Control of cytokine production in vivo

Last week I attended a talk by Dr. Markus Mohrs where he introduced the idea of a dual-reporter mouse model that they had developed some years ago to measure cytokine production in vivo. It fascinated me so much that I decided to go through his research a bit and read about their exciting findings, especially because they used parasites such as Heligmosomoides polygyrus, Toxoplasma gondii and Schistosoma mansoni as their infection models.

Th2 immune response is highlighted by IL-4 production. But when looking in vivo, it is impossible to find the source of the IL-4 that is present in the biological fluids. So in their study published in Immunity in 2005, the authors used an intestinal infection by nematode  H. polygyrus  as their model. This worm resides solely in the intestine and induces a robust Th2 response. they engineered a GFP sequence preceded by  an internal ribosome entry site (IRES) after the final exon of the IL-4 gene. In this way, when the IL-4 mRNA gets transcribed, the ribosome can bind separately to the IRES and translate GFP. Therefore, cells that are producing IL-4 mRNA could be identified. What they surprisingly found was that, CD4+ GFP+ T cells were not always producing IL-4 unless secondarily stimulated. So, they replaced the IL-4 allele on the other chromosome with a Human CD2 gene. Presence of huCD2 on cell surface would now be indicative of IL-4 production.

They therefore showed that CD4+ Tcells could be either ”IL-4 competent” or ”IL-4 producing”. They observe a very strong post-transcriptional control and selective secretion of IL-4 . Various types of cells start producing IL-4 mRNA but only select ones in certain areas actually produce the cytokine after receiving the proper secondary signal. This idea was tried on the key Th1 cytokine IFN-gamma in another study using influenza virus and Toxoplasma as infection models and similar results were found. Interestingly, the iconic pro-inflammatory cytokine IL-1beta also takes a similar route, but in this case, both control levels are post-translational: First signal induces pro-IL-1beta production and second signal induces its maturation via the inflammasome complex. These key cytokines have receptors on plenty of cells all through the body and their aberrant release can cause serious problems, no wonder multiple controls levels are set for their release.  Studies of this kind should really alert us on interpretation of microarray or qRT-PCR data.

Mohrs K, Wakil AE, Killeen N, Locksley RM, & Mohrs M (2005). A two-step process for cytokine production revealed by IL-4 dual-reporter mice. Immunity, 23 (4), 419-29 PMID: 16226507
ResearchBlogging.org

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Parasites to help fight cancer

Posted by Kasra

Research on parasites is important, even if most of them are not direct health concerns to the developed world! Millions of years of coevolution of parasites along with their hosts have made them masters in manipulation of the immune system and in coexistence with it. Many parasitic infections such as those with Toxoplasma, Trypanosoma and some Leishmania sp. elicit innate and adaptive immune responses that can result in life-long immunity to reinfection. However, the parasite might be carried chronically for life, keeping the antibody titers up.

The research team of Ricardo Gazzinelli have taken advantage of the stealth yet stimulatory property of parasites to target cancer. For their purpose, they expressed a cancer antigen in a strongly attenuated strain of Trypanosoma cruzi, the causative agent of Chagas disease in the Americas. This live vaccine showed great protection against melanoma in both prophylactic and therapeutic models.

T. cruzi was chosen as the vector for many reasons: it intrinsically possesses TLR ligands and thus induces a strong proinflammatory response; like many other parasites, it has ways of staying inside the body for a very long time; and it propagates inside the cytoplasm, therefore it can induce a Th1 type immune response and activation of cytotoxic CD8+ T cells, which are very important against cancer. It is interesting to me that although they did not observe any disease or parasitemia, such strong immune response and protection was observed. I think it is important to see if and how many residual parasites are sticking around and where and how long do they stay in the body. This might also teach us more about how this model actually works.

The study shows that if vaccinated before tumor induction, mice are completely protected from cancer. It is also shown that this antigen-carrying vector can delay tumor growth and lethality if given after. One might ask: what advantages does this vector provide over vaccination with traditional recombinant protein, or other vectors such as attenuated viruses? The authors compared the protection and immune response resulted from recombinant T. cruzi to the canonical recombinant antigen (NY-ESO1) with Alum or CpG adjuvants. Recombinant T. cruzi showed better protection and stronger immune response compared to canonical vaccination strategies. Doing a quick search through pubmed, I didn’t find other strategies such live or viral vectors rather than variations of recombinant protein being used so far. I might be wrong, but still this is a novel idea and this recombinant parasite deserves a chance of being further studied, especially for prophylactic uses. There are plenty of other cancer or maybe even infectious antigens that can be targeted by this method. But of course, there are plenty of biohazard issues that also need to be addressed with injecting an attenuated form a dangerous pathogenic parasite.

Here is the link to the article abstract on Pubmed.

Junqueira C, Santos LI, Galvão-Filho B, Teixeira SM, Rodrigues FG, Darocha WD, Chiari E, Jungbluth AA, Ritter G, Gnjatic S, Old LJ, & Gazzinelli RT (2011). Trypanosoma cruzi as an effective cancer antigen delivery vector. Proceedings of the National Academy of Sciences of the United States of America, 108 (49), 19695-700 PMID: 22114198

ResearchBlogging.org

In silico prediction of the human-malaria parasite interactome

Posted by Kasra

In silico prediction of protein-protein interactions within a species is an advancing field. Especially now that relatively large amounts of empirical data are available for training and validation, more and more in silico methods are being presented. However, as a host-parasite interactions enthusiast, I always had the question if the interactions between the host and pathogen proteins can be predicted. Although already done a few times for viruses such as HSV, HCV, HIV and Influenza, creation of an empirical inter-species interactome is not an easy and always affordable task. Still having an interactome database provides very valuable data in host-pathogen research. It not only reveals systemic overviews about the nature of the interaction occuring, but it can also open doors for more accessible and feasible research by suggesting a shorter list of proteins, pathways or interactions to focus on.

With this in mind, I had great pleasure in reading this single author paper by Stefan Wuchty published recently in PloS ONE that provides a computational interactome of Plasmodium falciparum and Homo sapiens. This paper actually led me to a whole body of research that has been done on experimental and computational determination of host-pathogen interactomes, albeit mostly on viruses.

In order to map P. falciparumH. sapiens interactome, Wuchty used experimentally determined host-parasite interactions plus orthologous protein groups between the two species as starting point. The false-positives were removed with various filtering methods that are beyond the reach of my knowledge of mathematics and informatics. Biological criteria such as co-expression of the interacting proteins in the host cell and specific parasite phase were also required.  Interstingly, the pattern of interactions he found, showed similarity with what was already observed with viral pathogens. In order to take control of the cell, intracellular pathogens appear to attack the host both at the protein as well as the pathway level. Hub proteins – proteins that interact with many other proteins and are envolved in multiple metabolic/siganling pathways – have been found to be an attack target not only by P. falciparum but also other pathogens. In addition, Wutchy saw that a relatively small number of human proteins interact with a big number of parasite proteins, suggesting that the parasite utilizes all it has got to take over the key proteins of the host.

This study and other works of the same style certainly provide precious knowledge about host-parasite interactions both in terms of systems biology and also hints for hands on research. I look forward to seeing these interactomes created and expanded for other pathogens and also their experimental validation and usage. Furthermore, an online database of these host-parasite interactomes would definitely make them more accessible.

 

Wuchty S (2011). Computational Prediction of Host-Parasite Protein Interactions between P. falciparum and H. sapiens. PloS one, 6 (11) PMID: 22114664

ResearchBlogging.org

Blood invasion of Plasmodium falciparum is dependent on a single receptor on the surface of red blood cells

Posted by Kasra

Plasmodium falciparum parasites invade different groups of cells during their life cycle. Upon injection into humans, sporozoites pass through the skin and travel in the blood to be picked up by hepatocytes. After completion of the liver phase, merozoites come back to the blood and invade red blood cells. Finally, there is another sort of invasion happening inside the midgut of the mosquito, different from the vertebrate host. The invasion of red blood cells by merozoites is the most accessible of the three for studying. Of great scientific and theraputic interest are the proteins that allow for this binding and internalization of the parasites to occur. Blocking the blood stage of malaria would essentially abrogate the majority of its pathological complications.

Many surface proteins of Plasmodium and red blood cells had been previously proposed to be involved in this host-parasite interaction and binding. However, in almost all cases, great redundancy was shown in the protein-protein interactions; meaning that knocking out one surface protein or blocking one interaction would only replace it by another one. In some cases this would result in a change in the tendency of the Plasmodium parasites to bind mature vs. Immature red blood cells, but invasion would still happen. But now, recent work has found a definitive receptor for invasion of red blood cells by P. falciparum merozoites. The beauty of this work is not only in finally finding a ligand and receptor for this stage of P. falciparum life-cycle, but also in my opinion in making great use scientific knowledge already available for making this discovery. So here is the brief story for those of you who do not feel like reading this short yet elegant letter to Nature:

In search for a definitive receptor, Crosnier and colleagues decided to study a surface protein of P. falciparum that was found by another group to be essential for parasite growth: PfRh5. In order to find its binding protein, they went through the already published proteome of the red blood cell and picked up the secreted and surface expressed proteins. Using an ‘Avidity-based extracellular interaction screen’ (AVEXIS) they screened for binding of PfRh5 to recombinantly produced secreted or surface proteins of the red blood cell and luckily they got a single hit: Basigin or BSG. They then validated this direct interaction using Surface Plasmon Resonance and showed that the interaction occurs independently from glycosylations. Next, they showed that adding soluble BSG, blocking it by a specific antibody or shRNA knockdown inhibits invasion of red blood cells by all clinical and lab strains of P. falciparum. As I previously mentioned, inhibition of invasion was never seen before for any of the receptor-ligand pairs suggested.

Finding such a well-fit receptor for invasion of P. falciparum brings up an evident question: Are people with mutations in the bsg gene resistant to malaria? The authors found very few nonsynomymous single nucleotide polymorphisms (meaning SNPs that lead to a different protein sequence) in some populations in the databases. Blood donations from some of those SNP carriers showed actually resistance to P. falciparum invasion. Unfortunately, population genetics data is seriously lacking in areas afflicted with malaria, so whether this gene has been through positive selection or not cannot be determined at the moment. The authors are hopefull to be able to answer this question after some genome projects currently in progress in Africa are complete.

This ligand-receptor interaction appears to be specific to P. falciparum and other Plasmodium species have not been mentioned in the paper (I am pretty sure they have been checked). This makes in vivo drug testing difficult as P. falciparum is not used in the murine malaria infection model. Nontheless, P. falciparum is the most prevalent and lethal malaria-causing species. The discovery of BSG as a receptor for invasion opens many doors towards a generation of theraputics and prophylaxis, bringing us hopefully one step closer to its elimination.

Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC, & Wright GJ (2011). Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature, 480 (7378), 534-7 PMID: 22080952

ResearchBlogging.org


TWIP: The ultimate podcast about parasites

Posted by Kasra

It has been more than a year since my last post. Apparently doing science has taken over writing about science. I am going to try to put more frequent updates which means that I get to explore research being done on parasites again! I would also like to state again that parasite diary would gladly accept your diaries as well. May it be your own research, or work by somebody else that you find fascinating. It doesn’t need to be recently published either. I am pretty sure nobody has read everything about everything. Therefore, all stories about parasites (whether eukaryotic or not) can be exciting and informative to read. If you are interested in participating, send us your diaries to  parasitediary AT gmail DOT com and we will publish them under your name.

As a start to the new era of the parasite diary, I would like to introduce a podcast  that I think anyone with the slightest interest in parasites should not miss. This Week in Parasitism (TWIP)  is narrated by Dr. Vincent Racaniello and Dr. Dickson Despommier from Columbia University. This podcast teaches you  about ecology, physiology and behaviour of eukaryotic parasites and tells you stories that you have never heard before about their history and impact on human life.  Their enthusiasm  for research and for parasites pumps up your energy to continue doing your boring benchwork while listening! I need say no more. Check out TWIP and its sisters (or brothers?) TWIV (Virology) and TWIM (Microbiology).

P.S. The picture in the logo of TWIP is of the nematode Trichinella spiralsis sitting comfortably inside its nurse cell in the muscle tissue.