Tag Archives: #cell

The Researchers Who Look Into The Tiniest Part Of a Cell (Biology)

It is a cold, grey November day in 2018 when we meet the researchers from Lund University at MAX IV, a research facility with the world’s brightest and most focused X-rays. Researchers from all over the world travel here to investigate things at the atomic level and see how molecules bind to one other; knowledge that is valuable when developing new drugs, for example.

Months of preparation have gone into today’s experiments, and now medical researchers Anders Malmström, Emil Tykesson and their team hope to obtain greater knowledge about how an enzyme affects processes in our cells. They don’t know it yet, but the experiments they are doing this morning will map a previously unresolved structure of the human protein they are studying. Their research was published in the scientific journal Chemical Science in late 2020.

What happens at the atomic level is significant for our health. The enzyme that the research team will investigate on this day in November 2018 is a protein that increases or decreases the rate of chemical reactions in our cells. Proteins are one of the most vital molecules in terms of the body’s functioning, and researchers are therefore interested in understanding how they work, what they look like and what they do. If they succeed in solving the structure of this specific protein they are studying, they will be one step closer to designing drugs that fit into and control the protein’s function.

Anders Malmström, professor of matrix biology at Lund University, has been hunting for the enzyme since he was a doctoral student in the 1960s. He explains that the enzyme, which has the somewhat complicated namedermatan-sulfate epimerase, is important in terms of how cancer cells move through the body. This had led to interest among researchers in investigating whether cancer treatments can be improved if the enzyme’s activity is reduced. Would this lead to the cancer cells moving more slowly in the body and could this reduce the spread of tumours?

Anders Malmström, professor of matrix biology at Lund University, has been hunting for the enzyme since he was a doctoral student in the 1960s. (Photo: Tove Smeds)

But before this question can be answered, researchers need to learn more about the enzyme, which is why they’re now visiting one of the world’s brightest microscopes: MAX IV.

“Many years of work have gone in to bringing us this far, and we are very excited about the results of the experiments”, says Anders Malmström.

“Our hope is that when we learn more about what the enzyme looks like and how it works, it will open up opportunities to reduce the activity of the enzyme with the help of small, specially designed molecules. Then we may be able to produce extra medication for cancer treatments and fibrosis, atherosclerosis, blood clotting diseases and infections, where the enzyme complicates the problem”, Anders Malmström explains. 

Some of the equipment needed to separate the enzyme by size (Photo: Tove Smeds)

In the early 2000s, the researchers succeeded in purifying the enzyme, which is an important step in the research. Put simply, this entails removing all other proteins and molecules that obscure the view of, or contaminate, the enzyme – to find out its structure and construction at a more molecular level.

“Here at the lab, we can obtain a small estimate of how big the enzyme is and we know which gene in the body carries the recipe for the enzyme. But to see it in detail we need MAX IV, which is like a giant microscope through which you can examine things at atomic level.”

BUILDING CRYSTALS

In order to be able to irradiate the enzyme at MAX IV, the researchers need to make crystals out of the enzyme; a process that takes time to develop. In fact, it was not entirely certain that it would be possible to do this, as proteins are not as easy to crystallise as minerals and small molecules, for example.

Since the researchers know which human gene can produce the enzyme, they have been able to produce large volumes of it in cell culture, enabling the preparation of crystals needed to conduct research at MAX IV.

It takes about three months to produce the crystals and though it may sound simple, it is one of the biggest challenges for researchers ahead of the day’s experiment at MAX IV.

“Not all proteins can be crystallised and they must also be so stable that they retain their structure all the way from production to analysis of the crystals. We have probably gone through a thousand different conditions in which we have altered parameters such as pH and salinity in the liquid in which the crystals grow”, explains Emil Tykesson.

The enzyme must be well organised so that we can see all the atoms and where they are located.

“Each individual protein molecule must match all the adjacent protein molecules and form a large symmetrical crystal lattice.”

The first opportunity to see how good the crystals are is at MAX IV when the X-ray is focused onto them. It turned out that the majority of the crystals were of such low quality that the researchers could not see clear details in the structure of the enzyme.

“However, we were lucky and produced a couple of crystals that provided very good data”, adds Tykesson.

All data collected by the researchers during the experiment at MAX IV is converted into a three-dimensional structure in which the researchers can see the atoms in detail and understand different parts of the protein. The most interesting aspect is what is known as the “active site” – where the chemical reaction takes place. By designing molecules, or inhibitors, the researchers hope to be able to prevent the enzyme’s function. (Photo: Tove Smeds)

HUGE AMOUNTS OF DATA

All collected data is analysed after the experiments conducted at MAX IV, which in turn can take weeks or months. And now, two years after the experiments were carried out, the research has been published in the scientific journal Chemical Science.

“Researchers attach extremely high demands to the truth and expect what we publish in scientific articles to be correct. This is why we test in every conceivable way to strengthen or falsify our hypothesis. That takes time, but we want to rule out every possible inaccuracy. This would have been faster to solve if a very large number of researchers had focused on this particular enzyme, but we are a small research team and have finite resources”, notes Tykesson.

Now that the researchers have the structure of the enzyme, they want to go further and investigate whether it can be significant in the treatment of glioma, a type of brain cancer, in which it has been noted that patients have increased activity of this particular enzyme.

“By enhancing knowledge about the enzyme through cell experiments and other methods, we believe that we can have an effect on diseases such as infection, atherosclerosis and musculoskeletal diseases in which the enzyme has changed”, explains Malmström.

MAX IV is a large synchrotron facility, a ring in which electrons are made to spin. After a while they lose their energy and disappear, which means that you need to add new electrons as the experiment progresses. If you have no electrons, you lose the beam needed to carry out the experiment. In order for the enzyme not to be destroyed by the X-rays, they are cooled down with the help of liquid nitrogen (200 degrees below zero). Here, Johan Unge, who at the time of the experiment worked at MAX IV, cools down the researchers’ crystals before the experiment.
A drop of liquid loaded with crystals. Emil Tykesson fishes out the crystals that will be used for today’s experiments. (Photo: Tove Smeds)

This is what it looks like on the screen when one of the crystals has been irradiated. The more black dots and the further out in the image they are, the sharper the image. On this day, the research group is testing 30 different crystals.

“Once the experiment is completed, we start working to produce the structure of the enzyme based on the black dots. And this can take anywhere from a couple of weeks up to several months”, explains Tykesson.

The enzyme dermatansulfat-epimeras is important in terms of how cancer cells move through the body. Emil Tykesson has studied the enzyme since he started his PhD at Lund University in 2010.

Publication

The study The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms is published in Chemical Science, December 2020. DOI: 10.1039/D0SC05971D

Financing

The authors from Lund were supported by the LMK Foundation, the Medical Faculty at Lund University, Lund University, the Swedish Research Council, the Swedish Cancer Society, the Mizutani Foundation for Glycoscience, the Greta and Johan Kock foundation, the FLAK Research School, the Albert Österlund foundation, the Royal Physiographic Society and the Lars Hierta Foundation. HK was supported by the Swiss National Science Foundation (early Postdoc mobility grant no. P2ZHP3_191289). LM was supported by the Knut And Alice Wallenberg Foundation (grant no. KAW 2016.0023). The glycoproteomic analysis was supported by the Swedish National Infrastructure for Biological Mass Spectrometry (BioMS), funded by the Swedish Research Council (VR).

Featured image: The researchers discuss whether to change the position of the crystal.


Provided by Lund University

Tracking the Formation of the Early Heart, Cell by Cell (Biology)

Richard Tyser and colleagues have mapped the origins of the embryonic mouse heart at single-cell resolution, helping to define the cell types that make up the heart in the earliest days of development.

© Gettyimages

Their techniques allowed them to identify for a first time a pool of progenitor cells that contributes to the formation of heart muscle cells as well as the early epicardium, the outermost layer of the heart. This layer provides cells and other proteins that guide the development and repair of heart tissue, so a better understanding of its origins could better inform regenerative heart therapies as well as improve our understanding of congenital heart defects.

Tyser et al. performed a micro-dissection of a portion of the embryonic mouse heart to observe a very early stage streak of cells called the cardiac crescent transform into the linear heart tube. Combining single-cell RNA sequencing to identify cell types with high-resolution imaging and time-lapse microscopy, the researchers were able to follow the development of distinct populations of progenitor heart cells over about 12 hours of development.

Reference: Richard C. V. Tyser, Ximena Ibarra-Soria, Katie McDole, Satish A. Jayaram, Jonathan Godwin, Teun A. H. van den Brand, Antonio M. A. Miranda, Antonio Scialdone, Philipp J. Keller, John C. Marioni, Shankar Srinivas, “Characterization of a common progenitor pool of the epicardium and myocardium”, Science 07 Jan 2021:
eabb2986 DOI: 10.1126/science.abb2986 https://science.sciencemag.org/content/early/2021/01/07/science.abb2986

Provided by AAAS

Research Reveals Why Some Tumors Have Different Makeup of Cells (Medicine)

Molecular changes in cells called fibroblasts, which help provide support for tissues throughout the body, may explain why one type of colon cancer doesn’t respond to therapy, according to a team of researchers from Weill Cornell Medicine. Targeting these cells may be a way to make treatment more effective.

Image of a colon tumor in mice. Cancer-associated fibroblasts (red) help the tumor grow more aggressively and help block the immune system response against it. ©Weill Cornell Medicine

In a study published Nov. 17 in DevelopmentalCell the investigators examined cells called fibroblasts in CMS4, the most aggressive and difficult-to-treat form of colorectal cancer, to determine how these cancer-associated cells acquire traits that allow them to support malignancy in neighboring cells. CMS4 affects about a third of all colorectal cancer patients.    

“There are two important components to this study,” said co-senior author Dr. Jorge Moscat-Guillen, Homer T. Hirst III Professor of Oncology in Pathology and Vice-Chair for Experimental Pathology. “First, we have shown in a mechanistic way how these cancer-associated fibroblasts acquire the characteristics that they have. Second, we confirmed that what we discovered in our lab models also applies to patients, which begins to suggest how these findings could be useful in the clinic.”

Tumors are made up not only of cancer cells, but many other kinds of cells as well. These other cell types can influence how a tumor behaves and can have a profound effect on whether it responds to therapy, including treatment with immunotherapy.

Fibroblasts are present throughout the body. In the colon, they are part of the contractile system that is key to digestion. Researchers have known that the presence of fibroblasts inside a tumor can impact its behavior, especially influencing whether tumors respond to immune checkpoint blockade drugs. These are drugs that take the brakes off the immune system and allow it to attack cancer. “Until now, a pending question in the field has been what causes these tumors full of fibroblasts to develop,” said co-senior author Dr. Maria Diaz-Meco, a professor in the Department of Pathology and Laboratory Medicine.

Graphical abstract by Hiroaki et al.

Previous research from Dr. Moscat and Dr. Diaz-Meco reported the creation of a mouse model that mimics CMS4 colorectal cancer. As in patients with this type of colorectal cancer, a high concentration of cancer-associated fibroblasts is found within the tumors of these mice.

In the current study, the researchers used these mouse models, as well as cells grown in the test tube and organoids (tiny clusters of cells grown in a dish) to further study the relationship between cancer cells and cancer-associated fibroblasts. “We found that the cancer cells send out a signal that changes the fibroblasts,” Dr. Moscat said. “The fibroblasts that emerge make the tumor more aggressive and more stealthy to the immune system.”

That molecular switch depletes levels of a protein called PKCz, which in turn increases levels of another protein called SOX2 in the fibroblasts. These changes appear to make the fibroblasts more friendly to the promotion of cancer growth.

The researchers used animal models to test the behavior of the cells they had altered in the lab. They also performed a type of analysis called single-cell RNA sequencing to further confirm that their observations in the lab matched what is observed in patients samples.

Drs. Moscat and Diaz-Meco say now that they understand the communication between cancer cells and fibroblasts, they believe that drugs could be developed to block it. “The fibroblasts are acting in a way that protects the tumor from the immune system,” Dr. Moscat said. “We want to find ways to target the fibroblasts that deprives the tumors of this protective environment.” These drugs would likely be used in combination with immunotherapy, to first lower the defenses of the fibroblasts, allowing the immune cells that are activated by checkpoint blockade drugs to better do their jobs.

“Now that we understand the roles of SOX2 and PKCz,” Dr. Diaz-Meco said, “we have two good biomarkers that we could use to assign patients to trials with these drugs.”

The researchers noted that the findings from this study could also apply to other cancers, especially pancreatic cancer. Pancreatic tumors also contain large numbers of fibroblasts that appear to make these tumors less receptive to treatment.

References: Hiroaki Kasashima, Angeles Duran, Anxo Martinez-Ordoñez, Eduard Batlle, Maria T. Diaz-Meco, Jorge Moscat, “Stromal SOX2 Upregulation Promotes Tumorigenesis through the Generation of a SFRP1/2-Expressing Cancer-Associated Fibroblast Population”, Developmental Cell, 2020. https://www.cell.com/developmental-cell/fulltext/S1534-5807(20)30834-0 DOI: https://doi.org/10.1016/j.devcel.2020.10.014

Provided by Sanford Burnham Prebys

Space Travel Can Adversely Impact Energy Production in a Cell (Planetary Science)

Studies of both mice and humans who have traveled into space reveal that critical parts of a cell’s energy production machinery, the mitochondria, can be made dysfunctional due to changes in gravity, radiation exposure and other factors, according to investigators at Georgetown Lombardi Comprehensive Cancer Center. These findings are part of an extensive research effort across many scientific disciplines to look at the health effects of travel into space. The research has implications for future space travel as well as how metabolic changes due to space travel could inform medical science on earth.

Georgetown researcher Evagelia C. Laiakis, PhD, and dozens of other scientists described recent findings about the impact of space travel on health as part of a large compendium of work that appears concurrently in Cell, Cell Reports, Cell Systems, Patterns, and IScience. ©Jerry Angdisen/Georgetown University

The findings appeared November 25, 2020, in Cell and are part of a larger compendium of research into health aspects of space travel that appears concurrently in Cell, Cell Reports, Cell Systems, Patterns, and iScience.

“My group’s research efforts centered around muscle tissue from mice that were sent into space and were compared with analyses by other scientists who studied different mouse tissue,” says Evagelia C. Laiakis, PhD, an associate professor of oncology at Georgetown. “Although we each studied different tissue, we all came to the same conclusion: that mitochondrial function was adversely impacted by space travel.”

In addition to studying the effects of space travel on cellular function, the scientists used a trove of data from decades of NASA human flight experiments to correlate their outcomes in animals with those from 59 astronauts. They were also able to access data derived from NASA’s repository of biospecimens that had flown in space to do further comparisons. Data from NASA’s Twin Study of Mark and Scott Kelly was particularly informative as it allowed for a comparison of the health effects seen in an astronaut in space, Scott, with his earth-bound brother, Mark, who is a retired astronaut. Comparing their studies of mice with human data, Laiakis and the team of researchers were able to determine that space travel led to certain metabolic effects:

  • Isolated cells were adversely impacted to a higher degree than whole organs
  • Changes in the liver were more noticeable than in other organs
  • Mitochondrial function was impacted

Because space travel almost always exposes people to higher levels of radiation than would be found on earth, the scientists knew that such an exposure could harm mitochondria. This aspect of radiation exposure translates to health outcomes here on earth for cancer patients who undergo radiotherapy. With this knowledge of radiation’s impact on mitochondria, clinicians might tailor radiation therapy in different ways in the future to protect normal tissue. The implications for travel to Mars are especially concerning, the researchers say, as that would involve a much longer time in space and hence lengthy exposure to radiation.

“The launch of SpaceX earlier this month was very exciting,” says Laiakis. “From this, and other planned ventures to the moon, and eventually Mars, we hope to learn much more about the effects that spaceflight can have on metabolism and how to potentially mitigate adverse effects for future space travelers.”

Provided by Georgetown University Medical Center

Computational Approach To Optimise Culture Conditions Required For Cell Therapy (Medicine)

Collaboration by researchers in Singapore and Australia lead to first-of-its-kind computational biology algorithm that could enable more effective cellular therapies against major diseases.

The scientists used EpiMogrify, an innovative computational biology algorithm, to predict molecules needed to control the cell state and fate of cardiac muscle cells (left) and astrocytes (right). ©Joseph Chen, Monash University

Cellular therapy is a powerful strategy to produce patient-specific, personalised cells to treat many diseases, including heart disease and neurological disorders. But a major challenge for cell therapy applications is keeping cells alive and well in the lab.

That may soon change as researchers at Duke-NUS Medical School, Singapore, and Monash University, Australia have devised an algorithm that can predict what molecules are needed to keep cells healthy in laboratory cultures. They developed a computational approach called EpiMogrify, that can predict the molecules needed to signal stem cells to change into specific tissue cells, which can help accelerate treatments that require growing patient cells in the lab.

“Computational biology is rapidly becoming a key enabler in cell therapy, providing a way to short-circuit otherwise expensive and time-consuming discovery approaches with cleverly designed algorithms,” said Assistant Professor Owen Rackham, a computational biologist at Duke-NUS, and a senior and corresponding author of the study, published today in the journal Cell Systems.

In the laboratory, cells are often grown and maintained in cell cultures, formed of a substance, called a medium, which contains nutrients and other molecules. It has been an ongoing challenge to identify the necessary molecules to maintain high-quality cells in culture, as well as finding molecules that can induce stem cells to convert to other cell types.

The research team developed a computer model called EpiMogrify that successfully identified molecules to add to cell culture media to maintain healthy nerve cells, called astrocytes, and heart cells, called cardiomyocytes. They also used their model to successfully predict molecules that trigger stem cells to turn into astrocytes and cardiomyocytes.

“Research at Duke-NUS is paving the road for cell therapies and regenerative medicine to enter the clinic in Singapore and worldwide; this study leverages our expertise in computational and systems biology to facilitate the good manufacturing practice (GMP) production of high-quality cells for these much needed therapeutic applications,” said Associate Professor Enrico Petretto, who leads the Systems Genetics group at Duke-NUS, and is a senior and corresponding author of the study.

The researchers added existing information into their model about genes tagged with epigenetic markers whose presence indicates that a gene is important for cell identity. The model then determines which of these genes actually code for proteins necessary for a cell’s identity. Additionally, the model incorporates data about proteins that bind to cell receptors to influence their activities. Together, this information is used by the computer model to predict specific proteins that will influence different cells’ identities.

“This approach facilitates the identification of the optimum cell culture conditions for converting cells and also for growing the high-quality cells required for cell therapy applications,” said ARC Future Fellow Professor Jose Polo, from Monash University’s Biomedicine Discovery Institute and the Australian Research Medicine Institute, who is also a senior and corresponding author of the study.

©Uma kamaraj et al.

The team compared cultures using protein molecules predicted by EpiMogrify to a type of commonly used cell culture that uses a large amount of unknown or undefined complex molecules and chemicals. They found the EpiMogrify-predicted cultures worked as well or even surpassed their effectiveness.

The researchers have filed for a patent on their computational approach and the cell culture factors it predicted for maintaining and controlling cell fate. EpiMogrify’s predicted molecules are available for other researchers to explore on a public database: http://epimogrify.ddnetbio.com.

“We aim to continue to develop tools and technologies that can enable cell therapies and bring them to the clinic as efficiently and safely as possible,” said Asst Prof Rack

“The developed technology can identify cell culture conditions required to manipulate cell fate and this facilitates growing important cells in chemically-defined cultures for cell therapy applications,” added Dr Uma S. Kamaraj, lead author of the study and a graduate of Duke-NUS’ Integrated Biology and Medicine PhD Programme.

References: Uma S. Kamaraj, Joseph Chen, Khairunnisa Katwadi, Jose M. Polo, Enrico Petretto, Owen J.L. Rackham, “EpiMogrify Models H3K4me3 Data to Identify Signaling Molecules that Improve Cell Fate Control and Maintenance”, Cell Biology, 2020. DOI: https://dx.doi.org/10.1016/j.cels.2020.09.004

Provided by Duke-NUS Medical School

This Unique Tool Predatory Bacteria Use To Help Escape The Cell They Have Invaded (Biology)

Predatory bacteria, capable of invading and consuming harmful bugs such as E .coli and Salmonella, use a unique tool to help them escape the cell they have invaded without harming themselves, according to a new study.

Salmonella bacteria

Researchers at the Universities of Birmingham and Nottingham have identified a particular enzyme used by the bacteria to rupture the cell wall of its prey bacteria and exit without damaging its own cell wall. Their findings are published in Nature Communications.

The bacterium, called Bdellovibrio bacteriovorus, is important because the types of cells they attack – Gram negative bacteria – are responsible for many infections that are resistant to currently available antibiotics. This means predatory bacteria could have the potential to be harnessed as a therapy against these infections.

Discovering precisely how Bdellovibrio bacteriovorus succeeds in invading, and then escaping its prey cells is an important step in this process.

The enzyme they discovered seems like a well-known enzyme called a lysozyme- one of the earliest- ever studied enzymes and found in human tears and saliva; but this one has a twist where it has changed to do something surprising.

“Bdellovibrio bacteriovorus is known for its ability to invade prey bacteria and stay inside the cell for a few hours, effectively eating the bacteria alive,” explains Dr Andrew Lovering, of the University of Birmingham’s School of Biosciences.

“At the end of this process, the predator is able to break the prey open and escape. Because the walls of both the predator and prey cells are made of very similar molecules, we wanted to find out how the predator was able to cut the cell wall material of the prey cell and get out without damaging itself in the process.”

The team already knew part of the answer lay in an early action by the predator bacteria to remove a particular molecule, from the cell wall of the prey. This created a ‘marker’ identifying the prey wall material as different to the predator. This suggested subsequent action by a particular type of enzyme known as a lysozyme might be at work. Lysozymes are a family of enzymes which are known to play a role in the breakdown of cell walls of certain bacteria. This particular lysozyme had evolved and been changed in the predatory bacterium to take on the task of rupturing the uniquely modified cell wall to enable the escape to take place.

Identifying precisely which lysozyme had been diversified for this role was the result of painstaking work by PhD students Hannah Somers and Chris Harding, working with Swiss National Science Foundation Fellow Dr Simona Huwiler.

Professor Liz Sockett, from the University of Notttingham’s School of Life Sciences and co-lead author of the paper, said: ”Checking the timing of when the Bdellovibrio used each of its lysozymes in predation, and hours at the microscope seeing what happened to prey-escape when each lysozyme was missing, gave us strong hunch which might be the important one for escape.”

“When we looked closely at the lysozyme it was clear we were on the right path,” says Dr Lovering. “It looked like a conventional lysozyme but with a warped active site, which meant it was unable to recognise the wall material unless it had been modified and marked by the Bdellovibrio bacteria.”

The next step was to confirm that the lysozyme was only active against the modified cell wall and the team worked with Dr Patrick Moynihan, also in the School of Biosciences at the University of Birmingham, on tests to verify this. This showed it to be a novel lysozyme with a different target to all those lysozymes previously studied in science.

Dr Simona Huwiler, in Professor Sockett’s lab then carried out a series of experiments with the predator bacteria showing clearly how adding this novel lysozyme during the predation process led to the predator falling out from the prey cell early before finishing its meal. So the novel lysozyme is the key to exit.

“Understanding the mechanism and actions of this novel lysozyme may help us use it directly against pathogens which modify their own cell walls to resist the lysozymes in saliva and tears. It is also an important step towards being able to use predatory bacteria themselves in new therapies against problematic bacteria,” adds Professor Sockett.

Researchers Discovered Potential Cause Of Immunotherapy-Related Neurotoxicity (Neuroscience)

New research has uncovered the previously unknown presence of CD19—a B cell molecule targeted by chimeric antigen receptor (CAR) T cell immunotherapy to treat leukemia, lymphoma, and multiple myeloma—in brain cells that protect the blood brain barrier (BBB).

This discovery may potentially be the cause for neurotoxicity in patients undergoing CD19 directed CAR T cell immunotherapy, according to the research team led by Avery Posey, Ph.D., an assistant professor of Systems Pharmacology and Translational Therapeutics in the Perelman School of Medicine at the University of Pennsylvania and Research Health Science Specialist at the Corporal Michael J. Crescenz VA Medical Center in Philadelphia, PA. The study was published today in Cell.

“Our work has revealed that there is CD19 expression in a subset of cells that are, one, not B cells, and two, potentially related to the neurotoxicity we observe in patients treated with CAR T cell therapy targeting CD19,” Posey said. “The next question is, can we identify a better target for eliminating B cell related malignancies other than CD19, or can we engineer around this brain cell expression of CD19 and build a CAR T cell that makes decisions based on the type of cell it encounters—for instance, CAR T cells that kill the B cells they encounter, but spare the CD19 positive brain cells?”

As so often happens in scientific endeavors, the path to this discovery was made somewhat by chance. Kevin Parker, a Ph.D. student at Stanford and co-author on the paper, was at home analyzing previously published single cell sequencing data sets in his spare time. He found CD19 expression in a data set of fetal brain samples that looked odd, because the accepted wisdom was that CD19 only existed in B cells. So his lab reached out to the pioneers of CAR T cell immunotherapy, Penn Medicine.

“I suggested we test this as a preclinical model. When we treated the mouse model with CAR T cells targeting the mouse version of CD19, we found what looks like the start of neurotoxicities,” Posey said.

The team observed an increase in BBB permeability when mouse CD19 was targeted by CAR T cells, even in mice that lack B cells, but not when human CD19 was targeted as a control treatment (mice do not express human CD19).

“Even more interesting, this BBB permeability was more severe when the CAR T cells were fueled by a costimulatory protein called CD28 than when the CAR T cells used 4-1BB,” Posey said “This difference in the severity of BBB permeability correlates with what we know about the clinical observations of CAR T cell-related neurotoxicities—the frequency of patients experiencing high-grade neurotoxicity is lower for those that received the 4-1BB-based CAR T cells.”

His team sought to investigate the higher incidence of neurotoxicity in CD19-directed immunotherapies, compared to treatments targeting other B cell proteins, such as CD20. Notably, CD19 CAR T cells are sensitive to even low levels of CD19 antigen density, emphasizing the importance of identifying any potential reservoir of CD19 other than B cells.

The researchers’ discovery of CD19 molecules in the brain provides evidence that this increase in neurotoxicity is due to CD19-directed CAR T cell immunotherapies. Posey said, though, that generally this neurotoxicity is temporary and patients recover.

This research also highlights the potential utility of developing a comprehensive human single-cell atlas for clinical medicine. Sequencing is an unbiased, genome-wide measurement of gene expression that can capture even rare populations of cells. These rare cell types might otherwise be missed in measurements of bulk tissue due to their low frequency, but as this study demonstrates could be critically important in understanding the clinical effects of targeted therapy. While current CAR T cells recognize only a single antigen, future generations of CAR T cells may be able to discriminate between unique combinations of target antigens to improve thei

r cell-type specificity. The researchers envision that a comprehensive database of gene expression across all human cell types will enable the precise identification of cell type-specific target antigens which can be used to design safe and effective cellular immunotherapies.

“That’s what we think one of the biggest take-home messages is,” Posey said. “The incredible usefulness of single cell atlas or single cell sequencing technology to determine whether a potential immunotherapy or drug target is going to be present somewhere in the body that we would not normally expect it based on conventional thought and whether this expression may lead to toxicity.”

CD19 is thought to be a lineage-restricted molecule—behaving in a functionally and structurally limited way. But this study shows that some small percentage of brain cells also express CD19.

“We would not have identified that through bulk sequencing, where we’re looking at a population of cells versus a single cell type,” Posey said. “It’s only through single cell sequencing that we’re able to identify that there’s this very small percentage of cells in the brain that also contain this molecule, contrary to popular thought.”

References: Kevin R. Parker et al. Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies. Cell. Published: September 21, 2020. DOI:doi.org/10.1016/j.cell.2020.08.022 link: https://www.cell.com/cell/fulltext/S0092-8674(20)31013-8?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867420310138%3Fshowall%3Dtrue

This Is How You Can Remove Unwanted Components From The Cell Nucleus (Biology)

Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores. During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix. When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores. These pores have a size limit of 39 nanometres, which raises the question of how larger cytoplasmic molecules are cleared from the nucleus.

This fluorescence image shows a dividing cell with segregated chromosomes (magenta) that are tightly clustered at the cell poles by the protein Ki-67 (green). Credit: Sara Cuylen-Häring/EMBL

Now, the research team from IMBA and the European Molecular Biology Laboratory in Heidelberg has now shown that large components such as ribosomes are in fact removed from the forming nucleus before the nuclear envelope is assembled again. This exclusion process requires the protein Ki-67, which was the focus of an earlier publication in Nature by Sara Cuylen-Haring, group leader at EMBL Heidelberg and the other joint first author of this study.

In this older study it was discovered that Ki-67 was responsible for keeping chromosomes separated in early stages of mitosis by acting as a surfactant. Remarkably, they have now found that it changes its properties at the end of mitosis and performs the opposite function, namely clustering of chromosomes. By coming together into a dense cluster at the end of cell division, chromosomes are able to exclude large cytoplasmic components before the nuclear envelope reforms.

They showed that the exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, their study revealed that chromosome mechanics help to re-establish the compartmentalization of eukaryotic cells after open mitosis.


References: Cuylen-Haering, S., Petrovic, M., Hernandez-Armendariz, A. et al. Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly. Nature (2020). https://doi.org/10.1038/s41586-020-2672-3 link: https://www.nature.com/articles/s41586-020-2672-3