This spring has brought rare but tangible moments of progress against the devastating lung disease idiopathic pulmonary fibrosis (IPF), which afflicts millions of people worldwide. Two drugs recently showed promise in clinical trials, and now a study offers both an unprecedentedly deep explanation of how the disease progresses and introduces another potential therapeutic avenue.
The new study features a central figure: an evolutionarily ancient protein called ‘chitinase 3-like-1’ (CHI3L1). The authors implicate it as the ‘master regulator’ of what appears to be a tragically errant repair response to the mysterious lung injuries that give rise to the disease. In describing how CHI3L1 works in IPF, the research also points to a strategy for treatment.
The report demonstrates that CHI3L1 is produced to help in response to the injury. It feeds back to protect injured cells from dying and simultaneously stimulates tissue repair to patch the damage that has occurred. But the study also shows how this dual role contributes to the ultimate problem. If IPF resulted from a single injury, like a paper cut, CHI3L1 would decrease the injury and cause local scarring while it restored tissue integrity. In that case, the amount of scarring would not be excessive and tissue function would not be significantly altered. But in IPF lungs, cells undergo ongoing injury, so CHI3L1 is chronically elevated and scar tissue accumulates. As CHI3L1 rescues cell after cell, the scarring builds up, eventually compromising the lung’s ability to breathe. In IPF, 70 percent of patients die within five years.
‘The CHI3L1 is doing exactly what it is supposed to do — it is designed to shut off cell death and decrease injury,’ said Dr. Jack A. Elias, a co-senior author of the study and dean of medicine and biological sciences at Brown University. He is joined on the paper by a host of his former colleagues and students at Yale University where the research occurred. ‘But at the same time it is decreasing cell death it is driving the fibrosis. You’ve got this ongoing injury so you’ve got these ongoing attempts to shut off injury which stimulate scarring.’
　
　
They compared tissues and serum from normal patients, outpatients with IPF, and patients with an acute exacerbation (AE) of IPF. In AE, widespread lung injury is superimposed on the pulmonary fibrosis, which frequently occurs before patients die. In lung biopsies and serum, they found that CHI3L1 levels are elevated in both tissue compartments in the outpatients with IPF and that the levels of CHI3L1 correlated with their disease progression. In the patients with AE, elevated levels of CHI3L1 were not noted, showing that the levels of CHI3L1 decrease right before the patients die.
‘This demonstrates that the CHI3L1 plays a key role in controlling lung injury in this setting,’ Elias said.
After documenting that elevated levels of CHI3L1 correlate with ongoing fibrosis and scarring and that a lack of the protein associates with widespread cell death, the team engaged in several manipulations of CHI3L1 in mice to see how levels and the clinical outcomes might be related. (In mice, CHI3L1 is also called BRP-39.)
Scientists can induce an IPF-like response in mice using a drug called bleomycin. In mice given bleomycin, the researchers found that the levels of CHI3L1 declined at first and then surged. At the times when the protein levels were low, cell damage occurred, and when the protein surged, the excessive scarring set in.
In previous research the team had engineered several lines of genetically modified mice. Some were transgenic and can produce CHI3l1 on chemically delivered command. Other mice were engineered to never produce BRP-39 — the mouse version of CHI3L1 — at all.
Using these mice, the researchers found that if they triggered CHI3L1 production early after administering bleomycin, the mice fared well, experiencing less injury, less damage and less scarring than controls. If they waited several days after bleomycin to trigger CHI3L1, the mice fared very poorly and scarring and mortality went up.
Mice who couldn’t produce CHI3L1/BRP-39, had acute lung cell damage, somewhat like AE patients who have a relative deficiency of CHI3L1. However, without CHI3L1 they did not generate much scarring.
All of these findings were supplemented with several other experiments that were designed to learn how CHI3L1 interacts with other cells involved in the tissue repair response in both human and mouse lungs. The experiments, including studies conducted in a bioengineered 3-D model of lung tissue seeded with relevant cells, showed that CHI3L1 regulates a pathway that recruits cells such as macrophages and fibroblasts that produce the scarring, or fibrosis.
In all, the results show that CHI3L1 plays a fundamental role in the course, if not the origin, of IPF. An ongoing buildup of it results in excessive scarring. Too little and cells die much more frequently.
‘To my knowledge this is the first comprehensive paper that’s been able to explain the many facets and presentations of IPF,’ Elias said. ‘It explains and links the injury and the repair responses that are critical in the disease. It also provides an explanation for the slowly progressing patients and the patients that experience acute exacerbations.’ Brown University

University of Delaware researchers have identified a protein, hiding in plain sight, that acts like a bodyguard to help protect and stabilize another key protein, that when unstable, is involved in Crohn’s disease. The fundamental research points to a possible pathway for developing an effective therapy for the inflammatory bowel disease.

The research was conducted by Catherine Leimkuhler Grimes, assistant professor of chemistry and biochemistry at UD, and Vishnu Mohanan, doctoral student in biological sciences,

As the scientists point out, our immune system provides the first line of defence against invading pathogens, a task even more challenging in the human gut, where over a trillion commensal bacteria live — resident microorganisms that help convert food into protein, vitamins and minerals.

To distinguish “bad” versus “good” bacteria, our bodies rely on a complex network of receptors that can sense patterns that are unique to bacteria, such as small fragments of bacterial cell wall. The receptors recognize and bind to these fragments, triggering an immune response to take out the “bad guys” or control the growth of the “good guys.”

However, when one of these receptors breaks down, or mutates, an abnormal immune response can occur, causing the body to mount an immune response against the “good” bacteria. Chronic inflammatory disorders, such as Crohn’s disease, are hypothesized to arise as a result.

The UD team focused on a protein called NOD2 — nucleotide-binding oligomerisation domain containing protein 2. More than 58 mutations in the NOD2 gene have been linked with various diseases, and 80 percent of these mutations are connected specifically to Crohn’s disease, according to Grimes.

In experiments to unveil NOD2’s signalling mechanisms and where they break down, “we stumbled on this chaperone molecule,” says Mohanan, who was the lead author of the scientific article.

The chaperone molecule was HSP70, which stands for “heat shock protein 70.” It assists with the folding of proteins into their correct three-dimensional shapes, even when cells are under stress from elevated body temperatures, such as a fever.

Grimes said she was a little sceptical at first about pursuing studies with HSP70 because it is a commonly known protein, but she found Mohanan’s initial data intriguing.

“Vishnu found that if we increased the expression level of HSP70, the NOD2 Crohn’s mutants were able to respond to bacterial cell wall fragments. A hallmark of the NOD2 mutations is inability to respond to these fragments. Essentially, Vishnu found a fix for NOD2, and we wanted to determine how we were fixing it.”

In further experiments, Mohanan created a tagged-wild-type NOD2 cell line in which NOD2 levels nearly matched the levels found in nature (versus “super” levels that might stimulate an artificial response) and found that NOD2 became more stabilized and degraded more slowly when treated with HSP70. In fact, HSP70 increased the half-life of NOD2 by more than four hours.

“Basically, HSP70 keeps the protein around — it kind of watches over and protects NOD2, and keeps it from going in the cellular trash can,” Grimes explains.

The researchers tested three human cell lines in their study: kidney cells, colon cells and white blood cells. In the next phase of the study, patient tissue will be examined through a collaboration with Nemours/A.I. duPont Hospital for Children to determine if NOD2 levels can be controlled via HSP70 expression.

“We want to figure out why the mutation in NOD2 results in an increase in inflammation,” says Mohanan. “Right now, we have limited knowledge. Once the signalling mechanism is figured out, we will have the keystone.” University of Delaware

The fungal infection invasive aspergillosis (IA) can be life threatening, especially in patients whose immune systems are weakened by chemotherapy or immunosuppressive drugs. Despite the critical need for early detection, IA remains difficult to diagnose. A study compared three diagnostic tests and found that the combination of nucleic acid sequence-based amplification (NASBA) and real-time quantitative PCR (qPCR) detects aspergillosis with 100% accuracy.

IA is caused by the fungus Aspergillus fumigatus, which is considered by many pathologists to be the world’s most harmful mold. ‘Traditional diagnostic methods, such as culture and histopathology of infected tissues, often fail to detect Aspergillus,’ comments lead investigator Yun Xia, PhD, of the First Affiliated Hospital of Chongqing Medical University, Chongqing, China.

In this retrospective study, scientists evaluated the diagnostic performance of two nucleic acid amplification assays (qPCR and NASBA) and one antigen detection method (galactomannan enzyme-linked immunosorbent assay [GM-ELISA]) using blood samples collected from 80 patients at high risk of IA. Of the 80 patients, 42.5% had proven or probable IA. The patients came from intensive care, haematology, neurology, nephrology, geriatrics, and other hospital departments.

The tests were evaluated singly and in combination. Individually, NASBA had the highest sensitivity (76.47%) whereas qPCR offered the highest specificity (89.13%). NASBA also was the test that best indicated that a patient did not have the infection (negative predictive value). NASBA and qPCR each had a high Youden index, a measure of the effectiveness of a diagnostic marker.

Combining the tests improved the outcomes. The combination of NASBA and qPCR led to 100% specificity and 100% positive predictive value (the probability that subjects truly have the infection).

‘Because each test has advantages and disadvantages, a combination of different tests may be able to provide better diagnostic value than is provided by a single test,’ says Dr. Xia. The combination of NASBA and qPCR should be useful in excluding IA in suspect cases, thus reducing both suffering and expense for immunocompromised patients. On the other hand, the combination of NASBA and qPCR could be more suitable for screening patients suspected of infection, because this assay had the highest sensitivity.’

The authors note that NASBA offers the advantages of rapid amplification (90 minutes) and simple operation with low instrument cost compared with qPCR and GM-ELISA. They caution that although GM-ELISA is widely and routinely used for aspergillosis diagnosis, this study indicates that it had low sensitivity (52.94%) with reasonable specificity (80.43%), making it ‘inferior to both NASBA and qPCR.’  EurekAlert

Dartmouth Institute for Quantitative Biomedical Sciences (iQBS) researchers developed a new gene expression analysis approach for identifying cancer genes. The study results challenge the current paradigm of microarray data analysis and suggest that the new method may improve identification of cancer-associated genes.

Typical microarray-based gene expression analyses compare gene expression in adjacent normal and cancerous tissues. In these analyses, genes with strong statistical differences in expression are identified. However, many genes are aberrantly expressed in tumours as a byproduct of tumorigenesis. These ‘passenger’ genes are differentially expressed between normal and tumour tissues, but they are not ‘drivers’ of tumorigenesis. Therefore, better analytical approaches that enrich the list of candidate genes with authentic cancer-associated ‘driver’ genes are needed.

Lead authors of the study, Ivan P. Gorlov, PhD, Associate Professor of Community and Family Medicine and Christopher Amos, PhD, Professor of Community and Family Medicine and Director of the Center for Genomic Medicine described a new method to analyse microarray data. The research team demonstrated that ranking genes based on inter-tumour variation in gene expression outperforms traditional analytical approaches. The results were consistent across four major cancer types: breast, colorectal, lung, and prostate cancer.

The team used text-mining to identify genes known to be associated with breast, colorectal, lung, and prostate cancers. Then, they estimated enrichment factors by determining how frequently those known cancer-associated genes occurred among the top gene candidates identified by different analysis methods. The enrichment factor described how frequently cancer associated genes were identified compared to the frequency of identification that one could expect by pure chance. Across all four cancer types, the new method of selecting candidate genes based on inter-tumour variation in gene expression outperformed the other methods, including the standard method of comparing mean expression in adjacent normal and tumour tissues. Dr. Gorlov and colleagues also used this approach to identify novel cancer-associated genes.

The authors cite tumour heterogeneity as the most likely reason for the success of their variance-based approach. The method is based on the knowledge that different tumours can be driven by different subsets of cancer genes. By identifying genes with high variation in expression between tumours, the method preferentially identifies genes specifically associated with cancer. This same feature, tumour heterogeneity, may reduce the ability to identify critical gene expression changes when comparing mean gene expression in adjacent tumor and normal tissues, as tumors of the same type may have different sets of genes differentially expressed.

The results of the study challenge the model that comparing mean gene expression in adjacent normal and cancer tissues is the best approach to identifying cancer-associated genes. Indeed, the team identified high variation in adjacent ‘normal’ tissue samples, which are typically used as control samples for comparison in analyses based on mean gene expression. The study suggests that methods based on variance may help get the most from existing and future global gene expression studies. Dartmouth Institute for Quantitative Biomedical Sciences

Understanding eye diseases is tricky enough. Knowing what causes them at the molecular level is even more confounding.

Now, University of Iowa researchers have created the most detailed map to date of a region of the human eye long associated with blinding diseases, such as age-related macular degeneration. The high-resolution molecular map catalogues thousands of proteins in the choroid, which supplies blood and oxygen to the outer retina, itself critical in vision. By seeing differences in the abundance of proteins in different areas of the choroid, the researchers can begin to figure out which proteins may be the critical actors in vision loss and eye disease.

“This molecular map now gives us clues why certain areas of the choroid are more sensitive to certain diseases, as well as where to target therapies and why,” says Vinit Mahajan, assistant professor in ophthalmology at the UI and corresponding author on the paper. “Before this, we just didn’t know what was where.”

What vision specialists know is many eye diseases, including age-related macular degeneration (AMD), are caused by inflammation that damages the choroid and the accompanying cellular network known as the retinal pigment epithelium (RPE). Yet they’ve been vexed by the anatomy: Why does it seem that some areas of the choroid-RPE are more susceptible to disease than others, and what is happening at the molecular level? The researchers set about to answer that question with non-diseased eye tissue donated by three deceased older individuals through the Iowa Lions Eye Bank. From there, Mahajan and Jessica Skeie, a post-doctoral researcher in ophthalmology at the UI, created a map that catalogues more than 4,000 unique proteins in each of the three areas of the choroid-RPE: the fovea, macula, and the periphery.

Why that’s important is now the researchers can see which proteins are more abundant in certain areas, and why. One such example is a protein known as CFH, which helps prevent a molecular cascade that can lead to AMD, much like a levee can keep flooding waters at bay. The UI researchers learned, though the map, that CFH is most abundant in the fovea. That helps, because now they know to monitor CFH abundance there; fewer numbers of the protein could mean increased risk for AMD, for instance.

“Now you can see all those differences that you couldn’t see before,” explains Mahajan, whose primary appointment is in the Carver College of Medicine. University of Iowa