands were used for the test because it was postulated to be more sensitive to somatosensory feedback. The vibration was then applied to areas of the palm, back of the hand, and inner wrists an attempt to increase sensitivity in the finger pads. The intensities of the vibration varied from 0% to 120% of the threshold.

At 60% of the threshold, the imperceptible white-noise vibrations significantly improved finger tactile sensation in the thumb and index finger when compared to 0%. At 80% of the threshold, no significant changes were seen, and at 120% of the threshold degraded sensory feedback was observed in the fingers, conforming to stochastic resonance behavior.

While the vibration stimuli would quickly decrease as it moved away from the application site, not reaching the fingertips, each site reacted similarly to the same intensities. According to the study, these results indicate that neuronal activity in the fingers is influenced by the vibration.

“This finding suggests a potential for a sensory orthotic that can be worn at the wrist,” says Seo. “It would provide minute vibration and enhance patients’ touch sensation and dexterity, improving their ability for activities of daily living.” 

This study suggests that patients who have experienced an injury or a stroke could gain access to the neuronal network in their hands through imperceptible white-noise vibrations. Wearing a vibrating device on their wrists could help these patients gain back some of the control they may have lost.

photo fo pill bottle and paperworkThe FDA approved alirocumab (Praluent; Sanofi and Regeneron Pharmaceuticals) for patients with heterozygous familial hypercholesterolemia (HeFH) and for patients with clinical atherosclerotic cardiovascular disease, according to a press release from the FDA July 24.

HeFH is an inherited disease that causes high levels of low-density lipoprotein (LDL) cholesterol in patients. Alirocumab is designed to lower this “bad” cholesterol and is an injectable monoclonal antibody and is the first in its class of PCSK9 inhibitors.

"This new class of medications can significantly and effectively lower LDL cholesterol in very high-risk patients who could not otherwise achieve acceptable LDL cholesterol levels,” said MUSC Health cardiologist Pamela B. Morris, M.D., who is the principal investigator for multiple trials of PCSK9 inhibitors at MUSC. “Long-term outcomes trials in progress will determine if this dramatic reduction in LDL-C results in a reduction in cardiovascular events," says Morris. (To learn more about PCSK9 inhibitors, their mechanism of action, and clinical trials with these inhibitors at MUSC, click here.)

This new drug is most successful when combined with a healthy diet and maximum-tolerated statin therapy. It is not indicated, however, for statin-intolerant patients without established clinical atherosclerotic cardiovascular disease.

Clinical trials for this drug in children have not yet been performed, so it is only prescribed to patients 12 years of age and older.

The side effects of this drug include itching, swelling, pain, or bruising at the injection site, nasopharyngitis, and the flu. There have also been reported cases of allergic reactions to this drug, causing hypersensitivity vasculitis and hypersensitivity reactions requiring hospitalization.

It is expected the FDA will approve a second PCSK9 inhibitor called evolocumab (Repatha; Amgen) by August 27.

photo of a box of fruits and vegetablesA recent study led by MUSC professor David P. Turner, Ph.D. finds that lifestyle habits such as diet and exercise could affect the progression of cancer and the rate of survival, but so could race. According to the study published in Cancer Research in May, our bodies have to metabolize food to obtain the sugars we need, thus leaving behind a reactive-metabolite waste product. These leftovers are referred to as advanced glycation end-products (AGE), and this study addresses the apparent correlation between AGE levels and the prevalence of age-related diseases among non-Hispanic whites and African Americans. 

High levels of AGE are associated with diabetes, cardiovascular disease, Alzheimer’s, and cancer. These levels are highest in African American men with prostate cancer—they are 1.5 times more likely to be diagnosed with this cancer and twice as likely to die from it than non-Hispanic whites. Consumption of sugar and processed food can contribute to AGE levels. Food preparation (i.e., browning) also plays a large role in these levels. They are higher in the West, where the diet commonly consists of red meat, refined grains, and high sugar and fatty foods.

 When analyzing serum from cancer patients, Turner found that AGE levels were significantly higher in patients with cancer than those without. Breast and prostate immortalized cancer cell lines grew more, migrated farther, and invaded more when treated with AGE. In conjunction with higher AGE levels, African Americans have more C-reactive protein (CRP), making them more susceptible to chronic inflammation. Chronic inflammation is one of the key factors implicated in the development of cancer, along with oxidative stress, an increased immune response, and the presence of AGE.

AGE cannot be completely eliminated, but levels of circulating AGE can be lowered. Simply changing lifestyle habits can slow down the accumulation of AGE in the body. Avoid food with high protein, sugar, and fat, as well as processed foods. Then increase your intake of natural grains, fruits, and vegetables. Change the way you prepare your food by cooking meats at a lower temperature for a longer period of time, skipping the browning step of a dish. You can also replace high-sugar, oil-based marinades with lemon juice, vinegar, and tomato juice. The last big step of lowering your AGE levels is exercise. A sedentary lifestyle only allows for more AGE to accumulate.

sub-cutaneous icd imageThe clinical promise of the subcutaneous implantable cardioverter-defibrillator (S-ICD System®; Boston Scientific, Natick, MA), the first ICD in which the leads are placed under the skin of the chest and are not connected to the heart or vasculature, was confirmed by longer-term (median of 22-month) safety and efficacy data reported in the April 28, 2015 issue of the Journal of the American College of Cardiology (JACC). The S-ICD was approved by the FDA in September 2012 to provide an electric shock to the heart (defibrillation) when the patient’s heart is beating chaotically (ventricular fibrillation) or abnormally fast (ventricular tachyarrhythmia). Because the S-ICD is not implanted in the vasculature or heart, major complications, such as device/lead displacement or failure and pneumothorax, are very rare. Because the S-ICD lacks pacing capacity, it is contraindicated in patients who require a pacemaker or pacing therapy. The JACC article, coauthored by MUSC cardiologist Michael Gold, M.D., Ph.D., is a pooled analysis of 882 patients implanted with the S-ICD who were either participants in the investigational device exemption (IDE) study that led to FDA approval or members of the European EFFORTLESS S-ICD registry, created to evaluate the long-term efficacy and safety of the S-ICD.  Of the 111 events of ventricular fibrillation/tachyarrhythmia reported in the study, 90% (100) were terminated with one shock and 98% (109) within the five shocks available with the S-ICD.  This is similar to reported shock termination rates with traditional ICDs. Device-related complications occurred in 11.1% of patients at three years, with generator pocket infections and inappropriate shocks due to oversensing being the more prominent. Rates of both decreased as providers gained experience with the S-ICD, infection control techniques were implemented, and dual zone programming was favored. The infection rate was reduced more than 3-fold in the latter half of these trials and supraventricular arrhythmias by about 70%. Improvement in this technology will occur in future iterations of the device that should be available in the summer of 2015.  MUSC, the leading center in South Carolina for implantation of this device, participated in many of the early clinical studies that led to the approval of the S-ICD.

Statins can lower low-density lipoprotein cholesterol (LDL-C) levels by 25%-50%, but for many patients that is not enough to reach target levels, leaving them at residual risk for cardiovascular events. Some patients experience muscle aches when taking statins and must discontinue therapy or take a suboptimal dose. The search has been on for agents that can provide additional benefit in patients already taking statins or that can provide an alternative therapeutic option for those who do not tolerate them, but with largely disappointing results.

That is why the dramatic reduction in LDL-C levels achieved by proprotein convertase subtilisin–kexin type 9 (PC3K9) inhibitors are being met with such excitement. The interim results of the Osler-1 and -2 trials (NCT01439880 and NCT01854918) and the Odyssey Long Term trial (NCT01507831) were published online on March 15 in the New England Journal of Medicine (Osler: http://dx.doi.org/10.1056/NEJMoa1500858; Odyssey: http://dx.doi.org/10.1056/NEJMoa1501031). The results showed a more than 60% decrease in LDL-C levels (from a median of 119-120 mg/dL to 48 mg/dL; P<.001) and a significant decrease in cardiovascular events in patients taking the PC3K9 inhibitor evolocumab (Amgen; Osler) or alirocumab (Sanofi/Regeneron; Odyssey) in addition to standard therapy vs those receiving standard therapy alone. Those decreases were maintained over time.  The FDA has scheduled a target action date for evolocumab and alirocumab for August and July, respectively, and could approve both for certain indications as early as September of this year.

“PCSK9 inhibitors are the most exciting thing going on right now in the field of lipids. They are rocking the lipid world,” says MUSC Health cardiologist Pamela B. Morris, M.D., who is the principal investigator for the MUSC site of two trials of these inhibitors: GAUSS III, which is testing the efficacy of evolocumab in patients who have been verified as being statin intolerant, and FOURIER (NCT01764633), which is seeking to definitively establish whether the dramatic decreases in LDL-C seen with evolocumab indeed reduce the risk for cardiovascular events long term in patients already receiving statin therapy.

Side effects of PCSK9 inhibitors include  minor injection-site reactions and a few cases of memory deficit that were at first thought to due to excessively low LDL-C levels; the Osler interim results showed that this was not the case.

PCSK9 inhibitors are monoclonal antibodies that must be subcutaneously injected, and it is still being assessed whether better efficacy and patient adherence can be achieved with a smaller dose every two weeks or a larger dose once a month.

If approved, PCSK9 inhibitors will offer a promising new treatment option for patients who could not take statins or who did not reach target LDL-C levels despite taking the highest dose of statins they could tolerate.

How They Work: The body clears LDL-C via LDL receptors on the surface of liver cells, which bind to LDL-C and target it for degradation. Depending of the body’s needs, the LDL receptor is then itself degraded or is recycled (as many as 200 times) to clear more LDL-C. PCSK9 binds to the LDL-C/LDL receptor complex and targets the receptors for degradation rather than recycling. When PCSK9 levels are decreased, more LDL-C receptors are recycled to the cell surface to clear LDL-C. Indeed, patients with a loss-of-function PCSK9 mutation tend to have very low levels of LDL-C and very low rates of cardiovascular disease, an observation that helped spark interest in PCSK9 inhibitors. 

baby earCongenital cytomegalovirus (CMV) infection, the most frequently transmitted intrauterine infection, is the leading nongenetic cause of hearing loss, accounting for 21% of cases at birth and 24% at 4 years of age. It is also the leading viral cause of mental retardation. An article in the March 5, 2015 issue of the New England Journal Medicine co-authored by Sandra L. Fowler, M.D., MSc, Director of Pediatric Infectious Diseases at MUSC Children’s Hospital, and other members of the National Institute of Allergy and Infectious Diseases Collaborative Antiviral Study Group (CASG), reported the results of a double-blinded, randomized, placebo-controlled trial of oral valganciclovir in neonates with symptomatic CMV infection (NCT00466817).1 A 2003 trial by the CASG of intravenous (IV) ganciclovir in neonates with CMV infection and central nervous system involvement showed that 6 weeks of ganciclovir therapy resulted in better hearing at 6 months but suggested that those gains were not durable.2 The recently completed CASG trial randomized neonates to either 6 weeks or 6 months of treatment with oral valganciclovir, a prodrug of ganciclovir, and assessed changes at 6, 12, and 24 months in better-ear and overall hearing and performance on a neurodevelopmental test, particularly as it related to linguistic and communication ability. No significant differences in hearing were noted between the two groups at 6 months, but improved hearing was 3 times as likely at 12 months and 2.6 times as likely at 24 months in study patients receiving 6 months vs 6 weeks of therapy. Those receiving 6 months of therapy also scored higher on the Bayley III assessment of neurodevelopment in the language-composite component and the receptive-communication scale, with low average results vs the borderline results seen in neonates receiving only 6 weeks of therapy. Neutropenia (i.e., a reduced number of neutrophils), which can be a concern in neonates taking IV ganciclovir, posed less of an issue with the oral administration of valganciclovir over the course of 6 months. According to Fowler, these results represent “a big step forward in our ability to improve outcomes in infants with congenital CMV and will establish a new standard of care. Our next step will be to determine whether infants who are asymptomatically infected with CMV—but still at risk for developing hearing loss—will benefit from antiviral therapy, as well.”

fowler - terriThe MUSC College of Nursing’s online graduate nursing program was recently ranked number one in the nation by U.S. News & World Report. Ninety six nursing programs were scored on factors such as graduation rates, academic and career support services offered, and admissions selectivity. MUSC earned an overall score of 100.  College of Nursing Dean Gail Stuart, PhD, says the achievement “is a reflection of the dedication of our faculty and staff.” 

The College of Nursing began its asynchronous online program in 2006 with the Master of Science in nursing (MSN) and the Doctor of Philosophy (PhD), followed by the Doctor of Nursing Practice (DNP) in 2009, and the Registered Nurse to Bachelor of Science in nursing (RN-BSN) in 2014. There are currently almost 300 students enrolled in the online graduate programs.  

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