A new blood test has been developed by a team of researchers from the ETH Zurich and Swiss Tropical Institute, led by Peter Seerberger that enables the detection of circulating antibodies against the toxic sugar molecules known as glycosylphosphatidylinositol (GPIs) presented on the surface of the malaria pathogen.
Malaria is transmitted via the bites of infected mosquitoes, by a parasite called Plasmodium. The infection is initiated when Plasmodium sporozoites are injected through the saliva of the feeding mosquito, these are then carried in the circulatory system to the liver and invade hepatocytes. Through a complex life cycle, the intracellular Plasmodium parasite eventually invades the erythrocytes where another round of replication is initiated. It is this stage that is responsible for the pathology associated with malaria.
Symptoms of the disease include fever, headache and vomiting, usually appearing between 10 and 15 days after the mosquito bite. If not treated, malaria can rapidly become life threatening by disrupting blood supply to the vital organs.
Every year, more than 500 million people become severely ill with malaria, and approximately 40% of the world’s population is at risk of infection. Most cases are in sub-Saharan Africa, although Asia, South America, the Middle East and some parts of Europe are also affected.
Following many years of work to develop a sugar-based vaccine, this latest chip-based test demonstrates the ability of the malaria pathogen to trigger a specific immune response in adults. The chip has been developed by Faustin Kamena, a post-doctor in Seeberger’s laboratory, for the detection of specific antibodies against various GPIs. The malarial glycans are produced synthetically and in large amounts in the laboratory. In total, 64 pads, each containing several tiny clusters of different GPIs in varying concentrations, are spotted onto a glass slide. Only a minute quantity of blood serum from the patient is administered onto the chip, and antibodies specifically bind to complementary GPI molecules. Dyes then unveil the GPIs to which antibodies have bound.
In regions of Africa where malaria is endemic, blood tests from the general adult population have revealed the presence of specific antibodies against particular GPIs. Upon infection, although the side effects are not entirely eliminated, they are greatly reduced. Europeans, on the other hand, lack these anti-GPI antibodies and, upon infection, the number of circulating antibodies greatly increases, generating a positive correlation between the amount of antibodies and protection against the disease.
Information obtained from this chip is enabling scientists to produce specific GPI molecules that can be recognized by the immune system, and the natural resistance that appears to be present in individuals within endemic malarial regions is crucial to developing a sugar-based malaria vaccine.
Infants under the age of 5 years are the prime targets of malaria infection, due to their inability to develop antibodies to the malaria pathogen’s toxic GPI molecules. Seeberger states: “This evidence is another important step towards finding a malaria vaccine because we now know which antibodies protect adults.”
Sources: Kamena F, Tamborrini M, Liu X et al. Synthetic GPI array to study antitoxoc malaria response. Nat. Chem. Biol. 4(4), 238–240 (2008); World Health Organisation www.who.int/topics/malaria/en
More obstacles for inhaled protein therapeutics
Joining Pfizer’s product Exubera®, Novo Nordisk has discontinued the development of its fast-acting inhaled insulin product, AERx iDMS, which was in Phase III development.
Exubera was set to revolutionize diabetes care through the easier management of patients’ blood sugar levels. Its market failure stemmed from the fact that it was impractical to use; patients still required an injection of longer-acting insulin to maintain a basal level, blood sugar levels still had to be measured and periodical lung function tests were mandatory.
Various companies, including MannKind and Alkermes are continuing to develop inhaled insulin products.
One of the greatest challenges to using the lungs as a delivery route for peptides and protein-based drugs is dosage unreliability. Peter Byron, an aerosol drug delivery systems expert at the Virginia Commonwealth University School of Pharmacy, VA, USA, states that, “many simple devices deliver rather more to the mouth than the lung… [and] people have different sized mouths and throats” resulting in variable dosage delivery.
Developers are now designing more effective inhalers, including powders, crystals and liquids. Alkermes is developing a powdered insulin inhaler where the particles are large, with low density. MannKind’s product, Technosphere, uses an absorption enhancer, although its potentially toxic effects have yet to be fully established.
Abnormalities in lung function as a result of these inhalers continue to raise concerns. Data revealed that Exubera did cause some lung impairment, indicating that one of the greatest challenges faced by novel drug delivery systems are the potentially harmful biological side effects on the lung or other therapeutic target organs.
Source: Opar A. Another blow for inhaled protein therapeutics. Nat. Rev. Drug Discov. 7, 189–190 (2008)
New Alzheimer’s disease protein target
Recent research carried out by scientists at the Scripps Research Institute has unveiled a new protein candidate that may serve a preventive role in the development of Alzheimer’s disease (AD).
Alzheimer’s disease is a degenerative brain condition characterized by a progressive decline in memory, thinking, comprehension, calculation, language, learning capacity and judgment. Although AD can occur at any age (even as young as 40 years), its occurrence is much more common in the aging population. There is an exponential increase in the rate of incidence with age, resulting in very few affected individuals between the ages of 40 and 50 years, an increased incidence in individuals aged between 60 and 65 years, and a high incidence among individuals over 80 years.
A large number of neurodegenerative disorders are associated with the misfolding of proteins, suggesting that neurons are particularly sensitive to the pathogenic effects of aggregates of misfolded proteins. Various mutations in the gene that encodes transthyretin (TTR) lead to the production of misfolded proteins forming insoluble aggregates that deposit in the heart, causing amyloidosis and subsequent heart failure. This same process occurs in the brain of AD individuals; the proteins that stick together are amyloid-β (Ab). The TTR protein is produced in the liver and functions in transporting vitamin A and the thyroid hormone through the blood stream. It is rarely associated with brain tissue, although studies have found that it can bind with Ab.
The study, led by Joel Buxbaum, investigated the effects of varied levels of the TTR protein on an AD mouse model. Transgenic APP23 mice carry a gene encoding a dominant Alzheimer’s mutation. These mice develop brain plaques and suffer from cognitive deficits. When bred with mice that overexpressed the human TTR protein (hTTR), a substantial improvement in their cognitive function was observed. Buxbaum states that, “Carrying the hTTR gene was associated with a less severe AD-like brain pathology and smaller amounts of Ab compared with what was found in the brains of control APP23 mice.” This can be explained by the assumption that the “hTTR protein bound to Ab either before or when it was most toxic.”
APP23 mice were also bred with mice missing both copies of their TTR genes (mTTR) and hence lacking any endogenous TTR protein. In these mice, increased Ab deposition was observed. Furthermore, in contrast to the usual age of development of AD in mice, which is 9–10 months, mice in this experiment developed the disease at approximately 5 months. A lack of the natural TTR protein in these mice resulted in an earlier onset of AD with increased severity.
Buxbaum and colleagues have concluded that the TTR protein binds Ab “In a manner that prevents both toxicity and plaque formation… by interfering with aggregation of the [form] of Ab that is most likely to stick together and causing neurological and behavioral deficits…” It is possible that a similar phenomenon may be occurring in the human brain. In later life, it may be that the levels of the TTR protein are insufficient to inhibit aggregate formation, and this opens up the gateway to new potential therapeutic targets. It may be that increasing TTR production in the human brain, or supplementing the fall in TTR levels is the key to developing novel treatments in the prevention or deceleration of AD.
Source: Buxbaum JN, Ye Z, Reixach N et al. Transthyretin protects Alzheimer’s mice from the behavioral and biochemical effects of Aβ toxicity. Proc. Natl Acad. Sci. USA 105(7), 2681–2686 (2008).
Use of Dymension 2D-gel analysis software in analysis of antiaddictive alkaloid treatment
Dymesion is a powerful software for the analysis of 2D protein gels. Its manufacturers, Syngene (Cambridge, UK), recently announced that it is being used at the Open Mind Institute (OMI) in Slovenia to determine the effect that the antiaddictive alkaloid ibogaine has on the brain. The OMI is a new center where research is focused around effects of medicinal plants.
In collaboration with scientists at the University of Ljubljana, Slovenia, researchers at the OMI are using Dymension to ascertain any postibogaine treatment differences in silver-stained rat brain proteins run on 2D gels. The protein profiles generated are used to determine which proteins are up- or down-regulated, potentially providing a greater understanding of the pharmacodynamics of antiaddiction therapies.
The director of the OMI, Roman Paskulin states, “We are relatively new to proteomic analysis so we chose to install Dymension software because we want to rapidly perform complicated analysis [and this software package] was visually easier to navigate and simpler for everyone to use.” He also added: “Using Dymesnion to analyze our 2D-gel images in association with MALDI-TOF on the resulting protein spots, we have seen differences in four metabolic enzymes after ibogaine treatment and are now looking at the significance of these findings.”
This software has proven to facilitate the rapid analysis of complex proteomic data. Scientists at the OMI have published a paper on their work already and Syngene’s Divisional manager, Laura Sullivan, states that this is “Testament to how quick and simple it is to integrate and work with Dymension software in any proteomics project.”
Source: Syngene www.syngene.com
A step closer to developing toxin-specific viper antivenoms
Snakes of the viper family are one of the most deadly species of snake and are found in the majority of countries around the world. They are quick to strike, and use their long-hinged fangs to inject their venom deep into the flesh. Although the bites are extremely painful, they are not necessarily fatal, particularly when prompt treatment is administered to the victim.
The viper species Vipera ammodytes ammodytes is native to Croatia, Slovenia, Serbia and western Bulgaria, whereas Vipera ammodytes meridionalis is found predominantly in Greece, Turkey, Albania, Romania and eastern Bulgaria. The venoms of both these subspecies were studied by Christian Betzel from the University of Hamburg, with colleagues affiliated to the University Medical Centre Hamburg-Eppendorf, the Helmoltz Centre for Environmental Research-UFZ in Leipzig and the Bulgarian Academy of Sciences.
Venoms were first fractionated by fast liquid protein chromatography (FPLC) on an anion-exchange column. In both venoms, proteins not retained on the columns were collected separately and subjected to SDS-PAGE. This fraction contained L-amino acid oxidases, toxins involved in envenomation, with the V. a. meridionalis fraction also containing ammodyoxin I2, a basic phospholipase A2 (PLA2) – a known neurotoxin. The other FPLC factions were collected separately, and those displaying PLA2 activity were subjected to a further FPLC step with a second anion exchange column. The resulting fractions were subjected to SDS-PAGE. Isolated protein spots were excised from gels and the proteins were digested with trypsin and analyzed by LC/tandem MS. Protein identification was carried out using the GenBank sequence database at NCBInr.
Results showed that although there were some similarities between the two venoms, there also existed some significant differences. Both contained the strong neurotoxin vipoxin, which is a complex between a toxic base PLA2 and a nontoxic acidic PLA2-like protein. The subspecies V. a. meridionalis also contained a monomeric catalytically active PLA2, in which the 20 N-terminal amino acids were identical to those of the nontoxic vipoxin acidic component. Scientists proclaim that this supports the theory of evolution of the toxic and enzymatic functions into the nontoxic inhibitory functions.
V. a. ammodytes venom proved to be the more complex and was composed of 139 proteins compared with the 104 proteins found in V. a. meridionalis. Shared protein families between the two subspecies were serine proteases, monomeric and dimeric Group II PLA2s, Group I, II and III metalloproteinases, cysteine-rich secretory proteins, disintegrin and growth factors.
Understanding the composition of the difference venoms is critical in the development of toxin-specific antivenoms, where the major toxins can be used as antigens. The properties of the enzymes extracted from the viper venom in these experiments account for some of the effects observed, such as breathing difficulties, clotting disorders, hemorrhage, apoptosis and paralysis. In contrast to the antivenoms that are currently available and overpotent, these targeted antivenoms should elicit fewer side effects.
Source: Georgieva D, Risch M, Kardas A, Buck F, von Bergen M, Betzel C. Comparative analysis of the venom proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis.J. Proteome Res. 7(3), 866–886 (2008).