High Fiber Diet in Graft Versus Host Disease
Itamar Greenfield
Mentors: Melissa Docampo, and Christoph Stein Thoreinger
Memorial Sloan Kettering Cancer Center
Lab of Marcel van den Brink
March 5, 2018
Abstract:
One possible way to treat blood cancers and other blood disorders is with a bone marrow transplant, which replaces the blood system of a host with that of a donor. A possible consequence of this treatment is Graft Versus Host Disease (GVHD), a disease that occurs when the immune cells of the donor attack the body of the host. This disease is very common after a bone marrow transplant, and it accounts for about 19-23% of the deaths after a bone marrow transplant. Because this disease has a very strong effect on the intestines of a patient, the effects of this disease can be dramatically influenced by the bacteria in the intestines, known as the intestinal microbiota. A more diverse microbiota is known to correspond with less severe GVHD, while a less diverse microbiota is known to correspond with more severe GVHD. A number of factors affect the diversity of the intestinal microbiota, and one that has a particularly profound impact is diet. Recent studies have found that a high fiber diet has a positive effect on the intestinal microbiota, and leads to better gut health. This project will feed mice a high fiber diet and investigate the resulting changes to their intestinal microbiota, thus contributing to a greater understanding of how diet plays a role in the severity of Graft Versus Host Disease.
Background:
A bone marrow transplant (BMT) is a common treatment for those afflicted with blood cancer or other blood disorders (Van den Brink, 2016). This treatment replaces all of the host’s blood system with that of a donor. This is done by irradiating a host killing the blood cells in his or her system. The host is then given blood cells extracted from a donor, including immune cells (which protect the body and attack invaders) and hematopoietic stem cells (which create new blood cells). The new cells are given through an injection into the host. One possible complication of such treatment is Graft Versus Host Disease (GVHD) (Van den Brink, 2016). GVHD occurs when the immune cells of the donor, which normally protect the body from invaders, attack the system of the host (Van den Brink, 2016). The results of GVHD are catastrophic; patients with GVHD often experience weight loss, lack of appetite, and diarrhea (Van den Brink, 2016). The digestive system can be severely impacted by GVHD, experiencing swelling of the intestines and many other forms of intestinal damage (Van den Brink, 2016). GVHD often causes death, and accounts for about 19-23% of all deaths following a bone marrow transplant (Docampo).
The intestinal microbiota has a strong influence on the overall health of the digestive system, and therefore, has the potential to mitigate the impact of GVHD (Van den Brink et al., 2016). The intestinal microbiota, consists of millions of bacteria living in the gut and can be affected by diet, genetics, environment, and drugs (i.e.antibiotics) (Van den Brink, 2016). Generally, a larger and more diverse intestinal microbiota can help protect the gut from diseases and promotes better digestion. Studies have shown that a more diverse intestinal microbiota leads to less severe GVHD (Van den Brink, 2016).
Recently, many studies have been published about high fiber diets. One study done on school children found that a high fiber diet leads to increased gut health, and that a low fiber diet leads to problems in gut health (Brauchla et al., 2013). Other studies, compiled in the review article of Simpson et al. have found that a high fiber diet has a diversifying effect on the intestinal microbiota and leads to a better intestinal health (Simpson et al., 2015). However, the precise effects of a high fiber diet on GVHD have not yet been successfully studied and there is no literature published about it yet. Studying this would give more information to improve diets for patients undergoing a bone marrow transplant. This proposed project will feed a high fiber diet to mice, and view its effect on their intestinal microbiota. Then, the mice will receive BMT’s with T-cells to induce GVHD. From there, the project will investigate how a high fiber diet effects the mice’s immune response to GVHD.
Project Description:
The overall aim of this project will be to investigate the effect of a high fiber diet on GVHD in a mouse model. The first objective of this experiment will be to determine the effect of a high fiber diet on the intestinal microbiota. After introducing five BALB/C (albino lab mice) mice to the high fiber diet and feeding a normal diet to five other BALB/C mice, fecal samples will be collected weekly from the high fiber group and the control group. The samples will then be tested to determine and track the changes in the intestinal microbiota between the two groups. The testing will be done by extracting deoxyribonucleic acid (DNA) from the feces of the mice and running a Polymerase Chain Reaction (PCR) test on the resulting DNA. This test will indicate the diversity of the DNA in the intestinal microbiota in the mice; the extracted DNA from the mice feces will be the DNA of the bacteria in the gut of the mouse.
The second objective of the experiment will be to determine the effect of the high fiber diet on the response to the GVHD. This will be done by first inducing GVHD to both the high fiber diet group and the control group through a bone marrow transplant. The two groups will be carefully observed to determine their reaction to the GVHD. They will be assessed based on their weight, posture, fur, skin, and activity.
This project will be deemed an overall success if the high fiber group outperforms the control group in the reaction to the GVHD and in their survival numbers.
Research Strategy:
The first step of this project will be to breed BALB/C mice. Ten of the resulting baby mice will be randomly split into a control and high fiber group with five mice in each group. The reason for breeding mice and not ordering them is so that they will be able to be introduced to the high fiber food as soon as they are able to eat solid food (at the age of two weeks). If they were to be ordered, the mice will have been on a normal diet in the facilities and during shipping; and their microbiota will be adjusted to the normal diet before being introduced to the high fiber diet. By breeding the mice in the lab, they will have more time to become acclimated to the food, and more time for the effects of the high fiber foods to take hold.
The next step of this project will be to collect fecal samples from the mice after 2 weeks and 1 month of eating the high fiber or normal diets. DNA will be extracted from the fecal samples using a DNA extraction kit. The extracted DNA from the feces will be the DNA of the intestinal bacteria of the mice. A PCR test will be conducted to measure diversity of the intestinal microbiota. This will be done by putting a special dye on the DNA and loading it into a gel. A different dye (known as a ladder) that corresponds to possible genes of the bacteria will be loaded in the gel next to the DNA samples of all the mice. An electric current will be run through the gel, and the DNA will move to different parts of the ladder corresponding to the diversity of bacterial DNA in them. By doing the PCR test of the DNA, it will be possible to assess the impact of the high fiber diet on the diversity of the intestinal microbiota.
After a month of eating the high fiber diet, the mice will be induced with GVHD. This will be done by first irradiating the mice in an irradiator. This exposure to radiation will kill the blood cells in the mice, allowing them to become replaced by a bone marrow transplant. The donor blood cells will come from a B6 (black) mouse, given by a tail vein injection. The donor B6 mouse will be a randomly selected healthy mouse (who ate a normal diet). Because BALB/C and B6 are very different mice, there will be a mismatch between the cells. The immune cells from the B6 mice will not recognise the cells of the recipient mice. Some of the donor cells will then attack the cells of the host mice, and will thus induce GVHD.
The mice in the high fiber and control groups will be observed to determine the effects of the GVHD. They will be assessed based on their weight, posture, fur, skin, and activity. For each category, they will be given a score between 0 and 2 (0 being no effect and 2 being very severe). The mice will be assessed every week for a month after the transplant, and will be euthanized with CO2 if they exceed a compiled score of 5 (to ensure that the mice are humanely treated and do not go through too much pain). The mice that die will have their date of death recorded, and the survival rates of the mice will factor into the analysis of the effect of the GVHD. After a month the experiment will be over and all remaining mice will be euthanized. The average score of each group of mice will be compared with each other, along with the survival rates of the mice in order to determine the effect of the high fiber diet on the reaction to GVHD.
Bibliography:
One possible way to treat blood cancers and other blood disorders is with a bone marrow transplant, which replaces the blood system of a host with that of a donor. A possible consequence of this treatment is Graft Versus Host Disease (GVHD), a disease that occurs when the immune cells of the donor attack the body of the host. This disease is very common after a bone marrow transplant, and it accounts for about 19-23% of the deaths after a bone marrow transplant. Because this disease has a very strong effect on the intestines of a patient, the effects of this disease can be dramatically influenced by the bacteria in the intestines, known as the intestinal microbiota. A more diverse microbiota is known to correspond with less severe GVHD, while a less diverse microbiota is known to correspond with more severe GVHD. A number of factors affect the diversity of the intestinal microbiota, and one that has a particularly profound impact is diet. Recent studies have found that a high fiber diet has a positive effect on the intestinal microbiota, and leads to better gut health. This project will feed mice a high fiber diet and investigate the resulting changes to their intestinal microbiota, thus contributing to a greater understanding of how diet plays a role in the severity of Graft Versus Host Disease.
Background:
A bone marrow transplant (BMT) is a common treatment for those afflicted with blood cancer or other blood disorders (Van den Brink, 2016). This treatment replaces all of the host’s blood system with that of a donor. This is done by irradiating a host killing the blood cells in his or her system. The host is then given blood cells extracted from a donor, including immune cells (which protect the body and attack invaders) and hematopoietic stem cells (which create new blood cells). The new cells are given through an injection into the host. One possible complication of such treatment is Graft Versus Host Disease (GVHD) (Van den Brink, 2016). GVHD occurs when the immune cells of the donor, which normally protect the body from invaders, attack the system of the host (Van den Brink, 2016). The results of GVHD are catastrophic; patients with GVHD often experience weight loss, lack of appetite, and diarrhea (Van den Brink, 2016). The digestive system can be severely impacted by GVHD, experiencing swelling of the intestines and many other forms of intestinal damage (Van den Brink, 2016). GVHD often causes death, and accounts for about 19-23% of all deaths following a bone marrow transplant (Docampo).
The intestinal microbiota has a strong influence on the overall health of the digestive system, and therefore, has the potential to mitigate the impact of GVHD (Van den Brink et al., 2016). The intestinal microbiota, consists of millions of bacteria living in the gut and can be affected by diet, genetics, environment, and drugs (i.e.antibiotics) (Van den Brink, 2016). Generally, a larger and more diverse intestinal microbiota can help protect the gut from diseases and promotes better digestion. Studies have shown that a more diverse intestinal microbiota leads to less severe GVHD (Van den Brink, 2016).
Recently, many studies have been published about high fiber diets. One study done on school children found that a high fiber diet leads to increased gut health, and that a low fiber diet leads to problems in gut health (Brauchla et al., 2013). Other studies, compiled in the review article of Simpson et al. have found that a high fiber diet has a diversifying effect on the intestinal microbiota and leads to a better intestinal health (Simpson et al., 2015). However, the precise effects of a high fiber diet on GVHD have not yet been successfully studied and there is no literature published about it yet. Studying this would give more information to improve diets for patients undergoing a bone marrow transplant. This proposed project will feed a high fiber diet to mice, and view its effect on their intestinal microbiota. Then, the mice will receive BMT’s with T-cells to induce GVHD. From there, the project will investigate how a high fiber diet effects the mice’s immune response to GVHD.
Project Description:
The overall aim of this project will be to investigate the effect of a high fiber diet on GVHD in a mouse model. The first objective of this experiment will be to determine the effect of a high fiber diet on the intestinal microbiota. After introducing five BALB/C (albino lab mice) mice to the high fiber diet and feeding a normal diet to five other BALB/C mice, fecal samples will be collected weekly from the high fiber group and the control group. The samples will then be tested to determine and track the changes in the intestinal microbiota between the two groups. The testing will be done by extracting deoxyribonucleic acid (DNA) from the feces of the mice and running a Polymerase Chain Reaction (PCR) test on the resulting DNA. This test will indicate the diversity of the DNA in the intestinal microbiota in the mice; the extracted DNA from the mice feces will be the DNA of the bacteria in the gut of the mouse.
The second objective of the experiment will be to determine the effect of the high fiber diet on the response to the GVHD. This will be done by first inducing GVHD to both the high fiber diet group and the control group through a bone marrow transplant. The two groups will be carefully observed to determine their reaction to the GVHD. They will be assessed based on their weight, posture, fur, skin, and activity.
This project will be deemed an overall success if the high fiber group outperforms the control group in the reaction to the GVHD and in their survival numbers.
Research Strategy:
The first step of this project will be to breed BALB/C mice. Ten of the resulting baby mice will be randomly split into a control and high fiber group with five mice in each group. The reason for breeding mice and not ordering them is so that they will be able to be introduced to the high fiber food as soon as they are able to eat solid food (at the age of two weeks). If they were to be ordered, the mice will have been on a normal diet in the facilities and during shipping; and their microbiota will be adjusted to the normal diet before being introduced to the high fiber diet. By breeding the mice in the lab, they will have more time to become acclimated to the food, and more time for the effects of the high fiber foods to take hold.
The next step of this project will be to collect fecal samples from the mice after 2 weeks and 1 month of eating the high fiber or normal diets. DNA will be extracted from the fecal samples using a DNA extraction kit. The extracted DNA from the feces will be the DNA of the intestinal bacteria of the mice. A PCR test will be conducted to measure diversity of the intestinal microbiota. This will be done by putting a special dye on the DNA and loading it into a gel. A different dye (known as a ladder) that corresponds to possible genes of the bacteria will be loaded in the gel next to the DNA samples of all the mice. An electric current will be run through the gel, and the DNA will move to different parts of the ladder corresponding to the diversity of bacterial DNA in them. By doing the PCR test of the DNA, it will be possible to assess the impact of the high fiber diet on the diversity of the intestinal microbiota.
After a month of eating the high fiber diet, the mice will be induced with GVHD. This will be done by first irradiating the mice in an irradiator. This exposure to radiation will kill the blood cells in the mice, allowing them to become replaced by a bone marrow transplant. The donor blood cells will come from a B6 (black) mouse, given by a tail vein injection. The donor B6 mouse will be a randomly selected healthy mouse (who ate a normal diet). Because BALB/C and B6 are very different mice, there will be a mismatch between the cells. The immune cells from the B6 mice will not recognise the cells of the recipient mice. Some of the donor cells will then attack the cells of the host mice, and will thus induce GVHD.
The mice in the high fiber and control groups will be observed to determine the effects of the GVHD. They will be assessed based on their weight, posture, fur, skin, and activity. For each category, they will be given a score between 0 and 2 (0 being no effect and 2 being very severe). The mice will be assessed every week for a month after the transplant, and will be euthanized with CO2 if they exceed a compiled score of 5 (to ensure that the mice are humanely treated and do not go through too much pain). The mice that die will have their date of death recorded, and the survival rates of the mice will factor into the analysis of the effect of the GVHD. After a month the experiment will be over and all remaining mice will be euthanized. The average score of each group of mice will be compared with each other, along with the survival rates of the mice in order to determine the effect of the high fiber diet on the reaction to GVHD.
Bibliography:
