The Stem Cells Technology In Diabetes Therapy Biology Essay

Biotechnology Program School of chemestry & molecular biosciencesEmerging Biotechnologies

Biot7009

Assignment 1 Portfolio 1

Carolina Alejandra Salazar Ruales s43043068

15th April, 2013

Contents

Week 2:

"Mesenchymal Stem Cells Cooperate with Bone Marrow Cells in Therapy of Diabetes" (1)

Description of the Stem Cells Technology in Diabetes Therapy

(How this technology works?)

The high levels of glucose determined in the blood leads to a disease known as Diabetes Mellitus (2). In this disease the hormone insulin is not properly produced by the β cells in the pancreas (3). The energy needed for the metabolism of the body comes from glucose and Insulin takes glucose into the cells (2). Due to the exposure of high glucose concentration levels, Diabetes can leads in retino, neuro and nephropathies, and other sickness associated with fat metabolism like atherosclerosis, cardiovascular diseases and including gangrene’s feet (4), making diabetes one of the major cause of death in the world . Worldwide an average of 346 million people are affected with Diabetes and the percentage is increasing (5). This increasing number directs research into new treatments to eradicate the disease (6).

There are two types of diabetes: Type I and Type II, in both of them the main characteristic is the absence, absolute or partial, of β cells (3). In Type I (TI), the autoimmune mechanism of T cells destroys β cells (6) which difficult to find a cure due to the incapacity of auto controlling this mechanism (7), whereas, defecting action of insulin cause the Type 2 Diabetes (TII) and it may be preventable (8) At the moment, the cure for Diabetes TI is unknown, and people with this disease need to take insulin permanently (Svendsen and Ebert 2008). Stem cells are multipotent and they can be renewed by themselves, they offer a big opportunity to cure several diseases and one of them is Diabetes (6). Using Stem cells technology the damage of β cells caused by the autoimmune mechanism of T cells can be controlled (7).

The regeneration of β Cells in diabetics can be induced by stem cells (1). Stem Cells can change immune activity therefore; they can repair tissues in the pancreas and "inhibit β Cell specific T-cell response" (1). Stem cells can also gum up the monocytes dendritic cells maturation to avoid the activation of T-cells, this allows the correct function of the remaining β Cells and also to produce new ones (9). Many studies have been developed to stop the auto-destruction of β Cells by the immune system using drugs (10). However, most of the studies that try to regenerate β Cells require the administration of insulin, stem cell technology will repair the tissue to produce insulin. The achievement to ease hyperglycemia in diabetic patients of stem cells, including Mesenchymal and bone Marrow, has raised research in the probability to definitely repair and repopulate pancreatic tissue (6).

Mesenchymal Stem Cells and Bone Marrow Cells to solve Diabetes problems

(How it was applied to solve a particular problem)

Some authors suggested that Bone Marrow cells could revert diabetes Type I, while others think that Mesenchymal Stem Cells could regenerate β cells and pancreatic tissue in diabetic models (11). In this study Mesenchymal Stem Cells and Bone Marrow Cells were administrated alone and together into induced diabetic mice to prove the regeneration of β cells due to the reduction of blood glucose (1). The animal model used was female induced diabetes mice with high blood glucose level. Bone Marrow and Mesenchymal Stem Cells -BMCs and MSCs respectively- were isolated from healthy male mice and injected to induced diabetic mice, 14 and 15 days after diabetic induction, to revert the disease (1) Figure 1.

Figure 1: Experimental design and results from Mesenchymal Stem Cells Cooperate with Bone Marrow Cells in Therapy of Diabetes (1)

The alone transplantation of Bone Marrow cells were not enough to reverse diabetes, blood glucose levels were not significantly reduced compare with non BMCs injected into control mice. Reduced insulin levels and regeneration of pancreatic tissue were found in mice with BMCs and MSCs injection. "MSCs might inhibit cell-specific immune response, contributing to the restoration of endogenous pancreatic islet integrity" (1), this prompt that MSCs could stop the β specific T cell response and allow the production of insulin and new tissue in the pancreas.

In conclusion, this technology combines two types of cells: Bone Marrow and Mesenchymal Stem Cells to reduce glucose levels in blood and repair pancreas tissue therefore it allows the recuperation and extends the life of diabetic induced mice, promoting new research in diabetes cure for humans.

Strengths and Weakness of Stem Cell technology relative to actual treatment for diabetes.

The actual treatment for Diabetes is the permanently supply of insulin. Even thought, insulin injections saved many lives they don’t represent a cure for this disease, and it will lead in other diseases if it is not well administered (3). In the other hand, stem cell technology may cure Diabetes but, it has not been proved in human models. The comparison of Strengths and Weakness between stem cell and actual treatment for diabetes is described in the following chart.

Comparison of Strengths and Weakness between stem cell and Insulin supply for diabetes treatment (3, 6)

Stem Cell

Insulin Supply

Strengths

Weakness

Strengths

Weakness

Can regenerate β cells

Not available for treatment of diabetes now days

Has been saving many lives for decades

Overdoses of insulin is dangerous, it could lead in Hypoglycemia and coma.

Self renewing

The exact doses or concentration of stem and bone Marrow cells is unknown

Easy access to insulin and to administrate

Make patients dependent of insulin

Can revert the disease

Need to be tested and developed more. This technology has not been proved on human models

Easy to apply. Prefilled pens are available

Treatment does not work in insulin resistant patients

Patients will not be dependent of stem cells to reduce glucose blood levels

Not many sources of β cells from stem cells or donors.

Suitable for all types of diabetes

Do not restore damaged cells in pancreas.

Few transplantation of stem cells will be required instead of daily doses of insulin

The disease will not revert back to normal

Difficult to storage. Needs special storage temperature

"Transdermal Influenza Immunization with Vaccine-Coated Microneedle Arrays" (1)

Description of the Technology : Transdermal Vaccine delivery

(How this technology works?)

Transdermal delivery is a safe and noninvasive technology in which a substance of interest –antigens for example- is delivered on the skin (1, 2). Transdermal delivery technology opens the way to develop vaccine delivery systems (3) without the use of needles (Lawson, AU - Freytag, & AU - Clements, 2007), avoiding pain and requiering no trained personel to apply the them (4).

Oral delivery may be one of the first and easiest routes to administrate drugs and vaccines but, this route can reduce the dose and/or eliminate the effect of them due to the metabolism of the liver and gastrointestinal degradation (4, 5) . Instead, transdermal delivery is a most appropriate route (4).

The way Transdermal vaccine delivery works is by directly application of the antigen in the skin. The skin has antigen presenting cells, like Langerhans cells, macrophages, dentritic cells, keratinocytes, which take the antigens and present them to the B and T cells to start the immune response (1, 6). Skin B cells needs minimal quantity of antigens to secrets antibodies and give protection, this make transdermal vaccine delivery a potential and effective technique to reduce doses of vaccines used in intramuscular syringes vaccination (4).

Due to the skin natural barrier, stratum corneum, does not allow the entrance of macro molecules like vaccines (7) microneedles are used together with transdermal patches, they make microscopically holes in the skin barrier and leave the coated vaccine deeper in the skin so that, the vaccine will dissolve and be available to start the immune response (4). Microneedles are pressed directly in the skin to allow the coated vaccines enter and after this, the patches containing microneedles can be discarded and the vaccine will start working (1). In conclusion, transdermal vaccine delivery uses patches and microneedles to deliver macromolecules, antigens, through the skin. The skin cell receptors take these macromolecules and present them to the B and T cells to start the immune response and give protection against infectious diseases.

Transdermal Vaccines Coated Microneedles Arrays for Influenza vaccination

(How it was applied to solve a particular problem)

One of the major causes of death and illness are infectious diseases (8). A cheaper, faster more effective and easy method to apply vaccines could be found in transdermal vaccination, Influenza, as well as other diseases, could be benefit of this technology (9).

In this study inactivated influenza virus was coated in microneedles to develop a vaccine against influenza. This technology was used to prove that transdermal delivery has the same or better effect than traditional vaccination system (intramuscular) (1).

First electropolished microneedles were developed in stainless steel; as a result 700µm long microneedles were coated with 5 mg of inactivated influenza virus. The animal model used in this study was female mice. Mice were immunized with transdermal microneedle patch, pressing the transdermal patches directly to the skin for two minutes, in low and high concentration of the vaccine and compared with the intramuscular vaccination, which is the actual method for this vaccine, with the same tow concentrations (1).

4, 14 and 28 days after the immunization the efficacy of the transdermal vaccine delivery was tested by "hemoaglutination inhibition and antibody responses" (1), the results demonstrated that single immunization with transdermal microneedles has the same levels of antibodies as the intramuscular injection. Virus IgG were higher in the tow concentrations of transdermal method. However, intramuscular method IgG were elevated only in the high concentration of vaccines, this means that lower concentration of vaccines in transdermal delivery are sufficient to produce the same effect as the intramuscular vaccination.

Several investigations have shown that the improving of immune response with transdermal delivery along with microneedles in Influenza vaccination is because this technique provides a more effective and faster way to present the inactivated virus to the antigen presenting cells and start the interactions with the lymphatic system than the traditional intramuscular vaccines (4).

Strengths and Weakness of Transdermal Vaccines Coated Microneedles Arrays for Influenza vaccination relative to Intramuscular vaccination.

The current method for influenza vaccination is intramuscular vaccination which needs the use of needles and syringes. Reutilization of needles could lead in hazard problems associated with infectious diseases. The strengths and Weakness between transdermal vaccine delivery and the actual treatment for Influenza are given in the table below.

Strengths and Weakness between Transdermal Vaccine Delivery and Intramuscular Vaccination (1, 4, 10, 11)

Transdermal Vaccine Delivery

Intramuscular Vaccination

Strengths

Weakness

Strengths

Weakness

The fabrication of transdermal vaccine delivery could be made at low costs when is production in massive

Expensive to produce low in quantities

Only method currently used which has saved many lives.

Insufficient production to reach all the world population

One simple administration of the vaccine can be assembled in a transdermal patch

Transdermal delivery needs microneedles for macromolecules

Difficult to transport, storage and distribute to remote villages

Can be applied by patients themselves

Needs clinical facilities to be produced

Difficult application, requires trained personnel

No trained, medical or specialized personnel will be requires to apply the vaccine

The cost of energy could be higher on its production

Existing risk of transmitting diseases due to the re-use of needles

Painless

Could induce B cells memory using fewer doses of vaccines. Thus, more amount of vaccines can be produced

Reduced efficiency and waste of vaccine can occur if it is not well applied. Microneedles need to be completed inserted in the skin

Expensive to keep in cold chain supply because of their size and liquid presentation

Small size, easy to transport and storage, the costs of these resources will be lower.

The skin barriers of humans are different compared with animal models. Therefore, is difficult to export the results obtained in other species to humans

Needs more storage space making it more expensive.

Reduce the costs of cold chain which is useful in developing countries

Eliminate the risks associated with hazard needle disposal, and re-use of them

No need of reconstitution to be applied

"Evaluation of potato-processing wastewater treatment in a microbial fuel cell" (1)

Description of the Technology: Microbial Fuel cell

(How this technology works?)

The microbial fuel cell or MFC is a technology that allows microorganisms to use an organic matter as a substrate and produce electricity. MFC produce electrons by degradation of the organic matter. Many enzymes of the microbial cells act on the electrons to produce energy, This technology is a revolutionary method for the production of bioenergy, as bacteria replicates by itself, this system becomes self-sustaining (2).

This technology is a system of communication between biological energy and electrodes (3). MFC can use wastewater as a substrate to produce energy and treat the water at the same time (4), so that MFC is an alternative to produce energy and treat many effluents of wastewaters.

To produce electricity, microorganisms only need one electron acceptor, in this case the anode, other electron acceptors will inhibit the production of energy (5). Oxygen is an electron acceptor present in the MFC system. However, bacteria and oxygen must be kept in different chambers to produce electricity. In the MFC, electricity will be produced between the two chambers (cathode and anode) connected by a cable with a load that can be a resistor (2). Microorganisms are used as catalysts for the reduction and oxidation reactions that fuel cells need in order to produce energy (1).

The biofilms formed by microorganisms in MFC convert the organic matter, that is used as substrate, to produce carbon and electrons, the last ones are transported to an external circuit to produce electricity (6).

Treatment of waste water in microbial fuel cells

(How it was applied to solve a particular problem)

Two of the biggest environmental problems for industries are: the large amount of wastewater produced in their processes and, the high energy consumption which is not renewable. The application of MFC will find the solution of these tow problems at the same time (7).

Wastewater is produced daily all around the world, agricultural industries produce tons of residues and wastewater containing organic loads (1). The treatment of wastewater is expensive but extremely necessary, contaminated water -including water with high nutrient and organic matter levels- pollute the environment, to avoid this contamination wastewater discharges are regulated (5)

MFC is a new technology which differs with the traditional technologies of wastewater treatment. This method allows treating the water and producing energy that could be employed in other process or in the same process, making it self-sustainable (2). This technology could convert the organic residues in water of industries in electricity (7).

In this study, Durruty et al., applied MFC technology in order to produce electricity and test this method as a replace or a complement of the anaerobic process used in a potato processing industry for wastewater treatment. To treat wastewater with MFC, bacteria need to degrade the organic load in the water to produce electrons required to electrical production. This inoculum of bacteria was obtained from the residual sludge of the potato processing industry (1).

The tests loaded to know if the MFC could replace anaerobic wastewater treatment or complement it, reveal that more efficiency could be obtained as a complement of the actual treatment. The volatile acids produced in the treatment of the potato wastewater increase the growing of a biofilm in the MFC, leading in a higher electricity production and the decrease of the chemical oxygen demand as well, reducing it in 80%. Both processes, anaerobic and MFC could treat wastewater. However, the combination of both increases the efficiency of the treatment and produce electricity necessary to run the process (1).

After this study, the efficiency of MFCs was proved, they could produce electricity after the anaerobic treatment of potato waste water, with fast reducing of COD (1) making this technology viable to reduce the problems of wastewater.

Strengths and Weakness of waste water treatment with Micro fuel cells relative to Aerobic and anaerobic treatments.

The current methods for industrial wastewater treatment are aerobic and anaerobic processes; the advantage of this method is that it allows many industries to reduce de organic loads of the residual water before it is release to the environment (1, 2). Anaerobic treatment of wastewater could recuperate energy to the system as hydrogen and methane. However, this mechanism doesn’t work at room temperature and organic loads with low carbon content, becoming an expensive disadvantage in terms of energy consumption. In order to make the wastewater treatment more efficient, both aerobic and anaerobic treatments could work together. Nevertheless, having the tow processes working at the same time is very expensive and requires high costs of energy. More energy is required in Aerobic process due to the need of mechanical aeration (1)

In the other hand, MFC is an auto sustainable technique; it could supply power for the system of the wastewater the treatment plant (2). The major advantage of the MFC is the ability of its microorganisms to convert wastewater organic matter into electricity (7). Producing electrical energy due to the oxidation of organic matter makes MFC a sustainable cheaper system that saves energy and produce electricity at the same time (1). MFC could be applied in the treatment of many wastewaters such as domestic, industrial and agricultural (7).

However, there are some obstacles such as cathode limitations that prevent this technology to be applied at the moment (6). MFC still an expensive technology that require high costs investments to be produced in large scale, this technology has worked at laboratory scale since no large scale test have been developed. As in current treatments of wastewater, the microorganisms used in MFCs requires adaptation to the system, this implies time and, the selection of the appropriate microorganisms to improve the electricity production (1).

"A fast and sensitive immunoassay of avian influenza virus based on label-free quantum dot probe and lateral flow test strip" (1)

Description of the Technology: immunoassay based on label free quantum dot probe and lateral flow test strip

(How this technology works?)

Recently advances in technology allows the analysis of biological and chemical agents in an easier and faster way, new diagnostic tools are developed to be more sensible, specific, efficient and to minimize the waiting time for the results of the test (1, 2). Most of the diagnostic tools used nowadays are based in molecular techniques (2). Some of these tools for detection of antigens are quantum dots and lateral flow test strips.

Quantum dots (QDs) have been recognized as an essential fluorescent tool for diagnostic in biosensing (3). This technology works by producing fluorescent signal when the antigen binds the QDs (1). QDs are semiconductors that emit fluorescent and could substitute dyes and fluorescent proteins because of their stability to photo bleaching and their capacity to be brighter. These characteristics of QDs lead in a variety of applications especially in DNA and diagnostics detection (4). Besides these properties, QDs are more stable than other molecules in hostile environments with changes in pH or temperature, making them a better model to be used as label in diagnostics (5).

Other useful tool in diagnostic is lateral flow test strips (LFTS), as its name says, this method works by the flow of the sample to be analysed through a complex of molecules and membranes to make conjugates and produce a signal visually detected as a line (6). This technology can be applied as a point of care diagnostic to analyse antigens, antibodies and many other proteins with qualitative and/or quantitative results in a shorter time than the common tests (1, 6-8).

The application of the two methods, described above, will add the advantages of each one to a new system, improving the sensitivity and the time applied for the test. Moreover, these advantages can reduce the cost of the test and could be a promising method for point of care diagnostics (1).

Treatment of waste water in microbial fuel cells

(How it was applied to solve a particular problem)

H5N1Influenza is a contagious avian disease that could be transmitted to humans by birds leading in a world pandemic (1, 9). Avian Influenza has caused the death of hundreds of people and animals (10), the virus could mutate, making this disease dangerous to health with an economical impact (9). The difference between avian influenza and other types of influenza is the probability of death is not only in older people but young, healthy people as well (1). This disease is still established in many countries in Asia as well as the possibility of a new pandemic (11). For those reasons, is important to develop accessible, affordable and quick methods to diagnose the disease in early and preventable stages (1).

A combined technology between QDs and LFTS has been developed in this study. The method consists in LFTS followed by fluorescent tests. The LFTS contains a conjugate in membranes, when the strip is in contact with the avian virus it will make a reaction line. QDs were bind in microplates to reveal a fluorescent reaction when contact with the antigen occurs, showing a high specificity and reactivity along with cero false negatives (1). After this experiment, it was possible to apply the method cheaper and faster than traditional methods. The time since the sample is taken to the presentation of the results is less than 30 minutes, the detection is sensitive and easy to reproduce, thus no trained people is required for this test allowing the reproduction of this technology in points of care to have a fast detection of the disease and properly prevent pandemics (1).

Strengths and Weakness of quantum dot probe and lateral flow test strip relative to Aerobic and anaerobic treatments.

QDs and LFTS method has the advantages of the both methods separately. This method is easy to reproduce thus, it will be easier to develop as point of care diagnostic without trained personnel, also no expensive or very advanced equipment is required for this technology, the time that takes to do this test is less than 30 minutes, compared with more than four hours that the current methods for detecting avian influenza take. The current methods to diagnostic avian influenza are ELISA and PCR, these methods are complicated, require expensive laboratory equipment and trained personnel. The sensibility of the method applied in this study is higher than ELISA . Reagents for PCR and ELISA are more expensive than the whole method of QDs and LFTS, approximately 0.3 US dollars per test. One major advantage is that QDs and LFTS method "increase 100 fold" the results from ELISA (1).

In the other hand, DQs cannot be used directly in living organisms due to the toxicity of the material that QDs are made of, so the use of this technology is limited to be used with samples (4). To reproduce the QDs and LFTS technology a spectrometer is required (1) which increments the costs of equipments. In order to reduce costs the method should be produced in large scale (1).

The current methods, as ELISA, applied to diagnostic avian influenza has an important strengths, this method make a detectable and effective signal reaction to detect the virus (1). However, ELISA has a low sensibility due to the colimetric reactions that limits the detection of the disease (1).

"Glycoprotein Microarrays with Multi-Lectin Detection: Unique Lectin Binding Patterns as a Tool for Classifying Normal, Chronic Pancreatitis and Pancreatic Cancer Sera" (1)

Description of the Technology : "Glycoprotein Microarrays with Multi-Lectin Detection"

(How this technology works?)

The glycosylation of protein is an important process for the development of some diseases as well as other biological functions like cell growth, and can be used for the detection of diseases associated with cellular abnormal growth (1, 2). The differentiation between glycan in normal and cancer cells is necessary to diagnose prostatic cancer in early stages (1). The variation in glycosylation can develop in cancer. This variation can avoid the function of the immune system, thus the detection of cancer should be detected in advanced stages (3). The changes in glycosylation have been seen in a variety of cancer types, this leads in the development of a new technologies for early detection of cancer (1).

New technologies for the detection of pancreatic cancer are glycoprotein arrays and lectin detection. The microarray technology is an important screening method for the detection of the structure of the glycan and determination of glycosylation. Enzyme levels, involved in glycosylation process, could have a significant change in tumour cells and this property can be detected by microarrays (4, 5). This method is extendedly applied as biomarker for the multiple analyses of samples (2, 6). The application of glycoprotein assays is not only useful to determine alterations in glycoproteins; it could be also used to increase the knowledge about the progression of diseases as well as their early identification (2, 7). In microarrays technology, glycoproteins are immobilized in membranes to be detected with a variety of agents like lectins (1).

Lectin detection is used to make the most appropriate bind of specific glycoproteins with lectins in order to eliminate the unnecessary glycoproteins. Only the intact glycoproteins can bind the lectins to give a sensitive fingerprint of the changes on the glycoproteins structure (2), this allows to look for glycosylation between different samples classify them and allow the detection of cancer in early stages (1).

Classifying Normal, Chronic Pancreatitis and Pancreatic Cancer Sera

(How it was applied to solve a particular problem)

During the year 2012 in the United States, 43 920 new cases of pancreatic cancer were detected and 37 390 deaths were reported due to this disease (8). Early detection of pancreatic cancer is not available and it is critical for an adequate treatment, since usually pancreatic cancer is detected at later stages when the disease is advanced and complicated to treat (1, 9). To decrease mortality due to this kind of disease a simple detection method for pancreatic cancer is needed (9).

In this study "Glycoprotein Microarrays with Multi-Lectin Detection" were used as a novel method to identify and classify sera samples from normal, cancer or chronic pancreatitis cells based in the variants of glycosylation. First the glycoproteins from the samples were enriched with lectins, glycoprotein arrays were printed with many lectins to look for glycosylation in the three kinds of cells samples (1).

As the glycoproteins secreted in normal and cancer cells are different in the surface of the cells, glycosylation variants, associated with cancer, can be determined in early stages. First the samples were immunodepleted from abundant proteins, then they were enrichment with lectins and ran into a glycoprotein microarrays, the last step was the multi-lectin fluorescent detection and after a tryptic digestion a glycopeptide mapping was developed in order to classify the samples according their glycosylation structure determined (1).

After the development of the method, the results were analyzed with peptide mapping, showing different clusters between the normal, chronic pancreatitis and pancreatic cancer cells. This method allows us to know that certain glycoproteins were present in cancer samples while in healthy and no cancer samples they were absent, determining the effectiveness of the method to detect early pancreatic cancer and clearly differentiate it from other diseases related to the pancreas as well as inflammatory complications. (1)

Strengths and Weakness of Glycoprotein Microarrays with Multi-Lectin Detection relative to current methods for cancer detection and classification between Normal, Chronic Pancreatitis and Pancreatic Cancer Sera.

The current methods for detection of cancer are biomarkers such as CA19-9, spectrometry mass and traditional arrays. The specificity of existing biomarkers for pancreatic cancer is low, this biomarkers are also able to detect inflammatory disease in pancreas and do not differentiate between cancer. "Glycoproteins microarrays with Multi-Lectin Detection" gives the opportunity to classify several samples of sera from healthy to pancreatic cancer patients, covering related sickness like chronic pancreatitis. Glycosylation allows to differentiate between cancer and inflammatory diseases in pancreas, this property gives glycosylation and advantage from the current biomarker used nowadays (1).

The new technology presented in this study, have access to the structure of proteins glycosylation in complex samples with easy data analysis. However, current methods can access to glycosylation information, the analysis of data is complicated and trained and experienced technicians are required (2). The main weakness of Glycoprotein Microarrays with Multi-Lectin Detection is that this method cannot detect the complete glycans in a sample and the technology is not well developed to start working in big scale since only lad tests have been done (1).

One strength of Biomarkers is that they can detect pancreatic cancer. But, the higher the stage cancer is the higher concentration of the biomarker is present. Thus, pancreatic cancer detection with actual biomarkers is diagnosed in advance stages. In the other hand, "Glycoproteins microarrays with Multi-Lectin Detection" can detect cancer in very early stages due to it is not dependent of the biomarker concentration, but can identify pancreatic cancer biomarkers as well. The time to develop the methods is other important difference between both techniques. Current methods take longer time to analyze samples, while "Glycoproteins microarrays with Multi-Lectin Detection" takes less time (1).

In conclusion, glycoprotein arrays are a tool to detect cancer and differentiate it from healthy cell and other diseases related with pancreas and inflammatory processes and can be used in pancreatic cancer early detection as well as in other kinds of cancer.