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July 9, 2008, Howard Hughes Medical Institute – After a malaria parasite invades a red blood cell, it sends a crew of proteins inside to do some major remodeling.

But this rehab job isn’t mean to refinish the floors. The proteins gut the red blood cell, transforming it from a vital oxygen delivery system into a nest for new parasites. These renovations are what make malaria so dangerous: they cause infected red blood cells to harden and stick inside blood vessels. When this happens in the brain or in the placenta of a pregnant woman, the results are deadly.

Alan F. Cowman, Ph.D.

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Professor Cowman received his Ph.D. in parasitology from the University of Melbourne. For his work on Plasmodium falciparum, Professor Cowman is the recipient of many awards, including the 1990 Burnet Prize, the 1992 Glaxo Award for Advanced Research in Infectious Diseases, the 1993 Gottschalk Medal for Medical Science and Biology of the Australian Academy of Sciences, the 1994 Boehringer-Mannheim Medal, the 2001 Royal Society of Victoria Research Medal, the Centenary Medal from the Governor-General of Australia, and appointment as a Fellow of the Australian Academy of Science. Professor Cowman is currently a researcher in the Division of Infection and Immunity at the Walter and Eliza Hall Institute of Medical Research. He was first named an HHMI International Research Scholar in 1992. This is his third HHMI award.

RESEARCH ABSTRACT SUMMARY:

Alan Cowman wants to know how Plasmodium falciparum, which causes the most lethal form of human malaria, invades mature red blood cells. This information will be important in determining the potential of the proteins involved in the process as vaccine and drug candidates

“What this paper has done is identify new proteins that are different from anything we’ve seen, that are absolutely essentially for different functions in the red blood cell.”
Alan Cowman

That’s why HHMI international scholar Alan Cowman and his colleagues set out to understand the proteins that oversee this destructive remodeling. Cowman, from the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia, and his colleagues present the first systematic examination of remodeling proteins from the most deadly species of the malaria parasite, Plasmodium falciparum, in a paper published online July 11, 2008, in the journal Cell. Brendan Crabb, another HHMI international scholar at the institute, is a co-author of the paper.

“What this paper has done is identify new proteins that are different from anything we’ve seen, that are absolutely essentially for different functions” in the red blood cell, Cowman said.

The researchers have figured out the roles of 56 proteins involved in this remodeling. This includes eight proteins that act like painters, putting down a sticky layer on the cell’s surface, and two more that act like carpenters, building knobs on the surface of the cells. They also identified several carpenter-like proteins that change the cell structure so it’s rigid.

Perhaps more important, they have found an additional 30 proteins that are essential to the parasite’s survival inside red blood cell. If the research team can understand what these proteins do, they may eventually be able to design a drug that could stop the parasite or make it less likely to kill.

Malaria parasites go through a series of steps on their way to causing human disease. They travel from a mosquito bite on the skin to the liver, where they hunker down and multiply. They then fan out into the bloodstream, where they invade red blood cells both in an attempt to evade the immune system and to remodel them for their own use. “This is key to the parasite’s survival in the host and the key to its pathogenesis,” Cowman said.

To identify the role of each protein involved in red blood cell remodeling, the research team had to create a parasite without the gene that creates it. This modified parasite is called a knockout. While red blood cell rehabilitation requires a work crew of as many as 400 proteins, Cowman’s team started their analysis with 200 of these, focusing on those that seemed unique or that had been linked to important roles before. This Cell paper describes the first 83 genes, many found only in P. falciparum.

The research has taken five years because identifying the function of genes in P. falciparum isn’t easy, Cowman explained. They have to knock out the genes one by one, then run a number of tests to find out what has changed about the parasite and the red blood cell it attacked. “It’s very difficult, and no one has every attempted anything on this scale before,” he said.

What they found was worth the effort. The team has identified two proteins responsible for building porcupine-like protrusions on the walls of red blood cells; without these knobs, infected cells don’t stick to vessel walls. Several other proteins were responsible for turning a flexible red blood cell into a rigid sphere that clogs up small blood vessels. The most interesting of the newly-identified proteins may be those responsible for placing a glue-like adhesive called PfEMP1 on the outer walls of the red blood cell. This adhesive, called a virulence protein, is the primary factor that sticks these rehabbed red blood cells to blood vessel walls. Cowman expects they will find more proteins involved in creating PfEMP1.

But the biggest part of the researchers’ job is just beginning. They want to understand the role of the 30 proteins that the parasite can’t live without. These essential proteins will require a new set of approaches, because they have found that a traditional gene knockout kills the parasite. While this proves the proteins are essential, it doesn’t explain what they do inside the parasite. Cowman suspects that many may be involved in the parasite’s uptake of nutrients.

Eventually, the team hopes to identify targets for new treatments against malaria. Targeting these essential proteins might kill the parasite, which is a good solution. But even better might be targeting proteins that would weaken the malaria parasite rather than killing it. This would leave enough of the parasite to stimulate the immune system to respond, but prevent most of the major illness caused by the parasite. “In some ways, it would be better than actually killing it,” Cowman said.

This study is the first step toward figuring out what proteins to target in a vaccine. “The blood stage causes all of the infection and the disease. That is the reason we are trying to understand how it interacts with the host,” he said.

Fighting Malaria On His Home Turf
by Shelley DuBois

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Surface protrusions, called knobs, on Plasmodium falciparum-infected red blood cells display aggregates of PfEMP1, a protein that shuts down part of the immune system alarm pathway. The smooth appearance of the red cell on the right is due to a mutation in another protein, KAHRP, required for knob formation.

For Louis Schofield, the malaria problem in Papua New Guinea is more than a passing interest.

When he was a boy, his family lived on the Pacific island, where his father worked as a physician treating patients with tropical diseases such as dengue fever, typhoid, and malaria. Schofield says, “I got both metaphorically and literally exposed to some of those infections. I have no doubt that it planted some seeds in my mind.”

Now at the Walter and Eliza Hall Institute of Medical Research (WEHI) in Melbourne, Australia, Schofield has merged his laboratory studies with field trials involving children in Papua New Guinea to better understand how molecules in mosquito-borne protozoa make malaria so difficult for the human immune system to fight.

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“Malaria causes more than a million fatalities every year, mainly in kids,” Schofield says. “I believe strongly that inappropriately regulated immunological reactions are responsible for a lot of those fatalities. So the thing is to identify the parasite molecules that alter the immune response.”

Most recently, Schofield and Alan Cowman, both HHMI international research scholars at WEHI, found the molecule used by Plasmodium falciparum—the protozoa that causes the deadliest form of malaria—to turn off the body’s immune response.

Typically, a protein known as interferon-gamma (INF-gamma) alerts white blood cells when a pathogen enters the body. But when P. falciparum protozoa infect a red blood cell, they send a molecule called PfEMP-1 to the surface of the cell. PfEMP-1 shuts down the INF-gamma alarm pathway.

In 2002, Schofield discovered how another malaria parasite molecule called GPI triggers an inflammatory response in the body, sometimes with fatal side effects. He and his team suspected that other parasite molecules also contributed to skewing the balance of the immune system. They set their sights on PfEMP-1 because they knew it mediates contact between parasites and white blood cells.

To study the function of PfEMP-1 in malaria infection, Schofield needed a parasite with an inactive form of the molecule to compare its effects on the immune system with those caused by unaltered protozoa. Schofield collaborated with Cowman, a parasite molecular biologist who had been studying the biology of PfEMP-1, to design his experiment.

“Alan’s always been interested in gene function and knocking genes out and knocking them in to examine the contribution of the different parasite molecules,” says Schofield. PfEMP-1 had been difficult to investigate because every malaria parasite contains 50 to 60 slightly different alleles, or variants, of the gene that encodes PfEMP-1. The protozoa’s ability to switch these gene variants on and off allows it to escape detection by the immune system.

Cowman could not knock out all 60 genes. He did, however, engineer a P. falciparum mutant that switched off all PfEMP-1 expression.

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Schofield exposed isolated adult white blood cells to unmodified, or wild-type, protozoa, and to Cowman’s mutant P. falciparum. The mutant protozoa prompted a normal inflammatory immune response while the wild-type protozoa down-regulated INF-gamma and remained undetected by the host immune system. Schofield and Cowman published their results in the August 2007 issue of Cell Host and Microbe.

Next, Schofield intends to identify PfEMP-1’s receptor in white blood cells. Then he hopes to design a field study that examines the response of white cells to wild-type P. falciparum or Cowman’s mutant protozoa in children with different levels of susceptibility to disease. Schofield will incorporate this study into his ongoing program at the Papua New Guinea Institute of Medical Research.

At the Institute, Schofield’s team works both on hospital-based studies with patients and on a longitudinal, population-based study with a group of children in Papua New Guinea. The researchers look at genetic variation within the group to learn how it might affect the children’s immune function and their risk of developing malaria.

Schofield has been studying malaria for nearly three decades, since he was 21 years old. He says that he’s kept his focus because of malaria’s widespread impact. Although there is practically no malaria in Australia, the disease thrives in neighboring countries—along with Papua New Guinea, it is epidemic in Indonesia and East Timor. “That’s one of the motivations,” adds Cowman, “It’s a big problem. But also, scientifically it’s incredibly interesting.” Your browser may not support display of this image.

-4.jpgD. Louis Schofield, Ph.D.

Dr. Schofield received his Ph.D. in biochemistry from the Queensland Institute of Medical Research in Brisbane, Australia, in 1986. From 1986 to 1988, he conducted postdoctoral research in the Department of Medical and Molecular Parasitology of the New York University School of Medicine in the United States, where he subsequently held the position of junior faculty instructor until 1990. From 1990 to 1994, he was staff scientist at the Medical Research Council National Institute for Medical Research in London. In 2002, he shared in the Robert P. Goldberg Grand Prize from the Massachusetts Institute of Technology; in 2003 and 2006, he received the National Health and Medical Research Council (NHMRC) Support Enhancement Award from the Australian government. He is a cofounder of Ancora Pharmaceuticals, Inc., in Boston, Massachusetts. He is currently an NHMRC Principal Research Fellow, a Bio21 Industry Fellow, and head of the Laboratory of Malaria Immunology at the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia. This is his second HHMI international research scholar award.

RESEARCH ABSTRACT SUMMARY:

Louis Schofield is investigating the role of innate immunity and the parasite toxin in susceptibility and resistance to severe malaria. He hopes that determining the role of the toxin and innate responses in disease and the role of antitoxin antibodies and counter-regulatory mechanisms in clinical immunity to malaria will provide a rational basis for the development of interventions that prevent malaria fatalities.

Summary: Louis Schofield is investigating the role of innate immunity and the parasite toxin in susceptibility and resistance to severe malaria. He hopes that determining the role of the toxin and innate responses in disease and the role of antitoxin antibodies and counter-regulatory mechanisms in clinical immunity to malaria will provide a rational basis for the development of interventions that prevent malaria fatalities.
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Plasmodium falciparum malaria is a major public health problem, infecting 5–10 percent of the global population and killing two million children annually. Those affected by and dying of malaria may variously suffer single-organ, multi-organ or systemic involvement, including cerebral malaria (CM), renal failure, pulmonary edema, acute respiratory distress, metabolic acidosis, hypoglycemia, coagulopathy, shock, and severe malarial anemia (SMA). Particularly among African children, the three most serious syndromes are CM, metabolic acidosis, and SMA. Despite their profound importance for global public health, the molecular etiology of these syndromes remains obscure.

Nonetheless, disease syndromes and fatalities are understood to result from the intersection of four fundamental processes: (1) the site-specific localization of parasites among target organs through vascular cytoadherence or sequestration; (2) the local and systemic action of bioactive parasite products, such as toxins, on host tissues; (3) the local and systemic production of pro-inflammatory and counter-regulatory cytokines and chemokines by the innate and acquired immune systems in response to parasite products; and (4) the activation, recruitment, and infiltration of inflammatory cells. I have been focusing on the role of innate regulatory mechanisms and a malarial toxin as key determinants of malarial pathogenesis, particularly in rodent models. And I am now extending my work to the role of the innate system and malaria toxin in pathogenesis and clinical immunity in human populations. This requires an integrated approach at the molecular, cellular, whole animal, and population biology levels.

Specifically, I am continuing to investigate the molecular basis of the CM and SMA syndromes using superior animal models, immunological and pathophysiological endpoints, microarray profiling, pure glycosylphosphatidylinositol (GPI), and anti-GPI vaccination during rodent malaria infection. As an example of this work, we recently developed a credible rodent model of SMA. Severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes. SMA is the most serious pernicious complication of malaria and may contribute to the majority of malarial deaths worldwide. The syndrome is thought to arise in part from increased destruction of uninfected red blood cells (RBCs); however, there is a paucity of experimental evidence concerning this process. In general, the study of SMA in animal models is confounded by susceptibility to excessive parasitemias that induce hemolytic anemia. Hemolysis due to hyperparasitization may not be informative with regard to the etiology of human SMA, which is typically associated with comparatively low parasite burdens. In this study we are describing Plasmodium berghei infections of semi-immune mice and naive rats with low parasite burdens that develop pronounced SMA, similar to that seen in semi-immune monkeys and experimental infections in naive humans. We found that SMA was independent of the level of peak or cumulative parasitemia but was linked temporally to the duration of patent infection. In animals with SMA, the entire blood compartment was turned over in approximately one week. The survival rate of both resident and transfused uninfected RBCs was markedly reduced in anemic animals, but reverted to normal upon transfer of RBCs from anemic donors to naive recipients, suggesting no lasting changes to target RBCs. Anemia was significantly alleviated by depletion of host phagocytic cells and CD4+ T lymphocytes and thus appears to result predominantly from accelerated reticuloendothelial phagocytosis of uninfected RBCs, a process that is under the control of the acquired immune system. We are currently developing similar models of erythropoietic suppression in malarial anemia. Models such as these should provide tractable systems to test prevailing hypotheses of the etiology of these important disease syndromes.

An integral part of the research program is to undertake translational research activities in human biology and field settings. Together with Dr. Ivo Mueller and Dr. John Reeder of the Papua New Guinea Institute of Medical Research (PNGIMR), I am coinvestigator responsible for human immunology in a longitudinal, prospective case-cohort study titled “Intermittent Preventive Therapy During EPI for the Prevention of Malaria and Anemia in Papua New Guinean Infants,” which was recently funded by the Bill and Melinda Gates Foundation in the context of the worldwide IPTi consortium (http://www.IPTi-malaria.org). Starting in January 2006, 1518 children are being followed in a three-year longitudinal cohort case-control study, with regular sampling for sera, blood, and peripheral blood mononuclear cells (PBMCs). We will examine disease associations with serological and cellular immunological parameters. Primary outcome measures will be the impact of IPTi on incidence and prevalence of clinical malaria, incidence of anemia, and Hb levels as a quantitative trait. Field-site capabilities include FACS and quantitative PCR. The study is nested in an ongoing demographic surveillance (DSS) catchment with case detection of 20,000 individuals. Also with Dr. Mueller of PNGIMR, I actively collaborate in two additional funded research projects. In the first, under the aegis of a Merit Award grant to Dr. Chris King (Case Western University) that is funded by the U.S. Veterans Affairs Administration, we are investigating serological and cellular correlates of immunity in a “time-to-reinfection” trial, in which a cohort of semi-immune primary school children have received radical antimalarial treatment and are being followed for a year with regular biweekly bleeds, clinical measurements and questionnaire, and both active and passive case detection. NK cell responses are being matched prospectively with risk of acute morbid episode and Hb levels. In the second project, I collaborate on the Papua New Guinea Institute of Medical Research’s grant number NIH RO3-AI63135, undertaking in Papua New Guinea’s Wosera region a two-year longitudinal study of 280 one- to four-year-olds, which involves bimonthly bleeds, fortnightly active morbidity surveillance, and comprehensive passive case detection. Plasma and PBMC samples from strictly defined cases and controls, and baseline and ongoing samples will be used to test positive and negative associations of GPI bioactivity, anti-GPI antibody responses, NK cell responses, and innate immune pathways with susceptibility and resistance to disease. These studies will ascertain the role of the toxin and innate responses in disease and the role of antitoxin antibodies and counter-regulatory mechanisms in clinical immunity to malaria. The findings may provide a rational basis for future interventions against key causal pathways in fatal pathogenesis.

Parasites destroying the red blood cells
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Definition

Malaria is a parasitic disease that involves high fevers, shaking chills, flu-like symptoms, and anemia.

Alternative Names

Quartan malaria; Falciparum malaria; Biduoterian fever; Blackwater fever; Tertian malaria; Plasmodium

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Female mosquito getting a blood meal

Causes, incidence, and risk factors

Malaria is caused by a parasite that is transmitted from one human to another by the bite of infected Anopheles mosquitoes. In humans, the parasites (called sporozoites) migrate to the liver where they mature and release another form, the merozoites. These enter the bloodstream and infect the red blood cells.

The parasites multiply inside the red blood cells, which then rupture within 48 to 72 hours, infecting more red blood cells. The first symptoms usually occur 10 days to 4 weeks after infection, though they can appear as early as 8 days or as long as a year later. Then the symptoms occur in cycles of 48 to 72 hours.

The majority of symptoms are caused by the massive release of merozoites into the bloodstream, the anemia resulting from the destruction of the red blood cells, and the problems caused by large amounts of free hemoglobin released into the circulation after red blood cells rupture.

Malaria can also be transmitted congenitally (from a mother to her unborn baby) and by blood transfusions. Malaria can be carried by mosquitoes in temperate climates, but the parasite disappears over the winter.

The disease is a major health problem in much of the tropics and subtropics. The CDC estimates that there are 300-500 million cases of malaria each year, and more than 1 million people die. It presents a major disease hazard for travelers to warm climates.

In some areas of the world, mosquitoes that carry malaria have developed resistance to insecticides, while the parasites have developed resistance to antibiotics. This has led to difficulty in controlling both the rate of infection and spread of this disease.

Falciparum malaria, one of four different types of malaria, affects a greater proportion of the red blood cells than the other types and is much more serious. It can be fatal within a few hours of the first symptoms.

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The Life Cycle of a Mosquito

Symptoms

Chills
Fever
Sweating
Headache
Nausea
Vomiting
Muscle pain
Anemia
Stools, bloody
Jaundice
Convulsion
Coma

Signs and tests

During a physical examination, the doctor may identify an enlarged liver and an enlarged spleen. Malaria blood smears taken at 6 to 12 hour intervals confirm the diagnosis.

Treatment

Anti-malarial drugs can be prescribed to people traveling to areas where malaria is prevalent. It is important to see your health care provider well in advance of your departure, because treatment may begin as long as 2 weeks before entering the area, and continue for a month after leaving the area. The types of anti-malarial medications prescribed will depend on the drug-resistance patterns in the areas to be visited.

According to the CDC, travelers going to South America, Africa, the Indian subcontinent, Asia, and the South Pacific should take one of the following drugs: mefloquine, doxycycline, chloroquine, hydroxychloroquine, or Malarone.

Malarone is a relatively new anti-malarial drug in the U.S. and is a combination of atovaquone and proguanil. It may be recommended over the other drugs mentioned, depending on your destination and the possibility of mefloquine resistance.

It is very important to know the countries and areas you will be visiting to obtain appropriate preventive support for malaria.

FOR ACTIVE INFECTIONS

Malaria, especially Falciparum malaria, is a medical emergency requiring hospitalization. Chloroquine is a frequently used anti-malarial medication, but quinidine or quinine, or the combination of pyrimethamine and sulfadoxine, are given for chloroquine-resistant infections.

Expectations (prognosis)

The outcome is expected to be good in most cases of malaria with treatment, but poor in Falciparum infection with complications.

Complications

Liver failure and kidney failure

Destruction of blood cells (hemolytic anemia)

Meningitis

Rupture of the spleen and subsequent massive hemorrhage

Calling your health care provider

Call your health care provider if you develop fever and headache after visiting the tropics.
Your browser may not support display of this image. Reviewer Info: Arnold L. Lentnek, MD, Division of Infectious Disease, Kennestone Hospital, Marietta, GA. Review provided by VeriMed Healthcare Network.; ADAM Health Illustrated Encyclopedia,

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There are about 2,700 species of mosquitoes, including more than 90 species of Anopheles mosquitoes, many of which carry malaria. Anopheles mosquitoes typically bite between dusk and dawn. Only female mosquitoes suck blood and, thus, spread malaria. This is the female Anopheles gambiae mosquito seen up close and personal under an electron micro-scope.
© 1998 SPL/Dr. Tony Brain, Science Photo Library/Custom Medical Stock Photo.