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Thu 27 Dec 2007 05:17 PM

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Research matters

News from the Harvard Medical School research community.

Endocrinology

Study identifies pathway required for normal reproductive development

Massachusetts General Hospital (MGH) clinical researchers, in collaboration with basic scientists from the University of California, Irvine, (UC Irvine) have identified a new molecular pathway required for normal development of the reproductive, olfactory and circadian systems in both humans and mice. In their report in the Proceedings of the National Academy of Sciences, the team describes defects in a gene called PROK2 (prokineticin 2) in human siblings with two different forms of infertility. The UC Irvine team had previously reported that mice lacking PROK2 had abnormal olfactory structures and disrupted circadian rhythm. The paper received early online release.

Mitochondria are vital for sustaining the health and longevity of a cell.

"We have demonstrated that PROK2 signalling is a novel pathway that is critical to the development of neurons that control the reproductive system, findings that should enable better understanding of human reproduction," says lead author Nelly Pitteloud, MD, of the Reproductive Endocrine Unit in the MGH Department of Medicine.

The current study is the latest in a series of investigations by the MGH group into the genetic basis of idiopathic hypogonadotropic hypogonadism (IHH), a rare condition in which puberty does not take place naturally. IHH occurs when a structure in the brain called the hypothalamus fails to develop neurons that secrete gonadotropin-releasing hormone (GnRH), a major controller of the reproductive system. Several genes involved in IHH have been discovered by the MGH investigators and others throughout the world; however, only 30% of IHH cases can currently be attributed to a known gene defect.

The investigation focused on PROK2, a protein known to regulate the development of the olfactory bulbs, the portion of the brain involved in the sense of smell, and to have a critical role in circadian rhythm in the mice. A form of IHH called Kallmann syndrome involves lack of both reproductive development and a sense of smell. PROK2's involvement in these systems led the researchers to investigate the protein's potential role in GnRH deficiency in human and mice.

The MGH team analysed the PROK2 genes of 100 study participants: 50 with Kallmann syndrome and 50 with IHH and a normal sense of smell. Three members from the same family in Portugal - two brothers and a sister - had identical defects in both copies of the PROK2 gene. Further study of this family revealed another brother with the mutation in only one PROK2 copy and a normal reproductive history. Five siblings of these individuals - now in their 70s - had died in infancy; similar early deaths have been seen in the PROK2-deficient mice. Interestingly, while the two affected brothers both had Kallmann syndrome, their affected sister had a normal sense of smell but did not experience normal puberty. "Until recently, IHH with a normal sense of smell and Kallmann syndrome with no sense of smell had been considered two distinct clinical entities," says Pitteloud, an assistant professor of Medicine at Harvard Medical School. "We now have described several kindreds in which different family members exhibit both syndromes yet harbour the identical mutation. So, it looks like additional gene defects or environmental cues modify how these syndromes develop in affected families.

The UC Irvine team was led by Qun-Yong Zhou, PhD, a professor of Pharmacology in its School of Medicine. His group has made fundamental contributions to the understanding of the neurobiological functions of prokineticin and its receptors. Their analysis of the reproductive status of mice lacking functional copies of Prok2 gene revealed that the animals' reproductive defect is due to the abnormal migration of neurons that secrete GnRH.

"Many recessive human genetic disorders, particularly the ones that have associated infertility symptom, are very difficult or almost infeasible to investigate using genetic analysis. The current study provides an elegant example how mouse studies can pinpoint the underlying genetic cause for human IHH disorders." says Zhou.

Genomics

Massive microRNA scan uncovers leads to treating muscle degeneration

Researchers have discovered the first microRNAs--tiny bits of code that regulate gene activity--linked to each of 10 major degenerative muscular disorders, opening doors to new treatments and a better biological understanding of these debilitating, poorly understood, often untreatable diseases. The study, published by the Proceedings of the National Academy of Sciences in November, was led by Iris Eisenberg, PhD, and Louis Kunkel, PhD, director of the Program in Genomics at Children's Hospital Boston and an investigator with the Howard Hughes Medical Institute.

The disorders, which cause muscle weakness and wasting, include the muscular dystrophies (Duchenne muscular dystrophy, Becker muscular dystrophy, limb girdle muscular dystrophies, Miyoshi myopathy, and fascioscapulohumeral muscular dystrophy); the congenital myopathies (nemaline myopathy); and the inflammatory myopathies (polymyositis, dermatomyositis, and inclusion body myositis). While past studies have linked them with an increasing number of genes, it's still largely unknown how these genes cause disease, and, more importantly, how to translate the discoveries into treatments.

For instance, most muscular dystrophies begin with a known mutation in a "master gene," leading to damaged or absent proteins in muscle cells. In Duchenne and Becker muscular dystrophies, the absent protein is dystrophin, as Kunkel himself discovered in 1987. Its absence causes muscle tissue to weaken and rupture, and the tissue becomes progressively non-functional through inflammatory attacks and other damaging events that aren't fully understood.

"The initial mutations do not explain why patients are losing their muscle so fast," says Eisenberg. "There are still many unknown genes involved in these processes, as well as in the inflammatory processes taking place in the damaged muscle tissue.

She and Kunkel believe microRNAs may help provide the missing genetic links. Their team analyzed muscle tissue from patients with each of the ten muscular disorders, discovering that 185 microRNAs are either too abundant or too scarce in wasting muscle, compared with healthy muscle. Discovered in humans only in the past decade, microRNAs are already known to regulate major processes in the body.

Therefore, Eisenberg believes microRNAs may be involved in orchestrating the tissue death, inflammatory response and other major degenerative processes in the affected muscle tissue. The researchers used bioinformatics to uncover a list of genes the microRNAs may act on, and now plan to find which microRNAs and genes actually underlie these processes.

The findings raise the possibility of slowing muscle loss by targeting the microRNAs that control these "cascades" of damaging events. This approach is more efficient than targeting individual genes. The team also defined the abnormal microRNA "signatures" that correspond to each of the ten wasting diseases. They hope these will shed light on the genes and disease mechanisms involved in the most poorly understood and least treatable of the degenerative disorders, such as inclusion body myositis. "At this point, it's very theoretical, but it's possible," says Eisenberg.

Pathology

Immune defect makes gut bacteria turn bad

In a striking new mouse model, an immune system glitch converts mild-mannered intestinal bacteria into a pathological army that inflames the host's colon and spreads the problem to normal mice.

The findings emphasize the importance of the interplay among the gut, its microbes, and its immune squad. The model opens new avenues to study inflammatory bowel disease and to test potential new therapies for people, said senior author Laurie Glimcher, the Irene Heinz Given professor of immunology at HSPH, and her colleagues. The study appeared in the Oct. 5 Cell.
"To my knowledge, this is the first paper to show that a host immune defect can shape the composition of microflora in a way that results in pathology," said Lora Hooper, assistant professor of immunology and microbiology at the University of Texas Southwestern Medical Center in Dallas.

"That's interesting in and of itself, but they went one step further and induced this inflammation in healthy mice by transferring the microflora," said Hooper, who was not involved in the study. "The implications of that are profound."

The researchers did not identify a specific disease-causing bacterial species, said lead author Wendy Garrett, a postdoctoral fellow in the Glimcher lab. They ruled out known troublemakers, such as Helicobacter pylori, Salmonella, and E. coli. Antibiotic experiments narrowed the probable menace down to an anaerobic species. Hooper speculates that a complex change in the community of bacteria likely unleashes renegade behaviour among several species. The Glimcher lab is trying to unmask the culprits in collaboration with Jeff Gordon at Washington University in St. Louis, whose group may be best known for finding evidence that intestinal bacterial composition may contribute to obesity.

Except for isolated reports, there is little evidence that inflammatory bowel disease is contagious among people, said Richard Blumberg, chief of gastroenterology at Brigham and Women's Hospital. In the study, the maleficent microbial mix may have passed from mother to pup during birth and by faeces-eating habits of unrelated mice.

No one knows exactly what causes inflammatory bowel disease. There is no cure, only treatment for symptoms, such as relieving the inflammation, and supportive therapies, such as nutrition, said Wayne Lencer, chief of gastroenterology at Children's Hospital Boston. Antibiotics may work in some patients for one major type-Crohn's disease-but not for the other-ulcerative colitis.

"We have pretty good genetic evidence that it's an abnormality of the immune system at one level and that the disease is due to a dysregulated immune response within mucosal tissues to bacteria normally resident in the intestines," said Blumberg, HMS professor of medicine. But the primary cause-immune system or bacteria-is debated, he said. This study knits together the two concepts.

"What's amazing here is that the microbe appears to be coming from the normal commensal bacteria population," said Lencer, HMS associate professor of paediatrics. "We live symbiotically with microbes. They're essential. We typically don't think of commensals as invasive. The study really makes poignant how important this dialogue is between commensals and our cells.

From many perspectives ranging from microscopic to physical examination, the mouse disease looks remarkably like human ulcerative colitis. In adults and children, the disease presents as continuous sores in the thin lining of the large intestine beginning at the often prolapsed rectum but limited to the colon. (Crohn's disease, in contrast, extends more deeply into the intestinal wall at sporadic locations and occurs throughout the gastrointestinal tract all the way up to the mouth, but mostly in the small intestine.

"We did not set out to establish a model of communicable ulcerative colitis," Glimcher said. Instead, researchers in her lab were systematically exploring the role of a transcription factor, T-bet, in adaptive and innate immunity. Discovered in her lab seven years ago, T-bet seems to single-handedly control the destiny of naive CD4+ T helper cells into type 1 and not type 2. The studies have found that this power is necessary for effective defence against pathogens and cancer cells; it is protective in asthma but pathogenic in the setting of autoimmunity.

About three years ago, Glimcher noticed her mouse facility bills going up and asked why. Co-author Geanncarlo Lugo-Villarino, a postdoctoral fellow now at the University of California, San Diego, told her that his mice had all become sick. For his thesis work elucidating the function of T-bet in the innate immune system, he was studying T-bet-deficient mice with no T cells or B cells, known in scientific shorthand as TRUC.

Postdoctoral fellow Graham Lord, now a professor of medicine at King's College London and a co-author, took on the task of determining what made the mice sick. A formal analysis showed a distinctive disease phenotype. "I've looked after plenty of patients with this," said Lord, a renal transplant physician who continues to collaborate with Glimcher. "It looks just like human ulcerative colitis.

Garrett, an HMS instructor in medicine at Dana-Farber Cancer Institute and Brigham and Women's Hospital, joined the lab two years ago and took over the project when Lord moved to London. She was able to detect the first signs of colonic permeability in mice as young as three and a half weeks. Electron microscopy showed holes in the colonic epithelium in mice at the same young age.

Together with colleagues, the team conducted a multitude of experiments that delineate the etiology in the mouse model with intriguing implications for human disease. They first narrowed down the innate immune cell that expressed T-bet, settling on dendritic cells after ruling out macrophages, mast cells, and natural killer cells. The transcription factor T-bet usually activates immune cells to produce factors-and contributes to T cell-mediated colitis. But in dendritic cells, T-bet binds to the promoter region of the TNF-alpha gene and suppresses production of the inflammatory cytokine. Human colonic dendritic cells also employ T-bet to suppress TNF-alpha.

In mice, both TNF-alpha antibodies and TNF-alpha antagonists can prevent and cure the disease. A recent paper from another group reported that TNF-alpha antagonists, approved to treat rheumatoid arthritis, seemed to alleviate ulcerative colitis in people. Unlike in people, antibiotics also cured the mouse colitis. That gave the researchers a major clue to what activated the dendritic cells.

"Dendritic cells can project their processes into the colonic lumen and sample the bacteria there," Garrett said. T-bet-deficient mice with an adaptive immune system did not get sick. The team identified a protective population of T regulatory cells, a lymphocyte that suppresses effector T cells and is in clinical development as a potential therapy for other conditions, Glimcher said.

The researchers turned again to the intestinal bacteria. All of the TRUC mice spontaneously suffered the disease. Normal mice with intact immune systems acquired a milder form of the disease by living in the same cage. "We could cure it with antibiotics, but it was more interesting to us that they got sick," Garrett said.

In people, resident bacteria outnumber human cells by as much as 100 to 1. Most of these microbes hunker down in the colon. Many of the estimated 500 unidentified commensal species perform essential symbiotic tasks of processing nutrients. The colonic epithelium layer-only one cell thick-separates the hordes of micro-organisms from the underlying mucosal epithelial cells, where dendritic cells lurk.

Now, Glimcher and her colleagues are seeking the identity of the rogue bacteria. "If you think about it, one thin lining is all that is protecting the body from the millions of bacteria teeming in the gut, and the peacekeeper at the gate is the immune system," Glimcher said. "Both the adaptive and innate immune systems want to keep the bacteria in the gut and out of mucosal organs."
Based on the mouse model, Glimcher and her colleagues think inflammatory bowel disease starts with genetic variations that predispose people to disease. Then, a hyperactive and misbehaving immune system leads to secretion of TNF-alpha, which injures the colonic epithelial lining. The new colonic environment somehow transforms the intestinal bacteria into a harmful mix that can cause disease in a healthy gut.

"Time will tell just how faithful a model of human disease this is," Glimcher said. "Just because it looks like human disease histologically and can be cured by agents that ameliorate the disease doesn't mean it's the same."

Cardiology

Study explains how exercise lowers cardiovascular disease risk

Researchers at Brigham and Women's Hospital (BWH) assessed a variety of cardiovascular disease (CVD) risk factors and exercise levels in over 27,000 women in the Women's Health Study and found their risk of CVD events decreased with higher levels of physical activity - a result that was substantially mediated by known risk factors such as inflammatory/hemostatic factors and blood pressure. These findings appeared in the November 6, 2007 print issue of Circulation.

"Regular physical activity is enormously beneficial in preventing heart attack and stroke," said Samia Mora, MD, lead author of the study and an attending physician in the Divisions of Preventive and Cardiovascular Medicine at BWH. "We found that even modest changes in risk factors for heart disease and stroke, especially those related to inflammation/hemostasis and blood pressure, can have a profound impact on preventing clinical events. This study is the first to examine the importance of a variety of known risk factors in explaining how physical activity prevents heart disease and stroke.

The women ranged from 45 to 90 years old (average age 55) and were assessed for a full range of risk factors and different levels of exercise. There was a 40 percent reduction in heart attack and stroke between the highest and lowest exercise groups.

The women self-reported physical activity, weight, height, hypertension and diabetes. The long-term benefits of exercise start at a relatively low level, 600 kilocalories per week, equivalent to about two hours of physical activity per week, Mora said, who is also an instructor of medicine at Harvard Medical School (HMS).

The study measured levels of a variety of traditional and novel risk factors to help understand the mechanisms that reduce risk for heart attack and stroke. Novel risk factors are emerging clinical, biochemical, and genetic markers that researchers have studied in order to better understand the development of a disease, to improve disease risk prediction, and to identify new targets for treatment.

Inflammatory and haemostatic biomarkers - fibrinogen, C-reactive protein and intracellular adhesion molecule-1 - together made the largest contribution to lower risk, 33 percent.

Blood pressure was the next major contributor to lower risk, 27 percent, followed by lipids, body mass index, glucose abnormalities, with minimal contribution from measures of renal function or homocysteine.

Inflammatory and haemostatic biomarkers are novel risk factors that relate to blood vessel function and inflammation of the arteries.

"Inflammatory and haemostatic factors as a group have overlapping functions and roles and, in our study, had the biggest effect in mediating exercise-related cardioprotection, more so than blood pressure or body weight," Mora said.

The study population was divided into four groups by levels of exercise:

• The highest level expended greater than or equal to 1,500 kilocalories per week (kcal/week) representing greater than five hours of moderately intense physical activity (such as brisk walking) per week.

• The next group expended from 600 to 1,499 kcal/week, which reflected about two to five hours of physical activity per week.

• A third group represented an expenditure of 200 to 599 kcal/week, which is about one to two hours of physical activity per week.

• The reference group had less than 200 kcal per week (less than one hr per week).

The risk of cardiovascular disease events decreased with higher levels of physical activity. Compared to the reference group, relative risk reductions were associated with =1,500, 600 to 1,499, 200 to 599 kcal/wk of 41 percent, 32 percent and 27 percent, respectively.

The American Heart Association (AHA) does not recognize the inclusion of inflammatory and haemostatic biomarkers as risk factors in assessing cardiovascular disease because they have yet to be clinically proven.
Neurology

Researchers develop targeted approach to pain management

Imagine an epidural or a shot of Novocain that doesn't paralyze your legs or make you numb, yet totally blocks your pain. This type of pain management is now within reach. As a result, childbirth, surgery and trips to the dentist might be less traumatic in the future, thanks to researchers at Massachusetts General Hospital (MGH) and Harvard Medical School, who have succeeded in selectively blocking pain-sensing neurons in rats without interfering with other types of neurons.

The pint-sized subjects received injections near their sciatic nerves, which run down their hind limbs, and subsequently lost the ability to feel pain in their paws. But they continued to move normally and react to touch. The injections contained QX-314, a normally inactive derivative of the local anaesthetic lidocaine, and capsaicin, the active ingredient in hot peppers. In combination, these chemicals targeted only pain-sensing neurons, preventing them from sending signals to the brain.

"We've introduced a local anaesthetic selectively into specific populations of neurons," explains Harvard Medical School Professor Bruce Bean, an author on the paper, which appeared in Nature on Oct. 4. "Now we can block the activity of pain-sensing neurons without disrupting other kinds of neurons that control movements or non-painful sensations.

"We're optimistic that this method will eventually be applied to humans and change our experience during procedures ranging from knee surgery to tooth extractions," adds Professor Clifford Woolf of Massachusetts General Hospital, who is senior author on the study.

Despite enormous investments by industry, surgical pain management has changed little since the first successful demonstration of ether general anaesthesia at MGH in 1846. General and local anaesthetics work by interfering with the excitability of all neurons, not just pain-sensing ones.

Thus, these drugs produce dramatic side effects, such as loss of consciousness in the case of general anaesthetics or temporary paralysis for local anaesthetics. "We're offering a targeted approach to pain management that avoids these problems," says Woolf.

The new work builds on research done since the 1970's showing how electrical signalling in the nervous system depends on the properties of ion channels, that is, proteins that make pores in the membranes of neurons.

"This project is a perfect illustration of how research trying to understand very basic biological principles can have practical applications," says Bean.

The new method exploits a membrane-spanning protein called TRPV1, which is unique to pain-sensing neurons. TRPV1 forms a large channel, where molecules can enter and exit the cell. But a "gate" typically blocks this opening. The gate opens when cells are exposed to heat or the chili-pepper ingredient capsaicin. Thus, bathing pain-sensing neurons in capsaicin leaves these channels open, but non-pain sensing neurons are unaffected because they do not possess TRPV1.

The new method then takes advantage of a special property of the lidocaine derivative QX-314. Unlike most local anaesthetics, QX-314 can't penetrate cell membranes to block the excitability of the cell, so it typically lingers outside neurons where it can't affect them. For this reason it is not used clinically.

When pain-sensing neurons are exposed to capsaicin, however, and the gates guarding the TRPV1 channels disappear, QX-314 can enter the cells and shut them down. But the drug remains outside other types of neurons that do not contain these channels. As a result, these cells fully retain their ability to send and receive signals.

The team first tested their method in the Petri dish. Alexander Binshtok, a postdoctoral researcher in Woolf's lab, applied capsaicin and QX-314 (separately and in combination) to isolated pain-sensing and other neurons and measured their responses. Indeed, the combination of capsaicin and QX-314 selectively blocked the excitability of pain-sensing neurons, leaving the others unaffected.

Next, Binshtok injected these chemicals into the paws of rats and measured their ability to sense pain by placing them on an uncomfortable heat source. The critters tolerated much more heat than usual. He then injected the chemicals near the sciatic nerve of the animals and pricked their paws with stiff nylon probes. The animals ignored the provocation. Although the rats seemed immune to pain, they continued to move normally and respond to other stimuli, indicating that QX-314 failed to penetrate their motor neurons.

The team must overcome several hurdles before this method can be applied to humans. They must figure out how to open the TRPV1 channels without producing even a transient burning pain before QX-314 enters and blocks the neurons, and they must tinker with the formulation to prolong the effects of the drugs. Both Bean and Woolf are confident they'll succeed.

"Eventually this method could completely transform surgical and post-surgical analgesia, allowing patients to remain fully alert without experiencing pain or paralysis," says Woolf. "In fact, the possibilities seem endless. I could even imagine using this method to treat itch, as itch-sensitive neurons fall into the same group as pain-sensing ones."

Research Matters brings together selected research being conducted at Harvard Medical School's 18 affiliated institutions. For more information, visit the Harvard Medical School website at www.hms.harvard.edu.

This article is provided courtesy of Harvard Medical International. © 2007 President and Fellows o ===College

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