Circadian rhythms control body’s response to intestinal infections

Circadian rhythms can boost the body’s ability to fight intestinal bacterial infections, UC Irvine researchers have found.

This suggests that targeted treatments may be particularly effective for pathogens such as salmonella that prompt a strong immune system response governed by circadian genes. It also helps explain why disruptions in the regular day-night pattern – as experienced by, say, night-shift workers or frequent fliers – may raise susceptibility to infectious diseases.

UC Irvine’s Paolo Sassone-Corsi, one of the world’s leading researchers on circadian rhythm genetics, and microbiologist Manuela Raffatellu led the study, which appears this week in the early online edition of Proceedings of the National Academy of Sciences. Marina Bellet, a postdoctoral researcher from Italy’s University of Perugia also played a key role in the experiments.

“Although many immune responses are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections has not been understood,” said Sassone-Corsi, the Donald Bren Professor of Biological Chemistry. “What we’re learning is that the intrinsic power of the body clock can help fight infections.”

Circadian rhythms of 24 hours govern fundamental physiological functions in almost all organisms. The circadian clock is an intrinsic time-tracking system in the human body that anticipates environmental changes and adapts to the appropriate time of day. Disruption of these normal rhythms can profoundly influence people’s health.

Up to 15 percent of human genes are regulated by the day-night pattern of circadian rhythms, including those that respond to intestinal infections.

In tests on mice infected with salmonella, the researchers noted that circadian-controlled genes govern the immune response to the invading pathogen, leading to day-night differences in infection potential and in the immune system’s ability to deal with pathogens.

Mice are nocturnal, with circadian rhythms opposite those of humans. While important differences exist in the immune response of mice and humans, Sassone-Corsi said, these test results could provide clues to how circadian-controlled intestinal genes regulate daily changes in the effectiveness of the human immune system.

“Salmonella is a good pathogen to study what happens during infection,” said Raffatellu, assistant professor of microbiology & molecular genetics. “We think these findings may be broadly applicable to other infectious diseases in the gut, and possibly in other organs controlled by circadian patterns.”

Sassone-Corsi added that it’s important to understand the circadian genetics regulating immunity. “This gives us the ability to target treatments that supplement the power of the body clock to boost immune response,” he said.

(Image: Stephen Sedam / Los Angeles Times)

What are stem cells?

In a paper published in Cell yesterday, scientists from the US and Thailand have, for the first time, successfully produced embryonic stem cells from human skin cells.

That sounds interesting, but what are stem cells and where do they come from?

If you take a limb from a rose tree, and put it in soil, it will grow into a thriving bush.

But you might say: “Plants are special. This won’t work with animals.” Or will it? If you cut off a lizard’s tail, a new tail may grow. A lobster can grow back a lost claw.

There is a special type of flatworm that can be cut in half, again and again hundreds of times, and each half grows back into a full worm.

Similarly, if you cut out half a human liver, it will grow back. The story of Prometheus, whose liver was eaten away by eagles and regrew each day, suggests that the Greeks of ancient times knew about regeneration of organs.

This sort of regeneration is attributed to special cells called “stem cells”.

Reprogramming the workers

Most of our cells are like many professional workers – they are hardened in their ways and can’t manage career changes.

Blood cells carry oxygen or fight disease, muscle cells expand and contract to move us around, nerve cells carry signals, skin cells form a protective layer over our bodies, and structures made up of kidney cells filter our blood.

The cells of most organs or tissues are referred to as “terminally differentiated” cells. They have specialised, and many won’t divide again. If they are damaged or die they will disappear. This is very important.

Although we feel like we grow a lot after we are born, we really only double in size two or three times and most of our cells don’t divide much.

If they did we would be at great risk from cancer – the uncontrolled doubling of cells at the wrong time.

We have a lot of cells and it is important that none of them run out of control.

But some cells can double to renew themselves and can also differentiate and give rise to specialised progeny.

These are the stem cells. We need them to produce new skin to replace damaged skin cells. Similarly, we need them in our guts to replace damaged cells on the surface of our intestines.

Our blood cells also get worn out as they race around our bodies so we have blood stem cells that divide and replace themselves. They also differentiate to form the different types of white and red blood cells we need.

Australian researchers identified stem cells in the breast that can proliferate and form a complete functioning breast. There are also stem cells in the brain and in the heart.

While stem cells tend to be very rare, they exist in many of our organs.

Types of stem cells

The ultimate stem cells are embryonic stem cells.

These cells are found in the inner cell mass of the early embryo and are referred to as “totipotent” since they have the ability to form every cell that is needed in the growing embryo.

They can be extracted from the early embryo and grown in culture dishes.

They can also be genetically modified by the addition of DNA, then injected back into other embryos or into adult animals where find their way into localities that suit them and replace themselves by duplication or differentiate into other cell types that may be needed. For a long time this type of work had been done primarily in laboratory mice.

The techniques in yesterday’s Cell paper involved injecting the nucleus from a human skin cell into a human egg (the nucleus of which has been destroyed) then growing the resulting embryo until the inner cell mass cells could be harvested.

The method may still be controversial because it uses unfertilised eggs, but many people will regard it as preferable to using human embryos. And there are other interesting methods for making stem cells.

Somatic cells to stem cells

It is also possible to convert skin cells, and indeed many different terminally differentiated cells, back into what are called “induced pluripotent stem cells” or iPS cells.

One uses the “magic four” or “OKSM” set of DNA-binding proteins that govern normal stem cell biology:

• Octamer-binding transcription factor 4 (OCT4)
• Kruppel-like factor 4 (KLF4)
• SRY (sex determining region Y)-box 2 (SOX2)
• cellular myelocytomatosis virus-like gene (MYC)

In 2012 Shinya Yamanaka won the Nobel Prize for discovering how to convert normal cells into iPS cells using the OKSM regulators to turn on and off the right genes and convert skin cells into stem cells.

Researchers are continuing to investigate whether iPS cells have the same therapeutic potential as embryo derived stem cells.

It is hoped that stem cells may provide therapies for people suffering from degenerative diseases.

Skin cells could be taken from a patient, converted to stem cells, and then these could be injected back into the damaged organ.

Ideally, they would repopulate the damaged organ with new cells.

So why doesn’t this happen in normal biology? Why aren’t our own heart stem cells busy trying to repair broken hearts?

They may be but our natural supply of stem cells is limited and presumably insufficient to tackle severe disease.

So why don’t we just have more stem cells in our bodies?

The down side of having too many stem cells may be cancer.

Stem cells share a number of features with cancer cells – both are able to self-renew and double without limit.

One theory about cancer holds that the disease most often originates not from terminally differentiated cells but from one of the small number of stem cells in the relevant tissues.

The obvious concern about using stem cells for therapy is that injecting too many could increase the chances that some of these cells would proliferate beyond control, and ultimately give rise to cancer.

Stem cell therapy for regenerative medicine is an exciting idea.

Every day we are learning more about stem cells – how to purify or make them, and how to grow them in culture and direct them down particular pathways to repopulate different organs.

Future research will assess the risks and how effective they can be in experimental systems and ultimately in human patients.

Human stem cells successfully cloned for the first time

A working process for cloning stem cells from existing human cells has finally been discovered by a team at Oregon Health & Science University.

These stem cells were created by reprogramming healthy skin cells, a goal that has eluded researchers around the world for years. It’s the first key step in developing medical procedures for replacing dying or injured cells with new ones to stave off disease and age. That could mean growing a new liver, or kidney or heart, in the lab for an organ transplant, or even repairing the brains of those suffering with diseases like Parkinson’s.

The team was led by Shoukhrat Mitalipov from the reproductive and developmental sciences department of the Oregon National Primate Research Centre. He said: “A thorough examination of the stem cells derived through this technique demonstrated their ability to convert just like normal embryonic stem cells into several different cell types, including nerve cells, liver cells and heart cells. Furthermore, because these reprogrammed cells can be generated with nuclear genetic material from a patient, there is no concern of transplant rejection.”

"While there is much work to be done in developing safe and effective stem cell treatments, we believe this is a significant step forward in developing the cells that could be used in regenerative medicine."

The technique Mitalipov and his team used is called “somatic cell nuclear transfer” — as you can see in the video, it essentially involves sucking out the DNA from an adult cell and inserting it into the empty nucleus of a donor egg. This creates a clone of the original cell, and is in fact the first step in the cloning method used to create animal clones like Dolly the sheep.

However, in its therapeutic mode, the new cells can be grown as replacements for the original type of cell. That objective hasn’t been reached until now as human eggs are extremely fragile compared to many of the animals which we have cloned. That Mitalipov and team have succeeded is down to research on primates, and adapting primate stem cell research to humans.

As a cell divides after fertilisation, it undergoes several transformations as it prepares to split and multiply. The metaphase is the moment just before a cell splits, as the chromosomes align alongside each other in the very centre of the cell so that, when it splits, one goes one way as another goes the other, each taking the full copy of the genetic code. The researchers managed to stall the metaphase while the cell underwent nuclear transfer, effectively giving the new chromosomes time to get settled before the metaphase finished and cell division proceeded.

An added bonus is that the eggs used have not been fertilised, so there won’t be any debates over the ethics of embryonic stem cells as we have seen in the US in the past. While the researchers placed skin cell nuclei into the receptor egg cells, the method is conceivably similar for any other kind of cell.

And, while it may sounds like the first step towards a practical method for cloning humans, the Mitalipov has made it clear that’s not the aim. “Our research is directed toward generating stem cells for use in future treatments to combat disease. While nuclear transfer breakthroughs often lead to a public discussion about the ethics of human cloning, this is not our focus, nor do we believe our findings might be used by others to advance the possibility of human reproductive cloning.”

The research has been published in the journal Cell.

Same musicians: brand new tune

A small ensemble of musicians can produce an infinite number of melodies, harmonies and rhythms. So too, do a handful of workhorse signaling pathways that interact to construct multiple structures that comprise the vertebrate body. In fact, crosstalk between two of those pathways—those governed by proteins known as Notch and BMP (for Bone Morphogenetic Protein) receptors—occurs over and over in processes as diverse as forming a tooth, sculpting a heart valve and building a brain.

A new study by Stowers Institute for Medical Research Investigator Ting Xie, Ph.D., reveals yet another duet played by Notch and BMP signals, this time with Notch calling the tune. That work, published in this week’s online issue of PNAS, uses mouse genetics to demonstrate how one Notch family protein, Notch2, shapes an eye structure known as the ciliary body (CB), most likely by ensuring that BMP signals remain loud and clear.

In vertebrates, the CB encircles the lens and performs two tasks essential for normal vision. First, it contains a tiny muscle that reshapes the lens when you change focus, or “accommodate”. And it also secretes liquid aqueous humor into the front compartment of the eye where it likely maintains correct eye pressure. Understanding CB construction is critical, as excessive pressure is one risk factor for glaucoma.

Eye development is a relatively new field for Xie, a recognized leader in the study of adult stem cells in the fruit fly: only recently did he branch out into mouse studies. “A few years ago I was asked to participate in a think tank-type meeting to discuss the potential application of cell therapy to treat glaucoma,” he says.
“I became interested in using retinal progenitor cells to treat diseases like glaucoma or macular degeneration. But I realized that first we needed to understand eye disease at the molecular level.” The new study is an important step in that direction.

Previously, investigators knew that once cells that form the CB are established in an embryo, the BMP pathway drives their “morphogenesis”, the term used by developmental biologists to describe the process of expanding and then sculpting a committed population of cells into a unique structure. “The Notch2 receptor was previously shown to be expressed in the developing mouse eye,” explains Chris Tanzie, M.D., Ph.D., a former graduate student in the Xie lab and the study’s co-first author. “But its function was unknown, and no one connected how various signaling pathways direct CB morphogenesis.”

To determine what Notch2 was doing in the developing eye, the Stowers team constructed a conditional knockout mouse, meaning that the Notch2 gene is deleted from the genome only in eye cells that give rise to the CB. In normal newborn mice a series of cellular “folds” that characterize the CB emerges over the first 7 days of life. But the mutant knockout mice showed a complete absence of folds, dramatic evidence that Notch2 is required to elaborate a CB.

Furthermore, in normal mice a protein called Jagged-1, which activates Notch2, was expressed in cells adjacent to Notch2-expressing CB cells during the same developmental period. Strikingly, the team’s collaborators in Richard Libby’s laboratory at the University of Rochester Medical Center, were able to demonstrate that just like the Notch2 mutants, Jagged-1 conditional knockout mice showed almost total loss of CB fold structures, a major hint that Notch2 was switched on by Jagged1 to drive CB formation.

Biochemical and microarray analysis provided further explanation for defects observed after Notch2 loss. Comparison of normal and Notch2-mutant eye cells revealed that not only did cells of mutant mice lose BMP signaling but that expression of two proteins known to interfere with BMP increased in those cells.

“Up-regulation of BMP antagonists following Notch2 loss is an important observation,” says Xie. “In other systems people often observe that Notch and BMP cooperatively regulate common targets by transcription factor collaboration at the transcriptional level, but this is a unique mechanism. We find that Notch2 keeps BMP signaling active by inhibiting its inhibitors.”

The study’s second co-first author is Yi Zhou, a University of Kansas Medical Center graduate student earning his Ph.D. in Xie’s lab. “Our work reveals a novel link between Notch and BMP pathways potentially involved in the pathogenesis of glaucoma,” says Zhou, noting one more tantalizing implication of the paper. “In addition, mutations in Jagged-1 and Notch2 are thought to underlie the human genetic disease known as Alagille Syndrome. Our work may lead to a better understanding of both.”

Alagille Syndrome is an inherited childhood disorder causing defects in organ systems including liver, heart and the skeleton. Xie is equally intrigued by potential connections between his group’s observations in the mouse eye and Alagille outcomes in humans. Nonetheless, he remains focused on nailing down how perturbation of the Jagged1-Notch2-BMP axis might cause eye disease.

“We now know how to build better mouse mutants to study CB development. In this work we show that Notch regulates BMP signaling but have not yet determined whether alterations in CB structure actually change interocular pressure,” he says. “Answering that question is our future goal.”

Animals in research: zebrafish

Zebrafish are probably not the first creatures that come to mind when it comes to animals that are valuable for medical research.

You might struggle to imagine you have much in common with this small tropical freshwater fish, though you may be inclined to keep a few “zebra danios” in your home aquarium, given they are hardy, undemanding animals that cost only a few dollars each.

Yet each year more and more scientists are turning to zebrafish to unravel the mechanisms underlying their favourite genetic or infectious disease, be it muscular dystrophy, schizophrenia, tuberculosis or cancer.

My (conservative) estimate is that zebrafish research is now carried out in at least 600 labs worldwide, including 20 in Australia.

So what is it about zebrafish that has taken them from the freshwater rivers and streams of Southeast Asia, beyond the pet shops and into universities and research institutes the world over?

A short history of zebrafish

A scientist called George Streisinger, working at the University of Oregon in Eugene, USA in the 1970s and 80s, recognised the vast potential of this organism for developmental biology and genetics research.

In contrast to fruit flies and worms, the other simple model organisms established at the time, zebrafish are vertebrates.

They have a backbone, brain and spinal cord as well as several other organs, including a heart, liver and pancreas, kidneys, bones and cartilage, which makes them much more similar to humans than you may have otherwise thought.

But as a vertebrate model, could they be as useful as mice?

Several things captured Streisinger’s imagination.

Most famously, zebrafish embryos, unlike mouse embryos, develop outside the mother’s body and are transparent throughout the first few days of life.

This provides unparallelled opportunities for researchers to scrutinise the fine details of embryonic vertebrate development without first having to resort to invasive procedures or killing the mother.

But this advantage is enhanced by the fact zebrafish reproduce profusely (each pair can produce 200-300 fertilised eggs every week); an ideal attribute for genetic studies. Again, the large, external embryos are a critical part of this success.

When just one or two cells old, zebrafish embryos can be easily microinjected with mRNA or DNA corresponding to genes of interest; undeterred, they then they go on to grow and reproduce, handing down the injected gene to the next generation.

From zebrafish to humans

A paper published last month in Nature unveiled the long-awaited sequence of the zebrafish genome, revealing that zebrafish, mice and human have 12,719 genes in common.

Put another way, 70% of human genes are found in zebrafish.

But even more notable is the finding that 84% of human disease-causing genes are found in zebrafish.

Perhaps not surprisingly then, when these genes are injected into zebrafish embryos, the growing animals are doomed to acquire the same diseases.

And while zebrafish are still used widely to answer fundamental questions of developmental biology, much current research is directed towards combining their many attributes in studies that are designed to improve human health.

This is especially true for cancer research where the expression of cancer-causing genes (oncogenes) can be directed to specific organs, virtually at will.

This process, known as transgenesis, is very straightforward in zebrafish and has allowed researchers to produce zebrafish models of liver, pancreatic, skeletal muscle, blood and skin cancers, to name but a few.

And when the genomic make-up of these zebrafish tumours is deciphered using the latest DNA sequencing technology, the patterns of mutations, or “gene signatures”, are found to overlap substantially with those in the corresponding human tumours.

Trialling cancer drugs

These parallels have encouraged researchers to exploit zebrafish in drug development – in particular for high throughput approaches such as chemical/small molecule screens.

Here, the ability to generate tens of thousands of zebrafish embryos harbouring the same disease-causing mutations is crucial.

Then, as the tumours grow in the synchronously developing larvae, the fish are transferred to small volumes of water containing chemicals that may stop the growth, or better still, kill the cancer cells.

Large collections of drugs can be screened relatively quickly for anti-cancer efficacy in this way.

One drug, Leflunomide, identified in such a screen is now in early phase clinical trials to kill melanoma cells.

The only other drug from a zebrafish chemical screen currently in clinical trials is dimethyl-prostaglandin E2 (dmPGE2).

There, the intent is not to kill cancer cells but rather to make mainstream leukaemia treatment more effective.

Studies of dmPGE2 increased the number of blood stem cells in zebrafish embryos and it is being trialled now as a way to expand the number of stem cells in human cord blood samples.

Human cord blood samples are a valuable commodity to restore bone marrow in leukaemia patients after high dose chemotherapy when a matched bone marrow transplant is unavailable.

But the success of this approach is currently limited by the scant number of stem cells in individual cord blood samples, requiring the use of two precious samples for each patient.

Tumour growth

As well as the transgenic zebrafish models of cancer described above, researchers are also transplanting cells derived from human tumours into zebrafish embryos and watching them grow and spread.

The creation of a transparent (non-striped) version of adult zebrafish (called casper, after the cartoon ghost) means the behaviour of tumour cells inside these living organisms can be followed for days at a time.

Coupled with the advent of high resolution live-imaging techniques, the birth, growth and spread of tumours can be scrutinised in movies that can be played over and over again.

These experiments are usually conducted in zebrafish that have been genetically modified to express genes that glow in specific body compartments, giving researchers the ability to pinpoint potentially critical connections between “host” cells and tumour cells that may determine whether the latter survive or die.

This type of experiment is revealing a complex interplay of potentially beneficial and detrimental components.

While the proximity of immune cells may instigate mechanisms capable of destroying the tumour, the stimulation of new blood and lymphatic vessel growth towards the tumour is more insidious, since it delivers the tumour with both the nutrients it needs to survive and a network to spread throughout the body.

These processes, once properly understood, are likely to provide opportunities for therapeutic intervention in the future.

The future of zebrafish

Cancer research is just one part of the zebrafish story. In Australia alone, investigators are also using zebrafish to study metabolic disorders such as:

• diabetes
• muscle diseases, including muscular dystrophy
• neurodegenerative disease
• the response of the host innate immune system to bacterial and fungal infections

Excitingly, research is also underway in this country to unravel the genetic mechanisms controlling heart, skeletal muscle and nervous tissue regeneration in zebrafish, in the hope that these processes can be one day recapitulated in humans to address the burgeoning socioeconomic problem of tissue degeneration in our ageing population.

So next time you peer into someone’s home aquarium, imagine the biomedical possibilities inherent in this lively and amiable little fish!