Intestinal use

The gastrointestinal tract is packed with up to a 100 trillion microbes, called the microbiota [1]. This life-sustaining microbiota assists in producing essential vitamins and the digestion of important nutrients. Perhaps the most promising discovery is that these microbes play an essen¬tial role in protection against pathogens and communication with our immune system [2]. Disturbances within the communication of the microbiota and our immune system, is associated with many different pathologies, such as Crohn’s disease, colon cancer and even obesity. A specific, reliable and rapid sensor for these diseases and biomarkers is still lacking. The development of our device can thereby be a first step towards such a diagnostic tool, and in the distant future even for treatment of these pathologies.
We have talked to the ICC (Initiative on Crohn and Colitis) and gastroenterologists from the Academic Medical Center (AMC) in Amsterdam about the feasibility of our sensor and the aspects in our system that can be a point to concern. Also Joost Drenth, head of the department Stomach-Colon and Liver diseases at the UMC St. Radboud in Nijmegen, and Alexandra Ginsberg, designer and writer in the field of synthetic biology, gave us their opinion and advice about our project.

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Current approach

Intestinal cancer

Intestinal cancer, also called colorectal cancer or colon cancer is the third most common cancer diagnosed in both men and woman in the U.S. [3]. The disease usually starts with the growth of polyps

Polyps are small clumps of cells formed in the lining of the colon. They are different variants and can turn into the formation of tumors. Benign polyps are not fatal and often termed as hyperplastic polyps. These polyps remain benign and the chance that these will turn into cancer is very low. Adenomatous and hamartomatous polyps are non-malignant variants; however, they have a chance of becoming cancerous if not removed. The last variant of polyps is an invasive variant and can turn into tumors. When polyps evolve into cancer, the uncontrolled tissue growth of the tumor can be seen through all tissue layers of the colon rectum. Even the spreading of cancer to other organs is possible in this stage.

in the mucosa, which is the innermost lining of the intestines. The mucosa consists of epithelium cells, loose connective tissue, and forms the first line of defense in the intestines.
Currently, intestinal cancer is diagnosed and screened by performing fecal occult blood tests (FOBT), a sigmoidoscopy or colonoscopy. A FOBT is the first screening method in which the feces are being checked for the presence of small amounts of blood that are not immediately visible. Even though FOBT is feasible, accepted and widely available, its sensitivity for polyps is low and it also has a relatively low specificity. This led to many false-positive results and repetitive screening is often necessary [4]. A FOBT screening is often combined with a sigmoidoscopy. This method of screening uses a lighted tube with a camera to examine the rectum and sigmoid colon. With a small biopsy tool, samples of tissue can be collected for further examination. Patients need to empty their bowel completely for the screening and therefore patients cannot eat solid food for several days beforehand to ensure accurate results. This manner of screening is invasive for patients and thus not preferred if it is not necessary [5]. Thirdly, a colonoscopy can be executed to detect and immediately remove precancerous polyps. A thin, flexible tube with a camera is used to examine the colon (large intestines) and the lower part of the small intestines. The only difference with a sigmoidoscopy and the benefit of a colonoscopy is that it can inspect the whole colon instead of only a small part [6]. Even though the colonoscopy is currently the most accurate and used screening method, patients often need to be sedated and before the test they need to empty the colon which means no solid food and taking laxatives for several days.

Inflammatory diseases: Crohn’s disease and Colitis

In both Crohn’s disease and Colitis, inflammations occur along the gastro-intestinal tract. The causes for these diseases are still unclear; however, it seems that it is triggered by an oversensitive or damaged immune system. Therefore these diseases belong to the immune mediated diseases, which means the body isn’t attacking its own substances but it’s triggered by external factors. For both diseases, first a physical examination is performed to see whether patients have the corresponding symptoms, such as blood in the stool. Afterwards, lab tests are performed to check for signs of inflammations and infections, for example to check white blood cells or blood protein levels. Then the decisive method is a colonoscopy and CT scans from the abdomen [7][8]. The disadvantage of diagnosing these inflammatory diseases is that there is no one single technique that gives a correct diagnosis, but the combination of multiple methods is needed for an accurate result.

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Future application: COMBs

Seen the problems with the current diagnostic tools, an accurate, non-invasive and early detecting sensor is needed. This can be realized by finding the right biomarkers. Currently the medical world is in search for a detecting mechanism that can sense human hemoglobin in the intestines. Blood’s main component is hemoglobin; therefore it is a crucial biomarker for various abnormalities in the intestines, such as colon cancer, Crohn’s disease, etc. However, human hemoglobin is a biomarker for diverse irregularities in the intestines, whereby still follow-up research has to be done after positive results. From gastroenterologists of the AMC Amsterdam we got the suggestion of COX-2, an enzyme that can be found in the intestines at an early stage of colon cancer. Both Joost Drenth and experts from the ICC saw opportunities of Calprotectin as another biomarker, which is a protein that is associated with inflammatory diseases, such as Crohn’s disease and Colitis. Calprotectin is specifically correspond to inflammatory diseases and thus can distinguish inflammatory diseases from irritable bowel syndrome, which has the same symptoms. By designing aptamers that specifically bind to human hemoglobin, COX-2 or calprotectin, the biosensor can be implemented in the intestines. It can sense the actual situation in the intestines and detect the biomarkers accurately and at an early stage instead of identifying it afterwards in the feces. The high specificity of aptamers is crucial, because other variants, such as bovine hemoglobin, can be found in the intestines as well. As mentioned above, the distinction between different intestinal diseases can be made directly within one test.
Our biosensor can be combined with auto fluorescence imaging. This imaging technique (combined with endoscopy) uses auto fluorescence to detect polyps whereby the intestinal tract lights up as green and the thickenings in the colon become purple. This current technique is not reliable as it doesn’t detect the polyps. By using fluorescent proteins on the intracellular side of our biosensor and the right biomarker, our sensor can accurately detect polyps and secrete a fluorescent signal that can be observed with this imaging technique. Furthermore, in a more distant future, the biosensor can release visible colors when it has detected a biomarker that for example can easily be observed in the feces. This would be easily for people to do at home and facilitates the large scale screening within a population. Besides detection, a more promising development is the secretion of medicine using COMBs when it has detected a biomarker. The detection of biomarkers can lead to the release of transcription factors which induces various signaling pathways in the bacteria causing the biosensor to produce medicine. There are some critical notions on this: the bacteria must be contained and cannot colonize within the intestines. This can be accomplished by bringing the bacteria into alginate beads. As described on our safety page.
Additionally, medicines have strict criteria, whereby each package needs to be equal and have the same dose. This is difficult to realize when working with living organisms. An option is to store the medicine in compartments in the bacteria. Another problem which has to be taken into account in future research is the presence of DNAses and RNAses in the intestines. These proteases are able to go through the pores of the alginate beads and will break down the aptamers.
Even though there are still many obstacles, it is believed to be the prospective manner of taking and secreting medicine. Therefore, permissions to do research with GMOs in the intestines are given more often these days. For example, the COGEM has given permission to perform an I-phase trial on a study about the secretion of IL-10 by GMOs in the intestines, to target treatment of Crohn’s disease. The first tests on humans are already done with promising results [9]. Another example is the study of programmable bacteria, which can detect and record an environmental signal in the mammalian gut [10]. These studies offer good prospects for our COMBs in the future.

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Q fever

Q fever is a zoonotic disease which is caused by Coxiella burnetii, a pathogen which usually resides in cattle and ticks and can be transmitted to humans through aerosols and raw animal products. The high susceptibility of humans for the pathogen was recognized by both the Soviet Union and the United States, who devised to use the organism as a biological weapon [1]. The high infectivity also resulted in numerous transient Q fever outbreaks in animals and humans within the European Union. Concerns were raised, however, in the European Union as the Netherlands was hit with the largest outbreak ever recorded with 4,026 human cases notified between 2007 and 2010 [2].

Transmission

We have talked to Dr. Roest, a veterinary microbiologist who is strongly involved in research to Q fever and Anja Garritsen, a scientist working at InnatOss, one of the biggest research institutes in the field of Q fever in the world, to get a better insight into the important aspects around Q fever. Besides that we spoke to Jeanette van der Ven, owner of a goat’s farm near Eindhoven and one of the board members of the ZLTO (association for entrepreneurs in the green sector), to get inspiration and feedback from people who were really involved in this problem. Additionally we spoke to René van den Brom, a researcher working for the national institute that focusses on animal well-being, who did his thesis about Q fever. He told us about the problems he faced in his research and showed us his results.

Current Approach

Pathology

The main challenge of diagnosis of Q fever is its atypicality. Contact with C. burnetii is followed by a 2-3 week incubation period. After the incubation period symptoms may occur, even though most cases of Q fever remain asymptomatic (60%) [7]. Even if the patient shows symptoms, these symptoms are often very atypical. The infection is characterized by the unpredictability and diversity of its symptoms. Examples of symptoms which may or may not occur within a single patient are fever, headaches, hepatitis and meningoencephalitis. This clinical polymorphism impedes an early and accurate detection of Q fever. This is very problematic, as failure to diagnose Q fever early and accurately can lead to the manifestation of chronic Q fever (see Figure 4). Figure 4: After contact with Coxiella burnetii, the pathogen enters the cell. After an incubation period of 2-3 weeks, 40% of the patients begin to show symptoms. These symptoms manifest in a wide range of forms, but pneumonia is by far the most common. The other 60% of the patients do not show acute symptoms. If the infection goes untreated, it may become chronic. This can lead to multiple complications, including abortion and endocarditis.

Diagnostics

Currently, diagnostic tools available for Q fever are aimed at a few biomarkers. The first biomarker is the DNA of C. burnetii. By amplifying a part of the genome of C. burnetii within the patient’s blood trough PCR acute Q fever infection can be detected [8]. The test is highly specific and very sensitive, but only as long as DNA remains detectable within the patient’s blood. The DNA can approximately be detected until two weeks after contact with the pathogen within blood samples of the patient. After these two weeks, the patient has typically become immune against the pathogen and the DNA of the pathogen can no longer be found within the patient’s blood. This is problematic as Q fever’s clinical polymorphism impedes doctors to rapidly recognize Q fever. Therefore, prevention of Q fever infection is often easier to accomplish than cure. The second biomarker is a direct result of this built up immunity. Two types of antibodies become detectable in the blood. In contrast to the DNA of C. burnetii, these antibodies remain detectable within the blood for longer periods (Figure 5) [9].A drawback of serological tests is the lag phase in antibody response: it takes up to two weeks before a patient’s blood gives detectable levels of Phase I-antibodies. Therefore, these tests can only be used to detect previous Q fever rather than acute Q fever. Additionally, serological tests and skin tests suffer from a higher variability and show long return times. Besides that, the success of skin tests relies on trained personnel. As an alternative to these tests, interferon gamma detection has been developed [10]. In this test, a blood sample from the patient is taken which is incubated with C. burnetii antigens. Rather than testing for the presence of antibodies, however, the immunity of the patient is tested by interferon gamma detection, a signaling molecule which is secreted by immune cells upon recognition of the pathogen. The developed test shows similar specificity and sensitivity as skin tests and serological tests. Data is, however, available within 24 hours and the test does not depend on training of personnel. A drawback in the diagnostics of Q fever is that the distinction between patients with an acute infection or patients which already have been through the disease process is hard to make. For microbiologists this is a very important thing, because they are only allowed to give antibiotics to patients with acute Q fever. At a later stage only treatment in psychological aspect can be done. Figure 5: After first contact with the pathogen, 40% of the patients fall ill. After the patient falls ill, DNA of Coxiella Burnetti becomes detectable in the blood. This DNA remains detectable for approximately 2 weeks. 1.5 weeks after the acute infection, Phase I-antibodies become detectable in the blood. In an acute infection, this is rapidly followed by detectable levels of Phase II-antibodies. In contrast to Coxiella Burnetii’s DNA, serological tests for antibodies remain positive for more than 12 months after the acute infection.

Future application: COMBs

Goat vaccination

The biomarkers available for Q fever have led to a wide range of diagnostical tools. Conventional tools to detect previous Q fever have been serological tests and skin tests. Both tests, however, have major drawbacks. Serological tests suffer from a higher variability and show long return times. Skin tests also suffer from a higher variability and success of skin tests relies on trained personnel [Specific Interferon gamma Detection]. As an alternative to these tests, interferon gamma detection has been developed. In this test, a blood sample from the patient is taken which is incubated with Coxiella Burnetti antigens. Rather than testing for the presence of antibodies, however, the immunity of the patient is tested by interferon gamma detection, a signaling molecule which is secreted by immune cells upon recognition of the pathogen. The developed test shows similar specificity and sensitivity as skin tests and serological tests. Data is, however, available within 24 hours and the test does not depend on training of personnel.

Both serology and the interferon gamma detection test have a major disadvantage, however, as they rely on immunology of the patient. This is a problem because of the lag phase in antibody response: it takes up to two weeks before a patient’s blood gives detectable levels of Phase I-antibodies. Therefore, these tests can only be used to detect previous Q fever rather than acute Q fever. An alternative test which can be used to detect acute Q fever infection is real-time PCR. During this test, part of Coxiella Burnetti’s genome within the patient’s blood is amplified through PCR. The test is highly specific and very sensitive, but only as long as DNA remains detectable within the patient’s blood. This is a disadvantage since DNA is only detectable for approximately two weeks after the acute infection.

The importance of prevention

Numerous serological tests have been developed to target Q fever. Whereas a wide range of tests has become available to detect previous Q fever accurately, these tests can only be used to detect previous Q fever. Therefore, these tests are very important to map Q fever epidemics by assessing whether a person should be vaccinated or not. These tests, however, fail to address acute infections of Q fever.
A test which can detect acute infections of Q fever, the real-time PCR test, can provide an early and accurate diagnosis of Q fever. The problem with this test, however, is that it relies on the presence of Coxiella Burnetii’s DNA in the blood of the patient. Since this DNA can only be detected for about two weeks after the patient falls ill, such a real-time PCR test should be conducted shortly after a patient falls ill. This is problematic as Q fever’s clinical polymorphism impedes doctors to rapidly recognize Q fever. Therefore, prevention of Q fever infection is often easier to accomplish than cure.

Goat vaccination

In 2009 at the peak of the Q fever outbreak, the Dutch government decided to offer risk groups who lived near animal farms vaccines against Q fever.[11]In addition to offering vaccines to risk groups, the Dutch government mandated the culling of pregnant goats at farms which were known to be infected, as pregnant goats presented a major threat to public health. All in all, tens thousands of pregnant goats were killed to stop the Q fever outbreak, even though it was not tested whether the pregnant goats were infected. The decreasing numbers of human cases of Q fever in 2010 might indicate that the measures taken by the Dutch government have been effective at limiting the Q fever epidemic [12].
In addition to culling of ten thousands of pregnant goats in 2009, the Dutch government imposed numerous regulations on goat farms to limit the outbreak. These measures include a mandatory vaccination of dairy sheep and dairy goats, mandatory tank milk monitoring for Q fever every 2 weeks and a ban on removing manure from the farm. Many of these regulations are still enforced: dairy goats and sheep have to be vaccinated yearly, there is a report duty for signs of Q fever and every dairy farm is obligated to take part in a biweekly bulk tank milk test for the presence of C. burnetii as the milk of infected goats contains DNA from the pathogen. Figure 6: The milk of goats infected with C. burnetii contains its DNA. In addition to the DNA of the pathogen, the milk usually contains the Phase I- and Phase II-antibodies which are active against C. burnetii. The tank milk test, however, consists of a real-time PCR test as the antibodies are present because of the vaccination of goats.
Jeanette van der Ven, owner of a goat’s farm near Eindhoven and one of the board members of the ZLTO (association for entrepreneurs in the green sector), told us that it is pretty hard for farmers to gain monsters for the testing of their goats. Protocols have to be followed and everything has to be disinfected, which is not pleasant for the animals. It would be a great improvement to make the milk tests easier to perform. With the use of COMBs the farmers will be able to test the milk at home by bringing our sensor system, encapsulated in alginate beads SAFETY, into the milk samples. These samples can be simply extracted from the milk tanks or even be built into the milk tanks. When antibodies are present, the beads shall turn into a color. This way, farmers don’t have to send milk samples to a research institute anymore and will get the result of the test immediately. Treatments can be started immediately and there will be no more risk of newly infected cattle in the meantime. Because all goats are vaccinated, detection of antibodies into the milk does not always signify infection. However, experts from the ICC told us that they vaccinate once a year, but often notice that previous vaccines are not working anymore. With the use of COMBs, real-time detection of antibodies is possible, which is really helpful by determine when the goats have to be vaccinated again. Also in this case the farmers are able to do these tests themselves. We hope that with the use of COMBs a main problem for the society can be solved, by detecting infections easily at an early stage and optimize the vaccination program.

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