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Discussing the Long Arc of Discovery with NIH’s Newest Nobelist

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Discussion with Dr. Harvey Alter

It’s been a tough year for our whole world because of everything that’s happening as a result of the coronavirus disease 2019 (COVID-19) pandemic. Yet there are bright spots that still shine through, and this week brought some fantastic news about NIH-supported researchers being named 2020 Nobel Prize Laureates for their pioneering work in two important fields: Chemistry and Physiology or Medicine.

In the wee hours of Wednesday morning, NIH grantee Jennifer A. Doudna, a biochemist at the University of California, Berkeley, got word that she and Emmanuelle Charpentier, a microbiologist at the Max Planck Institute for Infection Biology, Berlin, Germany, had won the 2020 Nobel Prize in Chemistry for developing the CRISPR/cas approach to genome editing. Doudna has received continuous NIH funding since 1997, mainly from the National Institute of General Medical Sciences and National Human Genome Research Institute.

The CRISPR/cas system, which consists of a short segment of RNA attached to the cas enzyme, provides the ability to make very precise changes in the sequence, or spelling, of the genetic instruction books of humans and other species. If used to make non-heritable edits in relevant tissues, such technology holds enormous potential to treat or even cure a wide range of devastating diseases, including thousands of genetic disorders where the DNA misspelling is precisely known.

Just two days before Doudna learned of her big award, a scientist who’s spent almost his entire career at the NIH campus in Bethesda, MD, received news that he too was getting a Nobel—the 2020 Nobel Prize in Physiology or Medicine. Harvey Alter, a senior scholar in the NIH Clinical Center’s Transfusion Medicine Department, was recognized for his contributions in identifying the potentially deadly hepatitis C virus. He shares this year’s prize with Michael Houghton, now with University of Alberta, Edmonton, and Charles M. Rice, The Rockefeller University, New York, who’s received continuous NIH funding since 1987, mainly from the National Institute of Allergy and Infectious Diseases.

In a long arc of discovery rooted in basic, translational, and clinical research that spanned several decades, Alter and his colleagues doggedly pursued biological clues that at first led to tests, then life-saving treatments, and, today, the very real hope of eradicating the global health threat posed by hepatitis C infections.

We at NIH are particularly proud of the fact that Alter is the sixth Nobel Prize winner—and the first in 26 years—to have done the entirety of his award-winning research in our Intramural Research Program. So, I jumped at the opportunity to talk with Harvey on NIH’s Facebook Live and Twitter chats just hours after he got the good news on Monday. Here’s a condensed version of our conversation, which took place on the NIH campus, but at a safe physical distance to minimize the risk of COVID-19 spread.

Collins: Harvey, let me start off by asking, how did you find out you’d won the Nobel Prize?

Alter: At 4:15 this morning. I was asleep and heard the telephone ringing. I ignored it. Five minutes later, I got another call. Now, I’m getting kind of perturbed. But I ignored it, thinking the call must be some kind of solicitation. Then, the phone rang a third time. I answered it, prepared to tell the person on the other end not to call me anymore. I heard a man’s voice say, “I’m the Secretary General of the Nobel Prize, calling you from Stockholm.” At that point, I just froze.

Collins: Did you think it might be a hoax?

Alter: No, I didn’t think it was a hoax. But I wasn’t expecting to win the prize. I knew about three years ago that I’d been on a Nobel list. But it didn’t happen, and I just forgot about it. Truthfully, I didn’t know that today was the day that the announcement was being made. The news came as a complete shock.

Collins: Please say a few words about viral hepatitis. What is it?

Alter: Sure. Viral hepatitis is an infection of the liver that causes inflammation and can lead to scarring, or cirrhosis. Early in my career, two viruses were known to cause the disease. One was the hepatitis A virus. You got it from consuming contaminated water or food. The second was the hepatitis B virus, which has a blood-borne transmission, typically from blood transfusions. In the 1970s, we realized that some other agent was causing most of the hepatitis from blood transfusions. Since it wasn’t A and it wasn’t B, we cleverly decided to call it: non-A, non-B. We did that because we hadn’t yet proven that the causative agent was a virus.

Collins: So, even though you screened donor units for the hepatitis B virus to eliminate tainted blood, people were still getting hepatitis from blood transfusions. How did you go about trying to solve this mystery?

Alter: The main thing was to follow patients prospectively, meaning forward in time. We drew a blood sample before they were transfused, and then serially afterwards. We saved those samples and also the donor samples to compare them. Using a liver function test, we found that 30 percent of patients who had open heart surgery at NIH prior to 1970 developed liver abnormalities indicative of hepatitis. That’s 1 in 3 people.

We then looked for the reasons. We found the main one was our source of blood. We were buying blood, which was then in short supply, from commercial laboratories. It turned out that their paid donors were engaging in high-risk behaviors [Note: like IV drug users sharing hypodermic needles]. We immediately stopped using these laboratories, and, through various other measures, we got the rate down to around 4 percent in 1987.

That’s when Michael Houghton, then at Chiron Corp. and a co-recipient of this year’s prize, cloned the virus. Think about it, he and his colleagues looked at 6 million clones and found just one that reacted with the convalescent serum of a patient with non-A, non-B. In other words, having contracted the virus, the patient already made antibodies against it that were present in the serum. If that one clone came from the virus, the antibodies in the serum would recognize it. They did, and Chiron then developed an assay to detect antibodies to the virus.

Collins: And that’s when they contacted you.

Alter: Yes, they wanted to use our panel of patient blood samples that had fooled a lot of people who claimed to have developed a non-A, non-B assay. Nobody else had “broken” this panel, but the Chiron Corp. did. We found that every case of non-A, non-B was really hepatitis C, the agent that they had cloned. Hepatitis C was the missing piece. As far as we could tell, there were no other agents beside hepatitis B and C that would result in transfusion transmission of the disease.

Collins: This story is clearly one of persistence. So, say something about persistence as an important characteristic of a scientist. You’re a great example of someone who was always looking out for opportunities that might not have seemed so promising at first.

Alter: I first learned persistence from Dr. Baruch Blumberg, my first NIH mentor who discovered the hepatitis B virus in 1967. [Note: Other NIH researchers identified the hepatitis A virus in 1977] The discovery started when we found this “Australian antigen,” a molecular structure that the immune system recognizes as foreign and attacks. It was a serendipitous finding that could have been easily just dropped. But he just kept at it, kept at it, kept at it. He had this famous wall where he diagrammed his hypotheses with all the contingencies if one worked or failed. Then, all of a sudden, the antigen was associated with hepatitis B. It became the basis of the hepatitis B vaccine, which is highly effective and used throughout the world. Dr. Blumberg won the Nobel Prize for his work on the hepatitis B virus in 1976.

Collins: Sometimes people look at NIH and ask why we don’t focus all of our efforts on curing a particular disease. I keep answering, ‘Wait a moment, we don’t know enough to know how to do that.’ What’s the balance that we ought to be seeking between basic research and clinical applications?

Alter: There is this tendency now to pursue highly directed research to solve a problem. That’s certainly how biopharma works. They want a payoff. The NIH is different. It’s a place where you can pursue your scientific interests, wherever they lead. The NIH leadership understands that the details of a problem often aren’t obvious at first. Researchers need to be allowed to observe things and then to pursue their leads as far as possible, with the understanding that not everything will work out. I think it’s very important to keep this basic research component in parallel with the more clinical applications. In the case of hepatitis C, it started as a clinical problem that led to a basic research investigation, which led back to a clinical problem. It was bedside-to-bench-to-bedside.

Collins: Are people still getting infected with hepatitis C?

Alter: Yes, hepatitis C remains a global problem. Seventy million people have contracted the virus, though the majority are generally asymptomatic, meaning they don’t get sick from it. Instead, they carry around the virus for decades without knowing it. That’s because the hepatitis C virus likes to persist, and our immune system doesn’t seem to be able to get rid of it easily.

However, some of those infected will have bad outcomes, such as cirrhosis or cancer of the liver. But there’s no way of knowing who will and who won’t get sick over time. The trick now is to identify people when they’re asymptomatic and without obvious disease.

That involves testing. We’re in a unique position with hepatitis C, where we have great tests that are highly sensitive and very specific to the virus. We also have great treatments. We can cure everybody who is tested and found to be positive.

Collins: People may be surprised to hear that. Here is a chronic viral illness, for which we actually have a cure. That’s come along fairly recently. Say a bit more about that—it’s such a great story of success.

Alter: For many years, the only treatment for hepatitis C was interferon, a very difficult treatment that initially had only about a 6 percent cure rate. With further progress, it got up to around 50 percent. But the big breakthrough came in the late 1990s when Gilead Corp., having the sequenced genome of the hepatitis C virus, deduced what it needs to replicate. If we know what it needs and we interfere with that, we can stop the replication. Gilead came out with a blockbuster drug that, now in combination with another drug, aims at two different sites on the virus and cures at least 98 percent of people. It’s an oral therapy taken for only 12 weeks, sometimes as little as 8 weeks, and with virtually no side-effects. It’s like a miracle drug.

Collins: What would you say to somebody who is thinking about becoming a scientist? How do you pick an area of research that will be right for you?

Alter: It’s a tough question. Medical research is very difficult, but there’s nothing more rewarding than doing something for patients and to see a good outcome like we had with hepatitis C.

The best path forward is to work for somebody who’s already an established investigator and a good teacher. Work in his or her lab for a few years and get involved in a project. I’ve learned not get into a lot of projects. Get into something where you can become the expert and pursue it.
The other thing is to collaborate. There’s no way that one person can do everything these days. You need too much technology and lots of different areas of expertise.

Collins: You took on a high-risk project in which you didn’t know that you’d find the answer. What’s the right balance between a project that you know will be productive, and something that might be risky, but, boy, if it works, could be transformative? How did you decide which of those paths to go?

Alter: I don’t think I decided. I just went! But there were interim rewards. Finding that the paid donors were bad was a reward and it had a big impact. And the different donor testing, decreasing the amount of blood [transfused], there were all kinds of steps along the way that gave you a reward. Now, did I think that there would be a treatment, an eradication of post-transfusion hepatitis at the end of my line? No, I didn’t.

And it wouldn’t have happened if it was only me. I just got the ball rolling. But it needed Houghton’s group. It needed the technology of Charlie Rice, a co-recipient of this year’s Nobel Prize. It needed joint company involvement. So, it required massive cooperation, and I have to say that here at NIH, Bob Purcell did most of the really basic work in his lab. Patrizia Farci, my closest collaborator, does things that I can’t do. You just need people who have a different expertise.

Collins: Harvey, it’s been maybe six hours since you found out that you won the Nobel Prize. How are you going to spend the rest of your day?

Alter: Well, I have to tell you a story that just happened. We had a press conference earlier today at NIH. Afterwards, I wanted to return to my NIH office and the easiest route was through the parking garage across the street from where we held the press conference. When I entered the garage, a security guard said, “You can’t come in, you haven’t been screened for COVID.” I assured him that I had been screened when I drove onto the NIH campus. He repeated that I had to go around to the front of the building to get screened.

Finally, I said to him, “Would it make any difference if I told you that I won the Nobel Prize today?” He replied, ‘That’s nice, but you must go around to the front of the building.’” So, winning the Nobel doesn’t give you immediate rewards!

Collins: Let me find that security guard and give him a bonus for doing a good job. Well, Harvey, will there be that trip to Stockholm coming up in December?

Alter: Not this year. I’ve heard that they will invite us to Stockholm next year to receive the award. But there’s going to be something in the US. I don’t know what it will be. I’ll invite you.

Collins: I will be glad to take part in the celebration. Well, Harvey, I really want to thank you for taking some time on this special day to reflect on your career and how the Nobel Committee came calling at 4:30 this morning. We’re really proud of you!

Alter: Thank you.

Links:

Hepatitis C (National Institute of Diabetes and Digestive and Kidney Diseases/NIH)

The Nobel Assembly at Karolinska Institutet has today decided to award the 2020 Nobel Prize in Physiology or Medicine jointly to Harvey J. Alter, Michael Houghton and Charles M. Rice for the discovery of Hepatitis C virus,” Nobel Prize announcement, October 5,2020.

Harvey Alter (Clinical Center/NIH)

The Road Not Taken, or How I Learned to Love the Liver: A Personal Perspective on Hepatitis History” Alter HJ, Hepatology. 2014 Jan;59(1):4-12.

Reflections on the History of HCV: A Posthumous Examination.” Alter HJ, Farci P, Bukh J, Purcell RH. Clinical Liver Disease, 15:1, Feb 2020.

Is Elimination of Hepatitis B and C a Pipe Dream or Reality?” Alter HJ, Chisari FV. Gastroenterology. 2019 Jan;156(2):294-296.

Michael Houghton (University of Alberta, Edmonton)

Charles Rice (The Rockefeller University, New York)

What is genome editing? (National Human Genome Research Institute/NIH)

Jennifer Doudna (University of California, Berkeley)

Emmanuelle Charpentier (Max Planck Institute for Infection Biology, Berlin, Germany)


Replenishing the Liver’s Immune Protections

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Kupffer cells
Credit: Thomas Deerinck, National Center for Microscopy and Imaging Research, University of California, San Diego.

Most of our immune cells circulate throughout the bloodstream to serve as a roving security force against infection. But some immune cells don’t travel much at all and instead safeguard a specific organ or tissue. That’s what you are seeing in this electron micrograph of a type of scavenging macrophage, called a Kupffer cell (green), which resides exclusively in the liver (brown).

Normally, Kupffer cells appear in the liver during the early stages of mammalian development and stay put throughout life to protect liver cells, clean up old red blood cells, and regulate iron levels. But in their experimental system, Christopher Glass and his colleagues from University of California, San Diego, removed all original Kupffer cells from a young mouse to see if this would allow signals from the liver that encourage the development of new Kupffer cells.

The NIH-funded researchers succeeded in setting up the right conditions to spur a heavy influx of circulating precursor immune cells, called monocytes, into the liver, and then prompted those monocytes to turn into the replacement Kupffer cells. In a recent study in the journal Immunity, the team details the specific genomic changes required for the monocytes to differentiate into Kupffer cells [1]. This information will help advance the study of Kupffer cells and their role in many liver diseases, including nonalcoholic steatohepatitis (NASH), which affects an estimated 3 to 12 percent of U.S. adults [2].

The new work also has broad implications for immunology research because it provides additional evidence that circulating monocytes contain genomic instructions that, when activated in the right way by nearby cells or other factors, can prompt the monocytes to develop into various, specialized types of scavenging macrophages. For example, in the mouse system, Glass’s team found that the endothelial cells lining the liver’s blood vessels, which is where Kupffer cells hang out, emit biochemical distress signals when their immune neighbors disappear.

While more details need to be worked out, this study is another excellent example of how basic research, including the ability to query single cells about their gene expression programs, is generating fundamental knowledge about the nature and behavior of living systems. Such knowledge is opening new possibilities to more precise ways of treating and preventing diseases all throughout the body, including those involving Kupffer cells and the liver.

References:

[1] Liver-Derived Signals Sequentially Reprogram Myeloid Enhancers to Initiate and Maintain Kupffer Cell Identity. Sakai M, Troutman TD, Seidman JS, Ouyang Z, Spann NJ, Abe Y, Ego KM, Bruni CM, Deng Z, Schlachetzki JCM, Nott A, Bennett H, Chang J, Vu BT, Pasillas MP, Link VM, Texari L, Heinz S, Thompson BM, McDonald JG, Geissmann F3, Glass CK. Immunity. 2019 Oct 15;51(4):655-670.

[2] Recommendations for diagnosis, referral for liver biopsy, and treatment of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Spengler EK, Loomba R. Mayo Clinic Proceedings. 2015;90(9):1233–1246.

Links:

Liver Disease (National Institute of Diabetes and Digestive and Kidney Diseases/NIH)

Nonalcoholic Fatty Liver Disease & NASH (NIDDK)

Glass Laboratory (University of California, San Diego)

NIH Support: National Institute of Diabetes and Digestive and Kidney Diseases; National Heart, Lung, and Blood Institute; National Institute of General Medical Sciences; National Cancer Institute


Can Organoids Yield Answers to Fatty Liver Disease?

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Liver Organoid
Confocal microscope image shows liver organoid made from iPS cells derived from children with Wolman disease. The hepatocyte cells (red) accumulate fat (blue). Credit: Cincinnati Children’s Hospital Medical Center

With advances in induced pluripotent stem cell (iPSC) technology, it’s now possible to reprogram adult skin or blood cells to form miniature human organs in a lab dish. While these “organoids” closely mimic the structures of the liver and other vital organs, it’s been tough to get them to represent inflammation, fibrosis, fat accumulation, and many other complex features of disease.

Fatty liver diseases are an increasingly serious health problem. So, I’m pleased to report that, for the first time, researchers have found a reliable way to make organoids that display the hallmarks of those conditions. This “liver in a dish” model will enable the identification and preclinical testing of promising drug targets, helping to accelerate discovery and development of effective new treatments.

Previous methods working with stem cells have yielded liver organoids consisting primarily of epithelial cells, or hepatocytes, which comprise most of the organ. Missing were other key cell types involved in the inflammatory response to fatty liver diseases.

To create a better organoid, the team led by Takanori Takebe, Cincinnati Children’s Hospital Medical Center, focused its effort on patient-derived iPSCs. Takebe and his colleagues devised a special biochemical “recipe” that allowed them to grow liver organoids with sufficient cellular complexity.

As published in Cell Metabolism, the recipe involves a three-step process to coax human iPSCs into forming multi-cellular liver organoids in as little as three weeks. With careful analysis, including of RNA sequencing data, they confirmed that those organoids contained hepatocytes and other supportive cell types. The latter included Kupffer cells, which play a role in inflammation, and stellate cells, the major cell type involved in fibrosis. Fibrosis is the scarring of the liver in response to tissue damage.

Now with a way to make multi-cellular liver organoids, the researchers put them to the test. When exposed to free fatty acids, the organoids gradually accumulated fat in a dose-dependent manner and grew inflamed, which is similar to what happens to people with fatty liver diseases.

The organoids also showed telltale biochemical signatures of fibrosis. Using a sophisticated imaging method called atomic force microscopy (AFM), the researchers found as the fibrosis worsened, they could measure a corresponding increase in an organoid’s stiffness.

Next, as highlighted in the confocal microscope image above, Takebe’s team produced organoids from iPSCs derived from children with a deadly inherited form of fatty liver disease known as Wolman disease. Babies born with this condition lack an enzyme called lysosomal acid lipase (LAL) that breaks down fats, causing them to accumulate dangerously in the liver. Similarly, the miniature liver shown here is loaded with accumulated fat lipids (blue).

That brought researchers to the next big test. Previous studies had shown that LAL deficiency in kids with Wolman disease overactivates another signaling pathway, which could be suppressed by targeting a receptor known as FXR. So, in the new study, the team applied an FXR-targeted compound called FGF19, and it prevented fat accumulation in the liver organoids derived from people with Wolman disease. The organoids treated with FGF19 not only were protected from accumulating fat, but they also survived longer and had reduced stiffening, indicating a reduction in fibrosis.

These findings suggest that FGF19 or perhaps another compound that acts similarly might hold promise for infants with Wolman disease, who often die at a very early age. That’s encouraging news because the only treatment currently available is a costly enzyme replacement therapy. The findings also demonstrate a promising approach to accelerating the search for new treatments for a variety of liver diseases.

Takebe’s team is now investigating this approach for non-alcoholic steatohepatitis (NASH), a common cause of liver failure and the need for a liver transplant. The hope is that studies in organoids will lead to promising new treatments for this liver condition, which affects millions of people around the world.

Ultimately, Takebe suggests it might prove useful to grow liver organoids from individual patients with fatty liver diseases, in order to identify the underlying biological causes and test the response of those patient-specific organoids to available treatments. Such evidence could one day help doctors to select the best available treatment option for each individual patient, and bring greater precision to treating liver disease.

Reference:

[1] Modeling steatohepatitis in humans with pluripotent stem cell-derived organoids. Ouchi R, Togo S, Kimura M, Shinozawa T, Koido M, Koike H, Thompson W, Karns RA, Mayhew CN, McGrath PS, McCauley HA, Zhang RR, Lewis K, Hakozaki S, Ferguson A, Saiki N, Yoneyama Y, Takeuchi I, Mabuchi Y, Akazawa C, Yoshikawa HY, Wells JM, Takebe T. Cell Metab. 2019 May 14. pii: S1550-4131(19)30247-5.

Links:

Wolman Disease (Genetic and Rare Diseases Information Center/NIH)

Nonalcoholic Fatty Liver Disease & NASH (National Institute of Diabetes and Digestive and Kidney Diseases/NIH)

Stem Cell Information (NIH)

Tissue Chip for Drug Screening (National Center for Advancing Translational Sciences/NIH)

Takebe Lab (Cincinnati Children’s Hospital Medical Center)

NIH Support: National Institute of Diabetes and Digestive and Kidney Diseases


Cardiometabolic Disease: Big Data Tackles a Big Health Problem

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Cardiometabolic risk lociMore and more studies are popping up that demonstrate the power of Big Data analyses to get at the underlying molecular pathology of some of our most common diseases. A great example, which may have flown a bit under the radar during the summer holidays, involves cardiometabolic disease. It’s an umbrella term for common vascular and metabolic conditions, including hypertension, impaired glucose and lipid metabolism, excess belly fat, and inflammation. All of these components of cardiometabolic disease can increase a person’s risk for a heart attack or stroke.

In the study, an international research team tapped into the power of genomic data to develop clearer pictures of the complex biocircuitry in seven types of vascular and metabolic tissue known to be affected by cardiometabolic disease: the liver, the heart’s aortic root, visceral abdominal fat, subcutaneous fat, internal mammary artery, skeletal muscle, and blood. The researchers found that while some circuits might regulate the level of gene expression in just one tissue, that’s often not the case. In fact, the researchers’ computational models show that such genetic circuitry can be organized into super networks that work together to influence how multiple tissues carry out fundamental life processes, such as metabolizing glucose or regulating lipid levels. When these networks are perturbed, perhaps by things like inherited variants that affect gene expression, or environmental influences such as a high-carb diet, sedentary lifestyle, the aging process, or infectious disease, the researchers’ modeling work suggests that multiple tissues can be affected, resulting in chronic, systemic disorders including cardiometabolic disease.


Snapshots of Life: The Hard Working Hepatocyte

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Human hepatocyte

Caption: Magnified image of a hepatocyte: nuclei in blue; actin fibers in red, yellow, orange, and green.
Credit: Donna Beer Stolz, University of Pittsburgh

The humble hepatocyte handles a lot of the body’s maintenance and clean up work. It detoxifies the blood, metabolizing medications and alcohol. It secretes important proteins that regulate carbohydrates and fats—including both the good and bad kinds of cholesterol. It’s also the most common cell in one of the few human organs that regenerate: the liver. When this organ is damaged, hepatocytes begin dividing to repair the tissue.

Its regenerative ability is just one reason that Donna Beer Stolz, a microscopist and cell biologist at the University of Pittsburgh, in Pennsylvania, has been studying the hepatocyte for more than 20 years. She captured this image while conducting one of her experiments. As she was carefully scanning a dish of cells, one particular hepatocyte caught her eye. It was perfectly round. Struck by its symmetry and beauty, Stolz snapped pictures of the cell at different layers and then used software to reconstruct and color the image.


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