A gene long presumed dead comes to life under the full moon of inflammation, Stanford University School of Medicine scientists have found.
The discovery may help explain how anti-inflammatory steroid drugs work. It also could someday lead to entirely new classes of anti-inflammatory treatments without some of steroids’ damaging side effects.
Chronic inflammation plays a role in cancer and in autoimmune, cardiovascular and neurodegenerative diseases, among others. Anti-inflammatory steroid drugs are widely prescribed for treating the inflammatory states that underlie or exacerbate these conditions.
‘Inflammation tells your body something is wrong,’ said the study’s senior author, Howard Chang, MD, PhD, professor of dermatology at Stanford and the recipient of an early career scientist award from the Howard Hughes Medical Institute. ‘But after it does its job of alerting immune cells to a viral or bacterial infection or spurring them to remove debris from a wound site, it has to get turned off before it causes harm to healthy tissue.’
That appears to be what the ‘undead’ gene does. Chang’s team, which identified it, has named it Lethe, after the stream in Greek mythology that makes the deceased who cross it forget their pasts.
　
The master regulator of inflammation inside cells — a bulky complex of several proteins, collectively called NF-kappa-B — is a transcription factor: It can switch on hundreds or even thousands of genes in a cell’s nucleus. When aroused by signals at the cell surface (typically delivered by circulating proteins or microbial components), NF-kappa-B activates pro-inflammatory genes, gearing that cell up to combat viral or bacterial assaults and respond to an injury.
Lethe, which the investigators found is activated by NF-kappa-B, subdues the master regulator’s massive influence on the genome, curtailing the inflammatory response.
NF-kappa-B also plays a key role in ageing. In a study published in 2007 in Genes and Development, Chang and his colleagues showed that old skin cells in which NF-kappa-B was temporarily inactivated began to act young. This finding has since been confirmed in other tissues and by other researchers.
To learn more about NF-kappa-B, Chang’s group decided to activate it and see which genes get turned on or off. But rather than ‘normal’ genes, which are essentially recipes for making proteins, they were curious about DNA sequences that generate long non-coding RNA molecules, or lncRNAs, which Chang helped to discover during the past decade.
RNA is best known as the intermediate material in classic protein production. Gene-reading machines in cells produce RNA transcripts, or copies, of protein-coding genes. These transcripts, known as messenger RNAs, are free to leave the cell nucleus for the cytoplasm, where they can transmit genes’ instructions to the protein-making machines situated there.
But lately RNA has been shown to play an increasing number of additional roles that have nothing to do with making proteins. The lncRNAs Chang studied are made by the same molecular machinery that protein-coding genes use to make a messenger RNA. Instead of heading for the cytoplasm to make proteins, though, lncRNAs can remain in the nucleus and directly regulate genes. More than 10,000 lncRNAs have now been discovered, although scientists are only beginning to understand what they do.
To see which lncRNAs were induced during inflammation, Chang and his colleagues exposed cultured fibroblasts from embryonic mice to TNF-alpha, an immune-signalling protein known to trigger NF-kappa-B. They found that levels of hundreds of lncRNAs inside the cells were driven either up or down by TNF-alpha stimulation.
Of those lncRNAs, a total of 54 were copied from so-called pseudogenes: DNA sequences that, while they closely resemble genes, don’t code for proteins. More than 11,000 pseudogenes — one for every two protein-coding genes — have been identified in the human genome. Scientists believe pseudogenes are copies of actual genes that, during the replication of some ancestral organism’s germ cell, were accidentally inserted into the genome and, redundant but harmless, came along for the evolutionary ride. Over the intervening eons, these genetic doppelgangers have roamed along the genome, mutated and decayed to the point where, it is believed, they no longer do anything at all.
‘Pseudogenes have been considered to be completely silent, ignored by cells’ DNA-reading machinery,’ Chang said. ‘But we got a real surprise. When a cell is subjected to an inflammatory stress signal, it’s like Night of the Living Dead.’
Equally surprising, Chang said, is that different signalling chemicals or microbial components (such as bits of bacterial cell walls or of viral DNA) wake up different groups of lncRNA-encoding DNA sequences, including pseudogenes. ‘They’re not really dead, after all. They just need very specific signals to set them in motion.’
Lethe was one such pseudogene tripped off by stimulation of NF-kappa-B. Lethe directly interfered with the complex’s ability to seat itself on appropriate DNA sequences, shutting down the pro-inflammatory genes the transcription factor ordinarily activates.
Several pseudogenes were activated in a selective manner. For example, TNF-alpha and another circulating signalling protein — but not microbial parts — activated Lethe.
Because some pseudogenes sit near protein-coding genes, some scientists have argued that the generation of RNA transcripts from the pseudogenes is simply an artifact of normal transcription of full-fledged protein-coding genes. ‘There’s a tendency to assume it’s some protein-coding gene that NF-kappa-B is really targeting, and to downplay the activation of a lncRNA as noise, a ‘ripple effect’ like the one you see when a boat goes by,’ Chang said.
But TNF-alpha failed to activate two nearby protein-coding genes on either side of Lethe. Reciprocally, stimuli that turned these two other genes on didn’t affect Lethe. Meanwhile, two other pseudogenes that very closely resemble Lethe were not activated by TNF-alpha, as Lethe was.
Another surprising finding was that dexamethasone, a commonly prescribed anti-inflammatory steroid drug, activates Lethe. Various other steroid hormones that are not anti-inflammatory in nature, such as vitamin D or oestrogen or a male steroid hormone, failed to boost Lethe levels.
‘We’re wondering whether there might be ways to artificially raise Lethe levels without steroids. These drugs have potentially deleterious side effects such as elevated blood pressure and blood sugar, thinning of bones and general suppression of the immune system,’ Chang said.
The study results suggest that not only Lethe but other pseudogenes undergo similarly selective awakenings to generate lncRNAs in response to different external inflammatory stimuli. ‘From the pattern of activated lncRNAs, you can tell what the cell has encountered — a virus, a bacteria or something else,’ Chang said. ‘These patterns of activation may be able to serve as an indicator of what kind of inflammatory situation or pathogenic invasion is responsible.’ Stanford University Medical Center

Huskies football defensive lineman Caleb Eidsvik takes up a lot of room as he sits on an examining table in the Huskies trainer’s room at Griffiths Field, patiently waiting for pharmacology student Hungbo Qudus to draw a small sample of his blood.

At six-foot three and 260 pounds, Eidsvik exudes strength and good health. And that’s the problem, according to researcher Changiz Taghibiglou, since Eidsvik is suspected of having a concussion.

‘There’s no easy way to conclusively diagnose concussion now. You need an MRI or a CT scan,’ he said. ‘Whether it’s car accidents, falls or sports injuries, we actually don’t have any simple tests.’

Taghibiglou is an assistant professor in the College of Medicine’s Department of Pharmacology. If he gets his way, testing for concussion will be so simple that a test kit will be a standard item in every medical bag, to be used by trainers and coaches at football fields and hockey arenas, and even by first responders and EMTs.

Diagnosis of concussion is critical. While short term symptoms such as vomiting, confusion and headache may be easy to spot, Taghibiglou explained that long-term effects can be more subtle and easier to brush off. This can be extremely dangerous: if the person suffers a second concussion before fully recovering from the first, they are at high risk of developing permanent brain damage, psychiatric problems or even dying. There are also risks of long term effects, including Parkinson’s and Alzheimer diseases, and post-traumatic stress disorder.

At the heart of Taghibiglou’s concussion test is a molecule that exists on the surface of brain cells. Through research carried out with scientists at the Canadian Department of National Defence, a link was found between the molecule and brain trauma. This research is ongoing and represents one of the agency’s many inquiries into the effects of battlefield blasts on soldiers.

‘Physical injuries are easy to spot but with a concussion a person can appear fine,’ Taghibiglou said. ‘In the worst case, there are no outward signs of injury so they are sent back out, re-injured, and suffer significant neurological issues later.’

Taghibiglou explains that head trauma – whether from an accidental blow to the head, a hard slam on the gridiron or a forceful check against the boards – can knock certain brain cell molecules loose. Once free, they circulate in the blood where they can be detected by a simple blood test (a patent for the test has been applied for through the U of S Industry Liaison Office).

Working with Huskie Athletics, Taghibiglou, Qudus and graduate student Nathan Pham are gathering blood samples from athletes pre- and post-injury. Taghibiglou praised Director of Huskie Athletics Basil Hughton and Huskies Head Therapist Rhonda Shishkin for arranging access, particularly during peak season.

‘We’re collecting from the football team and are also looking for concussion in other teams such as soccer and hockey,’ he said.

Since the test is so new, the research team also needs about 300 male and female volunteers to donate small blood samples to establish the normal level of the concussion-associated molecules in the blood.

‘There are no values in the reference books, simply because no one has gathered the data yet. Our ultimate goal is a simple diagnostic test, much like the blood sugar tests used by diabetics.’ The test would be particularly valuable for rural and remote communities that lack the medical equipment typically used for trauma diagnosis.

‘Small health clinics don’t have an MRI. It may help rural doctors refer their patients to larger centres and know what’s going on.’ University of Saskatchewan

Scientists have tracked down and quantified the diverse origins of cells that drive fibrosis, the incurable, runaway wound-healing that scars and ultimately destroys organs such as the lungs, liver and kidneys.
Findings are from research conducted at Beth Israel Deaconess Medical Center, Harvard Medical School and Massachusetts Institute of Technology in Boston and continued at The University of Texas MD Anderson Cancer Center.
‘Answering a fundamental question about the origin of these cells by identifying four separate pathways involved in their formation allows us to look at ways to block those pathways to treat fibrosis,’ said senior author Raghu Kalluri, Ph.D., M.D., MD Anderson chair and professor of Cancer Biology. ‘It’s highly unlikely that a single drug will work.’
‘In addition to being lethal in its own right, fibrosis is a precursor for the development of cancer and plays a role in progression, metastasis and treatment resistance,’ Kalluri said. ‘In some cancers, such as pancreatic cancer, up to 95 percent of tumours consist of fibrotic stroma.’
Working in genetic mouse models of kidney fibrosis, Kalluri and colleagues identified four sources of cells called myofibroblasts, the dominant producers of collagen. Collagen normally connects damaged tissue and serves as scaffolding for wound-healing. As healing occurs, myofibroblasts and collagen usually diminish or disappear.
In fibrosis, collagen production marches on. While inflammation-inhibiting drugs can sometimes slow its progress, fibrosis now is treatable only by organ transplant.
The researchers employed a fate-mapping strategy to track cells on their way to becoming myofibroblasts. In fate mapping, the promoter of a protein expresses a colour inside a cell that remains with the cell no matter what happens to it until it dies, Kalluri said.
This was particularly important because two of the four sources of myofibroblasts start out as another cell type and differentiate into the collagen-producing cells.
Their experiments showed:
Half of all myofibroblasts are produced by the proliferation of pre-existing resting fibroblasts.
Another 35 percent are produced by mesenchymal stem cells that originate in the bone marrow, migrate to the ‘wound’ site, and then differentiate into myofibroblasts.
An additional 10 percent are the products of endothelial to mesenchymal transition (EndMT), in which blood vessel cells change into mesenchymal cells, then become myofibroblasts.
The final 5 percent come from epithelial to mesenchymal transition (EMT), in which functional cells of an organ sometimes behave like mesenchymal cells and myofibroblasts.
‘These differentiation pathways provide leads for drug targets,’ Kalluri said. ‘Combining an antiproliferation drug with therapies that block one or more differentiation pathways could provide a double hit to control fibrosis. We hope to synergise these pathways for the most effective therapeutic response.’ MD Anderson Cancer Center

For the majority of cancer patients, it’s not the primary tumour that is deadly, but the spread or ‘metastasis’ of cancer cells from the primary tumour to secondary locations throughout the body that is the problem. That’s why a major focus of contemporary cancer research is how to stop or fight metastasis.
Previous lab studies suggest that metastasising cancer cells undergo a major molecular change when they leave the primary tumour – a process called epithelial-to-mesenchymal transition (EMT). As the cells travel from one site to another, they pick up new characteristics. More importantly, they develop a resistance to chemotherapy that is effective on the primary tumour. But confirmation of the EMT process has only taken place in test tubes or in animals.
In a new study Georgia Tech scientists have direct evidence that EMT takes place in humans, at least in ovarian cancer patients. The findings suggest that doctors should treat patients with a combination of drugs: those that kill cancer cells in primary tumours and drugs that target the unique characteristics of cancer cells spreading through the body.
The researchers looked at matching ovarian and abdominal cancerous tissues in seven patients. Pathologically, the cells looked exactly the same, implying that they simply fell off the primary tumour and spread to the secondary site with no changes. But on the molecular level, the cells were very different. Those in the metastatic site displayed genetic signatures consistent with EMT. The scientists didn’t see the process take place, but they know it happened.
‘It’s like noticing that a piece of cake has gone missing from your kitchen and you turn to see your daughter with chocolate on her face,’ said John McDonald, director of Georgia Tech’s Integrated Cancer Research Center and lead investigator on the project. ‘You didn’t see her eat the cake, but the evidence is overwhelming. The gene expression patterns of the metastatic cancers displayed gene expression profiles that unambiguously identified them as having gone through EMT.’
The EMT process is an essential component of embryonic development and allows for reduced cell adhesiveness and increased cell movement.
According to Benedict Benigno, collaborating physician on the paper, CEO of the Ovarian Cancer Institute and director of gynecological oncology at Atlanta’s Northside Hospital, ‘These results clearly indicate that metastasising ovarian cancer cells are very different from those comprising the primary tumour and will likely require new types of chemotherapy if we are going to improve the outcome of these patients.’ Georgia Tech

A 30-year-old woman with a history of upper respiratory infections had no idea she carried an immunodeficiency disorder – until her 6-year-old son was diagnosed with the same illness.
After learning she has common variable immunodeficiency (CVID), a disorder characterised by recurrent infections, such as pneumonia, and decreased antibodies, the woman, her husband, their three children and parents joined a multidisciplinary University of Utah study and researchers identified a novel gene mutation that caused the disease in the mom and two of her children. The researchers discovered that a mutation in the NFKB2 gene impairs a protein from functioning properly, which interferes with the body’s ability to make antibodies and fight infection. The children’s father did not have the mutation, nor did a third sibling or the woman’s parents.
Another 35 people with CVID were tested for the gene mutation, and one other unrelated person was found to have it. His father wasn’t tested, but no one else in his family immediate family had the mutation, so the researchers don’t know whether he could have inherited the disorder from his father or developed the gene mutation sporadically.
CVID typically doesn’t present with symptoms until adulthood and it’s not uncommon for someone to reach their 20s, 30s or beyond before being diagnosed, according to Karin Chen, M.D., co-first author of the study published Thursday, Oct. 17, 2013, in the American Journal of Human Genetics online. Identifying the NFKB2 mutation will make it easier to recognise and treat the disorder, particularly after a test developed in conjunction with the study by ARUP Laboratories becomes available as early as next May.
‘If we can screen patients for genetic mutations, we can identify disease complications associated with that gene, start looking for them and treating them sooner,’ says Chen, instructor of pediatric immunology at the University’s School of Medicine.
There’s no cure for CVID, but it can be treated with monthly infusions of antibodies at a cost of $5,000 to $10,000 per treatment.
Identifying the gene mutation and developing the test for it took approximately two years, a fast turnaround made possible because of the multidisciplinary research that the University of Utah Health Sciences encourages and is known for doing. The study involved researchers from the U School of Medicine’s Departments of Pediatrics, Pathology, Human Genetics and Program in Molecular Medicine and ARUP, which is a University-owned, nationwide testing laboratory.
Emily M. Coonrod, Ph.D., a research scientist with the ARUP Institute for Clinical and Experimental Pathology, is co-first author with Chen. Karl V. Voelkerding, M.D., also of the Institute for Clinical and Experimental Pathology and a U professor of pathology, is the senior author.
CVID probably is underdiagnosed, making it hard to know how common it is. But the disorder is estimated to occur in one in 10,000 people to one in 50,000 people, meaning it is one of more common types of immunodeficiency disorders, according to Chen. University physicians currently treat about 150 CVID patients in the Intermountain Region. Historically, CVID has been diagnosed clinically by doctors who are aware of the symptoms and then have individuals tested for low levels of antibodies.
No mutation had been identified in NFKB2 before this study. But Attila Kumánovics, M.D., assistant professor of pathology and co-author on the study, had perused the medical literature and found that a mouse model had been developed that carried a similar mutation in the NFKB2 gene and also had immunodeficiency. That was a key development, according to Voelkerding. ‘This meant that the finding in our patients could be correlated to literature.’ University of Utah Health Care