Monthly Archives: April 2010

Silencing human RSV infection

A new therapy based on RNA interference (RNAi) successfully reduces respiratory syncytial virus (RSV) infection in humans. The study, published free online in PNAS, demonstrates that therapeutic RNAi-based drugs are clinically effective, and suggests that similar ‘silencing’ therapies could be useful against other respiratory pathogens.

RSV is an RNA virus that infects the lungs to cause respiratory tract infections—especially in the lower respiratory tract. RSV infection can be severe in immunocompromised patients, the elderly and young children; in the US alone, the virus has a ten times higher mortality rate in young children than the influenza virus and is the most common cause of infant hospitalisation. No vaccine for RSV exists, and the only approved drug treatment, ribavirin, has limited use and effectiveness. Current treatment strategies for RSV infection are only supportive—namely oxygen and fluids until the infection naturally resolves. Previous work has shown that a small interfering RNA (siRNA) drug—called ALN-RSV01—effectively silences a RSV protein that is critical for virus replication, and has a considerable antiviral effect in a mouse model of RSV infection. Evidence for the clinical effectiveness of siRNA drugs to treat disease in humans is, however, lacking.

DeVincenzo and colleagues tested whether ALN-RSV01 was an effective antiviral drug in adult, human volunteers who were infected with wild-type RSV. The investigators enrolled 88 healthy participants into a double-blind, placebo-controlled trial. They randomly assigned the volunteers to receive a nasal spray containing either ALN-RSV01 or saline as a placebo control. This nasal spray was administered 2 days prior to, and 3 days after, inoculation with RSV. They found that treatment with the siRNA nasal spray decreased the number of people infected with RSV by 38%, with the greatest reduction in people’s symptoms between 4–7 days after RSV inoculation. This antiviral effect was not related to the concentration of proinflammatory cytokines (such as tumor necrosis factor and interferon α) in the nose or whether the participants had pre-existing antibodies against RSV. Furthermore, the scientists showed that intranasal ALN-RSV01 was well tolerated and safe to use in humans.

DeVincenzo et al. argue that their findings represent a “significant advance in the development of human therapies…[and] a definitive demonstration in humans of an RNAi effect using a synthetic siRNA.” More clinical trials are needed to determine whether intranasal ALN-RSV01 can reduce RSV infection in children and adults that are naturally infected by the virus, and to determine the optimal dose and frequency for ALN-RSV01 administration that produces the best antiviral effect and clinical outcome. The results from this study also demonstrate a “broader potential” for “locally delivered siRNAs as unique anti-infective drugs against other respiratory pathogens.”

ResearchBlogging.orgDeVincenzo, J., Lambkin-Williams, R., Wilkinson, T., Cehelsky, J., Nochur, S., Walsh, E., Meyers, R., Gollob, J., & Vaishnaw, A. (2010). A randomized, double-blind, placebo-controlled study of an RNAi-based therapy directed against respiratory syncytial virus Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0912186107

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A gene polymorphism helps protect against malaria, but makes you vulnerable to lupus

A polymorphism in the human gene FCGR2B is associated with susceptibility to systemic lupus erythematosus (SLE), but it is also associated with protection against malaria, according to a new study published in PNAS. The polymorphism was most common in people of Southeast Asian and African origin (i.e. populations from areas endemic for malaria), implying that its protective effects against malaria could provide a survival advantage and explain its prevalence in these populations despite the increased risk of SLE.

Systemic lupus erythematosus is a chronic and systemic autoimmune disorder whilst malaria is an infectious disease caused by Plasmodium parasites. These two disparate diseases are in fact linked by their association with the gene FCGR2B, which is involved in immune regulation and encodes the Fc gamma receptor IIb. A single nucleotide change in FCGR2B, termed rs1050501, codes for a threonine instead of isoleucine in the FCGR2B protein, which in turn leads to a non-functional Fc gamma receptor IIb (FcγRIIbT232) that has been shown to be important for resistance against Plasmodium. Furthermore, rs1050501 had already been associated with SLE and the frequency of this allele was found to vary between people of different ethnicities—it is more common in Southeast Asians or East Africans than Caucasians.

Lisa. C. Willcocks and colleagues analysed the genotypes of 819 people with SLE from Hong Kong compared with 1,026 ethnically matched controls, and 326 Caucasian patients with SLE compared with 1,296 controls in the “largest study of this FCGR2B SNP in SLE performed so far.” They found that SLE was strongly associated with FcγRIIbT232 in both ethnic groups. Next, the investigators genotyped rs1050501 in children from an area in Kenya endemic for malaria—they analysed control children as well as children who had suffered from either repeated episodes of mild malaria or a severe form of malaria. They found children homozygous for FcγRIIbT232 (the rs1050501 allele was present on each chromosome pair) were protected against severe malaria. In contrast, the same allele did not protect against bacterial infection.

Willcocks et al. state that “the high mortality from malaria has resulted in the strongest known force for evolutionary selection in the recent history of the human genome.” They argue that the protective effects of FcγRIIbT232 against malaria could explain why the mutant receptor is most common in Africans and Southeast Asians; the frequency of the rs1050501 allele, like sickle-cell disease and thalassemia, being related to malarial endemicity. “Malaria seems to have driven retention of a polymorphism predisposing to a polygenic autoimmune disease, and this may begin to explain the ethnic differences seen in frequency of that disease,” write the authors.

ResearchBlogging.orgWillcocks, L., Carr, E., Niederer, H., Rayner, T., Williams, T., Yang, W., Scott, J., Urban, B., Peshu, N., Vyse, T., Lau, Y., Lyons, P., & Smith, K. (2010). A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0915133107

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Defence against cytosolic pathogens…it’s all in the AIM2 baby

The DNA sensor AIM2 is crucial for host defence against cytosolic pathogens and DNA viruses; it not only senses these intracellular intruders but also promotes inflammatory mechanisms as part of our innate immune system.

In a research double whammy, Nature Immunology has published two studies online—Rathinam et al. and Fernandes-Alnemri et al.—that both discuss how the AIM2 inflammasome is essential for innate immunity against cytosolic pathogens.

The body’s innate immune system is the first line of defence against invading pathogens, it acts non-specifically and immediately in response to any infection but, unlike adaptive immunity, innate immunity is not long-lasting. Inflammasomes are multiprotein complexes that activate inflammatory processes (including the inflammatory cytokines IL-1β and IL-18) and pyroptosis (a type of programmed cell death that occurs during inflammation) that helps our body resolve an infection.

Both sets of researchers took advantage of AIM2-/- knockout mice to elucidate the role of AIM2 in innate immunity to cytosolic pathogens. First off, Rathinam and colleagues generated and bred mice that were deficient in AIM2. By comparing macrophages from AIM2-/- mice to macrophages from mice that expressed AIM2 they showed that AIM2 senses dsDNA in the host cell cytosol and is essential for the activation of the inflammasome—AIM2 was important for the caspase 1-dependent maturation of inflammatory cytokines (IL-1β and IL-18) and for regulating pyroptosis. The scientists then focused their efforts on whether AIM2 was important for the inflammatory response to microbial pathogens. Loss of AIM2 led to loss of inflammasome activation; macrophages lacking AIM2 had reduced caspase-1 activation and IL-1β in response to a number of intracellular microbes including Francisella tularensis, the vaccinia virus and the mouse cytomegalovirus. Finally, they showed in vivo that AIM2 was essential for the early control of cytomegalovirus infection in mice. This AIM-2 dependent response led to the production of IL-18 and natural killer cell-dependent production of IFNγ, which successfully decreased the viral load AIM+/+ mice.

Fernandes-Alnemri and colleagues focused their research efforts on understanding the importance of AIM2 in the innate immunity to a specific pathogen—Francisella tularensis, a highly infectious bacterium that causes tularemia, a disease that is high up on the biological warfare list. Again the researchers generated and bred AIM2-/- knockout mice and found that AIM2 was important for detecting cytosolic DNA and activating caspase-1 and inflammasome production. When comparing AIM2-/- and AIM2+/+ macrophages, the investigators found that AIM2 was required for sensing F. tularensis and subsequent activation of caspase-1 and proinflammatory responses, including IL-1β production and induction of pyroptotic cell death. On a mechanistic level, they found that activation of the AIM2 inflammasome by F. tularensis required depleted intracellular postassium levels, lysosomal acidification and IRF3 signalling (an intact type I interferon response). Finally, the scientists showed that AIM2-deficient mice were overwhelmed by infection with F. tularensis; all AIM2-/- mice infected with F. tularensis were dead 5 days after they were infected with the bacteria whilst 77% of wild-type mice were still alive at this timepoint and in fact 66% of AIM2+/+ mice survived for more than 20 days after F. tularensis infection. Moreover, mice lacking AIM2 had a higher bacterial burden in both their liver and spleen, and decreased serum levels of IL-18, compared with wild-type mice.

Both Rathinam et al. and Fernandes-Alnemri et al. conclude that AIM2 has an important role in the innate immune response to intracellular pathogens, and that future research on the AIM2 inflammasome could determine whether it also has a role in nucleic-acid dependent autoinflammatory and autoimmune diseases such as systemic lupus erythematosus.

ResearchBlogging.orgRathinam, V., Jiang, Z., Waggoner, S., Sharma, S., Cole, L., Waggoner, L., Vanaja, S., Monks, B., Ganesan, S., Latz, E., Hornung, V., Vogel, S., Szomolanyi-Tsuda, E., & Fitzgerald, K. (2010). The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses Nature Immunology DOI: 10.1038/ni.1864
Fernandes-Alnemri, T., Yu, J., Juliana, C., Solorzano, L., Kang, S., Wu, J., Datta, P., McCormick, M., Huang, L., McDermott, E., Eisenlohr, L., Landel, C., & Alnemri, E. (2010). The AIM2 inflammasome is critical for innate immunity to Francisella tularensis Nature Immunology DOI: 10.1038/ni.1859

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Sleeping sickness—a new cure for a neglected disease?

Scientists have validated a new drug target, the Trypanosoma brucei enzyme N-myristoyltransferase, in the fight against sleeping sickness, and have already identified and tested an inhibitor against this enzyme that successfully cures T. brucei infection in mice.

The study, published in the journal Nature, provides a much-needed boost to research into neglected tropical diseases, which are often associated with poverty.

Image provided by Wikipedia

Sleeping sickness—also known as African trypanosomiasis—is a disease caused by by the parasite T. brucei, which itself is transmitted to humans via the tsetse fly. African trypanosomiasis is endemic in regions of Sub-Saharan Africa and WHO estimates suggest that 50,000-70,000 people are currently infected with the parasite and 30,000 people die from the infection every year. The disease can cause significant morbidity and mortality and consists of two stages; stage 1, where parasites are present in the blood, lymph and interstitial fluid, and the more serious stage 2, with parasites present in the central nervous system (CNS). Without treatment, the severe neurological symptoms of the disease—confusion, extreme fatigue and sleep cycle disturbances—can ultimately lead to an irreversible and progressive mental decline ending in coma and death.

The few treatments available in our arsenal against African trypanosomiasis are out-dated and can have poor efficacy and serious side effects. Previous work has already proposed that N-myristoyltransferase is a potential target for the treatment of parasitics diseases, including African trypanosomiasis. This enzyme adds myristate (a common saturated fatty acid) to many eukaryotic and microbial proteins, a process which is required for their biological activity. Julie Frearson and colleagues, have now made a breakthrough in the discovery and development of effective, low toxicity drugs to treat sleeping sickness by investigating compounds that affect T. brucei N-myristoyltransferase.

The researchers first screened a library of 62,000 lead-based compounds to test their effectiveness at inhibiting N-myristoyltrasnferease and in preventing proliferation of the blood stage form of T. brucei. They found one compound—DDD85646—was very potent at inhibiting myristoylation and trypanosome growth during in vitro tests. The scientists then tested the efficacy of this compound in animal models of trypanosomiasis. They found that DDD85646 was well-tolerated and effectively cured acute trypanosomiasis in mice. Furthermore, DDD85646 is trypanocidal—it rapidly killed trypanosomes in both in vitro and in vivo assays. Finally, the investigators confirmed that DDD85646 truly acts “on target” against the T. brucei N-myristoyltransferase, and they also characterised the peptide pocket in which the inhibitor binds the target enzyme.

A possible drawback in using an inhibitor against N-myristoyltransferase as a trypanocidal drug is that humans also produce this enzyme. More research is needed into the inhibitor DDD85646 to improve its selectivity (ensuring that it is specific only for ­N-myristoyltransferase produced by trypanosomes) and to determine whether it can also penetrate the CNS and effectively kill parasites during the late-stage of trypanosomiasis. Crucially, clinical trials will be needed to ensure the compound is safe to use in humans. Only then will any future drugs for sleeping sickness, based on this research by Frearson et al., be seriously considered for production by big pharmaceutical companies.

ResearchBlogging.orgFrearson, J., Brand, S., McElroy, S., Cleghorn, L., Smid, O., Stojanovski, L., Price, H., Guther, M., Torrie, L., Robinson, D., Hallyburton, I., Mpamhanga, C., Brannigan, J., Wilkinson, A., Hodgkinson, M., Hui, R., Qiu, W., Raimi, O., van Aalten, D., Brenk, R., Gilbert, I., Read, K., Fairlamb, A., Ferguson, M., Smith, D., & Wyatt, P. (2010). N-myristoyltransferase inhibitors as new leads to treat sleeping sickness Nature, 464 (7289), 728-732 DOI: 10.1038/nature08893

*see also African trypanosomes just love social networking

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