Origins of Pain


Researchers identify pathway that drives sustained pain following injury

painful shoulder

Researchers have identified the nerve-signaling pathway behind the deep, sustained pain that sets in following injury.

A toddler puts her hand on a hot stove and swiftly withdraws it. Alas, it’s too late—the child’s finger has sustained a minor burn. To soothe the pain, she puts the burned finger in her mouth.

Withdrawing one’s hand to avoid injury and soothing the pain of that injury are two distinct evolutionary responses, but their molecular origins and signaling pathways have eluded scientists thus far.

Now research led by investigators at Harvard Medical School, published Dec. 10 in Natureidentifies the nerve-signaling pathway behind the deep, sustained pain that sets in immediately following injury. The findings also shed light on the different pathways that drive reflexive withdrawal to avoid injury and the subsequent pain-coping responses.

Clinical observations of patients with neurological damage together with past research have outlined the distinct brain regions that differentiate between the reflexive withdrawal from a skin prick, for example, and the long-lasting pain arising from tissue injury caused by the pinprick.

The new study, however, is the first one to map out how these responses arise outside the brain.

The findings, based on experiments in mice, put into question the validity of current experimental approaches for assessing the efficacy of candidate pain-relief compounds. Most current methods rely on measuring the initial, reflexive response that serves to avert tissue injury, rather than on measuring the lasting pain that arises from actual tissue damage, the researchers said. As a result, they said, some drug compounds that might have been successful in assuaging the sustained pain—the lasting sensation of pain that immediately follows injury—could have been dismissed as ineffective because they were assessed against the wrong outcome.

“The ongoing opioid crisis has created an acute and pressing need to develop new pain treatments, and our findings suggest that a more tailored approach to assessing pain response would be to focus on sustained pain response rather than reflexive protective withdrawal,” said study senior author Qiufu Ma, professor of neurobiology in the Blavatnik Institute at Harvard Medical School and a researcher at Dana-Farber Cancer Institute.

“All these years, researchers may have been measuring the wrong response,” Ma added. “Indeed, our results could explain, at least in part, the poor translation of candidate treatments from preclinical studies into effective pain therapies.”

Previous work by Ma and colleagues, as well as others, points to the existence of two sets of peripheral neurons—the nerve cells located outside the brain and spinal cord. One set of peripheral nerve cells send and receive signals exclusively to and from the superficial layers of the skin. As a first-line of defense against external threats, these peripheral nerve cells are geared toward preventing injury by triggering reflexive withdrawal—think pulling your hand after a pinprick or to avoid the hot tip of a flame. Another set of neurons are dispersed throughout the body and thought to drive the lasting pain that sets in after initial injury and induces pain-coping behaviors such as pressing a banged finger or licking a cut in the skin to sooth the damaged area.

Yet the existence of these neurons could not fully explain how the pain signal travels throughout the body and to the brain. So, Ma and colleagues proposed the existence of another critical player in this relay.

The team focused on a set of neurons called Tac1 emanating from the so-called dorsal horn, a cluster of nerves located at the lower end of the spinal cord that transmit signals between the brain and the rest of the body. The precise function of Tac1 had remained poorly understood so Ma and colleagues wanted to know whether and how these neurons were involved in the sensation of sustained pain.

In a series of experiments, the team assessed pain response in two groups of mice—one with intact Tac1 neurons and another with chemically disabled Tac1 neurons.

Mice with inactivated Tac1 neurons had normal withdrawal reflexes when exposed to a painful stimulus. They showed no notable differences in their withdrawal from pricking or exposure to heat and cold. However, when the researchers injected the animals with burn-inducing mustard oil, they did not engage in the typical paw licking that animals perform immediately following injury. By contrast, mice with intact Tac1 neurons engaged in vigorous and prolonged paw licking to assuage the pain.

Similarly, mice with disabled Tac1 neurons showed no pain-coping responses when their hind paws were pinched—something that induces sustained pain in humans. These animals did not engage in any paw licking as a result of the pinch. Such loss of sensitivity to a specific type of pain mimics the loss of sensation seen in people with strokes or tumors in a particular area of the brain’s pain-processing center—the thalamus—that renders them incapable of sensing lasting pain.

These observations confirm that Tac1 neurons are critical for pain-coping behaviors stemming from sustained irritation or injury but that they play no role in reflexive-defensive reactions to external threats.

Next, researchers wanted to know whether Tac1 neurons shared a common connection with another class of neurons, called Trpv1, present throughout the body and already known to drive the sensation of lasting pain induced by injury. Mice that had functional Tac1 but nonfunctioning Trpv1 neurons responded weakly to pinch-induced pain, showing minimal paw licking. The finding suggests that pain-sensing Trvp1 neurons connect to Tac1 neurons in the dorsal horn of the spinal cord to transmit their signals.

“We believe that Tac1 neurons act as a relay station that dispatches pain signals from the tissue, through Trpv1 nerve fibers all the way to the brain,” Ma said.

Taken together, the results of the study affirm the presence of two lines of defense in response to injury, each controlled by separate nerve-signaling pathways. The rapid withdrawal reflex is nature’s first line of defense, an escape attempt designed to avoid injury. By contrast, the secondary, pain coping response helps reduce suffering and avert widespread tissue damage as a result of the injury.

“We believe it’s an evolutionary mechanism conserved across multiple species to maximize survival,” Ma said.

Scientists Just Found a Previously Unknown Nerve Pathway For Pain


To ease the sting of a paper cut, most of us will instinctively pop the afflicted finger in the mouth and suck for a moment or two. We rub barked shins, cool blistered skin, and shake a crushed hand. And it might represent a completely different kind of pain.

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A pathway of nerves recently identified in mice appears to be responsible for the sustained ache we experience after the initial shock of pain has faded, and it could help explain why some painkillers aren’t living up to expectation.

Harvard Medical School researchers led a study on different ways painful stimuli travel from the site of trauma to the brain, seeking to better understand the various circuits responsible for creating acute and chronic discomfort.

Whether it’s a stubbed toe, a prickle in your foot, or grabbing a hot utensil, the basic response is the same – our body reflexes tell us to move away from the source of danger.

The sharp burst of agony known as nociception might last a brief moment, but it’s enough to force us to pull away and prevent the risk of further damage.

But after that initial burst, depending on the severity of the trauma, we can experience seconds, minutes, or even days of persistent discomfort. Sometimes we apply pressure, or cool the wound, to alleviate the pain.

Dana-Farber Cancer Institute neurobiologist Qiufu Ma has devoted years to researching the nervous routes of pain and itching. His studies have led him and his team to suspect those two different types of pain follow different paths.

It’s no secret that our perception of traumatic stimuli is the result of some pretty complex neurology involving sensory nerves called nociceptors and various pathways that carry signals to the spinal cord and areas of the brain.

But the details remain somewhat foggy. For example, is a ‘pain matrix’ of locations in the brain primarily responsible for our hurt, or is the story more complicated than it first appears?

Similarly, are different flavours of suffering mediated by different systems of nerves, or is it all in the processing?

To add to the evidence of distinct highways of nociception, Ma and his team looked to a category of spinal nerves that had been previously associated with noxious stimuli.

A gene expressed in these cells called Tac1 stood out as potentially having a key role in the neurons’ function. The natural way to see what a gene does is to switch it off and watch what happens. Doing this in humans is fairly problematic, but mice pose fewer practical or ethical dilemmas.

Strangely, mice engineered to have their Tac1 gene silenced still showed a response to pain. Having their feet pricked, pinched, or dosed with mustard oil still resulted in clear signs of aversion.

But unlike mice with their Tac1 intact, these tiny test subjects didn’t bother nursing their wounds by licking the afflicted skin, suggesting these spinal nerves might play a role in informing the brain of recent physical damage.

Tracing them to the periphery, the team found there was still a segregation of nerves, suggesting a completely distinct pathway all the way from the source.

Those nerves were already familiar thanks to their capsaicin receptors, called TrpV1. Not only do they respond to temperature, and spicy chemicals that trigger a sensation of heat, they become more sensitive in the presence of mediating chemicals released by inflammation.

Having distinct pathways for the initial burst of pain and for the persistent discomfort could explain why some potential pain-busting pharmaceuticals look good on animal trials, but don’t do much to alleviate ongoing aching, stinging, and burning sensations.

Many drugs are based on the initial responses of animals to pain – a clear withdrawal of a foot, for example. But fewer pay attention to what the team refers to as coping mechanisms that might demonstrate lingering discomfort.

It’s an interesting find, but the convenience of using mice also throws up the problem of accurately interpreting the results. Licking behaviours make a good proxy for ‘coping’ with pain, but it’s not like we can ask them.

“How can little mice tell us what they feel?” Ma admitted to Kelly Servick at Science Magazine.

“It’s forever a challenge.”

Still, this discovery is an important clue. And further research could help establish whether or not this new pain pathway might even present a new target for painkillers.

On behalf of people suffering chronic pain, we hope it’s a discovery that one day leads to relief.

The Problem With Probiotics


There are potential harms as well as benefits, and a lot of wishful thinking and imprecision in the marketing of products containing them.

A lot of trust has been put into the idea that gut bacteria can be a key to good health.

 

Even before the microbiome craze — the hope that the bacteria in your gut holds the key to good health — people were ingesting cultures of living micro-organisms to treat a host of conditions. These probiotics have become so popular that they’re being marketed in foods, capsules and even beauty products.

Probiotics have the potential to improve health, including by displacing potentially harmful bugs. The trouble is that the proven benefits involve a very small number of conditions, and probiotics are regulated less tightly than drugs. They don’t need to be proved effective to be marketed, and the quality control can be lax.

In a recent article in JAMA Internal Medicine, Pieter Cohen, an associate professor of medicine at Harvard Medical School, urges us to consider the harms as well as the benefits. Among immune-compromised individuals, for instance, probiotics can lead to infections.

Consumers can’t always count on what they’re getting. From 2016 to 2017, the Food and Drug Administration inspected more than 650 facilities that produce dietary supplements, and determined that more than 50 percent of them had violations. These included issues with the purity, strength and even the identity of the promised product.

Probiotic supplements have also been found to be contaminated with organisms that are not supposed to be there. In 2014, such a supplement’s contamination arguably caused the death of an infant.

Given all of this, what are the benefits? The most obvious use of probiotics would be in the treatment of gastrointestinal disorders, given that they are focused on gut health. There have been many studies in this domain, so many that early this year the journal Nutrition published a systematic review of systematic reviews on the subject.

The takeaway: Certain strains were found useful in preventing diarrhea among children being prescribed antibiotics. A 2013 review showed that after antibiotic use, probiotics help prevent Clostridium difficile-associated diarrhea. A review focused on acute infectious diarrhea found a benefit, again for certain strains of bacteria at controlled doses. There’s also evidence that they may help prevent necrotizing enterocolitis (a serious gastrointestinal condition) and death in preterm infants.

Those somewhat promising results — for very specific uses of very specific strains of bacteria in very specific instances — are just about all the “positive” results you can find.

Many wondered whether probiotics could be therapeutic in other gastrointestinal disorders. Unfortunately, that doesn’t appear to be the case. Probiotics didn’t show a significant benefit for chronic diarrhea. Three reviews looked at how probiotics might improve Crohn’s disease, and none could find sufficient evidence to recommend their use. Four more reviews looked at ulcerative colitis, and similarly declared that we don’t have the data to show that they work. The same was true for the treatment of liver disease.

Undaunted, researchers looked into whether probiotics might be beneficial in a host of disorders, even when the connection to gut health and the microbiome was tenuous. Reviews show that there is insufficient evidence to recommend their use to treat or prevent eczema, preterm labor, gestational diabetes, bacterial vaginosis, allergic diseases or urinary tract infections.

Reviews looking at the treatment or prevention of vulvovaginal candidiasis in women, pneumonia in patients hooked up to respirators, and colds in otherwise healthy people show some positive results. But the authors note that the studies are almost all of low quality, small in size, and often funded by companies with significant conflicts of interest.

Individual studies are similarly disappointing for probiotics. One examining obesity found limited effects. Another showed they don’t prevent cavities in teeth. They don’t help prevent infant colic, either.

None of this has prevented probiotics from becoming more popular. In 2012, almost four million Americans used them. In 2014, the global market for probiotics was more than $32 billion.

It’s not clear why. Even in specific diarrhea-focused areas, the case for them isn’t as strong as many think. As with nutrition research, much of this has to do with study design and how proof of efficacy doesn’t translate into real-world applications.

“Sometimes small studies suggest strains work, but when a larger more well-done study is performed, they no longer seem to,” Dr. Cohen said.

When research is done on probiotics, it usually involves a specific organism, defined by genus, species and even strain. When used in studies, they are pure and carefully dosed. But when we buy probiotics off the shelf, especially when they are in food products, we often have no idea what we’re getting.

Yet that’s how probiotics are often offered. They can be distributed in the United States as food, supplements or drugs. The regulations for each are very different. Most people looking for probiotics don’t see the distinctions. Ideally, the ways in which we use and consume probiotics would conform to the data and evidence that back them up. That rarely happens.

Further, there’s still a lot we don’t know. A recent study published in Cell compared how the microbiome of the gut reconstituted itself after antibiotic treatment with and without probiotic administration. The researchers found that probiotics (which might have improved diarrhea symptoms) led to a significant delay in microbiome reconstitution, if it occurred at all. And — again — this study was with purified strains of bacteria, which is not what you’re getting in probiotic-containing food.

Of course, people with no immune deficiencies should feel free to eat yogurt and sauerkraut, which can absolutely be part of a healthy diet. Eat them because they’re delicious, and most likely better for you than many other foods, not because of any health claims.

“It’s important that consumers understand that all those nicely labeled containers on store shelves are not vetted by the F.D.A.,” Dr. Cohen said. “They’re not carefully watching over the probiotic space, leaving consumers to be the guinea pigs for these science experiments.”

For too long we’ve assumed that probiotics are doing some good and little harm. That might be true for some, but it’s not assured in the current environment.

Guardian of the Cell


Scientists unravel the structure, key features of a human immune-surveillance protein, setting the stage for more-precise immune therapies

protein structure
Scientists have identified the key structural and functional features of a critical immune protein in humans that guards against cancer, viral and bacterial infections.

 

The human body is built for survival. Each one of its cells is closely guarded by a set of immune proteins armed with nearly foolproof radars that detect foreign or damaged DNA.

One of the cells’ most critical sentinels is a “first responder” protein known as cGAS, which senses the presence of foreign and cancerous DNA and initiates a signaling cascade that triggers the body’s defenses.

The 2012 discovery of cGAS ignited a firestorm of scientific inquiry, resulting in more than 500 research publications, but the structure and key features of the human form of the protein continued to elude scientists.

Now, scientists at Harvard Medical School and Dana-Farber Cancer Institute have, for the first time, identified the structural and functional differences in human cGAS that set it apart from cGAS in other mammals and underlie its unique function in people.

A report on the team’s work, published July 12 in Cell, outlines the protein’s structural features that explain why and how human cGAS senses certain types of DNA, while ignoring others.

“The structure and mechanism of action of human cGAS have been critical missing pieces in immunology and cancer biology,” said senior investigator Philip Kranzusch, assistant professor of microbiology and immunobiology at Harvard Medical School and Dana-Farber Cancer Institute. “Our findings detailing the molecular makeup and function of human cGAS close this critical gap in our knowledge.” Importantly, the findings can inform the design of small-molecule drugs tailored to the unique structural features of the human protein—an advance that promises to boost the precision of cGAS-modulating drugs that are currently in development as cancer therapies. “Several promising experimental immune therapies currently in development are derived from the structure of mouse cGAS, which harbors key structural differences with human cGAS,” Kranzusch said. “Our discovery should help refine these experimental therapies and spark the design of new ones. It will pave the way toward structure-guided design of drugs that modulate the activity of this fundamental protein.”

The team’s findings explain a unique feature of the human protein—its capacity to be highly selective in detecting certain types of DNA and its propensity to get activated far more sparingly, compared with the cGAS protein in other animals.

Specifically, the research shows that human cGAS harbors mutations that make it exquisitely sensitive to long lengths of DNA but render it “blind” or “insensitive” to short DNA fragments.

“Human cGAS is a highly discriminating protein that has evolved enhanced specificity toward DNA,” said co-first author Aaron Whiteley, a postdoctoral researcher in the Department of Microbiology and Immunobiology at Harvard Medical School. “Our experiments reveal what underlies this capability.”

Location, location, location

In all mammals, cGAS works by detecting DNA that’s in the wrong place. Under normal conditions, DNA is tightly packed and protected in the cell’s nucleus—the cellular “safe”—where genetic information is stored. DNA has no business roaming freely around the cell. When DNA fragments do end up outside the nucleus and in the cell’s cytosol, the liquid that encases the cell’s organelles, it’s usually a sign that something ominous is afoot, such as damage coming from within the cell or foreign DNA from viruses or bacteria that has made its way into the cell.

The cGAS protein works by recognizing such misplaced DNA. Normally, it lies dormant in cells. But as soon as it senses the presence of DNA outside the nucleus, cGAS springs into action. It makes another chemical—a second messenger—called cGAMP, thus setting in motion a molecular chain reaction that alerts the cell to the abnormal presence of DNA. At the end of this signaling reaction, the cell either gets repaired or, if damaged beyond repair, it self-destructs.

But the health and integrity of the cell are predicated on cGAS’ ability to distinguish harmless DNA from foreign DNA or self-DNA released during cell damage and stress. “It’s a fine balancing act that keeps the immune system in equilibrium. An overactive cGAS can spark autoimmunity, or self-attack, while cGAS that fails to detect foreign DNA can lead to tumor growth and cancer development,” said co-first author Wen Zhou, a postdoctoral researcher at Harvard Medical School and Dana-Farber Cancer Institute.

The current study reveals the evolutionary changes to the protein’s structure that allow human cGAS to ignore some DNA encounters while responding to others.

A foe, an accomplice

For their work, the team turned to an unlikely collaborator—Vibrio cholerae, the bacterium that causes cholera, one of humankind’s oldest scourges.

Taking advantage of a cholera enzyme that shares similarities with cGAS, the scientists were able to recreate the function of both human and mouse cGAS in the bacterium.

Teaming up with colleagues from the lab of Harvard Medical School bacteriologist John Mekalanos, the scientists designed a chimeric, or hybrid, form of cGAS that included genetic material from both the human and mouse forms of the protein. Then they compared the ability of the hybrid cGAS to recognize DNA against both the intact mouse and intact human versions of the protein.

In a series of experiments, the scientists observed activation patterns between the different types of cGAS, progressively narrowing down the key differences that accounted for differential DNA activation among the three.

The experiments revealed that out of the 116 amino acids that differ in human and mouse cGAS, only two accounted for the altered function of human cGAS. Indeed, human cGAS was capable of recognizing long DNA with great precision but it ignored short DNA fragments. The mouse version of the protein, by contrast, did not differentiate between long and short DNA fragments

“These two tiny amino acids make a world of difference,” Whiteley said. “They allow the human protein to be highly selective and respond only to long DNA, while ignoring short DNA, essentially rendering the human protein more tolerant of DNA presence in the cytosol of the cell.”

Plotting the genetic divergence on an evolutionary timescale, the scientists determined that the human and mouse cGAS genes parted ways sometime between 10 million and 15 million years ago.

The two amino acids responsible for sensing long DNA and tolerating short DNA are found solely in humans and nonhuman primates, such as gorillas, chimps and bonobos. The scientists hypothesize that the ability to ignore short DNA but recognize long DNA must have conferred some evolutionary benefits. “It could be a way to guard against an overactive immune system and chronic inflammation,” Kranzusch said. “Or it could be that the risk of certain human diseases is lowered by not recognizing short DNA.”

In a final set of experiments, the team determined the atomic structure of the human cGAS in its active form as it binds to DNA. To do so, they used a visualization technique known as X-ray crystallography, which reveals the molecular architecture of protein crystals based on a pattern of scattered X-ray beams.

Profiling the structure of cGAS “in action” revealed the precise molecular variations that allowed it to selectively bind to long DNA, while ignoring short DNA.

“Understanding what makes the structure and function of human cGAS different from those in other species was the missing piece,” Kranzusch said. “Now that we have it, we can really start designing drugs that work in humans, rather than mice.”

Other investigators included Carina de Oliveira Mann, Benjamin Morehouse, Radosław Nowak, Eric Fischer, and Nathanael Gray. The work was supported by the Claudia Adams Barr Program for Innovative Cancer Research, by the Richard and Susan Smith Family Foundation, by the Charles H. Hood Foundation, by a Cancer Research Institute CLIP Grant, by the National Institute of Allergy and Infectious Diseases grant AI-01845, by National Cancer Institute grant R01CA214608, by the Jane Coffin Childs Memorial Fund for Medical Research, by a Cancer Research Institute Eugene V. Weissman Fellow award, and by a National Institutes of Health T32 grant 5T32CA207021-02.

Relevant Disclosures: The Dana-Farber Cancer Institute and Harvard Medical School have patents pending for human cGAS technologies, on which the authors are inventors.

Harvard Medical School Harvard Medical School (http://hms.harvard.edu) has more than 11,000 faculty working in 10 academic departments located at the School’s Boston campus or in hospital-based clinical departments at 15 Harvard-affiliated teaching hospitals and research institutes: Beth Israel Deaconess Medical Center, Boston Children’s Hospital, Brigham and Women’s Hospital, Cambridge Health Alliance, Dana-Farber Cancer Institute, Harvard Pilgrim Health Care Institute, Hebrew SeniorLife, Joslin Diabetes Center, Judge Baker Children’s Center, Massachusetts Eye and Ear/Schepens Eye Research Institute, Massachusetts General Hospital, McLean Hospital, Mount Auburn Hospital, Spaulding Rehabilitation Network and VA Boston Healthcare System.

Impact Study


Understanding injuries on the basketball court

Wearable sensors will give researchers data on impact loading in real-world game and practice situations.

Whether players are crashing the boards on rebounds, sprinting down court on a fast break or taking a charge on defense, basketball is a physically demanding sport that can take a toll on athletes’ bodies.

In particular, basketball players experience a large number of high-impact loading events during their career and these repetitive loading events are thought to be related to the high incidence of lower-limb injury that elite players experience, including bone-stress injury. Unfortunately, the relationship between mechanical loading and injury is not clear because until now it has been difficult to measure impact loads during practice and games.

A new study led by Irene Davis, Harvard Medical School professor of physical medicine and rehabilitation and the director of the Spaulding National Running Center plans to examine the role of fatigue on landing impacts and bone-stress injuries in basketball players.

The research team will be using small devices, called wearable sensors, that can be secured to the lower leg to measure these loads while players are on the court. The relationship between load and injury is affected by other factors such as sleep, nutrition, bony anatomy and the current state of bone health. To prevent bone-stress injury and help athletes recover from bone-stress injury, we must first have a better understanding of these mechanical and biological factors and how they relate to bone health, the researchers said.

“Ultimately, we want to understand the factors that affect the foot-bone loading a basketball player sustains over the course of a season,” Davis said.

The study is funded by the National Basketball Association and GE Orthopedics and Sports Medicine Collaboration.

Davis and her team will focus on foot-bone stress injuries as these are the most common in basketball players. They plan to first collect baseline measures of foot-bone anatomy and bone health using various types of imagining techniques. They will also gather data on nutrition and diet, such as the amount of vitamin D in the blood and daily caloric intake. The study team will then monitor how hard the players are landing with each step across practices and games throughout the season and also monitor sleep over the course of the season. Finally, the team will image the foot again to determine if any injuries are developing.

“Bone-stress injuries can progress to small cracks in the bone, and ultimately, complete fractures. These injuries can be season ending and sometimes career ending. Therefore, preventing them from happening is the best approach. We believe this study will provide important information for sports medicine professionals who oversee these athletes’ care,” Davis said.

The advent of wearable sensors that can accurately measure and monitor impact loads provides an opportunity to characterize the physical demands of the game in unprecedented ways, Davis said. This new form of data has the potential to influence the understanding of lower-limb injury, as well as provide crucial insight on how to alter training to mitigate injury risk and how to improve return-to-play decisions.

Understanding the role of mechanical loading in maintaining bone health is particularly relevant to young elite basketball athletes whose skeletons are still undergoing a modeling process and to freshman college athletes who might receive a dramatic increase in impact load exposure during preseason training that their skeletons have not yet adapted to.

By including both college and elite basketball athletes in this cohort, Davis said that her research will address several interesting research questions: How does lower-limb load exposure due to training differ between college and elite level basketball athletes? What is the impact load exposure during a game and how does this compare to training? How do lower-limb impact loads change during a game and throughout the season (i.e., is there a fatigue-related change in impact load)? And ultimately, does impact load exposure predict bone health and possibly, bone-stress injury?

The findings of the study should also provide insight that will be helpful for the health of people who happen not to be elite athletes, Davis said.

“Ultimately, measuring and monitoring the mechanical loads experienced by musculoskeletal tissue is critical to improve our understanding of musculoskeletal injury and health,” Davis said.

Scientists have identified the immune cascade that fuels complications, tissue damage in Chlamydia infections


More than 2.8 million chlamydia infections make it the most common sexually transmitted disease in the United States.

Closing a critical gap in knowledge, Harvard Medical School scientists have unraveled the immune cascade that fuels tissue damage and disease development in chlamydia infection—the most common sexually transmitted disease in the United States.

Physicians have long known that complications of chlamydia are not caused by the bacterium itself but instead arise from inflammation in the reproductive organs. However, up until now, it remained unclear what drives this damaging inflammation.

Findings of the new research, conducted in mice and published Feb. 9 in PNAS, reveal the precise mechanism behind this phenomenon and identify the cast of immune cells involved in it. Further, the research shows that the body deals with chlamydia infection via two distinct and separate immune pathways—one driving the clearance of bacteria and one fueling inflammation and tissue damage.

More than 2.8 million chlamydia infections occur each year in the United States, according to the Centers for Disease Control and Prevention. Left untreated, chlamydia can lead to pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy, infertility and prostate inflammation.

Chlamydia infections can be cleared with prompt antibiotic treatment, but most people infected with the bacterium have silent infections, resulting in delayed treatment. Untreated infections that linger for months—and sometimes for years—can cause irreversible inflammatory damage to the reproductive organs.

“By the time the infection is identified, irreversible damage has often occurred so we urgently need therapies that prevent these devastating consequences,” said senior author Michael Starnbach, professor in the Department of Microbiology and Immunobiology at Harvard Medical School. “Our findings offer a roadmap for the development of vaccines that can stimulate immune protection against chlamydia-associated diseases.”

The study findings show that complications of chlamydia infections arise from inflammation that occurs when several types of protective immune cells rush to the reproductive organs after the bacterium invades the body. Remarkably, the research shows these immune cells are not involved in the clearance of bacteria, but rather that clearance is instead prompted by a different class of immune cells.

The existence of such separate immune responses is good news, the researchers said.

“This is a truly encouraging finding,” said study first author Rebeccah Lijek, who conducted the research as a post-doctoral fellow at Harvard Medical School and is now assistant professor of biological science at Mount Holyoke College.

“It means that if one class of immune cells is responsible for clearing the infection, while another class of immune cells causes tissue damage and subsequent disease, then we can develop treatments that precision target inflammation without exacerbating bacterial levels,” she said.

To understand how the bacterium damages urogenital tissue, the team started out by recreating symptoms that mimic human chlamydia infection in mice—the first successful instance of doing so.  Past failures to replicate human symptoms in an animal model have hampered the understanding of the chlamydia-driven diseases for decades, the team said.

Next, the team analyzed the reproductive tissue of infected mice, using a technique that identifies the presence of various immune cells. The researchers observed that in the first few days after infection, immune cells known as neutrophils—the body’s first responders—charge up to the urogenital tract and cause inflammation and damage.

To determine what happens in the absence of neutrophils, researchers used an antibody specifically designed to target these cells while sparing all others.

Chlamydia-infected mice that lacked neutrophils showed no tissue damage despite harboring the bacterium. The absence of neutrophils had no effect on bacterial levels—an indicator that neutrophils play no role in the clearing of the bacterium.

Further analysis showed that in the later stages of infection—a week or so after the bacterium enters the body—a different set of immune cells make their way to the urogenital tract.

Researchers observed dramatically elevated levels of two types of T cells. Known as the body’s elite assassins, T cells are “trained” to seek out and destroy pathogens. In this case, however, researchers identified two distinct subtypes of T cells: general-assignment, bystander T cells that drive inflammation and chlamydia-specific T cells, formed in response to the presence of this particular bacterium.

Previous research conducted by Starnbach’s team had shown that chlamydia-specific T cells are responsible for clearing the infection but up until now the scientists didn’t know whether the chlamydia-specific T cells might also spark damaging inflammation. They do not, the study showed.

To understand what attracts inflammation-inducing cells to infected urogenital tissue, researchers analyzed more than 700 inflammation-promoting and immunity-inducing genes.

During infection, these genes release signaling proteins known as chemokines, which call on immune cells to make their way to the site of infection. Researchers identified a trio of chemokines—CXCL 9,10,11—that were particularly elevated, compared with all others.

These very proteins are also known to play a role in the autoimmune conditions inflammatory bowel disease and rheumatoid arthritis.

In a final step, the researchers used a chemical compound to block the activity of the inflammation-inducing chemokines. Mice treated with the compound had markedly reduced levels of nonspecific, bystander T cells and markedly less inflammation and tissue damage, compared with untreated mice. Notably, the improvement occurred without any effect on bacterial levels.

Several compounds that target the receptor for inflammation-inducing chemokines CXCL 9,10,11 are currently being tested in clinical trials as a treatment for inflammatory bowel disease.

“Our data suggest that such therapies may also be beneficial in the treatment and prevention of pelvic inflammation following chlamydia infection,” Lijek said.

No Llamas Required


Researchers develop alternate method to uncover protein structures, design new drugs

https://player.vimeo.com/video/254936336?color=456d86&title=0&byline=0&portrait=0

Detouring around a major research roadblock, researchers have found a new way to create valuable antibodies without needing … llamas?

It is a little-known fact that llamas, alpacas, camels and other members of the camelid family make a unique class of antibodies that allow scientists to determine the structures of otherwise impossible-to-study proteins in the body, helping them to understand how those proteins malfunction in disease and how to design new drugs that act on them.

As one might imagine, there are downsides to taking advantage of this evolutionary happenstance.

First, not all researchers who need camelid antibodies for their experiments have access to llama (or alpaca or camel) facilities. Second, while the animals are not harmed, vaccinating them to generate the desired antibodies is expensive, takes as long as six months per attempt and often doesn’t work.

So, biochemists Andrew Kruse at Harvard Medical School and Aashish Manglik at the University of California, San Francisco, teamed up to create a llama-free solution: vials of specially engineered yeast.

The yeast method, described Feb. 12 in Nature Structural and Molecular Biology, can be done in a test tube in a researcher’s own lab. It has a higher success rate and faster turnaround time than both llama vaccination and previous attempts to circumvent camelids, the authors say.

It also marks the first time a camelid-bypass system has been made freely available for nonprofit use.

“There’s a real need for something like this,” said Kruse. “It’s low-tech, it’s a low time investment and it has a high likelihood of success for most proteins.”

“People who have struggled to nail down their protein structures for years with llamas are getting them now,” he said.

Lock and key

The active segments of camelid antibodies are often called nanobodies because they can be much smaller than regular antibodies. A llama nanobody might bind only to a particular conformation—for example, “open” or “closed”—of a particular protein. Nanobodies also can bind to challenging proteins, such as receptors that work in oily cell membranes.

Structural biologists like Kruse and Manglik want to find the exact nanobody that matches their protein of interest so they can lock the protein in one position and run tests to figure out its atomic structure. Learning the structure allows them to study how the protein works and provides a blueprint for designing drugs that target it.

Nanobodies have opened long-locked doors in biomedical science. For example, they have allowed researchers to see for the first time how neurotransmitters such as adrenaline and opioids bind to receptors in the brain.

Scientists just needed an easier way to find the keys.

Glowing success

Right now, a scientist who wants to study a difficult membrane protein has to laboriously generate several milligrams of it, inoculate a llama with it—usually done through a third-party service—and hope the animal’s immune system responds. Only then can she search for antibodies in a blood sample and hope there are enough to work with.

By contrast, Kruse and Manglik’s research team, led by first author Conor McMahon, a postdoctoral researcher in the Kruse lab, created a library of 500 million camelid antibodies using yeast cells.

Each yeast cell has a slightly different nanobody tethered to its surface, made by a slightly different piece of synthetic DNA.

The researchers mixed all the yeasts together and froze them for safekeeping. Anytime they want to run an experiment, they simply defrost a test tube’s worth: a miniature llama immune system. (The tube contains 10 to 20 times the amount needed to ensure that at least one of each unique antibody is included.)

The team developed a method where, instead of injecting a llama, scientists can now label their protein of interest with a fluorescent molecule and add it to the test tube. Yeast with surface nanobodies that recognize the protein will glow.

The researchers then use fluorescence-activated cell sorting, or FACS, to separate the glowing yeast from the rest.

They sequence the DNA of those glowing yeast cells to learn what the nanobodies are. They can then use E. coli bacteria to grow as many of those nanobodies as they need.

The whole process takes three to six weeks instead of three to six months.

Money for nothing, and yeast for free

The team tested its yeast system on two proteins: the beta-2 adrenergic receptor, linked to asthma, and the adenosine receptor, which is a gateway for caffeine to deliver its buzz. In both cases, the nanobody bound to the desired receptor, bound only to that receptor, and bound to it only when it was “on.”

“We found that the yeast-derived nanobodies can do everything llama-derived antibodies can,” said Kruse.

The team is now offering vials of the yeast mix and usage instructions free of charge to any nonprofit labs that want them. Commercial companies can license the yeast. “We made a big batch,” said McMahon, so there’s plenty to go around.

More than 40 labs requested vials before the paper was even published.

“Nanobodies are making it possible to develop drugs for biological targets that antibodies were simply too big to hit,” said Manglik. “By making nanobody discovery quick and easy, we hope our platform will dramatically accelerate the potential applications of this exciting technology.”

“I think we’ll see things that blow the animal immune system away,” added McMahon. “This is new technology. It’s only going to get better. Hopefully it will work as well or better so we won’t need llamas anymore.”

No Clear Benefit of Prazosin for Sleep-Related Symptoms of PTSD


Figure 1. Change in Scores of Primary Outcome Measures from Baseline.The three primary outcome measures were the change in score from baseline to 10 weeks on the Clinician- Administered PTSD Scale (CAPS) item B2 (scores range from 0 to 8, with higher scores indicating more frequent and more distressing dreams) (Panel A), the change in Pittsburgh Sleep Quality Index (PSQI) score from baseline to 10 weeks (scores range from 0 to 21, with higher scores indicating worse sleep quality) (Panel B), and the Clinical Global Impression of Change (CGIC) score at 10 weeks (range, 1 to 7, with lower scores indicating greater improvement and a score of 4 indicating no change from baseline; the CGIC assessed the participant’s ability to function in daily activities and the participant’s sense of well-being) (Panel C). Over the entire 26 weeks, the mean change from baseline on the CAPS item B2 was −2.0 (95% confidence interval [CI], −2.1 to −1.8) with prazosin and −2.1 (95% CI, −2.2 to −1.9) with placebo (P = 0.56); the mean change from baseline on the PSQI was −2.6 (95% CI, −2.9 to −2.3) with prazosin and −2.6 (95% CI, −2.9 to −2.3) with placebo (P = 0.99); and the mean CGIC score was 3.1 (95% CI, 3.0 to 3.2) with prazosin and 3.1 (95% CI, 3.0 to 3.2) with placebo (P = 0.86). I bars indicate 95% confidence intervals.

During my internal medicine residency, I was privileged to be the primary care physician for many veterans. Practicing in a Veterans Affairs (VA) medical center is unique in many ways. Besides the predominantly male population, many patients suffered from mental illnesses with post-traumatic stress disorder (PTSD) as a common culprit. One memorable young patient struggled with persistent sleep disturbance. When a consulting psychiatrist recommended treatment with prazosin, I thought it was a curious choice because alpha blockers sat firmly in my mental buckets of “not great anti-hypertensives” and “helps with urination.” I subsequently learned that alpha-1 antagonists can lower the high adrenergic activity that is thought to drive PTSD symptoms. Further, prazosin crosses the blood-brain barrier quite effectively, and evidence from small randomized clinical trials (RCT) gave us hope that it would work.

Consequently, I read with great interest the results from the PACT trial published in this week’s NEJM. In this largest RCT to date, investigators randomized 304 veterans with PTSD and frequent nightmares from 12 VA centers to receive prazosin or placebo for 26 weeks. During that time, existing therapies were continued but no new pharmacotherapy and psychotherapy could be added.

Unfortunately, the results of this trial did not show any benefit from prazosin for the three primary outcomes: Change from baseline at 10 weeks in the recurrent distressing dream component of the Clinician-Administered PTSD Scale (CAPS; score range, 0–8), the Pittsburgh Sleep Quality Index (PSQI; score range, 0–21), and the absolute score of the Clinical Global Impression of Change (CGIC; score range, 1–7). In general, higher scores indicate worse symptoms for all three scoring systems. The mean between-group differences in the change from baseline for the CAPS recurrent distressing dreams component and PSQI were 0.2 (95% CI, -0.3 to 0.8; P=0.38) and 0.1 (95% CI, -0.9 to 1.1; P=0.80), respectively. The mean difference in the CGIC was 0 (95% CI, -0.3 to 0.3; P=0.96). Of the adverse events, dizziness (34% vs. 21%), lightheadedness (34% vs. 20%), and urinary incontinence (12% vs. 4%) were more common in the prazosin group, and new or worsening suicidal ideation was less common in the prazosin group (8% vs. 15%).

With any clinical trial, the devil is in the detail. The authors offer some possible explanations for the lack of benefit of prazosin, primarily selection bias. At the time of the study, prazosin was already available for off-label use in the VA medical system and was often prescribed as an adjunct to first-line selective serotonin reuptake inhibitors (SSRIs), as with the patient I described above. Therefore, clinicians may have been hesitant to enroll patients with more severe symptoms and risk for clinical deterioration in a randomized trial with the possibility of receiving placebo when prazosin was available outside of the trial. Furthermore, patients treated with trazodone were excluded from the trial because of its alpha-1 antagonist activity, thus removing a population of potential responders.

In an accompanying editorial, Dr. Kerry Ressler from Harvard Medical School reminds us that PTSD is a heterogenous disorder with different manifestations of symptoms. Perhaps not every patient shares the same adrenergic hyperarousal response that is thought to be the driver of nightmares. He encourages the development of better biomarkers to identify the phenotype of patients who may respond to prazosin.

I left the VA before I had the chance to see whether my patient benefited from prazosin, but other patients have told me that prazosin is the only intervention that lets them sleep at night. Although the PACT trial did not show clear efficacy, prazosin may still have therapeutic benefit in select patients.

Can Apple Take Healthcare Beyond the Fax Machine?


Despite spectacular advances in diagnostic imaging, non-invasive surgery, and gene editing, healthcare still faces a lackluster problem: many patients can only get health records from their doctor if the fax machine is working. Even when records are stored electronically, different chunks of every patient’s health information sit in the non-interoperable, inaccessible electronic record systems in different doctor’s offices.

Anyone who needs her medical files gets them either printed or faxed, or has to log on into separate portals for each doctor and hospital, and even then getting view-only access. View-only apps can’t access data to help patients share information with family and healthcare providers, make decisions, monitor disease, stay on course with medications, or just stay well.

On the positive side, this is changing, sort of. Using the iPhone Health app, patients will soon be able to download and view health records on their phones. On the one hand, don’t get too excited–it will initially only work for patients at a handful of institutions, Android users are still out in the cold, and the data available will be limited. And, some dismiss the impact of Apple’s move because of others’ failures to give patients control of their records.

However, Apple’s move is a decisive and consequential advance in patients’ struggle to get a copy of their own health data. Apple wisely chose to use open, non-proprietary approaches that will float all boats–even for Android users.

Every patient deserves a ‘bank account’ of her health data, under her control, with deposits made after every healthcare encounter. After my colleagues and I demonstrated an open, free version of a “bank account” to companies in 2006, Google and Microsoft launched similar personally controlled health records — GoogleHealth and Microsoft Healthvault. Walmart and other employers offered our version, Indivo, as an employee benefit. Unfortunately, even these industry giants couldn’t shake loose data from the proprietary computer systems in doctors’ offices, or make the case to patients that curating the data was worth the effort.

But 12 years later, Apple’s product enters healthcare under different circumstances.  A lot more patient data is electronic after a $48 billion federal investment in promoting the adoption of information technology to providers. But those products, mostly older software and purchased at enormous expense, still don’t promote record sharing with doctors or patients.

Recognizing this unacceptable limitation and having received a generous grant comprising a tiny fraction of that federal investment, our team created SMART on FHIR. SMART is an interface to make doctors’ electronic health records work like iPhones do. Apps can be added or deleted easily. The major electronic health record brands have built this interface into their products.

Apple uses SMART to connect the Health app to hospitals and doctors offices. The good news for patients, doctors, and innovators is that Apple chose a standardized, open connection over a proprietary, closed one. This approach lets any other app, whether running on the web,  iPhone, or Android, use that very same interface to connect.

So Apple will compete on value and customer satisfaction, rather than on an exclusive lock on the data. Does Apple’s approach help Americans trying to stay well or manage their conditions? Yes. But only with follow-through by Apple, health systems, technology companies, patient groups, policy makers, and government regulators. The emerging ecosystem’s nuances must be appreciated.

First of all, the floodgates for patient information are at least a crack open and will be very hard to close. As patients gain access to their data, they will recognize it is incomplete and feel frustrated it’s not available everywhere. But, patients in need will drive demand for data access in their role as health consumers.

Secondly, the government is effectively using law and regulations to compel an open interface. By selecting SMART on FHIR, Apple and its healthcare launch partners mark the importance of standardization. A uniform approach is critical for scale. Imagine if every electrical product required a differently shaped 120V outlet. Understanding this, Google, Quest Diagnostics, Eli Lily, Optum, and many other companies are using the same interface to plug into healthcare.

Thirdly, Apple’s first version of health records brings data onto the phone, but from there, like the portals many patients are already familiar with, the data are still “view-only.”  In 2009, I had the chance to meet with Apple’s rockstar Bud Tribble and talk about how the iPhone could serve healthcare. We concluded that crucial data–like the medication list–had to be as easy for iOS developers to use in their apps as contacts and location are now.  I would not be at all surprised if this is the next step in Apple’s journey–making the health records available to iPhone app developers. Here too is an opportunity to chose open interfaces, and to allow patients to export the data to another device.

Lastly, competition in healthcare IT is hot. Amazon, Google, Apple and Facebook all have healthcare divisions.  Apple’s extraordinary hardware, including sensors in the phone and watch, will monitor patients at home.  Google’s artificial intelligence will lead doctors and patients to diagnoses and decisions.  Amazon is rumored to be eying pharmacy management. Facebook has sifted through posts to detect and possibly intervene when users may be suicidal.

There are so many opportunities to compete. Locking up a patient’s data should never be one of them.

Ken Mandl, MD, MPH directs the Boston Children’s Hospital Computational Health Informatics Program and is the Harvard Medical School Donald A.B. Lindberg Professor of Pediatrics and Biomedical Informatics.

Flip the Switch


Changes in fat metabolism may promote prostate cancer metastasis

Prostate tumors tend to be what scientists call “indolent”—so slow-growing and self-contained that many affected men die with prostate cancer, not of it. But for the percentage of men whose prostate tumors metastasize, the disease is invariably fatal.

In a set of papers published in the journals Nature Genetics and Nature Communications, researchers at Harvard Medical School and the Cancer Center at Beth Israel Deaconess Medical Center have shed new light on the genetic mechanisms that promote metastasis in a mouse model and implicated the typical Western high-fat diet as a key environmental factor driving metastasis.

“Although it is widely postulated that a Western diet can promote prostate cancer progression, direct evidence supporting a strong association between dietary lipids and prostate cancer has been lacking,” said first author Ming Chen, HMS research fellow in medicine in the laboratory of Pier Paolo Pandolfi, the HMS George C. Reisman Professor of Medicine at Beth Israel Deaconess.

Epidemiological data links dietary fats (and obesity) to many types of cancer, and rates of cancer deaths from metastatic cancers including prostate cancer are much higher in the United States than in nations where lower fat diets are more common. While prostate cancer affects about 10 percent of men in Asian nations, that rate climbs to about 40 percent when they immigrate to the U.S., mirroring the rates among the native-born U.S. population. That points to an environmental culprit that may work in concert with genetic factors to drive this aggressive, fatal disease.

“The progression of cancer to the metastatic stage represents a pivotal event that influences patient outcomes and the therapeutic options available to patients,” said senior author Pandolfi, who is also director of the Cancer Center and the Cancer Research Institute at Beth Israel Deaconess. “Our data provide a strong genetic foundation for the mechanisms underlying metastatic progression, and we also demonstrated how environmental factors can boost these mechanisms to promote progression from primary to advanced metastatic cancer.”

The tumor suppressor gene PTEN is known to play a major role in prostate cancer; its partial loss occurs in up to 70 percent of primary prostate tumors. Its complete loss is linked to metastatic prostate disease, but animal studies suggest the loss of PTEN alone is not enough to trigger progression. Pandolfi and colleagues sought to identify an additional tumor suppressing gene or pathway that may work in concert with PTEN to drive metastasis.

Looking at recent genomic data, Pandolfi and colleagues noticed that another tumor suppressor gene, PML, tended to be present in localized (nonmetastatic) prostate tumors but was absent in about a third of metastatic prostate tumors. Moreover, about 20 percent of metastatic prostate tumors lack both PML and PTEN.

When they compared the two types of tumor—the localized ones lacking only the PTEN gene versus the metastatic tumors lacking both genes—the researchers found that the metastatic tumors produced huge amounts of lipids, or fats. In tumors that lacked both PTEN and PML tumor suppressing genes, the cells’ fat-production machinery was running amok.

“It was as though we’d found the tumors’ lipogenic, or fat production, switch,” said Pandolfi. “The implication is, if there’s a switch, maybe there’s a drug with which we can block this switch and maybe we can prevent metastasis or even cure metastatic prostate cancer,” he added.

Such a drug already exists. Discovered in 2009, a molecule named “fatostatin” is currently being investigated for the treatment of obesity. Pandolfi and colleagues tested the molecule in lab mice. “The obesity drug blocked the lipogenesis fantastically, and the tumors regressed and didn’t metastasize.”

In addition to opening the door to new treatment for metastatic prostate cancer, these findings also helped solve a long-standing scientific puzzle. For years, researchers had difficulty modeling metastatic prostate cancer in mice, making it hard to study the disease in the lab. Some speculated that mice simply weren’t a good model for this particular disease. But the lipid-production finding raised a question in Pandolfi’s mind.

“I asked, ‘What do our mice eat?’” Pandolfi recalled.

It turned out the mice ate a vegetable-based chow, essentially a low-fat vegan diet that bore little resemblance to that of the average American male. When Pandolfi and colleagues increased the levels of saturated fats, the kind found in fast food cheeseburgers and fries, in the animals’ diet, the mice developed aggressive, metastatic tumors.

The findings could result in more accurate and predictive mouse models for metastatic prostate cancer, which in turn could accelerate discovery of better therapies for the disease. Additionally, physicians could soon be able to screen their early-stage prostate cancer patients for those whose tumors lack both PTEN and PML tumor suppressing genes, putting them at increased risk for progressing to metastatic disease. These patients may be helped by starving these tumors of fat either with the fat-blocking drug or through diet.

“The data are tremendously actionable, and they surely will convince you to change your lifestyle,” Pandolfi said.