Scientists at The Scripps Research Institute (TSRI) have identified the long-sought activating molecules for a rare but crucial subset of immune system cells that help rally other white blood cells to fight infection.

In the process, the team also uncovered a previously unsuspected link between the mammalian immune system and the communication systems of simpler organisms such as bacteria.

The findings could lead to novel therapeutic approaches for diseases such as type 1 diabetes that are the result of immune system over-activity, as well as new ways to boost the effectiveness of vaccines, according to study leader Luc Teyton, a professor in TSRI’s Department of Immunology and Microbial Science.

When a virus, bacteria or foreign substance invades the body, specialised cells known as dendritic cells present in the skin and other organs capture the trespassers and convert them into smaller pieces called antigens that they then display on their cell surfaces. White blood cells known as T and B cells recognize the antigens to launch very specific attacks on the invaders.

Dendritic cells also activate a specialized population of T cells known as natural killer T (NKT) cells. Once activated, NKT cells can commandeer the functions of dendritic cells to make them more effective and also recruit and coordinate the responses of T- and B-type cells.

“Because of their dual functions, NKT cells are a bridge between the body’s innate immunity, which is characterised by rapid but less specific responses to pathogens, and adaptive or acquired immunity, which is composed of specialised white blood cells that can remember past invaders,” Teyton said.

Previous studies indicated that NKT cells are activated by molecules known as glycolipids that dendritic cells produce and then display on their outer surfaces. It was widely assumed that the activating molecules were a class of glycolipids known as beta-glycosylceramides, an important component of nervous system cells.

However, this hypothesis had not been thoroughly examined, in part because there is no chemical test currently available to distinguish between two forms of the molecule that have slightly different configurations—beta-glycosylceramide and alpha-glycosylceramide. In addition, when scientists attempt to create either form synthetically for testing, there is always the possibility of small contamination of one by the other.

“When you’re making glycolipids, there is no completely faithful way of controlling the form that you’re making,” Teyton said. ‘You’re favouring the making of one, but you cannot say for sure that you don’t have a small amount of the other form.”

In their new study, Teyton and his colleagues, who included scientists from Brigham Young University, the La Jolla Institute for Allergy & Immunology and the University of Chicago, abandoned the chemical approach altogether. Instead, they combined a series of biochemical and biological assays to create a test that was sensitive enough to distinguish between the two different forms of glycolipids.

“Biological assays are exquisitely sensitive to low amounts of otherwise unmeasurable molecules,” said study first author Lisa Kain, a research technician in Teyton’s lab.

The scientists used custom antibodies to identify and eliminate alpha-glycosylceramides from their test batches. When the team was confident that their test batch contained only beta forms of the glycolipid, they tested it on NKT cells gathered from mice. To their surprise, however, nothing happened. Contrary to the conventional wisdom, the beta-glycosylceramides failed to activate the NKT cells.

“We were very skeptical about the early results,” Teyton said. “We thought we had used the wrong antibody.”

Next, the team combined enzymes designed to digest molecular linkages found only on beta-glycosylceramides with mice NKT cells inside test tubes. Surprisingly, the NKT cells were still being activated.

Finally, when the team used antibodies to disable alpha-glycosylceramides inside live mice, not only did the NKT cells fail to activate, they disappeared altogether from organs such as the thymus, where NKT cells are produced.

These multiple lines of evidence strongly indicated that it was the alpha form of the glycolipids that were the triggers for NKT cells. “What we thought was the contaminant turned out to be the activating molecule we were looking for,” Teyton said.

The results were surprising for another reason. Until that moment, scientists did not think mammalian cells were capable of producing alpha forms of the glycolipids. The molecules were thought to exist only in bacteria and other simple organisms, which use them primarily as a means of communicating with one another. The findings thus suggest that the roots of a crucial part of the mammalian immune response are even more ancient than previously thought.

“Nobody expected this,” Teyton said. “It’s like discovering that all languages share a common origin.”

Now that scientists know that alpha-glycosylceramides are made by our own body and activate NKT cells, they might be able to exploit it to create new therapies. For example, Teyton said, researchers could use enzymes to reduce alpha-glycosylceramide levels in order to suppress an overactive immune response, which happens with diseases such as type 1 diabetes. Or they could combine the molecules with antigens to create vaccines that elicit a faster and more efficient immune response.

“This opens up an avenue of new therapeutic approaches that we’ve never even thought about,” Teyton said. The Scripps Research Institute

Mice bred to carry a gene variant found in a third of ALS patients have a faster disease progression and die sooner than mice with the standard genetic model of the disease, according to Penn State College of Medicine researchers. Understanding the molecular pathway of this accelerated model could lead to more successful drug trials for all ALS patients.

Amyotrophic lateral sclerosis, commonly known as Lou Gehrig’s disease, is a degeneration of lower and upper motor neurons in the brainstem, spinal cord and the motor cortex. The disease, which affects 12,000 Americans, leads to loss of muscle control. People with ALS typically die of respiratory failure when the muscles that control breathing fail.

Penn State researchers were the first to discover increased iron levels in the brains of some patients with the late-onset neurodegenerative disorders Parkinson’s disease and Alzheimer’s disease. A decade ago, they also identified a relationship between ALS and excess iron accumulation when they found that 30 percent of ALS patients in their clinic carried a variant of a gene known as HFE that is associated with iron overload disease.

For this study, the researchers crossbred mice with the HFE gene variant with the standard mice used in ALS research.

‘When we followed the disease progression and the behaviour of our crossbred mice compared to the standard mice, we saw significant differences,’ said James Connor, vice chair of neurosurgery research and director of the Center for Aging and Neurodegenerative Diseases. The crossbred mice performed significantly worse on tests of forelimb and hindlimb grip strength and had a 4 percent shorter life span.

‘The disease progression was much faster in the crossbred mice than in the standard mice,’ Connor said. ‘What we found is that when ALS happens in the presence of the HFE gene variant, things go downhill more quickly.’

The lead investigator on this project, graduate student Wint Nandar, noticed that the HFE gene variant sped up disease progression and death in females but not males. Males with ALS die faster, on average, than females.

Connor said the variant may not have had time to accelerate the pace of the disease in male mice. An accelerated progression may show up in clinical trials in human males, who live longer with the disease than mice.

The researchers also studied how the HFE gene modified the pace of the disease in mice. The crossbred mice showed increased oxidative stress and microglial activation. Microglial cells normally help with repair in the body, but when over-activated they can promote unhealthy inflammation.

‘They can make things worse instead of better,’ Connor said.

The mice were also found to have disruption of the neurofilaments, the tiny cables that transport nutrients through nerve cells.

 ‘It’s a much worse environment when the gene variant is present,’ Connor said. ‘This makes it much easier for the disease to take off.’

The findings could help direct more successful clinical testing of new drug treatments, which have traditionally had disappointing results. Because patients with H63D HFE have an accelerated form of the disease, their results could skew study findings.

‘There might be drugs out there that work for 70 percent of the ALS population even though the studies don’t show that when all of the data are looked at without consideration of the genetic background,’ Connor said.

Separating the data out could help find effective treatments for both those with the gene variant and the rest of the ALS population.

‘How a drug is going to work on a carrier of the gene variant could be worse or it could be better, but it’s likely going to be different,’ Connor said. Penn State

An independent academic study provides a detailed analysis of deep well microplates and the significant levels of contamination found in more than 50% of the commercially available plates tested.
The study gives data on a large range of microplates from numerous manufacturers based in Europe, USA and China. Mass spectral data shows that persistent contamination from a range of compounds found in the raw polymer master batch continues to be evident in many of the microplates tested. The effect of extractables leached out of the deep well plates identified in this report depends on the exact application for which the plate was designed but is highly likely to significantly affect their performance and contaminate samples stored in them.
The authors of the report conclude it is likely that a low grade polypropylene was used in the production of a significant proportion of the deep well microplates that leached extractables when tested.

http://tinyurl.com/qaxn89a

A comprehensive analysis of the genomes of nearly 500 papillary thyroid carcinomas (PTC) – the most common form of thyroid cancer – has provided new insights into the roles of frequently mutated cancer genes and other genomic alterations that drive disease development. The findings also may help improve diagnosis and treatment. Investigators with The Cancer Genome Atlas (TCGA) Research Network identified new molecular subtypes that will help clinicians determine which tumours are more aggressive and which are more likely to respond to certain treatments. Their findings confirmed that PTCs are driven primarily by mutations in one of two cancer-associated genes: BRAF (and a particular mutation, V600E) or RAS. The work also detailed many differences between the two genetic types, particularly in signalling pathways that promote tumour development and growth.

The researchers developed a scoring system to reflect gene expression in the two PTC types, allowing them to characterize tumours and determine both the pathway a tumour uses to send signals and its relative aggressiveness. Where a tumour lies on a scale – called its thyroid differentiation score – can have important treatment implications because different tumor signaling properties can mean the cancer responds differently to particular therapies.

The study also showed that BRAF-driven tumors have a broader range of genetic complexity than previously thought, with distinct subtypes. The results suggest a need for a new classification system that more accurately reflects underlying genetic characteristics of the cancer.

Thyroid cancer is the fastest growing cancer in the United States, with more than 20,000 new PTC cases each year. Most thyroid cancers are slow-growing and treatable with surgery, hormone therapy and radioactive iodine. National Human Genome Research Institute

A study combining tumour cells from patients with breast cancer with a laboratory model of blood vessel lining provides the most compelling evidence so far that a specific trio of cells is required for the spread of breast cancer. The findings could lead to better tests for predicting whether a woman’s breast cancer will spread and to new anti-cancer therapies. The study was led by researchers at the NCI-designated Albert Einstein Cancer Center and Montefiore Einstein Center for Cancer Care (MECCC).

Maja Oktay, M.D., Ph.D.According to the National Cancer Institute, more than 232,000 American women developed breast cancer last year and nearly 40,000 women died from the disease. It is the most common cancer among women in the United States. Most breast cancer deaths occur because the cancer has spread, or metastasized, which means that cells in the primary tumour have invaded blood vessels and travelled via the bloodstream to form tumours elsewhere in the body.

In earlier studies involving animal models and human cancer cell lines, researchers found that breast cancer spreads when three specific cells are in direct contact: an endothelial cell (a type of cell that lines the blood vessels), a perivascular macrophage (a type of immune cell found near blood vessels), and a tumour cell that produces high levels of Mena, a protein that enhances a cancer cell’s ability to spread. Where these three cells come in contact is where tumour cells can enter blood vessels—a site called a tumour microenvironment of metastasis, or TMEM. Tumours with high numbers of TMEM sites (i.e., they have a high TMEM ‘score’) were more likely to metastasize than were tumours with lower TMEM scores. In addition, the researchers found that cancer tissues high in a form of Mena called MenaINV were especially likely to metastasize. (MenaINV refers to the invasive form of Mena.)

‘Those studies revealed new insights into how cancer might spread, but they didn’t necessarily show what is happening in patients,’ said study leader Maja Oktay, M.D., Ph.D., associate professor of pathology at Albert Einstein College of Medicine of Yeshiva University and attending cytopathologist at Montefiore.

Since then, the scientists have extended their research to include patients with breast cancer. In 2011, they published findings on 40 patients showing a correlation between high MenaINV levels and high TMEM scores. The present study combines results from those 40 patients plus an additional 60 patients. All 100 patients had been diagnosed with invasive ductal carcinoma and were being treated at MECCC. Invasive ductal carcinoma is the most common type of invasive breast cancer, accounting for 80 percent of cases. In this disease, the cancer has grown through the duct walls and into the surrounding breast tissue.
For the subset of more recent patients, the researchers assessed tumour cell behaviour—in particular, cancer cells’ ability to cross the endothelium (inner layer) of blood vessels. They obtained tumour cells using fine needle aspiration and placed them in a novel engineered tissue assay designed to replicate the endothelium of a blood vessel—the barrier that cells must cross so they can spread from a primary tumour to distant sites. Biopsied tumour tissue from all 60 new patients was fixed in formalin and embedded in paraffin so that TMEM sites in the tissue could be counted.
‘It’s critically important to learn more about the metastatic process so we can develop new ways to predict whether cancer will spread and identify new treatments.’

Breast cancer cells able to cross the endothelial layer in this assay were found to have higher MenaINV levels compared with the total population of patients’ aspirated cells. In addition, finding high levels of MenaINV correlated with finding high numbers of TMEM sites in paraffin biopsy specimens from the same patients. The TMEM ‘score’ for each biopsy specimen was calculated by counting the total number of TMEM sites observed within ten 400x magnification fields. Combining the results from all 100 patients showed that the findings were consistent across the three most common clinical subtypes of invasive ductal carcinoma.

‘These results confirm that TMEM sites and MenaINV are essential for the spread of breast cancer in humans,’ said Dr. Oktay. ‘They also imply that MenaINV expression and TMEM score measure related aspects of a commonly used mechanism that human breast cancers use to metastasize.’

Dr. Oktay noted that ‘the outcome for patients with metastatic breast cancer hasn’t improved in the past 30 years despite the development of targeted therapies. It’s critically important to learn more about the metastatic process so we can develop new ways to predict whether cancer will spread and identify new treatments.’ Albert Einstein College of Medicine