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).

On human health, interesting the public, and good scientific journalism...

from http://evidencebasedliving.human.cornell.edu/

In addition to the "pulled from the scientific journal headlines" research discussed here, I thought it would be interesting to periodically showcase fascinating research that I read elsewhere.  Awesome and interesting research about human health, disease and immunology is everywhere and sometimes, it's written about in a way that is clear, accurate and easy to understand!  That last point is exactly what Escaping Anergy is all about: a place to see, interpret, discuss and most importantly-UNDERSTAND- the scientific data behind the science stories we read about, the diseases we encounter and the medicines we take!

Today I came across a story published in The New York Times written by Laura Beil entitled: "In eyes, a clock calibrated by wavelengths of light".   It seemed apropos for me to include this NYT article after just posting about new data indicating a role for sleep in boosting vaccine efficiency!  Beil briefly, but accurately discusses the research from the University of Basel regarding how blue light (the kind strongly emitted by LED TVs, laptops, etc) enhances our alertness while inhibiting our ability to become sleepy.  In short, this happens because blue light (compared to reddish/orange light like older incandescent light bulbs) slows the production of melatonin, a light-sensitive hormone that induces sleep and is critical for the maintenance of circadian rhythm (why we humans sleep at night and are awake when it's light).  Biel also discusses how this new data may play a role understanding cancer progression and infection susceptibility as people are increasingly tied to electronic devices that utilize blue light.  

The lead author on this paper, Christian Cajochen, is the head for the Center for Chronobiology at the University of Basel and a quick pubmed search reveals that he also recently published in Proceedings of the National Sciences (PNAS) regarding the role of serum proteins in regulating the circadian rhythms of fibroblasts (cells that are important in wound healing and disease)! Although they still don't know which protein(s), the most common in serum include: antibodies, complement (important in killing invading microbes), and albumin, it is very exciting learning that our immune system is tightly connected with nearly every physiological process including sleep and circadian rhythm maintenance!

The idea that light affects the way we sleep and that sleep can in turn modulate disease is not a new revelation, however what remains to be clear is HOW exactly this all happens. The more we research, the more we will understand the underlying mechanisms that control human disease (progression, susceptibility, and host defense ability). The role of sleep and light is just now becoming of interest in the immunology and medical research fields, with many intriguing findings! It's exciting to live in a time when there's so much quality and new research available for the public to learn about and discuss!

What's a great science-related story you've read about recently?  What kind of research would you like to see more of discussed on Escaping Anergy?  I appreciate all your comments and suggestions! Thanks for reading!



Cajochen C, Frey S, Anders D, Späti J, Bues M, Pross A, Mager R, Wirz-Justice A, & Stefani O (2011). Evening exposure to a light-emitting diodes (LED)-backlit computer screen affects circadian physiology and cognitive performance. Journal of applied physiology (Bethesda, Md. : 1985), 110 (5), 1432-8 PMID: 21415172

How to get the most out of your next vaccine? A full night's sleep may enhance protection against viral infections

from gabby22197.glogster.com

Public Interest Note:

Let’s begin with a simple question: Why do we sleep?  To answer this, we can think of what happens when we don’t get enough or any sleep.  The day after a sleepless night, often we feel irritable, exhausted, unhappy and stressed.  If you are a parent, have a full-time job or a fellow graduate student you are acutely familiar with how precious even a few hours of sleep can be in redeeming your sense of sanity.  The side effects of sleep can become dangerous when the actions of a sleep-deprived individual affect those around him/her.  In March, an air traffic controller at Ronald Reagan Washington Airport fell asleep on the job, leaving two planes alone in the sky trying to land the planes alone.¹ Fortunately, in this case, no one was terrible injured, but the news magnified the public’s awareness and outrage of having to depend on sleep-deprived individuals for our safety.  It’s not surprising the single air traffic controller fell asleep on the job since it was reported this was “his fourth consecutive overnight shift, which runs from 10 p.m. to 6 a.m.” ¹ In fact, the American Academy of Sleep Medicine International Classification of Sleep Disorders classifies this condition as Shift Work Sleep Disorder (SWSD).  SWSD plagues nearly 70% of the 15 million Americans who work shifts between 10 p.m. and 6 a.m.²   It is further estimated that sleep deprived workers cost U.S. businesses $18 billion a year.³  

from cdc.gov

Importantly, sleepiness does not only affect the people who work the “graveyard shift”, but in an increasingly fast-paced society, coupled with a tough economy and technology-rich environment, many Americans are spending less time asleep.  The Center for Disease Control and Prevention (CDC) revealed that more than 35% of the 74,571 adult Americans surveyed get less than 7 hours of sleep a day and 37.9% reported “unintentionally falling asleep during the day at least once in the preceding month.  Alarmingly, nearly 5% of respondents admitted to nodding off while driving at least once in the past month. ⁴   Furthermore, it is estimated that sleep deprivation causes nearly 100,000 vehicle crashes and 1,500 deaths annually in the U.S.⁵ Perhaps, if people more strictly followed the CDC’s recommendation of getting 7-9 hrs of sleep (for adults), such scary statistics will improve and save our economy billions of dollars.  But why does the CDC discuss anything about sleep?  Does sleep have a role in disease prevention?

We know what happens to our bodies when we are sleep deprived: red eyes, achy joints, and sometimes we even catch a cold.  Besides behavioral problems that arise when we are sleep deprived, our immune system becomes compromised as well.  For that reason, the CDC warns, “persons experiencing sleep insufficiency are also more likely to suffer from chronic diseases such as hypertension, diabetes, depression and obesity…” The CDC refers to America’s sleep deprivation problem as an epidemic, a public health concern.  The problem of sleep deprivation in America is so great that since there is even a national organization called the National Sleep Foundation devoted to alerting the public, policy makers and healthcare providers of the consequences related to sleeping insufficiencies. Such agencies are worried that a continuously sleepy nation will result in a sick nation with increased susceptibility to infections and disease-which ultimately will cost the country billions and hamper productivity.  

Sleep Deprivation: The Immunology Behind It

EEG charts and amount of time spent in each sleep cycle, stages 3 and 4 constitue SWS which is also the time in sleep when pro-inflammatory cytokines, IL-1 and TNFalpha peak. Lange, et al. establishes that a full-night's sleep post vaccination BOOSTS your immunological memory, perhaps enhancing the vaccine's effectiveness. Image from seamist.hubpages.com

First, how does one define and measure sleep? There are two types of sleep: non-rapid-eye-movement (NREM) and rapid eye-movement (REM).    There are 5 main stages of sleep beginning with stage 1 in which you are just lying down to sleep, but still pretty conscious.  Stage 2 follows with a lower level of consciousness that develops into stages and 3 and 4.  Stages 3 and 4 are collectively called slow wave sleep (SWS).  Under SWS, the brain is at its lowest level of consciousness and is thought to be involved in restorative functions.  Lastly, REM is the final step in sleep and is when our brains are processing information and dreaming.  When asleep, we typically spend equal parts in REM and SWS.  Sleep stages can be monitored via EEG recordings as each stage has distinctly different frequencies and EEG wave amplitudes.⁶

 In the late 1980’s and early 1990’s, Toth and colleagues performed a series of infections on rabbits and monitored the animal’s sleep patterns during infections.  They tested a virus (influenza), bacteria (Staph. aureus and E. coli ), yeast (Candida albicans) and parasite (Trypansosoma brucei).  Each infection resulted in abhorrent sleep patterns, regardless of microorganism.  In general, the various infections increased the duration of slow wave sleep (SWS). ^6,7  The observation that infection increases SWS, but not other sleep stages, may mean that SWS, in particular, regulates our immune response while we are sleeping.  In humans, one of the most well studied infections to examine in regards to sleep is T. brucei, the parasite that causes Human African trypanosomiasis aka African sleeping sickness.  Infected individuals, usually by the second year of infection, lose the ability to manage their circadian rhythm resulting in the complete loss of sleep regulation and often leads to comatose.  In mice and rabbits, the release of parasites into the bloodstream is associated with acute increases in SWS, however this has not been evaluated in humans. ⁶

So what immune factors are modulated by sleep?  First, it is known that the majority of our immune cells, B and T cells and monocytes reach maximal levels in the blood during the night and are lowest when awake.⁸ In addition, the pro-inflammatory cytokines, TNFalpha and IL-1beta plasma levels also peak while we sleep, being the highest at the onset of SWS.⁸ An increase of either of these cytokines subsequently increases SWS duration, whereas decreases in TNFalpha or IL-1beta blocks SWS.⁶ This appears to be the clearest example of how our sleep patterns may regulate our immune response during infection. Both of these cytokines are critical in the clearance of infection and are some of the most abundant cytokines present during infectious diseases.  Therefore it seems to make sense why an increase in SWS during the diseases mentioned above occurs. 

Interestingly, infection is not the only thing that induces TNFa and IL-1beta release in the brain and plasma.  Sleep deprivation also promotes pro-inflammatory cytokine release, which then instructs your brain to increase the time it spends in SWS.  Alternatively, anti-inflammatory cytokines such as IL-10 is thought to inhibit SWS onset by antagonizing IL-1beta and TNFalpha.  It is also thought the induction of certain hormones such growth hormone (GH) and prolactin promote the release of IL-1beta, since inhibiting these hormones attenuates IL-1beta levels and SWS duration.  Conversely, the anti-inflammatory hormone, cortisol does the opposite, and decreases IL-1beta production.⁶ So it appears that our immune system mirrors our endocrine system in that they both regulate our sleep patterns by releasing pro- and anti- sleep mediators.  

TNFalpha and IL-1beta are generally thought of activators of the innate immune response, such as macrophages and dendritic cells.  Such cytokines act as an adjuvent, boosting the macrophage and dendritic cell's ability to be potent antigen presenting cells (APC).  The main job of an APC, is to display bits of processed pathogen (antigen) to a T cell.  Once a T cell recognizes a particular antigen, it then becomes activated and ready to fight the infection and help B cells secrete antibodies.  Realize that the next time you become ill and start to feel sleepy, your immune system is starting to work and that you should rest so that your body can adequately defend itself against infection.  Ignoring our body's demand for sleep affects the way our bodies fight diseases.  In a few human studies, it was revealed that the number of T helper cells decreased and the level circulating antibodies decreased with sleep deprivation.⁶ Not surprisingly, when the body is pushed to the extreme of total sleep deprivation, the immune system fails completely.  In one study, rats were sleep deprived for 21 days, until near death.  At the end of the 21 days, the rats had suffered from severe weight loss and septicemia with both opportunistic and pathogenic microbes in the blood.⁹ Furthermore, a recent survey of more than a million participants demonstrated a strong correlation between sleep of less than 7 hours a night and increased mortality.¹⁰

  

Sleep Deprivation and Disease: Why This Research Paper Matters

The evidence is clear: our immune system communicates with our brain while we sleep, but researchers have only begun scratching at the mechanisms behind this phenomenon.  This is very exciting and novel research that may provide not only insight into how the body operates, but also instigate new therapeutic approaches in fighting disease.  Although it is well established the role of certain cytokines in maintaining healthy sleep cycles and how the immune system responds to sleep deprivation, little is known about the effect sleep has on specific immune cell function.  The majority of what we know about the interplay between sleep and immune function is correlative.  For example, we know TNFalpha can promote APC function, and we know that TNFalpha levels increase during SWS, but it remains elusive whether sleep-induced TNFalpha promote APC function and thereby program and effective T cell response.  Sleep may represent a crucial part of the immune system that has been largely ignored by the immunological and medical fields.  Perhaps, the notion “Sick and Tired” can be immunologically explained and that greater insight into how sleep affects the body’s ability to clear infection is an underlying mechanism behind the increase of infections among infants and the elderly (acute vs chronic sleep deprivation).  How does sleep impact our ability to properly activate our immune system?  Can sleep play a role in designing better therapies and treatments for people fighting disease?   These questions motivate Jan Born’s research group at the University of Lubeck in Germany to find new ways to make more efficient vaccines…without even changing the vaccine components!  Therefore, as Lange, T. et al illustrates in their recent Journal of Immunology paper, catching a full night of ZZZ's  can make all the difference in getting the most out of your next vaccination.

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

Lange, T. et al. “Sleep after vaccination boosts immunological memory”. Journal of Immunology. 187 (2011).

There are two waves of antibody response: a slower, short-lived response during the initial infection followed by a quicker, longer-lived memory response . from click4biology.info

1) The essential point of vaccination is providing people with the antibodies needed to clear infections quickly and effectively.  Vaccines work by administering something that stimulates the adaptive immune response without causing disease.  Vaccines in the U.S. consist of purified proteins (not a whole, intact pathogen) from a pathogen that is injected or inhaled.  When APCs encounter the proteins in the vaccine, they cut the proteins into short peptide sequences.  Once processed, an APC presents the peptide on its surface to a T helper cell, which are named so because they help B cells make antibodies and help innate immune cells to destroy pathogens.  At the moment when the T helper cell recognizes a certain peptide sequence, the T cell begins to proliferate and secrete cytokines that help B cells secrete tons of antibodies- all of which can specifically target the protein that was used in the vaccine.  Vaccines help prevent infectious diseases by assisting our immune system in making the antibodies necessary to clearing an infection quickly.  When we become infected with something that we didn’t vaccinate against, our immune system has to take a lot of time (weeks) to produce the antibodies needed to fight the infection.  Some pathogens are extremely virulent and can cause a lot of harm, even death in the weeks it takes our bodies to properly protect itself against it.  Vaccines therefore help equip our immune system with the weapons (antibodies) it needs, before even heading into battle against a deadly pathogen. 
 2) Of course, this concept of a vaccine works best when the immune system can remember what the pathogen looks like when it encounters it a month, a year, or 10 years from the time of vaccination.  This is immunological memory.  An amazing aspect of your immune system is that when the adaptive immune cells, B and T cells, are activated and proliferate, some of the cells seem to last forever.  These cells are aptly named, memory B and T cells.  These memory cells will last you during your entire life time, always remembering what they are supposed to do when they encounter the specific protein or peptide that stimulated them to become active in the first place.  In order to maximize immunogenicity and achieve immunological memory you need a very good induction of the antigen-presenting cells by using adjuvants and booster shots to bolster the ability of your body to produce a reservoir of effective memory cells ready to kill a particular pathogen over the course of your life!

Compile this information together and we can infer that: Sleep following a vaccination, acts like an adjuvent promoting the development of memory cells that provides long-term immunological protection against a certain pathogen.  

Ready for an adventure? Read on for a guided-tour through the scientific data!

But first: A brief Q and A session regarding the overall experimental approach:

Q) What organism(s) was being tested?

A) All of the experiments presented here were performed on human volunteers.  The sample size consisted of 27 healthy, nonsmoking men with an average age of 26.  The group was on synchronized schedules with the same sleep-wake patterns for the 6 weeks prior to experimental testing.  All sleep-wake activity occurred in a sleep laboratory, and all participants had spent at least one night in the lab prior to experimental testing.  The group was randomly assigned to either the “sleep” or “wakefulness” group. 

Q) How did the “sleep” group differ from the “wakefulness” group?

A) The sleep patterns between the groups only differ the night following their vaccination (which occurred at 8am and occurred 3 times, one in Feb., March, and June. The “sleep” group had lights off at 11pm and lights on at 6:30am in the sleep laboratory.  During this same time, the “wakefulness” group stayed awake in bed watching TV, reading, listening to music or talking to a scientist (fun, no?)  The “wake” group was not allowed to sleep until 8pm the following day.  This may seem a bit extreme and rare that people would be awake for this long following a vaccine shot, but it does adequately distinguish the two experimental groups clearly. 

Q) What vaccine was used?

A) Hepatitis A vaccine (Twinrix, GlaxoSmithKline Biologicals)

Q) What did the researchers monitor throughout the trial period?

A) Four major conditions were tested:

1. Sleep activity via electroencephalography (EEG) to examine sleep stages.

2. Hormone analysis to investigate sleep-related hormone release via i.v. blood collection.

3. Hepatitis A Virus (HAV) - specific T helper cell response

4. HAV - specific antibody response

For immune response, peripheral blood cells (to look at T cells) and serum (where antibodies are found) was collected by drawing blood immediately before vaccination and then 1,2, and 4 weeks after each shot as well as a follow-up at 1 yr. after the first inoculation.

Now let’s dive into the results!

Because the whole basis of this paper rests on sleep activity it is first imperative that the authors of the paper show that the people in the “sleep” group slept normally.  Normal meaning that they spent time in each of the known sleep stages (see the above figure) that fit data that is already well established in the field, with nearly 50% of sleep spent in slow wave sleep (SWS; stages 3+4) and 50% in REM sleep.  There was no significant differences between the three sleep nights post vaccination. 

Over the course of the Hepatitis A vaccination period, blood was obtained to examine the T cell response. This is very important to look at because it is a standard read-out for the quality of a vaccine.  Recall that most vaccines, including the Hepatitis A vaccine, consist of purified proteins that contain immunogenic Hepatitis A viral proteins.  Once these proteins are injected, they enter the blood and tissue where they will be taken up by macrophages and dendritic cells.  Which is very convenient since these cells are antigen-presenting cells (APCs).  So then these APCs digest the viral proteins into smaller peptides and present these peptides on their surface so T cells can “see” the peptide.  If a T cell recognizes that particular peptide, the T cells become divides rapidly producing hundreds of more T cells that all are specific for that peptide.  Scientists can find out how well a vaccine is working by determining how many vaccine-specific T cells there are-the more there are, the more protection will be provided, the better the vaccine.   When a T helper cell encounters a peptide it’s specific for, it not only divides like crazy but it also upregulates an activation marker on its surface called CD40L.   For the purpose of this paper, it’s just important to understand that some proteins like CD40L can be used as a diagnostic tool to track the progression of T cell activation following vaccination.   In this experiment, Lange and colleagues, drew blood from the HAV-vaccinated men in both the “sleep” and “wakefulness” groups, then stimulated the blood cells with a pool of HAV peptides for 6 hours.  In this short amount of time, the only cells expressing CD40L are T helper cells that responded to and are therefore specific for HAV.  In both groups, the % of CD40L+ HAV-specific T helper cells increases.    It is important to note, that quickly following a shot, there is increase followed by a plateau of activated HAV-specific T helper cells and that with each vaccine shot, the T cell response to the vaccine increases; this is the purpose of booster shots.  The surprising result was that by the second HAV shot, there was a significantly lower T helper response in the “wakefulness” group than the “sleep” group.   Intriguingly, this difference lasted even 1 year after the initial vaccine shot with the “sleep” group having 2 times the number of activated, HAV-specific T helper cells than the “wakefulness” group.  Remember that the only major variable between the groups is that the “sleep” group went to sleep the night following the vaccination shots and the “wakefulness” group did not.  This one difference in sleep scheduling contributed to a two-fold difference in T cell activation and response to the HAV vaccine!

 Remember that T helper cells are called “helpers” because they produce cytokines that help other immune cells to become activated and better responsive to the vaccine leading to better protection against pathogens.  To make sure that the HAV-specific T helper cells they were detecting were in fact “helpers”, Lange, et al. measured various cytokines produced from CD40L+ cells in response to the HAV peptide pool.  In all the cytokines tested, the “sleep” group produced significantly more cytokines (Interferon(IFN)gamma, Interleukin(IL)-2, Tumor necrosis factor(TNF)alpha, and IL-4) than the group didn’t go to sleep after getting their vaccine shot.  

 In addition, T helper cells are “helpers” because they “help” B cells to produce and release antibodies (IL-4 helps this, for example).  Because antibodies are one of the major ways vaccines work to protect you against pathogens, the authors of this paper needed to assess whether antibody production is also affected by sleep.  To do this, they need to isolate the serum from the blood cells when they collect the blood samples.  Once they have the serum, which contains proteins and antibodies but not cells, they can look for HAV-specific antibodies-which is basically like searching for a needle in a haystack.  However, researchers can find HAV-specific antibodies in serum by performing an Enzyme-LinkedImmunosorbent Assay (ELISA).  The basis for an ELISA is using a culture plate coated with HAV proteins and then adding serum to the coated plate.  After a brief incubation period, the plate is washed thoroughly, and because antibodies bind extremely well to specific proteins, only the HAV-specific antibodies will stick to the plate, whereas the non-HAV antibodies floating in the serum will be washed away.  Finally, the amount of HAV-specific antibodies present (antibody titer) can be determined easily.  By this method, which is a very common, standard method used by immunologists and medical professionals, they found the people who slept the night following a vaccine shot produced significantly more HAV-specific IgG antibodies compared to those who didn’t get any sleep!

 What is very interesting is that the percentage of HAV-specific T helper cells greatly correlated with the sleep stage #4 compared to the other sleep stages, suggesting that something was present during sleep stage #4 might be regulating this phenomena.  Lange, et al. had found that hormones such as growth hormone (GH) and prolactin levels increased profoundly during SWS period.  Recall, that these hormones induce an inflammatory response, which has been studied extensively as a way the body regulates itself to become sleepy.  What is less known, is if these sleep-associated hormones have a role during the immune response as well.  What these data indicate is that GH and prolactin not only regulates the brain to induce sleep, but they also regulate the immune system to induce a highly activated immune response upon vaccination!  

Furthermore, during SWS, cortisol levels –which is immunosuppressive- is very low.  Given these data, the authors hypothesized that increase in GH and prolactin coupled with the decrease in cortisol levels during SWS provided a boost to the HAV vaccine that the “wakefulness” group did not receive.  In this way, these sleep-associated hormones behave like an adjuvant that further stimulates APCs to activate T helper cells.  By determining an “adjuvant factor” (GH x prolactin divided by cortisol levels), and correlating this “adjuvant factor” with the percent of HAV-specific T helper cells, Lange, et al. discovered the production of GH, prolactin and cortisol can eloquently predict the development of a strong HAV-specific immune response!

  As the authors admit, “the immunoregulatory functions of sleep are not well understood”.  Which makes this particular research so exciting and novel!  Of course, there are lots to expand upon and investigate to better understand how exactly these sleep-associated hormones boost the immune response to the HAV vaccine.  It would also be very interesting to know if sleep affects other vaccines besides HAV and if sleep cycles in women or people who are older or younger than the group tested in this paper provide similar results.  From an immunologist’s perspective, I think it would be valuable to know not only if the development of immune response to the HAV vaccine is significantly better, but also if the immune response is functionally improved because of sleep.  Which will be difficult to do in humans (people are ok with volunteering for a HAV vaccine, but to also volunteer to get infected with HAV? Probably not so great.) But perhaps this aspect could be assessed in mice or by looking at sleep patterns among people who have Hepatitis A and seeing if there is a smaller viral titer in people who sleep more.  It might seem ridiculous, that this paper rests all of its results on the one difference of either sleeping 7.5 hours or not sleeping at all, because it might appear that this data is only applicable if you are an insomniac.  It would be interesting to know what exactly the threshold of sleep (hours) is required to see the phenomena they illustrate in this paper.  However, what this research provides is strong evidence that little to no sleep has a profound effect on the development of your immune response during vaccination.  Interestingly, it would be important to test if sleep-associated hormones have a similar effect during infection with a live virus since this data might provide new insight into why newborns are highly susceptible to such infections (what newborn do you know that sleeps 7+ hours a night?) and why people who work 16+ hour days or graveyard shifts spend more time sick than people who work 9-5.  

Since starting graduate school, I’ve noticed that I have spent more days a year sick than I ever have, I used to think it was because of my increase use of public transportation, living in a bigger city, and interacting with more people (big lab, friends, students of mine, seminars, etc) than I had in my past.  But now, I think that I had been neglecting one of the major differences between life before graduate school and now: longer hours working in the lab generally means less sleep at home.  As if I needed another motivational reason to be done with graduate school!

Lange T, Dimitrov S, Bollinger T, Diekelmann S, & Born J (2011). Sleep after vaccination boosts immunological memory. Journal of immunology (Baltimore, Md. : 1950), 187 (1), 283-90 PMID: 21632713








References and Further Reading: 

1.    Hosford, M. et al. “Air traffic controller asleep on duty at Reagan National, NTSB says”. ABC News. (2011). 
2.    Beers, TM. “Flexible schedules and shift work: replacing the “9-to-5” workday?”. Monthly Labor Review. (2000). 
3.    Awake in Philly Community Education Group. “Fact sheet: shift work sleep disorder”. (2004). 
4.    CDC. “Insufficient sleep is a public health epidemic”. (2011). 
5.    US Department of Transportation, National Highway Traffic Safety Administration, National Center on Sleep Disorders Research, National Heart Lung and Blood Institute. “Drowsy driving and automobile crashes” (2011). 
6.    Bryant, PA., et al. “Sick and tired: does sleep have a vital role in the immune system?” Nature Reviews Immunology. 4:457-467. (2004). 
7.    Toth, LA., et al. “Alteration of sleep in rabbits by Staphylococcus aureus infection”. Infect. Immunity. 58: 1785-1791. (1988). 
8.    Born, J., et al. “Effects of sleep and circadian rhythm on human circulating immune cells”. Journal of Immunology. 158:4454-4464. (1997). 
9.    Everson, CA. “Sustained sleep deprivation impairs host defense”. Am. J. Physiol. 265:R1148-R1154. (1993). 
10. Kripke, DF., et al. “Mortality associated with sleep duration and insomnia”. Arch. Gen. Psychiatry.” 59:131-136. (2002).