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.

Blood Markers Point to Maladaptive LV Remodeling


Metabolites left from certain cellular processes may be markers of left ventricular (LV) remodeling and myocardial distress, as shown in patients undergoing transcatheter aortic valve replacement (TAVR) for severe aortic stenosis.

Blood samples taken immediately before and 24 hours after TAVR were used for metabolimic profiling via liquid chromatography and mass spectrometry. It turned out that baseline long-chain acylcarnitines, intermediates of fatty acid metabolism, were tied to maladaptive cardiac remodeling, according to a group led by Sammy Elmariah, MD, MPH, of Massachusetts General Hospital in Boston.

After adjusting for other factors (age, sex, diabetes status, renal function, and B-type natriuretic peptide), mean β values of acylcarnitines C16, C18:1, C18:2, C18, and C26 were independently associated with LV mass index, the authors described in their study published online in JAMA Cardiology.

“Similarly, with the exception of C18:2 acylcarnitine, each of the long-chain acylcarnitines distinguished between patients with and without severe LV hypertrophy,” Elmariah’s group reported. Circulating levels of C18:2 acylcarnitine were still associated with LV ejection fraction before and after multivariable adjustment.

Within 24 hours of TAVR, circulating levels of C16 decreased by 30.2%, C18:1 by 42.7%, C18:2 by 37.3%, and C18 by 38.3%, suggestive of an alleviation of cardiac pressure overload with the procedure.

“In symptomatic patients with severe aortic stenosis undergoing TAVR, circulating levels of long-chain acylcarnitines were independently associated with measures of maladaptive LV remodeling, and metabolic perturbations lessened after procedure completion,” the authors concluded.

“Clinical practice currently relies on clinical symptoms or the presence of overt LV failure to guide the timing of aortic valve replacement for aortic stenosis. This approach often identifies a late-stage cohort in whom maladaptive remodeling may be advanced and irreversible,” they suggested. “There is therefore an unmet clinical need for objective methods to identify early, reversible stages of maladaptive LV remodeling and to inform timely aortic valve replacement.”

 Blood metabolite levels did not independently relate to relative wall thickness.

The study included 44 consecutive patients undergoing transfemoral TAVR at a single institution (81.9 years old on average, 52% women).

Such a small sample size was a major caveat to the analyses performed, as was the lack of validation or complete statistical adjustment for the predictive value of plasma acylcarnitines.

How obesity contributes to, blocks treatment of pancreatic cancer


Investigators have discovered the mechanism by which obesity increases inflammation and desmoplasia — an accumulation of connective tissue — in the most common form of pancreatic cancer and also identify a treatment strategy that may inhibit the process.
 Massachusetts General Hospital (MGH) investigators have discovered the mechanism by which obesity increases inflammation and desmoplasia — an accumulation of connective tissue — in the most common form of pancreatic cancer. In their report published online in Cancer Discoverythe researchers describe how interactions among fat cells, immune cells and connective tissue cells in obese individuals stimulate a microenvironment that promotes tumor progression while blocking the response to chemotherapy. They also identify a treatment strategy that may inhibit the process.

“We evaluated the effects of obesity on numerous aspects of tumor growth, progression and treatment response in several animal models of pancreatic ductal adenocarcinoma and confirmed our findings in samples from cancer patients,” says Dai Fukumura, MD, PhD, of the Steele Laboratory of Tumor Biology in the MGH Department of Radiation Oncology, the study’s co-senior author. “Along with finding that tumors from obese mice or patients exhibited elevated levels of adipocytes or fat cells and of desmoplasia, both of which fuel tumor progression and interfere with treatment response, we also identified the underlying cause.”

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death worldwide, and more than half of patients diagnosed with PDAC are overweight or obese. Among patients with PDAC, obesity more than doubles the already high risk of death. Previous research by the MGH team and others has shown that PDAC is characterized by elevated desmoplasia — with an overproduction of extracellular matrix tissue by pancreatic stellate cells — which both promotes the survival and migration of cancer cells and blocks the penetration of chemotherapy drugs into tumors. Obesity itself is known to contribute to desmoplasia, with the expansion of fat tissue leading to inflammation and fibrosis and an accumulation of fat within the normal pancreas, which also causes inflammation.

The team’s experiments revealed that the elevated desmoplasia in obese mouse models of PDAC was caused by activation of pancreatic stellate cells through the antiogensin II type-1 receptor (AT1) signaling pathway. This activation was promoted by production of interleukin-1 beta (IL-1ß) both by fat cells and by the immune cells called neutrophils within and around tumors. Inhibiting AT1 signaling with losartan, which is used clinically to treat hypertension, reduced obesity-associated desmoplasia and tumor growth and increased the response to chemotherapy in the obese mouse model but not in normal weight animals. Analysis of tumors from human PDAC patients revealed increased desmoplasia and fat deposits only in samples from obese patients, and data from more than 300 patients showed that excess weight was associated with a reduction in patients’ response to chemotherapy.

João Incio, MD, PhD, of the Steele lab, lead author of the study, says, “Understanding the way that obesity affects pancreatic cancer may help us identify biomarkers — such as body weight and increased levels of tumor fibrosis — that could identify patients for whom AT1 blockers or IL-1ß antibodies would be most beneficial. Since FDA-approved versions of both agents are readily available, this strategy could be readily translated into the clinic. In addition, incorporating body weight into the design of preclinical studies could better account for the lack of response to conventional chemotherapeutical drugs.”

Co-senior author Rakesh K. Jain, PhD, director of the Steele Laboratory, adds, “With the majority of pancreatic cancer patients being overweight or obese at diagnosis, uncovering potential therapeutic targets within the mechanisms associating obesity with poor cancer prognoses is the first step towards developing remedies that could disrupt this association and significantly improve patient outcome. Targeting inflammation and fibrosis holds the promise to improve the clinical outcome of this major group of cancer patients.”

Blood test for patients on acne medication deemed unnecessary .


A study by researchers at Brigham and Women’s Hospital (BWH) has found that for young, healthy women taking spironolactone to treat hormonal acne, frequent office visits and blood draws are an unnecessary healthcare expense.

Dr Arash Mostaghimi

For the approximately 1,000 patients studied, blood tests to monitor potassium levels did not change the course of treatment, but the tests cumulatively totalled up to $80,000.

The research team suggests that routine potassium monitoring should no longer be recommended for this patient population in order to improve the patient care experience, decrease unnecessary office visits and reduce healthcare spending. Their results appear in JAMA Dermatology.

“The need for testing may be a deterrent for both physicians and patients alike,” said Dr Arash Mostaghimi, director of Dermatology Inpatient Service at BWH and senior author of the study. “By demonstrating that for young, healthy women, it’s safe to give spironolactone without close potassium monitoring, we hope that more patients will be able to benefit from this medication.”

Spironolactone, a generic drug that’s been used in the clinic since 1959, is commonly prescribed for treating hormonal acne – acne that tends to affect the jaw line most commonly around the time of the month when a woman gets her period. The medication addresses the root cause of these acne outbreaks by affecting the production of hormones as well as the way that hormones bind to the skin.

Spironolactone’s primary use is as a diuretic and antihypertensive treatment for patients with heart failure and liver failure. In these patients, spironolactone has been associated with an increased risk of hyperkalaemia – a dangerous elevation in potassium levels in the blood that can lead to abnormal heart rhythms.

Based on this risk, the US Food and Drug Administration recommends frequent potassium monitoring in patients with heart failure taking spironolactone, but it’s been unclear if these guidelines should apply to healthy patients taking spironolactone for the treatment of acne, and, if so, how frequently such patients should have their potassium levels tested.

Dr Mostaghimi and his colleagues evaluated clinical data from patients seen at Massachusetts General Hospital and Brigham and Women’s Hospital over a 15-year period. They calculated how frequently individual patients were tested and the rate of hyperkalaemia.

“Our goal was to understand the current screening practices of physicians taking care of young, healthy women on spironolactone for acne and to determine what the usefulness of the test was – what we found was that doctors checked potassium levels at different frequencies, suggesting an ambivalence about the tests,” said Dr Mostaghimi. “Of the more than 1,800 blood tests administered over that time period, we found no substantial problems or complications for these patients and no changes in their treatment based on what the tests showed.”

The researchers found just 13 cases of mild hyperkalaemia. When tested a second time, half of these patients had normal potassium levels, and none of the patients showed any signs or symptoms of hyperkalaemia.

The researchers note that a monthly dose of spironolactone costs $4.00; a serum electrolyte panel – frequently ordered along with potassium measurements – costs $43.51.

“There are two ways to think about cost,” said Dr Mostaghimi. “The first is in terms of money spent – that’s almost $80,000 worth of testing done without any benefit in terms of patient treatment. But there are also the time and psychological costs of being monitored while taking a medication. There’s time and lost work productivity to consider too. We hope that by presenting these data, we will begin to create a standard of practice that best serves patients and helps physicians who may be ambivalent about recommending these tests for otherwise healthy patients.”

Mass. Sees Steep Drop in Revascularization


Rates fell over the past decade for both PCI and CABG

Coronary revascularization has declined dramatically over the past decade, according to a population-based study in Massachusetts.

The age- and sex-adjusted rate fell 39% from 2003 through 2012, from 423 per 100,000 population to 258 (P<0.001), Robert W. Yeh, MD, MBA, of Massachusetts General Hospital in Boston, and colleagues reported online in a research letter in JAMA Internal Medicine.

The drop was significant for both percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG):
  • PCI: 318 to 200 per 100,000
  • CABG: 113 to 63 per 100,000

The exact reason for the trends wasn’t clear, but likely relates to dropping rates of myocardial infarction (MI) reported in multiple populations, “likely attributable to improved primary and secondary prevention.”

“These data have broad implications for regional health policy, training and provider accreditation, hospital resource allocation, and patient outcomes,” the researchers concluded.

The retrospective study included all 171,702 coronary revascularizations among Massachusetts residents seen at nonfederal hospitals from April 2003 through September 2012, whether inpatient or outpatient, and regardless of concomitant valve or aortic surgery. Only the first procedure per year per patient was counted.

PCI accounted for 76.9% of the procedures overall; CABG, 23.1%.

The biggest declines were in elective PCI (down from 206 to 109 per 100,000) and in isolated CABG (down from 90 to 45 per 100,000). All other categories except combined CABG and aortic or mitral valve surgery also declined significantly from 2003-2012.

From the American Heart Association:

Old enough to know better: how teenagers cope with a parent’s cancer


When his wife was undergoing treatment for breast cancer, Marc Silver admits he didn’t always consider how her illness affected their two teenagers. Now he – and his elder daughter – have some advice for other families

 

My daughter Maya is in the family room watching TV. I’m heading out to buy ginger sweets for my wife, Marsha, who’s upstairs in bed, feeling queasy after her latest round of chemotherapy.

“Going to get something for Mum; be right back,” I call to my 15-year-old.

“How is she doing?” asks Maya.

In my head, I think: “Why don’t you ask her yourself since she is just one flight of stairs away!” But I bite my tongue. I don’t want to add to the tension that cancer has already brought to our home.

Looking back, I realise that Maya wasn’t the only family member to avoid direct communication during the seemingly endless months of treatment for Marsha’s breast cancer. Consumed with all things cancer, my wife and I never asked her and her younger sister, Daniela, who was 13 at the time: “How are you doing?”

Many families find themselves in a similar situation: parent with cancer, teens in the house, not a lot of cross-generational conversation. Tens of thousands of children live with a parent who is a cancer survivor. Roughly a third of those children are 13 to 17 years old. While parents pay a lot of attention to the needs of younger kids, they may figure, as we did, that teens are old enough to cope.

“Adolescents are an unheard group,” says Shara Sosa, an oncology counsellor. Unfortunately, the nature of adolescence fights against openness of any kind, never mind the cancer in the family.

“With their kids locked behind a mask of teen indifference, parents are often intimidated and don’t know how to talk to them,” Ms Sosa says.

Teenagers are pulling away from the family, forging their own identity. The news that a parent has cancer yanks the adolescent back into the fold – exactly where they don’t want to be.

The reaction of a teen to a parent’s illness varies widely. Some respond with a disappearing act: after-school activities, shopping trips, sleepovers, you name it, they’ll do it to avoid the uncertain environment at home. It doesn’t mean they don’t love and care about the parent with cancer – it’s just their way of dealing with it all, says Maureen Davey, a family therapy Professor at the Drexel University College of Nursing and Health Professions, in Philadelphia.

Does that mean these kids are likely to turn to risky behaviour? Mental-health experts say that there are no data to quantify this and emphasise that most of the teens they work with do not act out. Yet typical teen temptations are always present.

Of the 100-plus teens who my daughter Maya and I interviewed for a book we wrote about teens and parental cancer, around 10 per cent confessed that they’d turned to drinking, drugs or vandalism as coping mechanisms.

Elissa Bantug, who was 12 when her mother was diagnosed with breast cancer 21 years ago, felt as if her mother had abandoned her. She drank, hooked up with an older boyfriend and forged her mother’s name 36 times on notes to get out of school. When the school asked her mother to come in for a conference, she felt too exhausted from her cancer treatments to turn up.

It’s impossible to say if Elissa would have acted out if her mother had been well. Still, looking back as an adult, Elissa says: “I felt like no one really talked to me.”

And she had lots of questions: would her mother be OK? What does it mean to be a cancer survivor? How would their family life change in the short run and the long run? Her rebellion, she says, was sparked by a lack of information.

Others respond by defying their developmental stage, assuming responsibilities that normally fall to the parents. Out of sync with their peers, these kids sometimes talk about their real age and their “cancer age”.

“I’m 16, and I have to act like I’m 40,” a teenager named Lyndsey told me. While her mother is in treatment for breast cancer, she says, “I have to cook, clean, make sure my mum eats, my brothers are fed.”

A “parentified” teen will inevitably feel frustrated. Teens may be “angry they have to take over everything and nobody appreciates that they’re doing so much more than they used to,” says psychiatrist Karen Weihs, medical director for supportive care at the University of Arizona Cancer Center in Tucson.

Stacy Hoover, a single mother, learned she had breast cancer when her daughters were 13 years old, and 18 months. She leaned on the older daughter, Megan, which took a toll. “Sometimes I wanted to go over to a friend’s house, but I didn’t want to leave my mum with the baby,” Megan recalls. When chemo made her mother irritable, Megan says: “It was hard not to yell back.”

No matter how the teenager responds, the parents can help shape the child’s frame of mind. That means sharing information, regardless of whether the news is good or bad.

Indeed, several studies establish the value of honest communication above all. Medical psychologist Stacey Donofrio looked at nearly 300 adolescents in the Netherlands who were coping with a parent’s cancer. She found that “the intensity of the parent’s treatment” for illness was not as important in influencing adolescent reactions as the way parents talked to the kids about it.

“Adolescents may feel especially uncertain if they feel their parents are not being entirely open,” she said.

Such an information gap elevated the tensions for Jackie Shmauch, a teenager whose father had leukaemia. One night, the 14-year-old fled her home in tears after eavesdropping on a call from her father’s oncologist. Jackie thought her father’s leukaemia was in remission, but she overheard a discussion of a bone-marrow transplant. After her parents found her at a friend’s house, they explained that the transplant was a preventive measure, not a sign that the cancer was back. That’s when Jackie delivered her ultimatum: “If there is information you have and you think you shouldn’t tell Jackie, that’s what I want you to tell me.”

Yet not every teen is like Jackie.

“If your child says, ‘Talking about this with you is not helpful to me’, it’s important to respect that,” says child psychiatrist Paula Rauch, who directs the Marjorie E Korff PACT Program (Parenting at a Challenging Time) at Massachusetts General Hospital in Boston.

It is critical for parents to remember that, cancer or no cancer, they still need to keep an eye on their teenagers – no easy task, especially when one of the parents is ill. The key, Ms Sosa points out, is listening closely even though “your head is in so many different places” because of the cancer diagnosis. That means asking follow-up questions, even challenging your teenager at times. If teens know you’re truly paying attention, she says, “they’re going to tell you all sorts of things”.

Some teenagers may just need a break from all the care-giving – perhaps by having other family members or friends shoulder the young person’s chores from time to time.

“Just to be 12 again, that was really quite a blessing,” recalls Bailee Richardson, now 19, who cared for her two younger sisters while her mother was being treated for breast cancer and her stepfather was working out of town.

A decade after my wife’s diagnosis, Marsha is in good health, but she and I are just beginning to understand how the experience affected our daughters. Maya tells me how uneasy she was with her mother’s bald head, courtesy of chemo, and that she found relief from the free-floating cancer anxiety that infiltrated our home by turning to friends, even if they didn’t quite understand what she was going through. And she’s sorry she didn’t help out more.

I, too, was sorry she didn’t step up. But I made the mistake of assuming that Maya and her sister could read my mind. I once exploded when my daughters didn’t rush to my aid as I dragged in bags of groceries after a day of errands.

“Can’t you give me a hand?” I yelled.

Maya calmly said: “We’d be happy to if you’d ask us.”

Genetic Test for Autism Refuted.


A team of Australian scientists claimed to have developed a genetic test that predicts a person’s risk of developing autism spectrum disorder (ASD) with 72 percent accuracy. Writing in Molecular Psychiatry, the team led by Stan Skafidis and Carlos Pantelis from the University of Melbourne said that their panel of 237 genetic markers could “correctly classify ASD from non-ASD individuals” and “may provide a tool for screening at birth or during infancy to provide an index of at-risk status.”

But a new study, led by Benjamin Neale from Massachusetts General Hospital, suggests that those claims were overblown. Neale’s team replicated the Australian group’s research in a larger sample, and found that the proposed panel of markers did not accurately predict ASDs.

“The claims in the original manuscript were quite bold. If they were true, it really would have been quite a major advance for the field, with serious ramifications for patients and other risk populations,” said Neale. “I think it’s important to ensure that this kind of work is of the highest quality.”

“This is a convincing refutation that calls into question the original results on specific technical grounds, rather than simply a non-replication that leaves a puzzling discrepancy between the two studies,” said Leonid Kruglyak, a geneticist from the University of California, Los Angeles, who was not involved in either study.

In 2012, Skafidis’s team compared the genes of 732 European people with ASD from the Autism Genetic Resource Exchange database, with those of 123 neurotypical people from a different cohort. They searched for single nucleotide polymorphisms (SNPs) that were linked to ASD, especially those in genes with roles in relevant cellular pathways.

They eventually settled on 237 SNPs in 146 genes, which they used to create a classifier for predicting ASD risk. When they tested the classifier on 243 cases and 42 controls from the same databases, it correctly predicted ASD with an accuracy of 85.6 percent.

The team then tested the classifier on an independent group of people—525 with ASD taken from the Simons Foundation Autism Research Initiative and 2,620 controls from the Wellcome Trust Birth Cohort. It identified the ASD cases with an accuracy of 71.7 percent.

But to other geneticists, these results seemed too good to be true. They implied that this small set of SNPs can explain around 11 percent of the variation in ASD risk—an unprecedented figure for any psychiatric condition. If the set truly had such strong effects, genome-wide association studies (GWAS) should have identified those SNPs by now—and they had not. It will likely take a sample of hundreds of thousands of people to find SNPs with such predictive power, as has been the case for other traits like height. “The magnitude of the study you need is dramatically larger than what was presented,” said Neale.

Neale wrote to Skafidis’s team asking for the full list of 237 SNPs, but did not receive it. (Skafidis told The Scientist that they offered the code that they used to generate their results, which should have been even better.) As such, they focused on the 30 most important SNPs, which were detailed in the published paper.

By comparing 5,417 cases and 5,417 controls from the Psychiatric Genomics Consortium, Neale’s team found that none of the 30 SNPs were significantly associated with ASD risk. The researchers also combined the SNPs into a classifier, using methods detailed in the original paper, and tested it on 4,623 cases and 4,623 controls from the same group. Again, the set failed to predict ASDs any better than chance. Finally, they also showed that the cellular pathways which the Australian team identified are not significantly associated with ASDs. The team’s results were published as a letter to the editor on 22 October, also in Molecular Psychiatry.

Several factors could explain the differences between the two studies. The Melbourne team initially tested the accuracy of their risk classifier on the same group of people whom they used to identify their SNP set. This is bad practice. “To appropriately assess the accuracy of a classifier, the sample which is used to develop it must be fully distinct from the sample on which it is tested,” said Kruglyak.

The Australian researchers also drew their cases and controls from separate populations with subtly different ethnic compositions. The SNPs they identified could have reflected reflect random ancestral differences between the two groups, rather than meaningful differences in ASD risk. Daniel Geschwind from the University of California, Los Angeles, made the same argument in a letter regarding Skafidis’s paper, which was published in the same journal this April.

Kruglyak added a third possible explanation: “batch effects, in which cases and controls are genotyped at different times and on different technology platforms,” he proposed. This problem also plagued a similar recently-retracted paper, which identified a panel of SNPs that could supposedly predict longevity.

But Skafidis said that Neale’s team may have come to different conclusions because the group did not use the full set of SNPs, nor the code that was provided. His team has submitted a response to the new study, which is in revision with Molecular Psychiatry (and does list the full set of 237 SNPs).

Meanwhile, Neale emphasized that other research into the genetics of autism are yielding stronger results. Several studies have identified loss-of-function mutations, and differences in the number of copies of certain genes, that are linked to ASD risk. Promising GWAS results have been presented at conferences and are making their way into published papers. “Autism genetics shouldn’t be tarnished by science that hasn’t been robustly proven,” he said. “There are successes beginning to emerge, and that’s really exciting and important.”

Estrogen, testosterone play role in development of hypogonadal symptoms.


In a study published in The New England Journal of Medicine, researchers suggest revisiting the approach to evaluation and management of hypogonadism due to data showing that declining levels of estrogen as well as androgens contribute to negative changes in body composition, strength and sexual desire in men.

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Researchers evaluated two cohorts of healthy men aged 20 to 50 years. All patients received goserelin acetate (Zoladex, AstraZeneca) 3.6 mg subcutaneously at weeks 0, 4, 8 and 12 to suppress endogenous gonadal steroids. Subsequently, patients in cohort 1 (n=198) were randomly assigned to placebo or 1.25 g, 2.5 g, 5 g or 10 g of topical 1% testosterone gel (AndroGel, Abbott Laboratories) daily for 16 weeks. In addition to goserelin acetate and placebo or the same doses of testosterone, patients in cohort 2 (n=202) received anastrozole (Arimidex, AstraZeneca) 1 mg daily to block aromatization of testosterone to estrogen, researchers wrote.

In cohort 1, body fat percentage increased significantly among patients who received placebo or testosterone 1.25 g or 2.5 g daily vs. those who received higher doses, according to data. Additionally, lean mass and thigh-muscle area decreased in patients who received placebo and in patients who received testosterone 1.25 g daily vs. higher doses. Leg-press strength was weakened in the placebo group only, according to data.

In cohort 2, body fat percentage rose in all groups, with similar increases seen in the placebo and testosterone 1.25-g, 2.5-g and 5-g groups. Significant decreases in total-body lean mass were observed in the placebo group vs. the testosterone 1.25-g, 2.5-g or 10-g groups. Researchers found reductions in thigh-muscle area in patients receiving placebo vs. any dose of testosterone. Similar to cohort 1, leg-press strength decreased in the placebo group, researchers wrote.

Overall, sexual desire declined with decreased testosterone doses in both cohorts, they added.

“By examining these relationships with and without suppression of estrogen synthesis, we found that lean mass, muscle size, and strength are regulated by androgens; fat accumulation is primarily a consequences of estrogen deficiency; and sexual function is regulated by both androgens and estrogens,” Joel S. Finkelstein, MD, from the endocrine unit in the department of medicine at Massachusetts General Hospital, and colleagues wrote.

In an accompanying editorial, David J. Handelsman, MB, BS, PhD, director of the ANZAC Research Institute and head of the department of andrology at Concord Hospital and the University of Sydney, Australia, wrote that longer studies are required to separate the effects of testosterone on bone density and fractures or on prostate growth and diseases.

“Nevertheless, this excellent study contributes to our expanding appreciation of the complex mechanisms of action of testosterone,” Handelsman wrote.

 

PERSPECTIVE

 

·         Part of the action of testosterone is actually mediated by aromatization to estradiol. Men with genetic aromatase deficiency cannot produce estrogens and show increased adiposity, decreased bone mass and reduced libido. This study shows that in normal men estrogens are important for body fat; androgens are important for muscle; and both estrogens and androgens are important for libido. These data explain why different men have differing sexual symptoms and metabolic risk profiles, despite equivalent systemic testosterone exposure and could lead to a rational approach to developing designer androgens, with varying aromatizability that would promote certain actions and not others. The full clinical implications of this important mechanistic study require the development of these novel androgens and estrogens.

o    Peter Y. Liu, MD, PhD, FRACP

o    Visiting professor in the division of endocrinology, metabolism and nutrition
Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center

Source: Endocrine Today

Blood pressure drug ‘fights cancer’


Losartan pill
Losartan has been prescribed as a blood pressure drug for decades

A commonly used blood pressure drug could help fight cancer by opening up blood vessels in solid tumours.

Used beside conventional cancer-fighting drugs, it could improve life expectancy, experts believe.

Following successful testing in mice, doctors plan to give losartan to patients with pancreatic cancer to see if it can tackle this hard-to-treat disease, Nature Communications reports.

Currently, only 5% of pancreatic cancer patients survive for at least 5 years.

This is partly because only one in 10 people with the disease has a tumour that is operable.

Future hope

Investigators at the Massachusetts General Hospital in the US are currently recruiting volunteer patients with inoperable pancreatic cancer to test out the new drug combination of chemotherapy plus losartan.

Although the treatment will not cure them, the researchers hope it will give the patients more months or years of life than they might otherwise get.

Losartan has been used for more than a decade as a safe blood pressure medication.

It works by making the blood vessels relax or dilate so that they can carry more blood, easing pressure.

The Massachusetts team found that the drug was beneficial in mice with breast and pancreatic cancer.

It improved blood flow in and around the tumours allowing more of the chemotherapy drugs to be delivered to their target.

Mice given this treatment, rather than standard chemotherapy alone, survived for longer.

Dr Emma Smith of Cancer Research UK said: “This interesting study in mice sheds light on why drugs for hypertension might improve the effectiveness of chemotherapy, but we don’t yet know if they work exactly the same way in people.

“The fact that these drugs are already widely used to treat high blood pressure will hopefully cut down the amount of time it will take to test their potential in treating cancer but they may not be safe for all patients or when combined with other cancer treatments, so we need to wait for the answers from clinical trials which are already under way.”

Safety of Midazolam for Sedation of HIV-Positive Patients Undergoing Colonoscopy.


Abstract

Objectives Because of concerns regarding interactions between midazolam and antiretroviral therapy (ART), alternative sedatives are sometimes used during procedural sedation. Our objective was to compare outcomes in patients on ART who received intravenous (iv) midazolam vs. iv diazepam, a second-line agent, during colonoscopy.

Methods We conducted a retrospective analysis of adult HIV-positive patients who underwent colonoscopy over a 3.5-year period. Primary outcomes were sedation duration, nadir systolic blood pressure (SBP), nadir oxygen saturation, abnormal cardiac rhythm, and change in level of consciousness using a standardized scale. We calculated rates of adverse events according to benzodiazepine use and identified risk factors for complications using univariate and multivariate analyses.

Results We identified 136 patients for this analysis: 70 received midazolam-based sedation and 66 received a diazepam-based regimen. There were no significant differences between the two groups with respect to sedation duration (mean 48.0 vs. 45.7 minutes for the midazolam and diazepam groups, respectively; P = 0.68), nadir SBP (mean 97.0 vs. 101.6 mmHgP = 0.06), nadir oxygen saturation (mean 94.6 vs. 94.8%; P = 0.72) or rate of abnormal cardiac rhythm (11.4 vs. 19.7%; P = 0.18). More patients in the midazolam group experienced a depressed level of consciousness (91% vs. 74% in the diazepam group; P = 0.0075), but no patient required reversal of sedation or became unresponsive.

Conclusions We did not find evidence that patients who received midazolam for procedural sedation had clinical outcomes statistically different from those who received diazepam. These findings should be confirmed in prospective studies or in a randomized controlled trial.

Introduction

Midazolam is a rapidly acting benzodiazepine widely used as a sedative and anaesthetic induction agent in patients undergoing colonoscopy. However, because of concerns regarding potential drug interactions between midazolam and antiretroviral therapy (ART), some clinicians use general anaesthesia instead in HIV-positive patients undergoing colonoscopy. Other providers have patients stop ART for 1 to 2 days before a planned procedure, although interrupting ART is not a recommended strategy from the perspective of HIV disease management.[1] Diazepam, an alternative agent, utilizes multiple metabolic pathways and is less likely to interact with HIV protease inhibitors (PIs); however, midazolam is favoured by most clinicians because of its more rapid onset of action, shorter half-life (1.5 h vs. 20 h for diazapem), amnestic effects, and lower incidence of pain on injection or phlebitis compared with diazapem.[2–4]

We sought to evaluate the safety of intravenous (iv) midazolam in HIV-positive patients on ART who received procedural sedation for colonoscopy. Our objective was to compare clinical outcomes and identify risk factors for complications in patients who received midazolam vs. diazepam.

HIV-positive patients who were 18 years of age or older and who underwent out-patient colonoscopy at Massachusetts General Hospital between 1 January 2007 and 30 June 2010 were identified through the Research Patient Data Registry (RPDR), a clinical database for the Partners Health-Care System. Inclusion criteria were: (1) completion of colonoscopy; (2) receipt of ART for a minimum of 4 weeks at the time of the colonoscopy; (3) receipt of midazolam- or diazepam-based sedation.

Data Collection

Gastrointestinal (GI) endoscopy procedure flow sheets were reviewed for each patient. Flow sheets included scores assigned by the providers for selected physiological parameters. Demographic data, past medical history, and medication data were documented for each patient and confirmed via review of the electronic medical record.

Outcomes

Primary outcomes were sedation duration, nadir SBP, nadir oxygen saturation, rate of abnormal cardiac rhythm, and change in level of consciousness. Level of consciousness was assessed using an institutional scale on which a score of 2 represents awake and oriented, 1 represents arousable or disoriented, and 0 represents unresponsive. Secondary outcomes included change from baseline in respiratory rate, heart rate, airway status, quality of respirations, skin perfusion, SBP, pain score, procedure time, and total monitoring time required as documented in the post-procedure report.

Statistical Analysis

Univariate analyses of associations between exposures and outcomes were performed using Student’s t-test for continuous variables and the Wald χ2 test or Fisher’s exact test for categorical variables. Multivariate analyses were completed using logistic regression models. Covariates included in the analyses were concomitant cytochrome (CYP) 3A4 inhibitors, chronic opioid use, chronic benzodiazepine use, liver disease, age and gender. In a separate analysis, outcomes were analysed by four treatment groups: PI/midazolam; PI/diazepam; non-PI/midazolam; non-PI/diazepam. A P-value < 0.05 was considered to be statistically significant.

 

Patient Characteristics

We identified 344 HIV-positive patients receiving ART who were scheduled for a colonoscopy. Of this group, 208 were excluded for a variety of reasons; the most common reason for exclusion was cancellation or postponement of the procedure by the patient (n = 127). The excluded patients were similar to the study group with respect to age (median 52 years), sex (82% male), and CD4 T-cell count (mean 571 cells/μL; P = 0.82). The final cohort consisted of 136 HIV-positive patients on ART who underwent out-patient colonoscopy with midazolam- or diazepam-based sedation (Fig. 1).

Demographic characteristics in the two groups were similar A greater proportion of patients in the midazolam group had liver disease (mostly viral hepatitis) compared with the diazepam group (31% vs. 17%, respectively; P = 0.04). Most liver disease was attributable to hepatitis C virus infection (72%; liver disease was mild to moderate in these patients). The remainder had a diagnosis of steatohepatitis or alcoholic hepatitis. The groups were similar with respect to physical characteristics, although there was a trend towards higher body mass index (BMI) in the midazolam group (mean 28 vs. 26 kg/m2 in the diazepam group; P = 0.05). No patient had significant renal disease. PI-based ART was used by 53% of patients in the midazolam group and 61% of patients in the diazepam group (P = 0.55).

Of the 136 patients in the cohort, 70 (51%) received midazolam-based sedation and 66 (49%) received diazepam-based sedation. Oxygen saturation was monitored in all patients and everyone received supplemental oxygen, as per institutional guidelines (the amount of supplemental oxygen administered was not collected). Colonoscopy procedures were performed by 34 different providers, 28 of whom used midazolam-based sedation in selected patients. Sedation was administered by a nurse under the supervision of an attending physician; most often a gastroenterologist. The mean total midazolam dose administered was 4.1 ± 1.2 mg (0.05 ± 0.02 mg/kg) with a mean initial bolus of 2 ± 0.2 mg. The mean diazepam dose was 7.7 ± 2.4 mg (0.10 ± 0.03 mg/kg) with a mean initial bolus of 3.4 ± 1.5 mg. Most patients received adjunctive opioids such as meperidine ± fentanyl. Meperidine was given to 69 (99%) of the patients who received midazolam-based sedation (mean total dose 79 mg meperidine), and to 57 (86%) of the patients who received diazepam-based sedation (mean total dose 89 mg meperidine).

Outcomes

There were no significant differences between the groups with respect to duration of sedation (length of time consciousness score < 2, mean 48.0 vs. 45.7 minutes for the midazolam and diazepam groups, respectively; P= 0.68), nadir SBP (mean 97.0 vs. 101.6 mmHg, respectively; P = 0.06), or nadir oxygen saturation (94.6 vs.94.8%, respectively; P = 0.72) Eight patients in the midazolam group experienced a newly abnormal cardiac rhythm vs. 13 patients in the diazepam group (P = 0.18); no patient had a newly abnormal cardiac rhythm requiring treatment. Sinus bradycardia was the most frequently observed abnormal rhythm, occurring in six patients (9%) in the midazolam group and eight patients (12%) in the diazepam group. No patient became unresponsive with a consciousness score of 0 at any time or required a reversal agent, but more patients in the midazolam group experienced a depressed level of consciousness at some point during the procedure (91.4 vs.74.2%; P = 0.0075). All patients remained responsive to verbal or tactile stimulation; no patient in either group was unarousable to painful stimulation.

In terms of other outcomes, the diazepam group experienced a significantly greater change from baseline in respiratory rate, which decreased by 3.5 breaths per minute vs. a decrease of 2.8 breaths per minute in the midazolam group (P = 0.04). There were no significant differences in the overall blood pressure score—which compares intra-procedure SBP with pre-procedure SBP—in the midazolam group compared with the diazepam group (P = 0.38). The time to nadir SBP following sedative administration was also similar between the two groups (44 vs. 47 min for the midazolam and diazepam groups, respectively; P = 0.46).

We also compared outcomes in patients on PI-based ART vs. non-PI-based ART. There were no significant differences between these groups in terms of age, sex, liver disease, baseline SBP, or concomitant use of CYP 3A4 inhibitors, chronic opioids or chronic benzodiazepines. There were no significant differences between the four treatment groups (PI/midazolam; PI/diazepam; non-PI/midazolam; non-PI/diazepam) in the outcomes of lowest SBP, heart rate, nadir oxygen saturation, nadir respiratory rate, consciousness score < 2, sedation duration, or newly abnormal change in cardiac rhythm.

Multivariate Analysis

We performed multivariate analysis for four discrete outcomes – attainment of consciousness score < 2, sedation duration, nadir SBP, and cardiac rhythm score – adjusting for possible confounders based on clinical relevance and significance in univariate analysis. In adjusted analyses, we found that midazolam use (vs.diazepam use) was significantly associated with a consciousness score < 2 (odds ratio = 3.80; 95% confidence interval 1.33; 10.88) but not with cardiac rhythm score, nadir SBP, or sedation duration (data not shown).

Discussion

Most endoscopic procedures are performed with the patient under moderate (‘conscious’) sedation, during which the patient is able to respond purposefully to verbal or tactile stimulation.[5] Adequate sedation can both facilitate successful completion of the procedure and increase patient tolerability by decreasing anxiety and pain.

In this retrospective analysis of 136 adult HIV-positive patients on ART who underwent a colonoscopy, we assessed the safety of midazolam vs. diazepam based on multiple physiological outcomes. We found that the patients in our cohort safely received midazolam-based sedation, with comparable haemodynamic, cardiac, and respiratory outcomes compared with patients who received diazepam. We did observe a difference in consciousness score between the midazolam and diazepam groups, consistent with the known interaction between midazolam and ART. However, no patient suffered an outcome indicative of over-sedation, such as requirement for reversal of sedation, hospitalization or unresponsiveness. It is notable that more patients in the diazepam group compared with the midazolam group (26% vs. 9%, respectively) were awake and oriented during the procedure. Had patients in the diazepam group been given more diazepam to achieve a level of moderate sedation, it is likely that the duration of sedation would have been longer in the diazepam group.

Concerns regarding the safety of midazolam use in HIV-positive patients on ART arise from the possibility of drug interactions resulting in increased levels of midazolam.[6–8] Metabolism of midazolam is almost entirely dependent on hydroxylation by CYP 3A4.[2] Guidelines indicate that iv midazolam can be co-administered with ART with close clinical monitoring.[9–11] In addition, it has been demonstrated in a number of small studies that, with careful titration, drugs such as fentanyl, meperidine and midazolam, can be administered safely for short-term procedures in HIV-positive patients on ART.[12–15]

Previous studies investigating the clinical impact of co-administration of midazolam and ART have yielded conflicting results. In a cohort of 143 ambulatory patients undergoing bronchoscopy and receiving midazolam (total doses ranged from 2 to 15 mg; average 7.5 mg), no patient suffered an adverse outcome or required intubation.[16] By contrast, in a recent study of 241 HIV-positive in-patients who received iv midazolam prior to bronchoscopy,[17] the incidence of prolonged sedation (defined as > 90 minutes) was 9.8% in patients receiving PIs compared with 1.58% in those not taking ART. One possible reason for the difference between these findings and our results is the fact that the previous cohort consisted of in-patients, in whom concomitant comorbidities may have predisposed patients to complications from sedation; indeed, 10% of the patients in the PI group had pre-existing respiratory distress or altered mental status prior to the procedure and 29% were diagnosed with Pneumocystis jirovecii pneumonia. By contrast, we examined only ambulatory patients and compared outcomes between patients on ART receiving iv midazolam and those receiving iv diazepam. It is important to acknowledge that the risks of iv midazolam may differ by procedure and the presence of medical comorbidities.

In our institution, based on pharmacokinetic principles and available evidence, we generally do not empirically reduce the dose of midazolam for HIV-positive patients undergoing procedural sedation. Peak benzodiazepine concentrations after a single dose or multiple doses over a very short period of time (e.g. 20 minutes) are not affected by drug half-life or clearance. Furthermore, in the context of short-term use, the recovery time for benzodiazepines is mainly dependent on redistribution kinetics rather than half-life.

Limitations of this study include its retrospective design and the limited sample size. To address the possibility of confounding, we conducted a multivariable analysis adjusting for variables such as age, sex, liver disease, and concomitant medications including CYP 3A4 inhibitors and chronic opioid use. We also examined outcomes in four different subgroups – midazolam- and diazepam-based sedation with and without concomitant PIs – and could not detect an association between the treatment groups and any of the outcomes. An additional limitation is that our institutional endoscopic anaesthesia scoring system may not be sensitive enough to detect the optimal level of sedation.

In conclusion, we demonstrated that HIV-positive out-patients undergoing colonoscopy who received iv midazolam for procedural sedation had similar clinical outcomes to those who received diazepam. Based on these findings, we conclude that the use of iv midazolam can be considered for HIV-positive patients on ART with close clinical monitoring. These findings should be confirmed in prospective studies or in a randomized controlled trial.

Source: Medscape.com