Eukaryon

Abstract
Type 1 Diabetes is an autoimmune disorder resulting in the destruction of beta-cells within the pancreas. The destruction of these cells means the body cannot produce insulin, and without treatment this condition is fatal. Current treatment includes artificial injection of insulin after ingesting glucose. Though this treatment has a high survival rate, no permanent cure has been found. Therefore, it is pertinent to research possible cures for the disease. Current research has outlined some improvement in the condition among individuals treated with an antibody for the complement molecule C3. Due to intense side-effects as well the fact that it only partially relieves disease status, these experiments have not been pursued in high-phase clinical trials. It is still necessary to find a highly effective method of curing Type 1 Diabetes without causing destructive side-effects. This paper outlines an experimental proposal, which would hopefully lead to clinical trials and come to the market with indication for curing Type 1 Diabetes. This proposal uses a NOD Murine model to exemplify safety and effectivity. Overexpression of the MW 60,000 protein would be executed through retroviral infection of the gene into the NOD Murine model. Confirmation of successful transfer of the gene of interest would then be performed through the use of PCR followed by Agarose Gel Electrophoresis. Succeeding confirmation, the mice would then be tested for presence or absence of C3 through the protein-quantifying methods monoclonal antisera and western blot. Finally, effect of treatment on Type 1 Diabetes would be measured through C-Peptide levels, allowing for analysis of β-cell survival in the pancreas 

Introduction
Type 1 Diabetes is an autoimmune disorder that affects 9.3% of the United States population (Center for Disease Control, 2012). This prevalent disease is characterized by the inability to naturally produce insulin. Current treatments for the disease include injecting one’s self with insulin after eating either through a syringe or an electronic pump. The individual with the disorder is responsible for controlling their blood sugar levels, which, in a healthy individual, is taken care of naturally by the body.
On a molecular basis, the initial cause of Type 1 Diabetes is unknown, but there is a lot that has been found out up to this point about the development of the disease. After the initial β-cell destruction, dendritic cells are recruited to the site of cell destruction to clean up the debris (Medline Plus, 2015). Dendritic cells will take the debris and display it externally, and when they reach the lymph nodes they can use the fragmented cell to activate the unspecified T-cells and initiate the β-cell specific immune response (Alberts, 2002). These newly initiated T-cells then will travel to the pancreas and target the β-cells that make up the islets for destruction. Once they have destroyed some of these islet cells, the dendritic cells are recruited again to clean up the cell debris, which perpetuates the cycle of auto-reactivity until all of the islet β-cells have been destroyed. Since these pancreatic β-cell clusters are responsible for the production and secretion of insulin, the cycle of auto-reactivity will eventually eliminate the body’s ability to create insulin (Alberts, 2002).
The body’s inability to create insulin is a highly problematic issue. Insulin in a normal body allows for the use of the glucose taken in by eating. When the pancreas releases insulin, the molecule binds to its receptor and activates individual cells (Medline Plus, 2015). This binding initiates the opening of GLUT4, a glucose transport protein (Joslin Diabetes Center, n.d.). Once GLUT4 is open, the glucose molecules in the blood stream and extracellular space can be transported into the cells and consequently used for energy to drive all cellular processes and maintain survival. However, if no insulin is being created, activation of the insulin receptor cannot occur, which, in turn, will cause the GLUT4 receptor to remain closed. If glucose cannot enter the cell it won’t be able to drive its cellular processes and will eventually die. Therefore, it is evident why the destruction of pancreatic islet β-cells can be dangerous and even life threatening, and thus why research for this disease is essential.
Current Research
Current research is working on illuminating the cellular basis for the destruction of these β-cells, as well as determining the initial cause of the auto-reactive response. There has been a lot of work done on identifying the specific molecules involved in the signaling cascades that recruit the immune system components. The more we understand about these specifics, the better the hypotheses for cures and long-term treatment will be.
Before working on treatments and therapeutic targets, researchers first needed to understand on a more molecular basis how the disease works so they can properly target further research. This focus elucidated a lot of specific immune mechanisms involved in the development of Type 1 Diabetes. Plasmacytoid Dendritic Cells (pDCs) are heavily involved in the onset of the disease, and represent a high proportion of dendritic cells in individuals recently diagnosed with the disease (Allen, 2009). Researchers found this by using flow cytometry to measure the relative proportions of specific types of dendritic cells in recently diagnosed Diabetes patients to that of healthy patients. It followed from qPCR of samples from mice that pDCs secrete IFN-alpha and are essential to the development of Type 1 Diabetes (Diana, 2013). Similarly, upon examination of blood samples from recently diagnosed Type 1 Diabetes patients, it was discovered that pDCs use immune complexes to drive the T-cell activation, which is what perpetuates β-cell targeting by the immune system (Allen, 2009). This lead to the question of what other immune molecules are involved in this perpetuation of β-cell death.
To further investigate this, Diana et al. utilized flow cytometry to determine relative quantities of known immune system molecules (2013). After finding that B1a cells were a part of the measured serum, they looked at how they might be interacting with pDCs. Through ELISA they found that B1a cells use IgG to activate pDCs (Diana, 2013). After immunohistologically analyzing the tissue, researchers found that neutrophils work in conjunction with B1a cells to initiate the immune response that causes β-cell death, through releasing CRAMP and IFN-alpha (Diana, 2013). They confirmed using ELISA that the most effective way to develop Type 1 Diabetes was with an equal mixture of B1a and neutrophil cells.

Interleukin-1 is another immune system molecule often implicated in Diabetes. Research is being done on it and its role in the development of the disease. IL-1 binds to its receptor and initiates a cascade of signals that promote β-cell dysfunction, which leads to apoptosis of the cell (Moran, 2013). Specifically, it promotes a signaling pathway involving Fas, which is a signaling molecule that leads to cell death. IL-1β specifically promotes creation and survival of the T-cells that are involved in memory of a foreign invader, as well as helping to differentiate the T lymphocytes (Dinarello, 2012). IL-1β, is a molecule that has been deeply associated with the regulation of the immune system response with respect to Type 1 Diabetes. Therefore, it seems likely that it would be a powerful target for therapeutic research. In preliminary experiments, 15 children with diabetes were treated with an IL-1 receptor inhibitor for 28 days to see how it would impact the development and progression of their disease after recent diagnosis. They found that these treatments didn’t cause many adverse side effects, and that their external insulin dependence decreased, implying that their insulin production was somewhat salvaged (Sumpter, 2011).
After Sumpter’s team of researchers did a successful initial trial on a group of children as described previously, it seemed only logical to launch clinical trials on this method with a larger sample of individuals with the condition. Thus, building off of Sumpter’s research, Antoinette Moran and her team of researchers investigated the possibility of IL-1β as a target for treatment among the masses. In a sample of 105 patients, they ran both tests on introducing an antibody to the IL-1 molecule, or by introducing an IL-1 receptor inhibitor into the system (Moran, 2013). In either case, they didn’t find any significant data that showed that the method was helping to fend off progression of the disease (Moran, 2013). Though this may seem disappointing, failed results are often equally as enlightening as successful results.
One group of researchers investigated 42 recently deceased Type 1 Diabetes patients to determine how many of their β-cells were destroyed through programmed cell-death. Meier used their pancreatic tissue to look at β-cell death using Capsase-3 as a marker for apoptosis (Meier, 2005). One might expect that these Diabetes patients would express high levels of β-cell death, as the disease functions via the destruction of pancreatic islet β-cells. They actually found that only 5 patients had extremely high levels of β-cell death, and the levels of cell death were two times higher in those patients than the majority of the Type 1 Diabetes patients (Meier, 2005). This prompted them to analyze the insulin secretion in these patients using immunofluorescences of the pancreatic tissue samples. Interestingly, Meier and his team found that 37 of the 42 patients had insulin positive cells remaining in their pancreas, which were found both in islets as well as free-floating β-cells within the pancreas. Only 5 of the 42 patients examined showed little to no insulin-secreting β-cells left in their tissue (Meier, 2005). This interesting information lead the scientific community to further explore the way that β-cell death occurs in patients, specifically with respect to age.
Oram and his team of researchers did some investigation on 924 Type 1 Diabetes patients during which they used a C-Peptide assay to measure insulin levels. C-Peptide is a relevant measure of β-cell activity because β-cells produce pro-insulin, which gets cleaved into C-peptide and insulin as the final step to production of insulin (Sima, 2001). In other words, the quantity at any given moment of C-peptide is directly proportional to the amount of insulin being produced (Figure 1). Thus, the measurement of C-Peptide within diabetic patient serum allowed Oram and his team to determine if there is a relationship between the amount of living β-cells and the age of the patient. After analyzing the 924 Type 1 Diabetes patients’ levels of C-peptide, they found that the highest C-Peptide values were associated with patients who had Type 1 Diabetes for 5-10 years in duration (Oram, 2015). In other words, 92% of individuals tested who had the disease 5-10 years had significant and detectable C-Peptide levels. As duration of the disease increased, the amount of patients with detectable C-Peptide levels decreased to only about 75% of the subjects (Oram, 2015).
This research combined with the results Meier and his team found suggest that even after β-cell destruction at the onset of the disease, there is a small subset of islet β-cells that remain alive and functional. In accordance to what Oram found, the longer the duration of the disease and the longer the individual treats with artificial insulin, the less β-cells can survive to micro-secrete insulin. Consequently, salvaging the surviving β-cells poses itself as a possible therapeutic target. Additionally, earlier in disease progression seems to be the time period for intervention that would produce the most notable results.
Keymeulen decided to attempt to limit the β-cell death in newly diagnosed Type 1 Diabetes patients. His team of researchers designed an antibody for CD3, which is the molecule in the complement system that is involved in perpetuating the cycle of β-cell destruction through T-cell activation. This antibody, called ChAglyCD3, was administered to 40 patients, while a placebo was administered to 40 other patients. Similar to Oram, they measured efficacy through C-Peptide levels in their patients’ serum. They also checked for side effects in blood fractions through flow cytometry and simple blood tests. Interestingly enough, Keymeulen found that 21 of the 40 treatment patients developed antibodies to the ChAglyCD3 antibody itself. They determined that by running a qPCR on the serum retrieved from the patients. After treatment for 18 months with the CD3 antibody they found that there was a 35% decrease from subjects’ initial diagnosis of β-cell function (Keymeulen, 2005). Similarly, there was a 50% increase in artificial insulin requirement (Keymeulen, 2005). They also found a large portion of the treatment group experienced adverse side effects, which they hypothesized was due to the release of cytokines in response to the autoantibody ChAglyCD3. What was notable, however, is that over a shorter period of time, specifically the first 3 weeks, the treatment showed a significant salvage of β-cell destruction and, compared to the control, a lower reliance upon insulin supplementation and a higher C-peptide level. Unfortunately, after that 3-week mark, all measured values became equal with or worse than the placebo control group. Therefore, it seems that the concept of protecting β-cells can be successful, but the details of the study caused adverse side effects, and over the long run didn’t produce significant enough results.
To build off of this study, Harold and his team of researchers chose to study a different antibody in Type 1 Diabetes patients that binds CD3 as well. The antibody, hOKT3gamma1(Ala-Ala), targets CD3 similar to ChAglyCD3, the antibody used by Keymeulen. The mechanisms for this group of antibodies is not well known, which disables researchers from understanding how, specifically, these antibodies differ. Regardless, it is a different antibody for the same target, and one of these antibodiesseemed to produce semi-effective results for Keymeulen’s investigation. For this round of research, 10 subjects were used to determine the efficacy of hOKT3gamma1(Ala-Ala) (Herold, 2009). To determine the influence of the antibody on the immune system, they used flow cytometric analyses to determine the coating of the CD4+ and CD8+ cells, and whether they were altered as compared to baseline. Similar to other studies, Herold and his team used C-Peptide assays to determine the way the antibody influences β-cell efficacy. They found there was no significant increase over 24 months in C-Peptide levels, but over a longer period of time they found a reduced use of insulin in the experimental group. They found limited statistically significant results, which they attributed to their small sample size. Therefore, the information related to this antibody does not necessarily support its use as a potential therapy.
Following from the current research and all of the recent discoveries regarding β-cell survival within the pancreatic islets of Type 1 Diabetes patients at different stages of their disease progression, the next step is to determine the most efficacious way of preserving β-cell function. Two different researchers discussed here studied antibodies for CD3, a complement system molecule. Though some promise can be pulled out of their results, in that over long enough periods of time there is a reduction in synthetic insulin dependence, or in some cases over the short term there is a reduction in synthetic insulin dependence, nothing has shown significant enough results to successfully get through clinical trials. And thus, there remains more research to be done in order to come up with an effective manner of reducing β-cell death through the immune system, while minimizing side effects for patients and maximizing the effectiveness of the treatment to salvage β-cells.
Experiment Proposal
In order to aid in the treatment of Type 1 Diabetes, it is essential to spend funding and time researching the right questions, and for them to be heavily based in a scientific hypothesis. Based on the scientific experiments outlined in the previous section, there is promising evidence for treatment of Type 1 Diabetes through elimination of immune system elements that promote the T-cell activation and destruction of β-cells. Previous research has demonstrated that administration of CD3 antibodies within a month of diagnosis of the disease is a semi-efficient way of slowing disease progression, but by no means eliminates disease progression or the dependence upon synthetic insulin injections. Therefore, an experimental procedure that further investigates a way to halt the immune response to β-cells and allows for their survival would be a beneficial way to build off of current research and strive toward the end goal of curing Type 1 Diabetes.
Since alteration of the complement pathway within Type 1 Diabetes patients shows some promise, it seems logical to stick with alteration of the complement pathway as opposed to trying to alter a different part of the immune response all together. But, it is clear from the research discussed in the previous section that introduction of an antibody for C3 is not sufficient and causes too many adverse side effects to be a viable option for treatment. Therefore, it seems logical to look at another way of stopping C3 from activating the immune system response. An obvious alternative
Eukaryon, Vol.13, March 2017, Lake Forest College
Review Article
to introducing an antibody for something is to inhibit the enzyme that the molecule interacts with. Therefore, it seems worthy to study the effect of increasing decay of C3 Convertase, the enzyme that cleaves C4 into C3, to prevent C3 from being created in the first place.
Model: Before exploring how inhibiting this enzyme might affect the progression of Type 1 Diabetes, we would first want to test its efficacy in a NOD mouse model. This model, as outlined in Diana et al., 2013, provides a mouse that develops Type 1 Diabetes in a progression analogous to that of human Type 1 Diabetes progression. This is essential before embarking upon a human study because it shows safety and efficacy on a very basic level. Additionally, it is important to find a homologous gene within the mouse model before experimenting upon humans. A murine membrane protein of MW 60,000 has demonstrated properties that enhance decay of C3 convertase (Kameyoshi, 1989). This acceleration of decay would diminish the ability of C3 convertase to cleave C3.
Experimental Group: To achieve this goal, one would need to create a retroviral transgenic mouse that contain and overexpress this MW 60,000 membrane protein. This will be done through retroviral infection of the murine organisms. The MW gene will be put into the virus which will then be inserted into the experimental murine group. The murine will then take up the gene of interest through the virus’s infection process, which generally consists of reverse transcriptase working through the virus to integrate the viral genome into the host’s genome. The transgenic NOD mice then will be sacrificed and tested using a few methods, which will be outline below.
Control Group: The control group will be littermates that are not transgenic. In order to ensure that they are close to replicates of the experimental group as they can be, they will be injected with a saline solution by the same protocol that the experimental group uses for injection of the retrovirus. Similarly, since they are littermates that will maintain as much genetic replication as possible.
Methods
PCR for Transgene Confirmation:
The first thing that needs to happen before proceeding with any experiments is confirmation that the retroviral transgenic method was successful. In order to do that, DNA samples from the experimental group of NOD mice must be taken. PCR will be used to amplify the DNA samples. This is generally done by mixing a primer, which is designed by the scientist to match the DNA sequence of interest, in with the DNA sample and denaturing the mixture. The denatured mixture is combined with the necessary enzymes to promote replication. Then, the temperature of the mixture is raised and lowered to cause elongation and annealing of the DNA fragments. Finally, to ensure that the DNA fragment was replicated and it is the correct DNA fragment, an agarose gel electrophoresis is necessary. This method confirms the existence and quantity of the amplified DNA fragments by using a ladder of known size as a control. This control helps to mark different size markers (in kDA), which allows for accurate prediction of where the band of DNA fragments should show up. When completing this experiment, one would expect to find the transgene band present at the predicted kDA location (Figure 2). Once confirmation of the desired transgene is completed, then experiments to analyze the effect on C3 in the organism.
Monoclonal Antisera for C3 Quantity:
In order to determine if this increased degradation of C3 Convertase has an effect on the levels of C3 in the NOD mice, it is essential to identify and compare the amount of C3 in the experimental and control groups. One way to do this is to use Polyclonal antisera. In order to use this, we need to sacrifice the NOD mice, and we need an antibody for the protein of interest (C3). According to previous literature, which was discussed in the Current Research section of this proposal, a previously confirmed and successful antibody for C3 is ChAglyCD3 (Keymeulen, 2005). Therefore, using this antibody, and a secondary rabbit antibody for tagging, polyclonal antisera will allow for the antibody to bind to the C3 molecules in the NOD mice pancreatic tissue sample. Then the technique calls for a purification of the protein by affinity chromatography, which allows for 

visualization of the difference between the experimental and control groups. After running this technique, one would expect to find more florescence in the control groups than in the experimental groups (Figure 3).

This would signify a higher quantity of C3 in the control group than in the experimental group.
Western Blot for C3 Quantity:
It is extremely important in scientific inquiries to have more than one confirmation of expected results. Therefore, it is necessary to appeal to another technique, so that the results found in experiment (b) can be confirmed. This is important also to make sure the results can be replicated, which is how scientists know the results were not an accident or due to another confounding factor. The natural technique to invoke when analyzing the amount of protein in a sample is Western Blot. This technique involves taking a tissue or serum sample from your organism, in this case it would be a pancreatic tissue sample from the sacrificed NOD mice, and lysing it. The lysed pancreatic tissue sample then is put into a centrifuge, and the supernatant is separated from the pellet. The supernatant contains the protein of interest, so that is what is run through the agarose gel. Similar to the gel electrophoresis described above, the wells are then filled with the supernatant and compared to a buffer of known size. From there, the wells are run and the proteins separated using electrical force. The end result is a agarose gel with bands representing sizes of the proteins loaded in the gel, which can then be used to compare relative amounts and sizes of proteins. In this case, it would be used to compare the relative amounts of C3 in the experimental and control groups. Given the set up was successful, one would expect to see a significant decrease in C3 quantity in the experimental group as compared to the control (Figure 4).
C-Peptide Activity in NOD Mice:
Finally, to assess how all of this impacts the end result of developing Type 1 Diabetes, the obvious technique based on modern research is to analyze C-Peptide release. This would need to be done in vivo over an extended period of time. In NOD mice, disease maturation occurs around 6 weeks (Diana et al., 2013). Some of the retroviral transgenic mice should be treated with the MW 60,000 and tested at 3, 6, and 12 weeks for C-Peptide activity. The amounts of C-Peptide can be analyzed against those of the control group at the same time intervals. The products that would show the desired results would be an increase in C-Peptide levels among the experimental group as compared to the control group (Figure 5). The increase in C-Peptide levels are coincidental with an increase in natural insulin production, which in turn is a signal for beta-cell activity and survival.
Conclusion
These experiments are designed to help in addressing the question of whether decreasing the quantity of C3 within a Type 1 Diabetes model is a viable route for curing the disease. The research up until this point has pointed to success in relief of the dependence upon artificial insulin through muting C3’s ability to function. The previous research all tried to utilize an antibody to mark C3 for degradation. Though the artificial insulin dependence did decrease with this method, it incited an immune response within the subjects. This immune response created many negative side-effects that made the success of the partial alleviation of the disease diminish. Researchers hypothesized that the side-effects were derived from a cytokine response to the antibody injected into the system. Therefore, it is important to find a way to get rid of C3 without inciting the immune response that causes these side effects.
This paper outlines the first step to testing a different method of C3 destruction that, hopefully, will evade an immune system reaction. By increasing the degradation of C3 convertase, you limit the amount of C3 that is able to be cleaved into an active immune system molecule, and therefore indirectly limit C3 concentrations. This can be executed through the retroviral infection of the NOD mice with the MW 60,000 gene, which is what increases the degradation of C3 convertase. This transformation of genetic material will be confirmed using PCR and Gel Electrophoresis. Then, the decrease in C3 concentration will be confirmed using Western Blotting and Monoclonal Antisera methods. Then, to determine the overall influence of the experiment on the individuals, C-Peptide values will be taken from subjects to analyze if an increase in insulin secretion is seen with a decrease in C3 quantity.
Hopefully, these results will demonstrate a statistically significant decrease in C3 concentrations among the experimental group. That is, we hope to see a decrease
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in C3 concentrations given that the subjects have been treated with MW 60,000 gene transfection. Additionally, we hope to see a statistically significant increase in C-Peptide quantities among the experimental group, representing an increase in natural insulin production within the islets of the pancreas. If these results are found after completion of these experiments, the next steps would include conducting similar experiments within human trials at a small sample size. These human trials should consist of 10-15 recently diagnosed (which, as defined by literature, consists of diagnosis less than 30 days previous to initial experimentation) Type 1 Diabetes patients. Furthermore, if these human trials didn’t show high levels of side-effects, the next steps would include increasing sample size to determine if there are any less common side-effects that weren’t found in a small sample size. From there, clinical trials should occur to show efficacy and safety of the treatment within the Type 1 Diabetes patients.
These experiments are crucial to curing a highly prevalent disease within our society. Though treatment is available, a cure is always the ultimate goal, and with 9.3% of the U.S. population being affected by the disease, it is important to continue researching possible cures to put this horrible disease to rest. Additionally, a better understanding of how to cure this autoimmune disease might trigger similar treatment trials among other autoimmune diseases, and in turn could benefit an even high percentage of the population. This research would be extremely beneficial to the public as well as the scientific community, and is absolutely worth the funding and time necessary to undergo experiments such as this.

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College.

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