Thinking Small To Save Big: new research combines nanotechnology with immunology to develop an inexpensive, accurate diagnostic tool for the developing world

SEM  image of HIV virons (green) infecting a CD4+ T cell (red) from CDC.gov.

Public Interest Note:

            Can you live on an annual salary of less than $2,000?  Even if you could somehow, manage to have some clothes, water, food and shelter, what happens when you get sick? These are the difficulties facing the millions of people living in resource-poor countries.  Resource-poor countries are often plagued by a combination of factors including: dense population, low GDP per capita, economic and political instability. ¹  Consequently, these countries often have weak health care systems and lack the finances and proper education in combating these countries’ biggest health challenges. Additionally, these countries are typically the most disease-ridden areas in the world. The most prevalent diseases that affect areas of the world such as Sub-Saharan Africa, Indonesia, and India are not the ones we, in America, see as threat, because despite our current economic woes we can afford valuable education and treatments for these diseases.  Thanks in part to huge scientific and medical advances in vaccination, education, and preventative strategies, the developed world has been able to lower the rate of new cases of HIV/AIDS, tuberculosis, and malaria. ²   

    From Tortora, B. "Sub-Saharan Africans Struggle Financially Even as GDP Grows". Gallup. (Jan. 2011)

Unfortunately, technology is generally expensive. Expensive to produce and subsequently expensive to distribute and purchase. Common diagnostic tests performed in U.S. hospitals can cost upwards of a few hundred dollars each. And that’s the cost for the patient, these prices do not incorporate what it costs the hospital to purchase the test from the drug manufacturing company, the cost in paying trained individuals to administer and interpret the test, or any external equipment needed such as electronics and labware!  Although, there have been recent successes in lowering the cost of antiviral drugs in developing countries, there remains a lack of affordable, by means of the developing world, to receive the same quality of accurate and effective diagnostic testing we receive in America.

The exciting thing is, is that despite this particularly difficult endeavor, the scientific community continues to strive to discover innovative ways to improve health in these areas. The newest published research from Samuel Sia’s laboratory at Columbia University exemplifies the ultimate goals, I hope every scientist seeks: to utilize both creativity and intellect to discover something meaningful that will better the world.    

Sia and colleagues present a novel diagnostic device the size of an index card that has the ability to screen for multiple diseases simultaneously! Besides the awesome science behind it (discussed below), the test, itself occurs on a small chip costing as little as $1 to produce, which then is quickly analyzed by a machine “as inexpensive and simple to use as a cellular phone”, and requires little to no training to interpret the results. Oh, did I mention the whole test requires a single drop of blood and only takes 15-20 minutes to complete? A patient can receive results from a single pinprick to results in hand in less than a half an hour! ³

There is no doubt that this technology will be beneficial to resource-poor countries and will help instigate a movement to develop more diagnostic tests in the future, the only problem is: who will fund this kind of production? Such developments need the public’s constant support by understanding the science, providing funding and enhancing communication between scientists and companies that manufacture and distribute these novel devices. Increased support within the public will likely result in providing these diagnostic tools to resource-poor countries while while keeping the cost as minimal as possible. 

 

Developing New Diagnostic Tools: Why This Research Matters

            Sia and his research team set out to design a new diagnostic tool that would be able to accurately screen people for two of the most common infectious diseases in developing countries: HIV and syphilis. The authors of the published paper explain they “chose an HIV-syphilis combination test because HIV and syphilis are treatable in diagnosed pregnant mothers, for whom short-course antiretroviral prophylaxis reduces transmission of HIV, and treatment with penicillin reduces congenital syphilis, which can be fatal for the newborn”.  ³ By focusing on diseases that are passively transmitted from mother to baby, the scientists hope to increase the rate of treating infected individuals and subsequently improving the overall healthcare in infectious disease ridden countries.  Sounds like a pretty amicable quest right? Not only would there be hurdles designing the world’s first-ever multi-diagnostic test for these diseases, but the goal was to achieve all of this with quickest results return rate as possible. 

            How do scientists think of ways to do this? They think small: small devices, small patient samples, small amount of reagents needed, etc.  All of this adds up, theoretically, to a small cost to the patient.  The most common testing technology that fits these criteria is categorized as point-of-care testing (POCT).  The purpose of POCT is to provide on-site immediate medical diagnoses that are portable for patients-no matter if they live in a big city, in the country, or in the most isolated, resource-poor habitats in the world. Commonly used POCTs include: individual blood-glucose tests, pregnancy test strips, and rapid agglutination tests for blood-typing. All of these examples are extremely valuable tools because not only are the tests reliable, but they are also inexpensive, easy to use, easy to interpret, and require the minimal amount of external equipment, thus keeping the cost low. All of the mentioned POCTs test for harmless molecules, but how do you keep the convenience of POCTs in mind when designing a tool that is going to detect highly infectious agents? 

            For a long time, it was believed that microscopy was the only way to detect pathogens in an infected person’s blood.  Because you can literally see the presence of a particular bacteria, virus, or parasite, thanks to a variety of dyes and light maneuvering microscopy techniques, you can confidently determine if someone was in fact infected with a deadly microbe. However, microscopes are expensive (as a point of reference, the cheaper student-microscopes used in the General Microbiology Labs I teach, are worth hundreds of dollars each and don’t offer any fancy optics!). Notably, microscopy done right is not as easy as one might believe, it requires extensive training which requires more money in addition to thousands of dollars spent for the microscope, lens, slides, dyes and reagents needed.  Furthermore, microscopes are generally not the most accessible devices, it seems quite challenging to lug a microscope into the middle of the Congo, for example, without somehow breaking one of the objective lens. According to a recent review article on the impact of POCTs on global health, “over a century of poor microscopy performance has contributed to the culture of mistrusts and undervalue of diagnostic test results by health care providers in low-resource settings”.  ⁴

            For these reasons, health care providers in developing countries have turned to immunoassays, which rather than seeing whole microbes, can quantify how much of pathogenic protein is present in a patient’s blood sample.

Screening for Infections in Developing Countries: Challenges and Advances

            POCTs represent a fantastic example of cooperation between scientists, engineers, businesses and public health personnel. There are the biological research laboratories that focus on identifying antigens, molecules that stimulate the immune response, associated with certain infectious diseases, the engineers who then figure out a way to detect these antigens in biological samples, and finally there is the mass-production and distribution of these diagnostic devices by businesses and global health agencies. 

            One of the biggest challenges in designing the most effective POCTs, that is the ones that can realistically be used in the field no matter the location or finances available, are ones that can operate with as little equipment as possible.  This is particularly important for resource-poor countries such as ones located in Sub-Saharan Africa.  Biosaftey and waste disposal tend to be quite poor in developing country clinical facilities, which is especially a concern for areas plagued by high infectious disease rates.  Improper handling of needles, culture plates, tubes, pipet tips, and any other equipment that stores biological samples, increases the spread of disease and further hinder a country’s overall health care. ⁴    For this reason, the most commonly used POCT to detect pathogenic infection currently is the lateral flow test.  

Lateral flow tests are also called immunochromatic strip (ICS) tests: “immuno” meaning antibodies are used to detect an antigen present in a sample and “chromatic” indicating that some sort of chromatography will be used to visualize the result of the test.  Lateral flow tests are very simple and consist of a small strip of nitrocellulose that has some antibody bound to it at one end of the strip. On the opposite end of the antibody spot, a small blood sample, obtained from a pinprick, is mixed with a buffer that will help carry the blood through the entire nitrocellulose strip simply by capillary action. If a person has the antigen present in their blood that the bound antibody recognizes, it will stick to the strip. As more antigen accumulates on the antibody strip, the strip will produce a visible line to indicate the presence of that antigen. This is the most common method to currently detect malaria and HIV in developing countries. As perhaps, a more easily understood example of lateral flow tests in action, this is the mechanism behind how take-home pregnancy tests work too!  So why invent something new? How could these screening for infectious diseases be improved upon for resource-poor nations?

The “mChip”: The Immunology Behind the Technology

Those were the questions that motivated Sia and his research team to create the “mobile Microfluidic chip for immunoassay on protein markers” or the "mChip" for short (a bit more catchy too right?!). Essentially, the mChip is a full laboratory-based assay shrunk down to fit on a plastic chip not much bigger than a credit card.  The strongest advantage the mChip has over lateral flow tests is that you can screen for multiple antigens or diseases at the same time!

            So how does the test work? Well, part of the name is immunoassay, so again, this test utilizes antibodies specific for certain antigens associated with disease. The laboratory-based test that is miniaturized is the Enzyme-linked Immunosorbant Assay (ELISA).  The ELISA, is perhaps the most commonly used immunological test in the world and is used everywhere from hospitals to academic research labs to undergraduate immunology courses. ELISAs are an extremely valuable test because: 1) you can test multiple samples simultaneously, 2) is very good for detecting even small amounts of antigen and 3) the results are typically colormetric, making the interpretation of results simpler.  You can detect very small amounts of antigen that usually is below the threshold of detection in other screening strategies, because of the Enzyme-linked detection antibodies used in the assay. 

Diagram illustrating an ELISA done commonly in labs in hospitals to test for the presence of disease-related antibodies and pathogens. The mChip miniturized this whole assay onto a polystyrene chip and used metal nanoparticles in place of enyzmes to simplify the technique. From signosisinc.com

          An ELISA begins by coating a 96-well polystyrene plastic plate with a capture antibody (that’s right you can test 96 samples at once!). The capture antibody is an antibody that is specific for a particular antigen, say gp41- a well-known HIV antigen.  Polystyrene plastic is used because it binds proteins extremely tightly, which is important when you need to wash the plate to remove unbound antigen.  By removing anything that doesn’t stick to the capture antibody, the level of background interference decreases dramatically.  So if a person has HIV, the gp41 will stick to the capture antibody on the plate, but how will you visualize this?  That’s where the enzyme-linked antibody comes into play.  In an ELISA, a second antibody is used to detect the bound antigen.  Think of this as a sandwich: where on a plate there’s an antigen trapped between two antibodies-both antibodies detect the same antigen. This second, detection antibody is covalently linked to an enzyme, so you can add a particular substrate that if the enzyme-linked antibody is stuck to the plate, will cleave the substrate revealing a color. Therefore in a typical ELISA, the darker the color means the more enzyme there is, thus the more antigen there is detected in the sample!

            By greatly reducing the volumes of reagents used, and shrinking the plate to a polystyrene chip with seven channels, Sia and his team effectively minimized the size of a typical laboratory-based ELISA. This sounds great, but based on personal experience, ELISAs take a long time to complete. Each step has to be incubated for about an hour and involves a lot of pipetting and waste, so this wouldn’t really work for a resource-poor environment. To circumvent these concerns, the mChip utilizes microfluidics and multistep reactions to significantly reduce the amount of time and labware needed to do the test!  The chips come pre-coated with specific capture antibodies so the only part that would have to be done in the field is running the sample.  All the pipetting and experimental techniques are replaced with a small tube that a field technician can easily prepare by simple syringe-induced vacuum so no reagents will contact the syringe itself.

             Another innovative difference between the classic ELISA and the mChip is that instead of using enzyme-linked detection antibodies, the mChip uses gold nanoparticles attached to secondary antibodies. After the secondary, gold-linked antibodies are loaded into the chip, a flush of silver nanoparticles flow through the chip.  The silver nanoparticles will react with any bound gold nanoparticles, resulting in the release of light-emitting silver ions.   

           These ions can then be exposed to an inexpensive, simple optical beam such as light-emitting diodes (LEDs) and generate a visible signal within-get this- 10-15 minutes!! Because the compact LED device has an imaging screen, a technician can quickly and easily see the results. 

What else makes the mChip advantageous over lateral flow tests? Another caveat to currently available screen tests, is the subjectivity of interpreting results. This is especially true for developing countries where efficient training is lacking. For example, it can be difficult for one to distinguish between a negative result and a positive result if the dye on the strip is not super strong.  When there is no clear threshold or value placed on an observation, it is up to the experience of the available technician to conclude the results of the test. To correct for this, the Columbia University bioengineering team developed a simple compact device costing less than $1 per unit hat converts the light emitted to an electronic signal. No calculations are required of the clinic-so by simply seeing a signal greater than that of background (a sample run without any blood), indicates a positive result.   

Pretty cool, right? But, does this new technology work in reality, in the field-say, for example in Rwanda?  That’s exactly where the team started to test their mChip, where nearly 3% of the nation is infected with HIV and turnaround times for on-site ELISA tests can take several weeks to obtain. By using only a pinprick of unprocessed whole blood, the mChip was used to screen the presence of HIV (gp41) and syphilis (TpN17).  According tothe study, “the assay took less than 15 minutes to complete.  Out of a total of 70 specimens with known HIV status, only one tested false, resulting in overall sensitivity of 100% (98.9-100) and specificity of 96% (88.7-100), rivaling the accuracy of lab-based HIV testing”.  Similar results were obtained for syphilis detection with 94% sensitivity and 76% specificity with samples collected in Project Ubuzima in Rwanda. ³  Importantly, the lab also tested the stability of reagents used for the mChip, and noted that they remain stable for at least 6 months at room temperature. This is especially important for it to be used in areas where refrigeration is hard to find.

What’s Next: From Experimental Research to Distribution

A prototype to model the mChip and detector that would like to be distributed and used in the future. 
From popsci.com

            By incorporating physics and microfluidics, the mChip has the potential to deliver advanced immunological diagnostic tests to resource-poor countries. Currently, the mChip is still in the early design phases, due to limited resources for such academic-based studies.  It will take more funding and people to actually design the mChip that Sia envisions that will actually be distributed throughout the world. It will take more funding and public support to cover production and distribution of the mChip. This research was supported largely by the National Institutions of Health and is a contender for a $14 million dollar grant sponsored by USAID, the Gates Foundation and other agencies as part of the “Saving Lives at Birth” challenge. Sia’s research team explains in the paper, that the “ultimate goal of this research is to develop a device for infectious-disease screening of pregnant women located in remote areas to prompt early treatment”. This technology has great potential in reducing infectious-disease prevalence in the world, but needs further support to develop this potential into reality.

Chin CD, Laksanasopin T, Cheung YK, Steinmiller D, Linder V, Parsa H, Wang J, Moore H, Rouse R, Umviligihozo G, Karita E, Mwambarangwe L, Braunstein SL, van de Wijgert J, Sahabo R, Justman JE, El-Sadr W, & Sia SK (2011). Microfluidics-based diagnostics of infectious diseases in the developing world. Nature medicine, 17 (8), 1015-9 PMID: 21804541






References and Further Reading: 
1: Quet, F., et al. "Challenges of epidemiological research on epilepsy in resource-poor countries". Neuroepidemiology. 30: 3-5. (2008) 
2: IB Times Staff Reporter. "Lab on Chip to Offer, Cheaper, Faster HIV Test". (Aug. 2011)
3:Chin, C., et al. "Microfluidics-based diagnostics of infectious diseases in the developing world". Nature Medicine. 17:1015-1019. (2011). 
4: Yager, P., et al. "Point-of-care Diagnostics for Global Health". Annu. Rev. Biomed. 10:107–44 (2008).

T cells Receive Molecular Ammo to Kill Cancer: new research highlights the great potential of immunotherapy to treat leukemia

T cells (Blue) attacking a cancer cell (white) in vitro. From The Center for Cancer Research

Spotted: Good Scientific Journalism!

The popular media has all been a buzz this week over an exciting new study about a new possible cancer treatment.  As a PhD Immunology Student, I have to begin by complementing NPR science reporter, Joe Palca for his post on the NPR Health Blog, Shots for 1) describing the basics behind immunotherapy in a way for the public to understand and 2) for "warning" the reader that the sample size is very small and that this is preliminary research.  Both points are important to state clearly, as often we, the public, reads a brief story about a new therapy and we instantly want to use it and have it work 100%.  Although, the sample size of this study was merely 3 individuals, the fundamental technique and findings offer great promise in the development of the most effective, least invasive treatments to combat cancer.

 

Gene Therapy: Engineering Cells to Cure Disease

Basic procedure behind gene therapy whereby a patient's own T cells are isolated then engineered to express a certain protein it didn't express before, then the engineered T cells are injected back into the patient to help fight the disease. From attack-cancer.org

A University of Pennsylvania research team, lead by renowned tumor immunologist, Carl June, tested a novel therapy to help treat three individuals with chronic lymphocytic leukemia (CLL).  The technique is an improvement in a molecular genetics technique called gene therapy.  The principle behind gene therapy is to equip cells with a function that that particular cell didn’t have before.   

One of the first human-based study that gene therapy was used to treat babies born with severe combined immunodeficiency (SCID).  SCID is named as such because people born with this genetic disease, have essentially no immune system making them incredibly susceptible to illnesses-think David Vetter aka “The Bubble Boy”.  One of the most common causes of SCID is a single genetic deficiency in a gene called adenosine deaminase (ADA).  ADA is important in the building of new nucleotides for DNA synthesis, and as you might imagine, without this ADA enzyme, DNA synthesis ceases and without the ability to generate new strands of DNA, cells die when they divide.  So ADA-deficiency largely affects highly proliferating cells, like your B and T cells.  To treat ADA-deficiency, stem cells are isolated from the patient and sent to a laboratory, where the cells will be transduced with a viral vector.  This virus is not immunogenic and is just used as a vector-something to deliver the ADA gene to the cell.  Once the virus gets in, it will insert the ADA gene into the DNA of the isolated patient’s cell.  The power of genetic therapy is great since what happens is “fixing” cells to express a necessary gene, like ADA, that the cells didn’t express before!  Once the cells express ADA, they are transplanted back into the patient, where theoretically the person is cured of their genetic deficiency.

However, there are many challenges to genetic therapy, which scientists have struggled with including: how to make the newly injected “fixed” cells last longer?  These cells don’t seem to survive very long in the patient post transplantation, requiring patients to undergo this treatment over and over.  Another concern is the lack of control of where the gene of interest inserts.  For example, sometimes the gene is inserted in an unstable location, and cells lose the expression of the inserted gene over time. 

Since the early 1990’s when gene therapy was used to treat ADA-deficiency, researchers have used this genetic technique as a means to treat a variety of human diseases including Parkinson’s Disease, myeloid lymphoma, and HIV.  With each trial, comes better understanding and innovations in perfecting the therapy to enhance its effectiveness.

The Molecular Immunolog:Re-engineering Our Immune System to Kill Cancer

Creating Chimeric Antigen Receptors (CARs) by piecing together signaling components from different proteins using molecular biology. CART19 would look similar with the anti-tumor extracellular domain to recognize CD19 with the intracellular TCR signaling domain. From DiscoveryMagazine.com

            The new study, published in two parts by June’s group in TheNew England Journal of Medicine and Science TranslationalMedicine, provide novel innovations to gene therapy as a potential treatment of CLL. CLL is a form of cancer in which B cells grow out of control and form tumors.  The goal of June’s latest published research was to figure out a way for a patient’s own body to find the B cell tumor and kill it.  Sounds pretty bizarre and extremely hopeful right?  It doesn’t seem so unreal when you begin to think of how your body fights off other harmful agents.  When pathogenic bacteria infect you, for example, your immune cells are able to detect the bacteria and recognize it as foreign.  In a matter of a few days your immune system is fully activated, innate cells are being rapidly recruited to the site of infection and sending signals to the rest of your body to alert that an infection is occurring.  Meanwhile your T cells become activated and divide like crazy and migrate rapidly to the site of infection to help exterminate the bacteria.  In addition, some of these responsive T cells live essentially forever as memory T cells, so that if and when that same bacteria infects you, you are better prepared with T cells that remember that bacteria and kill it more quickly than the first time. 

If you apply this same concept to eradicating a cancerous tumor, by having T cells that could recognize tumor cells and kill them, you could develop a therapy that would lessen the requirement of using painful chemicals and drugs to solely fight cancer and circumvent rejection issues since the treatment is using the patient’s own cells to kill the tumor (versus bone marrow transplant from another person, where graft-verse-host disease is a possible danger).

            So how did June and his research team do this?  They engineered a gene-construct in the lab consisting of an extracellular domain that recognizes a protein only expressed by B cells (CD19) fused to an intracellular signaling component of the T cell receptor (TCR).  With this strategy, T cells would be able to recognize B cells, become activated, proliferate, and subsequently kill their targeted B cell.  This sort of genetic engineering is a forte of June’s laboratory and is called chimeric antigen receptor (CAR) generation.  The clinical trial was appropriately titled "CART19" (CAR+ T cells for CD19). 

            By transducing the CLL patients’ isolated T cells with a viral vector containing this CAR, then injecting these CAR+ cells into the CLL patients, they found that the “engineered T cells expanded >1000-fold in vivo, trafficked to bone marrow, and continued to express functional CARs at high levels for at least 6 months …moreover, a portion of these cells persisted as memory CAR+ T cells…”  The authors of the study explain that these memory B-cell reactive T cells “may provide a mechanism for CAR memory by means of “self-vaccination/boosting” and, therefore, long-term tumor immunosurveillance”! This exciting research really puts forth the idea that we are on the right track to discovering the ultimate treatment for cancer patients- a treatment that consists of as little pain, money and tumor re-occurrence as possible.  Utilizing the body’s own defense system to fight cancer, with the potential ability to fight the tumor over and over again without further injections and drugs, may represent an ideal cancer therapy!

The patients who participated in this study all had “advanced, chemotherapy-resistant CLL”.  Two of three patients in the study are in remission 10 months post CART19 infusion; the third still has the disease. The researchers acknowledge that chemotherapy still plays in a role in fighting tumors, and that chemo is likely to have played a role in the success of their CART19 therapy.  It is important to consider, that this research-however exciting-is still in the very early stages of development and much is left unknown including: 1) how long these CAR+ memory T cells live for and if their effector function is still in tact, 2) how healthy B cells are affected by this therapy, since the targeted protein, CD19, is expressed by all B cells-tumor and healthy ones and 3) if there are any long-term side effects of using the particular virus vector used in this study.  Importantly, all three CART19 patients are still being monitored to further address these questions.

Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, & June CH (2011). T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Science translational medicine, 3 (95) PMID: 21832238






  Porter DL, Levine BL, Kalos M, Bagg A, & June CH (2011). Chimeric antigen receptor modified T cells in chronic lymphoid leukemia. The New England journal of medicine,   365 (8), 725-33 PMID: 21830940

Learning to live together: new research explains how bacteria's urge to survive in our gut promotes intestinal health

Get to know your insides and the benefits to having a symbiotic relationship with the microbial world! From Food Poison Journal

Public Interest Note:

The mere thought of microbes: bacteria, parasites, fungi and viruses tend to make us sick to our stomach.  We all know that when we catch a cold: a virus is to blame and when we have a nasty skin rash, we apply creams to kill off the infection-causing bacteria.  To think that our bodies are coated with an estimated 100 trillion microbial cells ¹ can be, at first, a very startling and threatening idea to believe.  100 trillion is a ridiculously large number to conceptualize, but let’s try to put this number into perspective: it is estimated that our Milky Way Galaxy contains 200-400 billion stars. ²  For those of you keeping score at home, that’s about a ten-fold difference in that for every star in our night sky, there are ten times more microbes colonizing a single person’s body.  Yes, that’s right- that estimated 100 trillion microbial cells is for ONE person.   In addition, the number of microbial cells found in and on our body outnumbers our human cells 10:1.  Needless to say, if we were to think of ourselves in terms of cellular content we would have to think of ourselves as being more microbial than human! Surprisingly, we know more about the stars millions of light years away than we do about the microbes living in and on our bodies!

from neuroanthropology.net

Whew! It can be very exhausting concentrating on such big numbers!  After all, this IS an immunology blog, not a mathematical one! Most of the microbes studied, thus being the ones that are best understood are the ones that cause severe diseases and make us sick.  However, the number of these virulent bugs doesn’t come close to the number of non-disease causing microbes that live with us everyday.  So why spend all our time and resources studying the things that we encounter rarely and that constitute a fraction of the total number of microbes we live with everyday?  One major reason is because, in order to study a microbe, it has been historically crucial to be able to isolate it and study it in the lab.  However, commensal bacteria, the microbes we live with that don’t cause disease, are very tricky bugs that don’t seem to cooperate with historical scientific practices.  They are usually very difficult to isolate and grow in the lab with usual laboratory medias.  One explanation for this is that the microbial species living inside our bodies are part of a complex ecosystem (aka microbiome/microbiota) that we do not fully understand yet, making it incredibly difficult to recreate in the lab and study.  

That is, until recently, when the National Institutes of Health launched a $140 million initiative in 2007 called “The Human Microbiome Project (HMP)”.  Through a large cooperation of the Nation’s leading microbiology, genetics, bioinformatics and immunology laboratories, the HMP mission is to generate “resources enabling comprehensive characterization of the human microbiota and analysis of its role in human health and disease”. ³   The HMP has undoubtedly spearheaded the advancement of genomic sequencing strategies to better understand what kinds of microbes live on our skin, in our nose and mouth, gastro-intenstinal and urogenital tracts.  With an enhancement of research technology advancement, and an increase in federal funding devoted to studying commensals, our understanding of our microbiome and how it relates to our overall health has greatly increased over the last couple years.

 In fact, earlier this year, fascinating research led by Peer Bork’s group at the European Molecular Biology Laboratory in Germany, discovered that humans can be classified based on three distinct gut microbiomes. ⁴ 

The research team analyzed genomic data obtained from human fecal matter (the least invasive (and least glamorous) method to analyze bacteria living in your guts) derived from people living in Denmark, France, Italy, Spain, Japan and the U.S. and discovered that three different clusters could be distinguished such that irregardless of sex, weight, height, age or geographic location, the balance of gut bacteria could be separated into 3 different groups-each differing in the bacterial contents that lived in their gut.  People who were Type 1 had a different balance of gut bacteria than people in Type 2 or 3.  Similar to bloodypes, that is that all people can be classified into one of 4 groups based on if they express A, B, AB or O antigens on their red blood cells, Bork suggests that this new biological classification, enterotypes (named for the collection of bacteria that live in the gut that distinguish the three groups) may be used to better tailor diets, drug regimines and antibiotics for an individual based on his/her microbiome. For example, someone of Enterotype 1 may respond better to particular antibiotics or diets than someone who is Enterotype 2 or 3. However, this is still a hypothesis that requires more research, funding and public interest to better understand the differences between these enterotypes and whether specific enterotypes are found in other highly colonized areas of the body such as the urogenital tract and skin, and importantly: if different microbial environments in our bodies play a role in disease susceptibility or resistance.

Probiotics: The Immunology Behind the Health Buzz

Most of the “microbial cosmos” living inside our body are bacteria, 70% of which take up residence in our digestive tract. ⁵  What are these bacteria doing there?  Perhaps the best way to answer that question is think of what happens when these bacteria aren’t there.  Researchers can discover answers to this question by using germ-free mice to model what it would be like for humans without intestinal flora.  To do this, mice are born via caesarian section and then immediately housed in a sterile environment by which all their bedding, cages, and food is autoclaved, preventing the colonization of any detectable bacteria.  Studies using such mice have revealed these germ-free mice have increased susceptibility to infections, reduced digestive enzyme activity, muscle wall thickness, and decreased vascularity and nutrient uptake.⁶  All of these observations are important for maintaining a healthy gut environment.  That’s right: bacteria can be healthy for us!  The commensal bacteria that constitute our intestinal microbiome largely include bacteria of the genera: Bacteroides, Clostridium, Escherichia and Lactobacillus.  Some of these genera might seem familiar, most likely because you might know of a common species within these genera that are pathogenic like Clostridium tetani (causes tetanus) and Escherichia coli O157:H7 (common to foodborne illness).  It’s important to re-iterate that commensal bacteria are part of your normal gut flora that you are essentially born with, and are NOT pathogenic. 

Molecules expressed by commensal bacteria that stimulate their growth are known as probiotics (as opposed to antibiotics, which kill bacteria).  Commensal bacteria express a variety of surface molecules that are an intense area of current research because of their potential to use these molecules to communicate to our cells-epithelial, neuron and immune cells to mediate their beneficial actions. The most commonly studied, and therefore advertised, are probiotics generated by Lactobacilli.  Lacto-meaning milk, is how this bacteria generally gets into your gut, therefore making Lactobacilli easy to culture and use as a medicinal tool, since it lives in fermented milk such as yogurt and cheese. Probiotics, like Lactobacilli have been clinically studied for its ability to attenuate gastrointenstinal infections, allergic symptoms, inflammatory bowel disease (IBD), decrease colon cancer severity ⁷ and alterations in behavior including autism ⁸. (Note: yogurts marketed specifically as “probiotic” contain enriched amounts of certain strains of priobiotic bacteria.) There are three main ways in which bacteria can be beneficial to the health of our guts:

1. Competing against invading pathogens by secreting antimicrobial peptides and lowering the gut pH

2. Increasing barrier protection and mucus generation to protect our guts from pathogen invasion and enhancing nutrient acquisition

3. Immunomodulation by affecting gut immune cells function and distribution.

                  For example, Lactobacillus acidophilus has been reported to inhibit pathogenic E. coli-induced inflammatory cytokine release by intestinal epithelial cells (IECs).  There is data to suggest that this happens due to a molecule expressed by common Gram-positive bacteria, called lipoteichoic acid (LTA).  It is thought that LTA, which looks structurally very similar to a molecule expressed by pathogenic bacteria, is able to outcompete and bind tighter to a receptor on IECs, thereby blocking inflammation. ⁹ Commensal bacteria have been shown to further induce an anti-inflammatory environment, by regulating the T and B cells, macrophages and dendritic cells in the intestines.  For example, Lactobacilli induces the production of the potent anti-inflammatory cytokine, IL-10, from dendritic cells and macrophages, while increases antibody secretion from B cells. ¹⁰ Although we generally know the beneficial effects of commensal bacteria, we don’t fully understand the biological mechanisms to explain how probiotic bacteria contribute to protection from such an array of diseases.

In 2005, the Nobel Prize for Physiology and Medicine was awarded to Australian Physicians/Microbiologists, Robin Warren and Barry Marshall. Warren and Marshall discovered “the bacterium Helicobacter pylori and its role in gastritis and peptic ulcer disease”.¹¹  Ann O’Hara and Fergus Shanahan recall the 2005 Nobel Prize and explain one crucial key in unlocking the mystery of how priobiotics prevent disease, stating that this “is a reminder that the solution to some human diseases does not reside solely within the host but rather might be found at the interface with the microbial environment”.  ¹²

Understanding Beneficial Bacteria: Why This Research Paper Matters

Although the field of probiotic research is expanding, there remain many questions left unanswered including:

1. Which molecules involved-expressed by both the bacteria and our immune cells?

2. How are commensal bacteria beneficial to the maintenance of healthy gut?

3. If bacteria express similar molecules, how does our immune system recognize the difference between commensal and pathogenic microbes?

The answers to these questions are among the primary foci of a recent paper led by  Sarkis Mazmanian’s research group at California Institute of Technology.  One of the many reasons why Mazmanian’s research is so interesting is because he unselfishly thinks about human disease from the perspective of the microbe.  In fact it seems he tries to stand up for the little guys, explaining that, “They [the bacteria] couldn’t care less about us except that we provide them a stable and nutrient-rich habitat”.  Mazmanian, whose primary research interest is to better understand why the “good” bacteria, the commensals, are good for us, believes that commensal bacteria are just trying to live another day in our guts, skin and mouth-and they can potentially evolve new ways to do this anyway they can, weather it be by preventing cancerous tumors to form, harsh inflammatory environments or pathogenic intruders from taking over their home.  These beliefs represent a compelling juxtaposition between microbiologists and medical professionals.  Most scientists studying immunology and medicine think of particular immunological phenomena from the perspective of the host (people) such that even though we know that some bacteria are good for us, they believe that we, the host, have evolved to live symbiotically with bacteria.  But perhaps, thinking of this from an entirely human-centered perspective is not the only way to understand the fundamental question: how and why are commensal bacteria in our gut?

Why not let Mazmanian explain in his own words the importance of investigating this bacteria-focused angle to help solve some the biggest concerns in human health?:

Perhaps, it is this refreshing and novel outlook that enabled Mazmanian to be named one of Discover magazine’s “20 best brains under 40” in 2008.  In that interview, he reasons “The potential of beneficial microbes appears to be limitless” and that “this symbiotic relationship between the human body and microbes [is] a gold mine of potential therapies for a number of illnesses”. ¹³ 

What the &*%$#! Does the Title Mean?!

Round, JL., et al. “The Toll-LikeReceptor 2 Pathway Establishes Colonization by a Commensal of the HumanMicrobiota”. Science. 332:974-977. (2011).

1. Toll-Like Receptors (TLRs) are one of the most extensively studied, most important signaling molecules in immunology.  They are named “Toll-LIKE”, because the first such receptor, Toll, was discovered in Drosphilla, a common fruit fly used as a model for genetics research.  Originally identified as an important gene that regulated dorsoventral axis formation in developing embryos, a Toll-deficient fruit fly revealed that these flies were highly susceptible to fungal infections.  Since that discovery, in the early 1990’s, Toll-Like receptors have been identified in every animal with an immune system.  The signaling cascade and inflammatory output is amazingly similar between the fruit fly and humans, thus making the discovery of Toll one of the most intriguing examples of how basic research and the use of model organisms provide powerful insight into understanding human disease. 

Great cartoon illustrating the function of TLRs as receptors on the surface of cells (TLR1/2,4,5,2/6) as well as within cells (TLR3,7,9) to detect microbial patterns not expressed by humans. Immune cells are particularly good at expressing these TLRs and using them to alert the body of infection. But how do commensal microbes differ from pathogenic ones with respect to TLR activation? This is the question that drives Mazmanian's research group. Image from www.invivogen.com

In humans, there are at least 10 TLRs that are capable of recognizing unique molecules expressed exclusively by microbes, and not us.  Examples of microbial-associated molecular patterns (MAMPs) include: lipopolysaccharide (LPS), flagellin, and single stranded RNA (ssRNA) which stimulate immune cells to become highly inflammatory.  The inflammatory response is very quick and offers a way to recruit immune cells to the site of infection to destroy pathogens with speed.  Like most biological processes, if there’s an “on” switch (pro-inflammation), there’s a regulatory “off” switch.  Regulation is paramount to a functioning immune system and survival of a host, for example endotoxic shock is caused by a significant dose of LPS, which is found on the surface of Gram-negative bacteria like the pathogenic strain, Escherichia coli O157:H7.  The “shock” derives from an overproduction of inflammatory cytokines induced by TLR stimulation that mediates high fever, shortness of breath and even death.  TLR4 is the TLR that recognizes LPS. 

The focus of the paper discussed below, is TLR2, which is known to bind primarily MAMPs associated with Gram-positive bacteria and yeast.  TLR2 is expressed on the surface of nearly every immune cell and has also been detected on epithelia. ¹⁴ However, a few years ago it was discovered that a MAMP located on the Gram-negative bacteria, Bacteroides fragilis called polysaccharide A (PSA) can also stimulate TLR2.  Usually, it isn’t terribly exciting news to read: “bacteria activate TLR!”; however things began to turn into a more thrilling adventure when you learn that this is a commensal bacteria that belongs to the genera, Bacteroides, which alone makes up 30% of your gut bacterial flora. Currently, we do not fully understand why B. fragilis expresses a molecule that binds TLRs-but we do know that purified PSA can regulate the inflammatory cytokine release by T cells and dendritic cells ¹⁵ and we know that purified PSA injected into mice, can prevent the onset of inflammatory bowel disease (IBD). ¹⁶

Both of these findings are very interesting, but one of the most critical aspects to support these phenomena remains unknown: if, according to Mazmanian commensal bacteria don’t give a damn about us; that they are living with us because we let them, why does B. fragilis express PS? Does PSA/TLR2 signaling do something positive for the bacteria, in addition to comforting us with a balanced gut?

Now, with the information above, we can infer: 

This paper will provide data regarding PSA as being beneficial to, B. fragilis because of its interaction with TLR2 allowing the commensal bacteria to survive and colonize in our gut, while regulating out intestinal inflammatory environment.

Ready for an adventure? Read on for a guided-tour through the scientific data!
                In this paper, the basic question that motivates Round and colleagues’ research is this: why don’t commensal bacteria activate an inflammatory immune response, but pathogenic bacteria do, given the fact that both express MAMPs that stimulate TLRs?  Given that this research is directed by Mazmanian, the team set out to investigate this question with the hypothesis that B. fragilis, evolved ways to block our inflammatory, anti-microbial defense system so that this symbiotic bacteria can essentially go unnoticed by our gut’s immune system. 
            To begin, they looked at inflammatory T cells in the gut.  But not just any T cell (that’s right, there are at least 5 different subsets), they assessed a subset of T cells called, Th17 cells, which populate the gut in response to infection and further the inflammatory immune response by produces loads of the inflammatory cytokine, IL-17.  In the gut of wild-type (WT) mice, they found that Th17 cells constituted 7.65%. Interestingly, the germ-free mice only had about a tenth of Th17 cells compared to WT.  This result indicates that total lack of commensal bacteria (germ-free mice) results in less inflammatory Th17 cells in the gut.  But, when they mono-colonized germ-free mice with only B. fragilis, so that the only bacteria living in these mice is this particular commensal, they found that the Th17 population increases slightly to 1.46%, and that when they genetically delete the PSA gene from B. fragilis, the percent of Th17 cells in the gut increases dramatically to levels similar to that found in WT mice!  This data suggests that B. fragilis is a relatively weak inducer of Th17 development since these germ-free mice colonized with B. fragilis has nearly 7 times less inflammatory T cells in their gut and that more importantly, by deleting PSA, B. fragilis looks like a pathogenic bacteria able to greatly induce Th17 development!  Collectively, this data indicates that B. fragilis actively blocks inflammatory Th17 development in the gut by expressing PSA!  I’d say that is one pretty impressive start to a paper!  Let’s see what else they show us!

            Next, Round, et al. set out to determine the mechanism by which PSA suppresses Th17 development.  It is widely known in the T cell field, that the anti-inflammatory cytokine, IL-10, is a potent inhibitor of inflammatory T cells such as Th17.  Also, based on their previously published research, that PSA limits IBD inflammation and that PSA can bind TLR2, the team wanted to know if TLR2 stimulation by PSA results in IL-10 production from immune cells.  As mentioned before, a number of cells express TLR2, but since they are looking in the gut-where else better to start than investigating T helper cells and dendritic cells (DCs), which are among the most numbered immune cells in the gut?  In an in vitro experiment, in which purified T cells are mixed together with cultured DCs and stimulated with purified PSA, IL-10 is produced.  But where is the IL-10 coming from: the T cell or dendritic cell?  To answer this question, the researchers repeated this in vitro experiment, using DCs from WT mice and T cells (CD4+, T helper cells) from TLR2-deficient mice.  When they cultured these cells with PSA, IL-10 production was significantly reduced.  To be thorough, they also performed a co-culture experiment with WT T cells and TLR2-deficient DCs stimulated with PSA.  Under these conditions, the amount of IL-10 detected was just as high as in the presence of WT T cells and WT DCs, suggesting that PSA was selectively stimulating TLR2 on T cells, not DCs, to induce IL-10 production!  Even more interesting, is that DCs aren’t even needed to get PSA-induced IL-10 secretion; that T cells can recognize PSA via TLR2 and make IL-10 intrinsically! 

This diagram represents the "old" model of how B. fragilis regulated gut inflammation. From Mazmanian's newest research, we no know that PSA can DIRECTLY induce Treg development by stimulating TLR2 on T cells, thus by-passing the requirement for DCs to process and present antigen to T cells!!
From www.rndsystems.com

            With this data, they wanted to go back and see if TLR2 is the missing link to explain how PSA blocks Th17 development in the gut, as shown in Figure 1.  When they mono-colonize germ-free mice that lack TLR2, with either the WT B. fragilis strain or the one without PSA, Th17 development is not impaired suggesting that TLR2 is critical to the mechanism by which B. fragilis is able to prevent inflammation in the gut! 

            Now the ultimate inquiry: So what?!  It’s cool to discover the details that explain how commensal bacteria can regulate gut inflammation, but from Mazmanian’s perspective, microbes don’t care if our guts in a knot, making us want to cry in pain.  So, why does B. fragilis express a molecule that blocks inflammation?  What is the benefit of expressing PSA to the bacteria? 
             To address this important question, Round and colleagues looked at microscopic images of colon sections to look for B. fragilis.  Using confocal microscopy and 3D digital reconstruction imaging, they discovered microcolonies of the commensal closely associated with the host intestinal tissue.  There was no B. fragilis found in the germ-free mice.  Further more, when the mono-colonized mice with PSA-deficient B. fragilis, they could hardly find any microcolonies, and injecting purified PSA recovered the bacteria’s growth in the intestinal tissue!  So it appears that not only is PSA inhibits inflammation, which is good for us, but PSA is good for the bacteria, because B. fragilis needs it to survive in our guts!

             The obvious follow-up question to this extraordinary finding is: how does inflammation affect commensal bacteria survival?  With the first bits of data indicating that PSA-deficient B. fragilis can’t survive in mice and that Th17 cells increases dramatically in mice colonized with B. fragilis lacking PSA, Round, et al. wanted to know if IL-17 was the reason why B. fragilis evolved to express PSA.  To test this theory, they mono-colonized mice with PSA-deficient B. fragilis and injected a blocking antibody to inhibit IL-17.  Only in the presence of the anti-IL-17 antibody, was the PSA-deficient B. fragilis able to colonize and live in the intestines! This research team finally unveiled the reasons why and how B. fragilis live mutualistically with us!

             Of course, this still plenty of biological investigation to be done in completely understanding the purpose of commensal bacteria.  For example, is PSA a universally expressed molecule on all Gram-positive commensal bacteria, or is PSA specific to B. fragilis?  Furthermore, how do other common commensals such as Lactobacillus spp. survive in our guts and is this TLR mediated? In terms of the immunological mechanism, it is still unclear how exactly IL-17 is anti-microbial, which would complete the story of why B. fragilis wants to inhibit IL-17 function.  Lastly, it remains to be understood how TLR2, which binds pathogenic ligands to induce inflammation can also recognize PSA on B. fragilis to block inflammation.  How does TLR2 on our immune cells know the difference between MAMPs on pathogenic vs commensal bacteria? It’s always good to leave a few unsolved mysteries at the end of a paper; it really keeps the field of immunology and microbiology exciting and interesting, right?!
            Lastly, it would be interesting to see if these same findings can be observed in humans.  If this were to be true, it is clear that this novel data may result in new therapies to treat an array of gut inflammatory conditions such as IBD, Crohn’s and Celiac Disease like taking PSA-supplements purified PSA to treat a number of diseases that plague thousands of Americans! 
On a side note: if you are interested reading some of the best critical scientific writing currently available, I suggest you click here to read the paper for yourself! I was blown away by the clarity and philosophical spin in the concluding statements from Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, & Mazmanian SK " The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota". Science (2011) discussion:

“It is historically believed that the micriobiota is excluded from the mucosal surface.  However, certain symbiotic bacteria tightly adhere to the intestinal mucosa and thus immunologic ignorance may not explain why inflammation is averted by the microbiota.  Our study provides new insight into the mechanims by which the immune system distinguished between pathogens and symbionts….On the basis of the importance of the microbiota to mammalian health, evolution appears to have created molecular interactions that engender host-bacterial mutualism.  In conclusion, our findings suggest that animals are not “hard-wired” to intrinsically distinguish pathogens from symbionts, and that microbital-derived mechanisms have evolved to actively promte immunologic tolerance to symbtiotic bacteria.  This concept suggests a reconsideration of how we define self versus nonself.”

This conclusion reads like an English literature paper, not a scientific one and left me wanting to push myself, when it comes time for me to write an article about my own research; to discuss it in a captivating manner that speaks to both the academic research community as well as the interested general public.

Round JL, Lee SM, Li J, Tran G, Jabri B, Chatila TA, & Mazmanian SK (2011). The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota. Science (New York, N.Y.), 332 (6032), 974-7 PMID: 21512004




References and Further Reading: 
1: Qin, J., et al. “A human gut microbial gene catalogue established by metagenomic sequencing”. Nature. 464:59-65. (2010).

2: Grant, Jo and Ben Lin. “The stars of the Milky Way”. ChView.
3. The NIH Common Fund. “The Human Microbiome Project: Overview”.
4: Arumugam, M., et al. “Enterotypes of the human gut microbiome”. Nature. 473:174-180. (2011).
5: Tlaskalova-Hogenova, H., et al. “The role of gut microbiota (commensal bacteria) and the mucosal barrier in the pathogenesis of inflammatory and autoimmune disease and cancer: contribution of germ-free and gnotobiotic animal models of human diseases”. Cellular & Molecular Immunology. 8:110-120. (2011).
6: Hentschel, U., et al. “Commensal bacteria make a difference”. Trends in Microbiology. 11:148-150. (2003).
7: Mohamadzadeh, M. et al. “Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid”. PNAS. 108:4623-4630. (2011).
8: Heijtz, RD., et al. “Normal gut microbiota modulates brain development and behavior”. PNAS. 108:3047-3052.(2011).
9: Vidal, K., et al “Lipoteichoic acids from Lactobacillus johnsonii strain La1 and Lactobacillus acidphilus strain La10 antagonize the responsiveness of human intestinal epithelial HT29 cells to lipopolysaccharide and gram-negative bacteria”. Infect. Immun. 70: 2057-2064. (2002).
10: Ng, SC., et al “Mechanisms of action of probiotics: recent advances”. Inflamm. Bowel Dis. 15:300-310. (2009).
11: The Nobel Assembly. “Press Release: The Nobel Prize in Physiology or Medicine for 2005”. Released on-line Oct. 3, 2005.
12: O’Hara, AM and Shanahan, F. “The gut flora as a forgotten organ”. EMBO. 7:688-693. (2006).
13: Grant, A., et al. “20 Best Brains Under 40”. Discover. Published online Nov. 20, 2008.
14: Wang, Q., et al. “A bacterial carbohydrate links innate and adaptive responses through Toll-like receptor 2”. J. Exp. Med. 203:2853-2863. (2006).
15: Mazmanian, SK., et al. “A microbial symbiosis factor prevents intestinal inflammatory disease”. Nature. 453:620-625. (2008).