ScienceDaily (Nov. 18, 2009) – The largest national stem cell study for heart disease showed the first evidence that transplanting a potent form of adult stem cells into the heart muscle of subjects with severe angina results in less pain and an improved ability to walk. The transplant subjects also experienced fewer deaths than those who didn’t receive stem cells.

In the 12-month Phase II, double-blind trial, subjects’ own purified stem cells, called CD34+ cells, were injected into their hearts in an effort to spur the growth of small blood vessels that make up the microcirculation of the heart muscle. Researchers believe the loss of these blood vessels contributes to the pain of chronic, severe angina.

“This is the first study to show significant benefit in pain reduction and improved exercise capacity in this population with very advanced heart disease,” said principal investigator Douglas Losordo, M.D., the Eileen M. Foell Professor of Heart Research at the Northwestern University Feinberg School of Medicine and a cardiologist and director of the program in cardiovascular regenerative medicine at Northwestern Memorial Hospital, the lead site of the study.

Losordo, also director of the Feinberg Cardiovascular Research Institute, said this study provides the first evidence that a person’s own stem cells can be used as a treatment for their heart disease. He cautioned, however, that the findings of the 25-site trial with 167 subjects, require verification in a larger, Phase III study.

He presented his findings Nov. 17 at the American Heart Association Scientific Sessions 2009.

Out of the estimated 1 million people in the U.S. who suffer from chronic, severe angina — chest pain due to blocked arteries — about 300,000 cannot be helped by any traditional medical treatment such as angioplasty, bypass surgery or stents. This is called intractable or severe angina, the severity of which is designated by classes. The subjects in Losordo’s study were class 3 or 4, meaning they had chest pain from normal to minimal activities, such as from brushing their teeth or even resting.

The stem cell transplant is the first therapy to produce an improvement in severe angina subjects’ ability to walk on a treadmill. Twelve months after the procedure, the transplant subjects were able to double their improvement on a treadmill compared to the placebo group. It also took twice as long until they experienced angina pain on a treadmill compared to the placebo group, and, when they felt pain, it went away faster with rest. In addition, they had fewer overall episodes of chest pain in their daily lives.

In the trial, the CD34+ cells were injected into 10 locations in the heart muscle. A sophisticated electromechanical mapping technology identifies where the heart muscle is alive but not functioning, because it is not receiving enough blood supply.

The study was supported by Baxter Healthcare Corporation. Losordo formerly was a paid consultant to Baxter.

Journal of Clinical Investigation  –  One approach being developed as a way to improve heart function following heart attack is the injection of heart stem/progenitor cells directly into the heart. Now, a team of researchers, at Tokyo Women’s Medical University, Japan, and Chiba University Graduate School of Medicine, Japan, has found that transplanting sheets of clonally expanded heart cells expressing the protein Sca-1 (cells that are heart stem/progenitor cells and that the authors term CPCs) improves heart function after a heart attack in mice.

The team, led by Katsuhisa Matsuura and Issei Komuro, found that CPCs not only formed heart muscle cells but also secreted a soluble molecule (sVCAM-1) that induced the migration of endothelial cells (which help form new blood vessels) and CPCs and prevented heart muscle cells dying from oxidative stress.

In the mouse model of heart attack, preventing sVCAM-1 from binding to the protein VLA-4 inhibited the formation of new blood vessels and blocked CPC migration and survival, leading to a decreased ability of the transplanted CPC sheets to improve heart function. The authors conclude that these data provide new insight into the mechanisms by which heart stem/progenitor cells improve heart function following heart attack.

Journal Reference:

  • 1. . Transplantation of cardiac progenitor cells ameliorates cardiac dysfunction after myocardial infarction in mice. Journal of Clinical Investigation, July 13, 2009

Adapted from materials provided by Journal of Clinical Investigation,



Children’s Hospital of Philadelphia, November 18, 2009  –  Heart function significantly improved in children and young adults with single-ventricle congenital heart disease who have had the Fontan operation following treatment with sildenafil, a drug used to treat erectile dysfunction and pulmonary hypertension, say researchers from The Children’s Hospital of Philadelphia. 

Single-ventricle defects are a collection of cardiac malformations that impair the heart’s ability to pump blood. Examples include tricuspid atresia, pulmonary atresia/intact ventricular septum and hypoplastic left heart syndrome. 

The Fontan operation is a procedure that redirects systemic venous blood directly to the pulmonary arteries, bypassing the heart. It is the third surgery in a staged palliation for single-ventricle heart defects.

Researchers hypothesized that sildenafil may help cardiac performance by directly improving the squeeze of the heart muscle and by allowing for better filling of the heart.

In this study, researchers randomized 28 children and young adults who had undergone the Fontan operation to receive placebo or sildenafil three times a day for 6 weeks. After a 6-week break, subjects were switched to the opposite treatment course. The researchers found significant improvement in heart performance during treatment with sildenafil.

“The enhanced heart performance may improve exercise performance and quality of life in these children and young adults,” said David J. Goldberg, M.D., pediatric cardiologist at The Children’s Hospital of Philadelphia, who presented the abstract on Nov. 17 at the American Heart Association Scientific Sessions in Orlando, Fla.

Grants from The Mark H. and Blanche M. Harrington Foundation and from Big Hearts to Little Hearts provided funding for this study.

Story Source:

Adapted from materials provided by Children’s Hospital of Philadelphia.

More About Sildenafil……………


Sildenafil (compound UK-92,480) was synthesized by a group of pharmaceutical chemists working at Pfizer’s Sandwich, Kent, research facility in England. It was initially studied for use in hypertension (high blood pressure) and angina pectoris (a symptom of ischaemic heart disease). The first clinical trials were conducted in Morriston Hospital in  Swansea.  Phase I clinical trials under the direction of Ian Osterloh suggested that the drug had little effect on angina, but that it could induce marked penile erections.  Pfizer therefore decided to market it for erectile dysfunction, rather than for angina. The drug was patented in 1996, approved for use in erectile dysfunction by the US Food and Drug Administration on March 27, 1998, becoming the first oral treatment approved to treat erectile dysfunction in the United States, and offered for sale in the United States later that year.  It soon became a great success: annual sales of Viagra in the period 1999-2001 exceeded $1 billion.

Mechanism of action 

The mechanism of action of Sildenafil citrate involves the release of nitric oxide (NO) in the corpus cavernosum of the penis. NO binds to the receptors of the enzyme guanylate cyclase, which results in increased levels of cyclic guanosine monophosphate (cGMP), leading to smooth muscle relaxation (vasodilation) of the intimal cushions of the helicine arteries, resulting in increased inflow of blood and an erection.  Robert F. Furchgott won the Nobel Prize in Physiology or Medicine in 1998 for his discovery and analysis of endothelium-derived relaxing factor, a key part of the NO mechanism of action.

Sildenafil is a potent and selective inhibitor of cGMP specific phosphodiesterase type 5 (PDE5), which is responsible for degradation of cGMP in the corpus cavernosum. The molecular structure of sildenafil is similar to that of cGMP and acts as a competitive binding agent of PDE5 in the corpus cavernosum, resulting in more cGMP and better erections.  Without sexual stimulation, and therefore lack of activation of the NO/cGMP system, sildenafil should not cause an erection. Other drugs that operate by the same mechanism include tadalafil (Cialis) and vardenafil (Levitra).

Sildenafil is metabolised by liver enzymes and excreted by both the liver and kidneys. If taken with a high-fat meal, absorption is reduced; the time taken to reach the maximum plasma concentration increases by around one hour, and the maximum concentration itself is decreased by nearly one-third. 

Pulmonary hypertension 

As well as erectile dysfunction, sildenafil citrate is also effective in the rare disease pulmonary arterial hypertension (PAH). It relaxes the arterial wall, leading to decreased pulmonary arterial resistance and pressure. This, in turn, reduces the workload of the right ventricle of the heart and improves symptoms of right-sided heart failure. Because PDE-5 is primarily distributed within the arterial wall smooth muscle of the lungs and penis, sildenafil acts selectively in both these areas without inducing vasodilation in other areas of the body. Pfizer submitted an additional registration for sildenafil to the FDA, and sildenafil was approved for this indication in June 2005. The preparation is named Revatio, to avoid confusion with Viagra, and the 20 milligram tablets are white and round. Sildenafil joins bosentan and prostacyclin-based therapies for this condition.


Spray-on skin: In a unique treatment for second-degree burns, surgeons harvest a small number of skin cells through a skin biopsy, suspend them in solution, and then spray the resulting mixture onto a burn wound. Once in place, skin stem cells, called basal cells, proliferate to create a new layer of skin.   Credit: ReCell


A new technique in burn treatment provides an alternative to skin grafts in the operating room.


MIT Technology Review, November 17, 2009, By Lauren Gravitz  —  Traditionally, treatment for severe second-degree burns consists of adding insult to injury: cutting a swath of skin from another site on the same patient in order to graft it over the burn. The process works, but causes more pain for the burn victim and doubles the area in need of healing. Now a relatively new technology has the potential to heal burns in a way that’s much less invasive than skin grafts. With just a small skin biopsy and a ready-made kit, surgeons can create a suspension of the skin’s basal cells–the stem cells of the epidermis–and spray the solution directly onto the burn with results comparable to those from skin grafts.

The cell spray is intended to treat severe second-degree burns, in which the top two layers of skin are damaged but the subcutaneous tissue is left intact. Third-degree burns, which are more severe, still require a skin graft. The spray, already approved for use in some countries, has garnered interest from the United States Army, whose Armed Forces Institute of Regenerative Medicine is funding a trial, slated to begin before the end of this year, of more than 100 patients.

The technology, developed by Australian surgeon Fiona Wood, relies on cells, such as skin progenitor cells and the color-imparting melanocytes, that are most concentrated at the junction between the skin’s top two layers. With a small step-by-step kit dubbed ReCell, surgeons can harvest, process and apply these cells to treat a burn as large as 50 square inches. The kit, marketed by Avita Medical, a United Kingdom-based regenerative-medicine company, is a tiny, self-contained lab about the size and shape of a large sunglasses case.

After removing a small swatch of skin near the burn site (the closer the biopsy, the better for precise matching of color and texture), the surgeon places it in the kit’s tiny incubator along with an enzyme solution. The enzyme loosens the critical cells at the skin’s dermal-epidermal junction, and the surgeon harvests them by scraping them off the epidermal and dermal layers and suspending them in solution. The resulting mixture is then sprayed onto the wound, repopulating the burn site with basal cells from the biopsy site.

“Currently, treating any burn that requires a skin graft is the same technology we were routinely using 30 years ago,” says James Holmes, a surgeon and the medical director of the Burn Center at Wake Forest University Baptist Medical Center. Current practice with larger burns requires grafts from donor skin that are anywhere from one-quarter to the complete size of the burn area. ReCell requires only as much as four square centimeters. “This allows you to take a very small skin biopsy and process it at the table there in the operating room using a fully prepackaged device,” Holmes says. “You’re able to cover an area that’s 80 times the size of your biopsy.”

Holmes is the lead investigator on an upcoming multicenter trial that will compare skin grafts and ReCell. Patients in the trial will act as their own controls: If a burn victim has a second-degree burn severe enough for surgeons to deem treatable by skin graft, half of the burn will be treated that way, while the other half will be treated with the cell spray.

Not everyone agrees that second-degree burns require grafts or other treatments to heal. “Most burns heal without a skin graft. They mostly heal with a Band-Aid,” says Robert Sheridan, a surgeon at the Shriners Burn Institute at Massachusetts General Hospital in Boston. “There’s a long history of autologous [derived from the patient] products for burn treatment, and they all suffer from high costs or neutral results.” The upcoming U.S. study won’t compare ReCell against no treatment, so this issue is unlikely to be resolved soon.

As a complete replacement for skin grafts, ReCell only works against severe second-degree burns–deeper, third-degree burns have destroyed the layer of skin that the ReCell solution would be able to repopulate. But the spray may be useful in treating more severe burns in conjunction with other approaches, as well as for treating existing scars. Wood, director of the burns unit at Royal Perth Hospital, uses ReCell in a process called scar remodeling, in which the cell spray helps repopulate scarred skin with melanocytes so that it more closely matches the patient’s original skin tone. She also uses it in combination with other treatments, such as the skin-growing scaffold Integra, to reduce scarring and improve healing time.

“I use this technology in combination with all the other traditional technologies, and I can improve the outcome and the speed of healing,” says Wood, who is also co-founder of the McCombs Foundation, a nonprofit dedicated to burn research and scarless healing. Royal Perth Hospital once had a long waiting list of patients for reconstruction surgery to fix the deep scars that accompanied third-degree burns. Now, she says, no waiting list exists. “Our reconstruction rates are going down because people don’t need it. Because we’re doing more at the beginning, they don’t need the secondary scar work.”

ScienceDaily (Nov. 18, 2009) – Infectious organisms that become resistant to antibiotics are a serious threat to human society. They are also a natural part of evolution. In a new project, researchers at the University of Gothenburg are attempting to find substances that can slow the pace of evolution, in order to ensure that the drugs of today remain effective into the future.

The resistance of infectious organisms to antibiotics is particularly serious in drugs against fungi. Fungal cells are similar to human cells, which means that it is difficult to develop effective drugs that can destroy them without also damaging human cells, i.e. without causing side effects. We must therefore safeguard the effectiveness of the few antifungal drugs that are available today. Resistance to these would leave many diseases without effective treatment.

However, drug resistance is a natural part of evolution. Evolution creates random variations in the characteristics of organisms, which results in some of them developing greater tolerance to drugs to which they are exposed. This leads eventually to completely resistant fungal strains, and the drug will become totally ineffective. The quicker these random variations appear, the greater the risk of resistance developing. One way of combating drug resistance is to slow down the pace of evolution.

Researcher Jonas Warringer at the Department of Cell and Molecular Biology is using advanced genetic experiments to try to find such “evolution-slowing” substances. In the first instance, this involves identifying the cell components that regulate the speed of evolution. Jonas Warringer and his colleagues are using ordinary brewer’s yeast as a model for their studies. A yeast has 6 000 genes, and destroying single genes in otherwise identical organisms enables Jonas Warringer and his colleagues to use the method of exclusion.

Looking for gold dust

“We stimulate the evolution of the yeast cell and observe it in real time. As our yeasts develop resistance to a particular drug, we measure how the survivability of the different strains changes during the process. Evolution progresses more slowly in some strains when a specific component is destroyed. These strains are like gold dust to us, because they tell us that these particular components are critical to the speed of evolution,” says Jonas Warringer.

“This is how we eventually found the genes that regulate evolution. If, in the next phase, we can find a substance that can attack one of these components, we will be able to delay the development of drug resistance and ensure that today’s drugs remain effective into the future.” The research project is funded by the Magnus Bergwall Foundation and other benefactors. Jonas Warringer hopes that evolution-slowing drugs will become available within the next 10-15 years.


The comeback of an old idea in immunology prompts a rethink of cancer progression and approaches to treatment.

The-Scientist.com, November 18, 2009, by Mark J Smyth  —   In a photo I often use in presentation slides of my work, I’m standing in nothing but my swim trunks and sunglasses, covered in mud up to my ears. Next to me is my long-time colleague Joe Trapani, in much the same state, with a raccoon-like pattern on his face where he has smeared the mud away. We’d just spent an hour lounging in the Israeli side of the Dead Sea, the deepest hypersaline lake in the world, full of rich mineral mud that attracted visitors for thousands of years. I explain to my audience that we’re grinning like fools in the photo in part because of the way we looked but also, perhaps, because we were feeling so clever for coming up with a new way to answer an old, nagging research question: Do cells of the immune system detect and kill cancerous cells?

We had just come from the 1993 EMBO Cell-Mediated Cytotoxicity meeting in Jerusalem where a Swiss group, led by Hans Hengartner, reported the phenotype of a knockout mouse lacking the ability to produce the pore-forming molecule, perforin. Released by the killer cells of the immune system, perforin damages target cells or pathogens by punching holes in their plasma membranes. I had worked on the transcriptional control of perforin during my postdoctoral studies at the National Cancer Institute (NCI) in Frederick, Md., in the late 1980s. Joe, then at Memorial Sloan-Kettering Cancer Center in New York, had studied the perforin gene and attempted to make his own perforin knockout mouse. The two of us listened to the presentation with rapt attention, with the same thought: might this model system reveal a role for perforin in early cancer elimination by immune cells?

With their mouse model, Hengartner’s group demonstrated how fundamentally important perforin was to the immune system’s ability to kill cells infected with bacteria or viruses. While most of the attendees were captivated with the immunological implications, such as which killing pathways remained in the absence of perforin, we wondered whether the model might also uncover cell-mediated killing of tumors, a process called immunosurveillance. Joe and I reasoned that if cancer cells are regulated via perforin-releasing lymphocytes, then mice lacking perforin should develop more cancers than wild-type mice with normal perforin function.

The idea that the immune system could survey the body for cancerous cells, killing the ones that looked abnormal, was not novel. It was suggested in passing by German scientist Paul Ehrlich at the turn of the 20th century, long before even rudimentary understanding of the immune system existed. Fifty years would pass before scientists had experimental evidence to support the idea. Even then, many doubted that the immune system was capable of detecting cancer. After all, tumors originate from our own cells, which the immune system is trained to ignore.

The first formal demonstration of the immune system’s ability to “see” cancer came with the discovery of tumor-specific antigens in the 1950s and 1960s. Lloyd Old, George Klein, and others used tumor transplantation models to show that cancer cells indeed express specific antigens that can be recognized by the immune system as different from healthy cells. In the 1960s, one of my most famous fellow countrymen, Frank Macfarlane Burnet, along with the American Lewis Thomas, drew upon their own research, as well as Old and Klein’s findings, to propose the term “cancer immunosurveillance.” They envisioned it as a process by which the immune system recognized and destroyed cancer cells very early in cellular transformation. Although this hypothesis was formulated without any direct supporting experimental data, it was so compelling that it was enthusiastically embraced by the medical and basic science communities.

In 1973-74, a serious challenge to the cancer immunosurveillance hypothesis arose when Osias Stutman, at Memorial Sloan-Kettering Cancer Center in New York, tested a central prediction of the theory: immunodeficient mice should develop more spontaneous and carcinogen-induced tumors than their wild-type counterparts. In a series of very nicely performed experiments, Stutman could not validate these predictions and his experiments seemingly sounded the death knell for the cancer immunosurveillance hypothesis.

Joe and I were well aware that this wouldn’t be a popular question to resurrect-most researchers had bid good riddance to the idea that the immune system might keep cancer at bay. But, with a healthy dose of youthful curiosity, we waded in.

One reason for my interest in the perforin knockout was my research on natural killer (NK) cells, which produce perforin as part of their killing arsenal. NK cells were first described by Ron Herberman at the NCI in 1975 as cells with a natural propensity for killing tumor cells in vitro (hence their name). No other immune cell had this spontaneous ability. Its effects were impressive: a few NK cells would kill many types of cancer cells in culture without any additional activation. Now I knew perforin was essential. It seemed simplistic to think that cytotoxic lymphocytes, such as NK cells, might behave the same way in the tissues of a live animal, but that was the idea that I wanted to test.

Most researchers had bid good riddance to the idea that the immune system might keep cancer at bay. But, with a healthy dose of youthful curiosity, we waded in.

By then a few researchers had realized several flaws in Stutman’s experiments. First, the immunodeficient mice that he had used weren’t actually devoid of NK cells, as he had thought at the time, leaving the possibility that immunosurveillance was intact in these mice. Second, the strain of mouse he used was overly susceptible to the carcinogen and therefore the immune system may well have been overwhelmed by cancer development, failing to reveal immune control.

Coming back to Australia after our trip to Israel, Joe and I started planning a convincing way to test the concept of cancer immunosurveillance using perforin-deficient mice at the Austin Research Institute in Melbourne. Several groups had made perforin knockout mice and Bill Clark from the University of California, Berkeley generously provided his strain. We wanted to cross the perforin knockout mouse with one we could be sure would develop cancer, so we acquired a mouse with a genetic predisposition for cancer-the p53 knockout mouse from Alan Harris at the Walter and Eliza Hall Institute. p53 mutations are found in over half of all human cancers, and mice that lack p53 develop a spectrum of sarcomas, lymphomas and, more rarely, adenocarcinomas over their life span. We decided to intercross mice that had one allele of p53 and one allele of perforin knocked out to generate progeny with nine different genotypes (see graphic). It took more than 50 breeding pairs to generate hundreds of mice. We coded the newborns and simply left them on the shelf of our pathogen-free animal house to age, without breaking the code.


The Comback of Immunosurveillance
To test for the role of the immune system in cancer detection and elimination, we inbred female and male mice with both a predisposition to cancer (a single allele p53 knockout) and immunodeficiency (a single allele for perforin-an immune cell-secreted molecule that kills pathogen-infected cells). The progeny of these breeders had nine different genotypes. All mouse genotypes lacking both perforin alleles developed cancer sooner than those with one or two intact perforin genes, showing for the first time that immune-mediated killing was involved in cancer surveillance and elimination.


Our greatest concern was whether we could age sufficient numbers of mice long enough to obtain statistically significant data, given that no one had reported aging the perforin-deficient mice beyond a year. Thus we obsessively watched these mice for signs of cancer development, keeping fingers crossed that no facility disaster would compromise our significant investment. Remarkably, some of the oldest mice in this study developed such a relationship with Kevin Thia-one of the PhD students monitoring them-that no one else could go near them without fear of being bitten!

Along the way we sacrificed mice that exhibited lumps, weight loss, or other signs of disease. Within the first 200 days, a number of mice fell ill, but after determining their genotype we saw that, as expected, all of these mice lacked both copies of p53. Interestingly, mice that lacked both copies of perforin and p53 developed lymphomas significantly sooner than the p53 double-knockouts that had functional perforin. This excited us, but we anticipated that the mice missing only one p53 allele would be even more revealing since the cancer development in these mice was slower, allowing any involvement of immunity to become more obvious. It was not until 600 days into the study that we had decoded and genotyped enough mice with associated disease to appreciate two remarkable facts. One, mice with one working copy of p53 and lacking perforin developed spontaneous B-cell lymphoma earlier and more frequently than their littermates with at least one perforin allele intact. Two, it did not matter whether the perforin knockout mice had one or both working copies of p53-both developed B-cell lymphomas at the same rate, making us wonder why we had bothered using the p53 knockout in the first place.

Joe, Kevin, and I were excited and relieved that perforin was an unequivocally important suppressor of B-cell lymphoma. We had gone some way to validating Burnet’s hypothesis and the study was the catalyst for many others of similar ilk. While these studies were ongoing, we started a collaboration with Dale Godfrey at the University of Melbourne to assess the importance of NK cells and a small population of specialized T cells, called NKT cells, in immune surveillance of cancer. Rather than using p53 mutant mice, we were studying a mouse model of fibrosarcoma induced by methylcholanthrene (MCA), a carcinogen component of the tar found in cigarettes. We showed that mice lacking either NK or NKT cells were more susceptible when exposed to the carcinogen than the wild type.1 This work was the first to clearly demonstrate a role for early (innate) immunity in control of carcinogen-induced tumors.

Seven years after that pivotal meeting in Israel, Joe and I submitted and published our perforin knockout aging study.2 At the onset of this first aging study, he and I relocated to the Peter MacCallum Cancer Hospital in Melbourne and I began to develop an interest in other death-inducing pathways that lymphocytes might use to suppress tumors. Many other experiments were underway in my new lab, but this publication was my first major breakthrough in this area and complemented a lot of in vitro work that Joe and I had published in the intervening years.


Our 2000 publication in the Journal of Experimental Medicine did not go unnoticed. Shortly afterwards, I was invited by the director of the Ludwig Institute, Professor Lloyd Old, to speak at the International Symposium of Cancer Immunosurveillance in New York, which was quite an honor. Lloyd has devoted his career to promoting the discipline of cancer immunology, and at that meeting he introduced me to many key people in the world of tumor immunology.

No one opportunity was more important than meeting with Robert Schreiber, who seemed to be the only other person in the world prepared to undertake long-term aging experiments of the kind necessary to characterize cancer immunosurveillance. Bob had a distinguished history in cytokine signaling, particularly with the inflammatory mediator, interferon gamma (IFNγ). In broadening my interest in cancer immune surveillance, I had noted Bob’s work on interferons, including a landmark paper in 1998 that largely supported the idea that IFNγ could protect the host from cancer development, much the same way that perforin did.3 On meeting, we appreciated the great opportunity that existed for us to join forces. For a few years, we each independently completed studies that further supported the cancer immune surveillance hypothesis.

Clearly, despite the immune system’s survey and rejection of cancer cells, tumors still develop in the presence of a functioning immune system. To account for this, Bob and his colleagues refined the concept of cancer immune surveillance into what they called “cancer immunoediting.” The origins of this term immunoediting came from his laboratories’ 2001 study published in Nature.4 They showed that tumors induced by carcinogens in wild-type mice were less immunogenic-less likely to spur an immune reaction-than those derived from immunodeficient RAG knockout mice that lack all T and B cells. They proposed that the tumors from the RAG-deficient mice were more immunogenic because presumably they had not been sculpted by T and B cells as the tumor developed, whereas the wild-type tumors had.

To explain this, Bob proposed three phases of immune system-tumor interaction: elimination, equilibrium, and escape. The elimination phase is essentially immune surveillance, in which the immune system detects and destroys tumor cells. Tumor cells that aren’t destroyed can enter a state of equilibrium with the immune system, in which the growth of the tumor is checked by immune cells killing a proportion of the cancer cells. During this period, however, tumor cells may continue to accumulate mutations, potentially generating variants that resist, avoid, or suppress the antitumor immune response. This leads to the escape phase, in which the tumor growth is unchecked by the immune system. Cancers that doctors see in the clinic have achieved this third phase.5

Bob based his theory on a number of observations. Still, while there was evidence for surveillance/elimination and escape phases, there was no real characterization of the equilibrium phase. Clinically, tumors can persist for years without developing into full-blown cancer; there have been cases of cancer inexplicably going into remission for many years. The equilibrium process was inferred largely from these clinical observations. Most striking were the reports of organ transplant recipients developing cancer that originated from the donor, despite the donor’s having been healthy and apparently cancer free. In such cases, it is possible that the tumor was being held in the equilibrium phase in the donor, in a dormant state, and that transplantation of the organ into an immunosuppressed host allowed tumor outgrowth. Although several mechanisms underlying tumor dormancy have been proposed, there was very little evidence that the immune system played an important role. We wanted to develop a mouse model that mirrored the experience of patients whose cancer resurfaced after many years of lying dormant.

Instead of trying to remove all vestiges of cancer, might it be possible to confine the cancer to a state in which the immune system controls it?

By around 2002, Bob’s group and ours were doing similar experiments in a mouse model of fibrosarcoma induced by methylcholanthrene (MCA). We had both noted that some mice treated with a limiting dose of this powerful carcinogen did not develop clinically apparent cancer but rather harbored tiny stable lesions that eventually disappeared. We realized that these mice might provide a model to test the concept of equilibrium. Bob’s lab began injecting large cohorts of 129 mice with low doses of MCA, and shortly thereafter we commenced an identical experiment in a different background strain of mouse (C57BL/6). Mice that developed progressively growing tumors were removed from the study, leaving mice that had small, nongrowing masses at the site of the injection. We saw that these “equilibrium phase” tumors were infiltrated with immune cells. When we depleted mice of either strain of T cells and removed the protective IFNγ with antibodies, the stable lesions rapidly progressed into aggressive cancers. Surprisingly to me, when we removed NK cells with antibodies, the small cancers did not progress. It suggested that NK cells, which had been important in the elimination phase, were not involved in keeping tumors in equilibrium.6

There is clearly much more work to be done in this mouse model of cancer, and in other models. Why do some tumors enter equilibrium and others do not? What genetic signature does a tumor have to have in order to enter equilibrium? By what mechanism does the immune system suppress a tumor’s outgrowth? It is also unclear whether this state actually exists in human patients, and if so, how this knowledge could be used to develop new approaches to improving patient outcomes. Instead of trying to remove all vestiges of cancer, might it be possible to confine the cancer to a state in which the immune system controls it?

Other than elevated cancer incidence in transplant recipients receiving immunosuppressive drugs, evidence of cancer immunoediting in humans is comparatively sparse. However, recent studies of human cancers suggest that the type of immune cells present in and around cancer predicts the outcome of the patient. Positive outcomes are correlated with the presence of activated effector cells, whereas the presence of the immune suppressing T regulatory cells are associated with a poorer prognosis. This strengthens evidence for the role of the immune system in cancer, but more evidence is needed.

Our work has taken many years. That is the price paid to study cancer in a biologically relevant context, where you look at tumors arising over the lifetime of a mouse. This has distinguished our work from others that have relied on short-term transplantable tumor models. However, some scientists rightfully argue that inbred mice don’t capture the complexity of the human disease and their cancer predisposition is low compared with humans. Lacking an alternative, we still rely on our mouse models to reveal new ideas about cellular and molecular mechanisms of disease.

Joe Trapani and I felt quite clever to attack a question that helped rekindle the debate over immune surveillance. Twenty years later, there are spots of clarity, where we’ve cleared the mud away, but the picture is still a bit blurred.


Disagreement over how older influenza drugs stop the virus is stymieing efforts to find new compounds

The-Scientist.com, November 18, 2009, by Elie Dolgin  —  A scientific spat over how drugs affect the structure of an influenza channel could be imperiling the development of new drugs targeting seasonal and pandemic flu viruses such as the H1N1 swine flu. 

In the fall of 2007, the scientific advisory board of Influmedix, a Radnor, PA-based biotech company aimed at developing novel anti-flu medications, convened a conference call to discuss an important flu protein, a proton channel called M2. Two different researchers associated with the company had just cracked the proton channel’s atomic-scale structure, but the two then-unpublished structural models came to strikingly different conclusions about how two flu drugs targeted this channel.

Influmedix founder William DeGrado of the University of Pennsylvania in Philadelphia, thought that the drugs amantadine and rimantadine prevented the channel from opening directly. Alternatively, scientific advisor James Chou of Harvard Medical School in Boston, Massachusetts, had evidence that the drugs bound at a site outside the pore that modulated the protein by changing its shape. The two were at an impasse.


Since most influenza viruses, including the H1N1 pandemic swine flu strain, are now resistant to amantadine, rimantadine, and other drugs that target M2 — one of only two proven anti-flu drug targets — the hope is that researchers can use a structure-based approach to find new, critically needed compounds that target the same proton channel and make once-resistant flu strains vulnerable again. But that’s contingent on settling the academic altercation, noted Lawrence Pinto of Northwestern University in Evanston, Illinois, who collaborates with DeGrado and advises Influmedix. “There has to be an end to it.”

DeGrado and Chou published their structures side-by-side months later, in January 2008, each claiming that their own model explained the drugs’ mode of action. The two men haven’t spoken since — although they have exchanged occasional emails — and Chou resigned from Influmedix’s scientific advisory board a few months later.

The dispute is now firmly stuck in the scientific literature as each researcher continues to publish follow-up papers that support his viewpoint. “Both sides have become increasingly rigid,” said Christopher Miller, an ion channel researcher at Brandeis University in Waltham, Massachusetts, who reviewed the original two papers and wrote an accompanying commentary.

When Chou started his Harvard lab five years ago, he decided to apply his expertise in nuclear magnetic resonance (NMR) imaging to solve the structure of membrane-gated ion channels, which no one had ever done before using this particular technique. He turned his attention to M2, a small channel that affects how the influenza virus replicates.

Like most people, Chou initially figured that the drugs that targeted this channel worked like corks in a bottle — stick something in the middle of the opening and nothing can get through. “That’s just intuition,” he said. “It’s common sense.” But something didn’t sit right about that idea. M2 blockers are tiny drugs, so how could several disparate mutations inside the larger pore all confer drug resistance if the compounds adhere to one particular spot, Chou wondered.

After a four-year effort, Chou and his postdoc Jason Schnell, now at the University of Oxford, UK, eventually concluded that the drugs didn’t block the pore directly. Rather, their NMR imaging showed that they bound on the outside of M2 and caused the channel to lock in a closed state. “We unambiguously found that the drug interacts at that binding site,” said Chou.

DeGrado’s results suggested otherwise. His postdoc Amanda Stouffer, now at the Swiss Federal Institute of Technology (ETH) in Zurich, spent a year crystallizing the transmembrane region of the protein using X-ray crystallography, and concluded that the drugs nestled right into the pocket of the channel’s pore. “It’s quite clear that we know where the drug binds,” said DeGrado.

Both can’t be right. DeGrado doesn’t doubt Chou’s data, but suspects that Chou is observing non-specific drug binding to a non-pharmacologically relevant part of the protein. Chou’s NMR structure was created with a “massive amount” of drug compounds, DeGrado said, so the drugs might just be getting stuck on the outside of the channel. The channel’s locked position is likely caused by a drug bound inside the pocket that Chou missed, DeGrado reasoned.

Chou stands by his results and counters that what DeGrado saw in the middle of the channel was not the drug at all. At DeGrado’s crystal resolution of 3.5 Angstroms, there’s no way to say definitively that the pocket wasn’t filled with leftover reagents from the crystallization protocol, rather than the drug, Chou argued.

Robert Lamb, a Northwestern University virologist and Influmedix scientific founder, concedes that the resolution would need to be better in both experiments to definitively prove either hypothesis. But he points to mounting evidence arguing in favor of the pore-blocking (DeGrado’s) model.

Last year, Lamb and Pinto mutated the sites that Chou had proposed were important for drug binding outside the pore and measured the electrophysiological properties of the channel in cell models and in live viruses. The channel was still sensitive to the drug, arguing against Chou’s model. Chou counters that he analyzed the same mutations using a different approach and found that the amino acid changes indeed altered the drug sensitivity.

The only other person actively publishing papers supporting Chou’s data is his father, Kuo-Chen Chou, the founder and president of the Gordon Life Science Institute, a non-profit research organization in San Diego, California. In July, Kuo-Chen Chou and his colleagues in China published two papers based on computational analyses that support James Chou’s model of the M2 structure.

James Chou stressed that he was not involved in his father’s research, and that he doesn’t trust conclusions based solely on computational modeling. “I want to make it clear to you that I had nothing to do with those papers,” said James Chou.

DeGrado and his colleagues want to wash their hands of the whole controversy. “I’m not saying there might not be another binding site, but the question is, ‘Is there a pharmacologically relevant one?’ And the answer is clearly no,” DeGrado said.