Pseudoislet system expected to advance pancreas and diabetes research

The multicellular, 3-D structure of human pancreatic islets—the areas of the pancreas containing hormone-producing or endocrine cells—has presented challenges to researchers as they study and manipulate these cells’ function, but Vanderbilt University Medical Center researchers have now developed a pseudoislet system that allows for much easier study of islet function.

A pancreatic islet is composed primarily of beta cells, alpha cells and delta cells, but also includes many supporting cells, such as endothelial cells, nerve fibers and immune cells, which act in concert as a mini organ to control blood glucose through hormone secretion. Insulin, secreted from beta islet cells, lowers blood glucose by stimulating glucose uptake in peripheral tissues, while glucagon, secreted from alpha islet cells, raises blood glucose through its actions in the liver.

Dysfunction of these islet cells is a primary component of all forms of diabetes, and a better understanding of this dysfunction can lead to improved treatment and management of the disease. Vanderbilt scientists and others around the world have identified potential targets for diabetes using both mouse models and human tissue, however the lack of a system to manipulate these pathways in human islet cells has limited the field.

The VUMC team led by Marcela Brissova, Ph.D., research professor of Medicine and director of the Islet Procurement and Analysis Core of the Diabetes Research and Training Center, began attempting a protocol for the pseudoislet system in 2016, performing countless trials. In late 2017, Rachana Haliyur, then a Vanderbilt MD/Ph.D. student, combined media containing factors that support vascular cells and endocrine cells into what the group named the Vanderbilt Pseudoislet Media. The team watched as the cells began reaggregating, or organizing themselves in a way that resembled native islets.

“A lot of things in science happen serendipitously, and this was one of those,” said Brissova. “We tried and failed many times, and basically it came down to the media we used for our cells. In our recent publication, we have provided all experimental details and our protocol so others can make the media and create pseudoislets in their own laboratories.”

Because of the complex structure of the human islet, it is difficult to introduce and manipulate cells past the first cell-layer of the islet sphere. The pseudoislet system allows investigators to separate the pancreatic islet into single cells, introduce a virus into the cells which allows genetic manipulation and then combine the cells back together again into a pseudoislet. This allows researchers to target certain cell types or replicate changes happening in disease and study them in the 3-D environment of the islet.

John “Jack” Walker, an MD/Ph.D. student in the Powers & Brissova Research Group, continued to refine the pseudoislet system protocol and was co-first author on a recent study based on the system published in JCI Insight, an open access journal published by the American Society for Clinical Investigation (ASCI).

The pseudoislet system allowed the VUMC investigators to more clearly examine intracellular signaling pathways, allowing genetic manipulation of those pathways to change their function and better understand how insulin and glucagon secretion are altered with that manipulation. They determined that activation of Gi protein signaling reduced insulin and glucagon secretion while activation of Gq protein signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion.

In addition, this approach allowed the scientists to introduce biosensors into the islet cells to measure intercellular signaling events within the cells and better understand how those are linked to hormone secretion.

Another advance was the combination of the pseudoislet system with a unique microfluidic device, developed by co-authors Matthew Ishahak and Ashutosh Agarwal, Ph.D., from the University of Miami, that allowed the investigators to simultaneously document the changes in both calcium ions and hormone secretion.

“The exciting thing about this approach is that we both deconstruct the islet for our manipulation and reconstruct it to understand functional consequences at a larger level,” Walker said. “Since we put the islet cells back together, we can look at both insulin and glucagon secretion, but in a coordinated manner. Both of the secretion profiles measured are reflective of intra-islet interactions that are happening as well.”

This work greatly benefited from the research environment and infrastructure at Vanderbilt, particularly the National Institutes of Health (NIH)-funded Diabetes Research and Training Center (DRTC) and the Vanderbilt Cell Imaging Shared Resource.

“Another research direction will be creating pseudoislets that replicate a specific disease state, such as pseudo-islets that look like native islets from an individual with type 1 diabetes,” Haliyur said.

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Scientists ‘re-train’ immune system to prevent attack of healthy cells

The body’s immune system can be re-wired to prevent it from recognizing its own proteins which, when attacked by the body, can cause autoimmune diseases like multiple sclerosis, a significant new study by UK scientists has found.

Autoimmune diseases are caused when the immune system loses its normal focus on fighting infections or disease within and instead begins to attack otherwise healthy cells within the body. In the case of multiple sclerosis (MS), the body attacks proteins in myelin—the fatty insulation-like tissue wrapped around nerves—which causes the nerves to lose control over muscles.

Led by a multi-disciplinary team from the University of Birmingham, scientists examined the intricate mechanisms of the T-cells (or white blood cells) that control the body’s immune system and found that the cells could be ‘re-trained’ to stop them attacking the body’s own cells. In the case of multiple sclerosis, this would prevent the body from attacking the Myelin Basic Protein (MBP) by reprogramming the immune system to recognize the protein as part of itself.

Supported by the Medical Research Council, the two-part study, published today in Cell Reports, was a collaboration between two research groups led by Professor David Wraith from the Institute of Immunology and Immunotherapy and Professor Peter Cockerill from the Institute of Cancer and Genomic Sciences.

The first stage, led by Professor Wraith showed that the immune system can be tricked into recognizing MBP by presenting it with repeated doses of a highly soluble fragment of the protein that the white blood cells respond to. By repeatedly injecting the same fragment of MBP, the process whereby the immune system learns to distinguish between the body’s own proteins and those that are foreign can be mimicked. The process, which is a similar type of immunotherapy to that previously used to desensitize people against allergies, showed that the white blood cells that recognize MBP switched from attacking the proteins to actually protecting the body.

The second stage, saw gene regulation specialists led by Professor Peter Cockerill probe deep within the white blood cells that react to MBP to show how genes are rewired in response to this form of immunotherapy to fundamentally re-program the immune system. The repeated exposure to the same protein fragment triggered a response that turns on genes that silence the immune system instead of activating it. These cells then had a memory of this exposure to MBP embedded in the genes to stop them setting off an immune response. When T cells are made tolerant, other genes which function to activate the immune system remain silent.

Professor David Wraith said: “These findings have important implications for the many patients suffering from autoimmune conditions that are currently difficult to treat.”

Professor Peter Cockerill, said: “This study has led us to finally understand the underlying basis of immunotherapies which desensitize the immune system”

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A new plant-based system for the mass production of allergens for immunotherapy

Allergies can significantly affect health and quality of life. While allergen immunotherapy provides long-lasting therapeutic relief to people suffering from environmental allergies, the therapy can last several years and requires large amounts of allergen. Now, researchers from the University of Tsukuba developed a novel system that enables the mass production of the major birch pollen allergen Bet v 1 in plant leaves in just a matter of days. In a new study published in Frontiers in Plant Science, they showed that their system not only produces large amounts of Bet v 1, but the purified protein was also highly reactive towards the IgE antibodies in sera from individuals with birch pollen allergy.

“The idea of allergen immunotherapy is to desensitize the body’s response to the allergen by exposing patients to it in gradually increasing amounts,” says corresponding author of the study Professor Kenji Miura. “Because a significant drawback is the difficult, expensive and low-yield production of allergens, our goal was to develop a new system that allows for the rapid and massive production of allergens that can be used in the clinical setting.”

To achieve their goal, the researchers turned to their previously established “Tsukuba system,” which makes use of a method called agroinfiltration. They first introduced the gene for Bet v 1 into a specific type of bacteria called Agrobacterium tumefaciens and let them grow. They then immersed leaves of the plant Nicotiana benthamiana into the bacterial solution to bring the bacteria into close contact with the plant, so the bacteria could transfer the Bet v 1 gene to plant cells, which in turn started producing the protein. To test the quality of their product, the researchers also produced the protein in Brevibacillus brevis, which is a standard bacterial host for protein production.

“We were able to purify 1.2mg of Bet v 1 protein from 1g leaves in just 5 days,” explains Professor Miura. “This is a relatively large amount that is otherwise difficult to achieve using standard methods. Our next goal was to test whether our protein was immunogenic, which is a prerequisite for immunotherapy.”

The researchers isolated sera from individuals with birch pollen allergy and mixed them with Bet v 1 protein purified from plants and bacteria. In both cases, the researchers were able to show that Bet v 1-specific IgE from the patients’ sera, which is the antibody causing the allergy, was strongly reactive to their proteins.

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