A new murine model of Giardia infection: linking pathogenesis to malnutrition

Posted by Kasra

Giardia is a very successful parasite.   It’s highly durable cysts enter the body via contaminated food. Once inside the small intestine, the cysts hatch and the trophozoites start swimming with their multiple flagella. They attach to the intestine’s surface and enjoy the nutrient rich environment of the small intestine.  Shortly after, they start producing cysts that leave the body via feces.  Their presence may or may not cause severe symptoms. In many cases people carrying Giardia in their body – thus shedding cysts –  might not feel anything. On the other hand, Giardia infection can lead to severe diarrhea that could stick around for weeks if untreated. Our immune system usually manages to control the infection and get rid of the parasite. But in any case, the parasite can come, live and go undetected.

Similar to many other parasitic diseases, since Giardia infection does not kill a significant number of its victims, is not rampant in industrialized countries and is more or less readily treatable, it is not funded and studied by many researchers. Unfortunately, despite unique biological features such as lack of mitochondria, presence of two nuclei and an anti-inflammatory host-pathogen interaction, Giardia remains largely understudied.  We don’t fully understand the host and pathogen factors that could lead to disease or just sub-clinical infection. Nor we know much about how Giardia gets detected by the immune system and what is the nature of the immune response that kicks the parasite out of the body.

A scanning electron micrograph of the surface of the small intestine of a gerbil infested with Giardia sp. protozoa. The intestinal epithelial surface is almost entirely obscured by the attached Giardia trophozoites. (Source Wikipedia)

One of the strong incentives for studying Giardia is its higher prevalence in children. Infectious diarrhea is still the most common of cause of child death (Cryptosporidium is another understudied parasite and causative agent of diarrhea in children, which I discussed in another post). A recent study by Bartelt et al. published in Journal of Clinical Investigation, presents a new model of Giardia infection, focusing on malnutrition and young age. They argue that many children in areas where Giardia infection is common are undernourished. This malnutrition could contribute to development of a persistent Giardia infection with severe symptoms rather than a shorter non-symptomatic infection. To study the effect of children malnutrition on Giardia infection, they set their model on 3-week old recently weaned (taken away from mother, eating solid food) mice. They show that although healthy mice are able to clear the infection, Giardia parasites manage to stay longer in the small intestine of malnourished mice and also cause more growth impairment. It can be thought that this is a vicious cycle, where infectious diarrhea causes further weakening of the individual and thus further difficulty in fighting the infection, leading to severe weight loss and persistence of the parasite. Interestingly, the load of parasite in the intestine remains unchanged when comparing healthy and malnurished mice. However, the authors describe their model by pointing to other differences such as immune response and small intestine pathology. More studies on this model can help us better understand and hopefully better treat Giardia infection in children.

Highlight in Nature reviews in Gastroenterology and Hepatology

Bartelt, L., Roche, J., Kolling, G., Bolick, D., Noronha, F., Naylor, C., Hoffman, P., Warren, C., Singer, S., & Guerrant, R. (2013). Persistent G. lamblia impairs growth in a murine malnutrition model Journal of Clinical Investigation, 123 (6), 2672-2684 DOI: 10.1172/JCI67294

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Exosomes derived from grapes communicate with intestinal stem cells

Posted by Kasra

I worked on exosomes for some time, so I have written about them and other secreted vesicles every now and then. I still try to follow up the research in the field and get excited with the new findings, methods and applications. Here is exciting work by by Ju et al.  where they study the interaction between grape (yes the fruit) exosomes with mouse intestinal cells.

They purified exosome-like vesicles from grapes that they bought from grocery stores. Given to mice, the exosomes appeared to be absorbed by intestinal stem cells present in the intestinal crypts. Interestingly, the authors show that picking up these vesicles induce the Wnt/β-Catenin pathway. Generally, activation of this pathway promotes proliferation of stem cells. Ju et al. show grape exosomes induce proliferation of stem cells by putting stem cells together with the vesicles ex vivo and looking at crypt formation in organoids. Finally, to provide a health-related application for their vesicles, the authors show that grape exosomes protect mice and delay death in a murine colitis model. This can be because the vesicles induce proliferation of the stem cells and thus enhance tissue regeneration to revert the damage caused by colitis.

Formation of crypts from a single intestinal stem cell ex vivo is quickened when exposed to grape exosomes. From Ju et al. Molecular Therpay 2013

Formation of crypts from a single intestinal stem cell ex vivo is quickened when exposed to grape exosomes. From Ju et al. 11 June 2013;doi: 0.1038/mt.2013.64

This study contains so many buzz-words, I could think of terrible ways by which it can be mis-interpretted by media: grapes heal gut disease, nano-particles in fruit protect against gut disease, … As the authors say in the first paragraphs of the paper, this study is a proof of concept and there is still a lot more to learn. Thankfully, they show enough evidence to tickle other scientists to look at application of plant-derived exosomes and exosome-like vesicles as means for drug delivery and therapy. For one thing, plants-derived products are available in higher abundance compared to their animal-derived counterparts and can be cheaper to purify in commercial quantities.  Also, humans have been exposed to them (maybe not in such high concentrations) for millions of years, so they are no strangers to the gut cells.

Ju S, Mu J, Dokland T, Zhuang X, Wang Q, Jiang H, Xiang X, Deng ZB, Wang B, Zhang L, Roth M, Welti R, Mobley J, Jun Y, Miller D, & Zhang HG (2013). Grape Exosome-like Nanoparticles Induce Intestinal Stem Cells and Protect Mice From DSS-Induced Colitis. Molecular therapy : the journal of the American Society of Gene Therapy PMID: 23752315

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Bacteriophages may protect us against pathogens

Posted by Kasra

Given the extremely large amount of bacteria in our gastrointestinal track, it is not surprising to think that the gut would be also swarming with pathogens of bacteria, that is bacteriophages as well. In their recent work published in PNAS, Barr et al. take a look at what impact these particles could have on the population of bacteria in mucosal surfaces and what could it mean for us. Their work actually turns out very interesting results.

Mucosal surfaces are the body’s points of contact  with the outside. Being highly populated with bacteria, they can be suitable points of infection as well. That is why they are heavily guarded with various immune barriers and mechanisms, both innate and adaptive. Barr et al. point to a possible mechanism of protection against infection which not innate nor adaptive. They start by comparing the amounts of bacteria and bacteriophages in different mucosal and non-mucosal surfaces in various mucus producing animals. They interestingly observe that the bacteriophage to bacteria ratio in mucosal sites is way larger than those in adjacent non-mucosal sites (from average about 40fold to average about 3fold). They verify this in both invertebrates and vertebrates and thus suggest that this could be a phenomenon in all mucus-producing metazoans.

Next, they point to a previous recent study by Minot et al. who had found immunoglobulin (Ig)-like domains in the total analyzed genome of human gut viruses (or so called human gut virome).  These domains that usually act as in recognition and binding (as an antibody would do); they show that the bacteriophages actually bind to mucus through these proteins.  Barr et al. also show that presence of bacteriophages on a mucosal surface significantly reduces Escherichia coli invasion in vitro.

bacteriophage

Model for how presence of bacteriophage on the mucosal surface can help in protection against bacterial infection. From Barr et al. PNAS 2013 PMID: 23690590

This is an incredible system where the benefit of the bacteriophages and their hosts actually match. It is not clear at this point whether the animal body would have to do something other than producing mucus to keep the bacteriophages where they are or that it is just enjoying this protection more or less free of charge.

Barr JJ, Auro R, Furlan M, Whiteson KL, Erb ML, Pogliano J, Stotland A, Wolkowicz R, Cutting AS, Doran KS, Salamon P, Youle M, & Rohwer F (2013). Bacteriophage adhering to mucus provide a non-host-derived immunity. Proceedings of the National Academy of Sciences of the United States of America PMID: 23690590

Minot S, Grunberg S, Wu GD, Lewis JD, & Bushman FD (2012). Hypervariable loci in the human gut virome. Proceedings of the National Academy of Sciences of the United States of America, 109 (10), 3962-6 PMID: 22355105

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Science is boring

Posted by Kasra:

I often think of how Richard Feynman so simply and truly describes the beauty or extra-beauty that science brings to everything around us. I believe what Feynman says is more or less obvious to scientists or people somehow involved in science. We enjoy asking questions and seeking answers that naturally lead to more questions. Then why is it that we can’t convey this feeling to more people? Why the general consensus is that “Science is boring?” Is it because it solves riddles and demystifies magic? Or is it because of the overused and misused jargon? Or mazybe it is because it replaces a preferred presumption with hard truth? With all the discoveries and advancements and technologies surrounding us, why would some people still think science is boring? Maybe science communication should not be just writing about cool stuff that happen in science, but also discussing why we find them cool and fascinating. Feynman elegantly describes the way a scientist would look at nature.

I tried to read only little bit about the interaction of pollinators and flowering plants and was overwhelmed with its complexity. At least one way that I find science fascinating is how it explains the complexity of nature by enhancing its details. It reveals and explains hidden patterns and interactions, at the end making it even more complex that it originally was, nevertheless more beautiful. Here is just a glimpse of this beauty, ultraviolet vision of butterflies and nectar guides of flowers.

Apoptosis, not so quiet after all

Posted by Kasra

Apoptosis has been conventionally regarded as a quiet and non-inflammatory event, compared to necrosis which results in release of alarmins and other danger signals inducing inflammation and immune cell recruitment. However, a recent report by Cullen et al. published in Molecular Cell suggests otherwise. They suggest that at least in one form of apoptosis, pro-inflammatory mediators are released by the apoptotic cells and can act as ‘find-me’ signals to the phagocytes to find and clear them.

Fas receptor or CD95 is among the famous apoptosis receptors. It is a member of the TNF receptor family and it induces apoptosis through Caspase-8 activation. Interestingly, Cullen et al. show that different cell types produce pro-inflammatory chemikones such as MCP-1, CXCL1 and MIP-2 when they go through apoptosis via Fas-pathway. They show that this chemokine release is NF-kappa-B mediated and independent of Caspase-8 activation. It is possible be that somewhere during the evolution, the apoptotic pathway cross-linked with the pro-inflammatory signaling pathway and found benefit in it. Accordingly, Cullen et al. show that the Fas-induced pro-inflammatory cytokine/chemokine production still occurs  even if the apoptotic pathway is inhibited showing that these pathways are separate.

Next the authors show that the supernatant from the apoptotic cells can induce migration of macrophages and neutrophils. They also pinpoint the responsible chemokine by depleting them one-by-one. They show that MCP-1 induces macrophage migration and IL-8 recruits neutrophils.

At this point it cannot be said really how inflammatory these apoptotic cells would be in vivo. There are parts of the body where apoptosis occurs constantly,  so this could potentially lead to an unwanted constant inflammation in those areas. Therefore, either different cells would have different levels of apoptotic-proinflammatory chemokine release, or local mechanisms would compensate and counteract the inflammation. More studies will help us understand how apoptotic cells can send their ‘find-me’ signals without causing too much turbulence in their tissue.

Schematic diagram of model proposed by Cullen et al. From

From Cullen et al. Molecular cell, 49 (6), 1034-48

Cullen SP, Henry CM, Kearney CJ, Logue SE, Feoktistova M, Tynan GA, Lavelle EC, Leverkus M, & Martin SJ (2013). Fas/CD95-induced chemokines can serve as “find-me” signals for apoptotic cells. Molecular cell, 49 (6), 1034-48 PMID: 23434371

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Cryptosporidium, the understudied killer

Diarrhea is the second major killer of children under the age of 5 in developing countries (second to pneumonia). We know much less than we should about the causative agents, severity, burden etc. of diarrhea in developing countries. Funded by Bill and Melinda Gates Foundation, A Global Enteric Multicenter Study (GEMS) picked up the task of learning more about diarrhea in children in developing countries with high incidence (Gambia, Mali, Mozambique, Kenya, India, Bangladesh and Pakistan)   and did an extensive 3 year-long case-control study. I won’t go to details about their magnificent work but just mention one rather surprising finding. They found the 4 top pathogens causing diarrhea in children under 5 to be: 1. Rotavirus 2. Cryptosporidium 3. Shigella 4. Enterotoxigenic Escherichia coli producing heat-stable toxin (ST-ETEC). Rotavirus has long been known as a major cause of diarrhea in children and there are effective vaccines against it who have significantly reduced its incidence in developed countries. Shigella and ST-ETEC were also previously known. But the high incidence of Cryptosporidium has come out as a surprise to everyone. Crypto is an apicomplexan protozoan parasite, kin to other famous parasites Plasmodium and Toxoplasma. It’s durable cysts are shed in the stool and can be ingested in contaminated food and water. Compared to the other top pathogens and with regards to the high mortality it is causing, Crypto is relatively unpopular and extremely understudied (even reflected in its Wikipedia page). Now GEMS calls for more research on Crypto and better therapies against it. Hopefully this would mean more funding for studying this bizarre parasite and more exciting knowledge learnt from its biology and pathophysiology. 

Find and share GEMS infographic about their findings from here.

Cryptosporidium trophozoite bound to the small intestine epithelium, inducing actin accumulation at its binding site

Cryptosporidium trophozoite bound to the small intestine epithelium, inducing actin accumulation at its binding site. From Elliott and Clarck, Infection and Immunity, 2000

Kotloff, K., Nataro, J., Blackwelder, W., Nasrin, D., Farag, T., Panchalingam, S., Wu, Y., Sow, S., Sur, D., Breiman, R., Faruque, A., Zaidi, A., Saha, D., Alonso, P., Tamboura, B., Sanogo, D., Onwuchekwa, U., Manna, B., Ramamurthy, T., Kanungo, S., Ochieng, J., Omore, R., Oundo, J., Hossain, A., Das, S., Ahmed, S., Qureshi, S., Quadri, F., Adegbola, R., Antonio, M., Hossain, M., Akinsola, A., Mandomando, I., Nhampossa, T., Acácio, S., Biswas, K., O’Reilly, C., Mintz, E., Berkeley, L., Muhsen, K., Sommerfelt, H., Robins-Browne, R., & Levine, M. (2013). Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study The Lancet DOI: 10.1016/S0140-6736(13)60844-2

Elliott, D., & Clark, D. (2000). Cryptosporidium parvum Induces Host Cell Actin Accumulation at the Host-Parasite Interface Infection and Immunity, 68 (4), 2315-2322 DOI: 10.1128/IAI.68.4.2315-2322.2000
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A systematic review in non-clinical research: a case of pathogen metabolites

Posted by Kasra

Doctors and scientists in the field of clinical research are well acquainted to systematic reviews and their importance in clinical research. The important difference between a normal review and a systematic review is that in the latter the authors make sure (or at least try very hard) to include and cover all the published research about the topic of review. Along with the review of the data, they should also publish the search strategy they used to make sure they get everything that has been published about their topic of study. Collecting all the data is extremely important especially when deciding about the beneficial effects of a certain drug, vaccine or public health intervention.  The Cochrane collaboration is a well-known organisation that collects and publishes systematic reviews in field of health research and health care.

Although they could be very useful in non-clinical research, systematic reviews are actually rarely written in these fields. During my graduate studies, I had to write a systematic review on innate receptors for a certain fungus. I realized then how diverse the experimental models are and how hard it is compare their controversial results due to small or big differences in experimental setup and strains used. Maybe that is why these papers are rare in non-clinical research. Still, no matter how hard, I was able to do it with as much time as a graduate student would put on a term paper and get a good grade for it ;). I am looking forward to reading more non-clinical systematic reviews.

Recent work of Bos et al. is an excellent example of how useful it could be to gather all the available data in a certain field, even if it is not all clinical trials. They point to most common abundant bacteria in sepsis Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), Enterococcus faecalis (EF), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), and Escherichia coli (EC). They argue that current strain detection methods are too slow and do not allow for efficient targeted antibiotic therapy. On the other hand, non-targeted therapy is not always successful. They argue that the unique and some-what well-identified metabolic pathways of these bacteria leads to production of certain volatile chemicals that are not produced by humans and could be used as rapid diagnostic markers. The diagram below shows the gram positive bacteria on the left and gram negative bacteria on the right, graphing unique and common volatile chemicals they produce. The blue circle in the center shows the chemicals produced by all bacteria. Therefore, their absence would exclude infection. The red (or pink as you may) circles highlight the unique products of each species which could help in targeted antibiotic therapy of sepsis.

Staphylococcus aureus (SA), Streptococcus pneumoniae (SP), Enterococcus faecalis (EF), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP), and Escherichia coli (EC)

Bos, L., Sterk, P., & Schultz, M. (2013). Volatile Metabolites of Pathogens: A Systematic Review PLoS Pathogens, 9 (5) DOI: 10.1371/journal.ppat.1003311

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