Type 2 odontoid fracture classification denis

Type 2 diabetes mellitus statistics in the philippines zip,yamaha m3000 ebay,has diab?te type 2 suivi grossesse - Plans On 2016

Type 2 diabetes is caused by a combination of genetic, behavioural and environmental factors. It is estimated that pre-diabetes affects roughly 5.0 million Canadians over the age of 20 years. Ethnicity has also been associated with certain behavioural risk factors for type 2 diabetes.
Type 2 diabetes occurs when your body’s cells resist the normal effect of insulin, which is to drive glucose in the blood into the inside of the cells. Extremely high blood sugar levels also can lead to a dangerous complication called hyperosmolar syndrome. Retinopathy — Tiny blood vessels in the retina (the back of the eye that sees light) can become damaged by high blood sugar. Obesity, especially abdominal obesity—a condition that greatly raises a person’s risk for type 2 diabetes. High blood pressure—a condition often present in people with type 2 diabetes, that together with diabetes greatly increases the risk of heart disease and strokes. Exercising regularly—like a brisk walk of 1-2 miles in 30 minutes—at least five times a week, even if that does not result in you achieving an ideal weight.
In most cases, type 2 diabetes treatment begins with weight reduction through diet and exercise.
Because type 2 diabetes develops when the pancreas cannot make enough insulin to overcome insulin resistance, about one of three people with this disease take some form of insulin injection. In advanced type 2 diabetes, or for people who want to tightly control glucose levels, insulin may be needed more than once per day and in higher doses. Treatment plans that include both very long-acting insulin and very short-acting insulin are frequently the most successful for controlling blood sugar.
Fortunately, these side effects are uncommon, so the benefits of treatment far outweigh the risks. In addition to medicines that help control the level of blood sugar, people with type 2 diabetes often take other medicines that reduce the risk or to slow the onset of the complications of diabetes. Slow the worsening of kidney disease—particularly drugs called angiotensin-converting enzyme (ACE) inhibitors, and angiotensin receptor blockers (ARBs).
After the first few years, the majority of people with type 2 diabetes require more than one medicine to keep their blood sugar controlled. In Diabetes Type 1 the body is not producing insulin, while in Diabetes Type 2 the cells are not responding properly to the insulin, and there is not enough insulin being produced. When ever food enter in our body ,Food get converted into the Glucose and because of insulin it enter and adsorb by the our body so the insulin is the main part and factor by which our body can absorb the glucose. Insulin, a hormone, is produced by Beta cells in the Islets of Langerhans, which are in the pancreas. So if you have diabetes then your body or bloodstream will not absorb Glucose properly or not at all absorb so this activity resulted high amount of Glucose and one the amount of glucose got high level than this situation called hyperglycemia. When the cell of body does not respond to insulin than this situation is called Diabetes Type 2.
So when body is not able to get proper energy and continuously increasing the level of Glucose than it a time people to get worry and rush to your doctor. So basically so cannot reduce Diabetes Type 1 through exercise because the beta cell has already destroyed. The major quantity of diabetes patient has Diabetes Type 2 (Approx 85 %) and patient usually seems  over weight and unfit.This kind of diabetes comes late in the life and it is very uncommon to find Diabetes Type 2 in 20s age people. Guys here we have written what we can but if you and your dear one is suffering from diabetes type 1 or diabetes type 2 than you must rush towards doctors and for you later on we will also publish the home remedies to cure diabetes.
Science, Technology and Medicine open access publisher.Publish, read and share novel research. 1995 Isolation and structural identification of 1,25-dihydroxyvitamin D3 produced by cultured alveolar macrophages in sarcoidosis.
1983 Metabolism of 25-hydroxyvitamin D3 by cultured pulmonary alveolar macrophages in sarcoidosis.
2003 Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting autoimmune diabetes. 2010 Glycemic changes after vitamin D supplementation in patients with type 1 diabetes mellitus and vitamin D deficiency.
2009 Genome-wide association study and a meta-analysis find that over 40 loci affect risk of type 1 diabetes. 1983 Specific high-affinity receptors for 1,25 dihydroxyvitamin D3 in human peripheral mononuclear cells: Presence in monocytes and induction in T lymphocytes following activation. 1995 Susceptibility to human type 1 diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene mini satellite locus.
1994 A polymorphic locus near the insulin gene is associated with insulin-dependent diabetes mellitus. 2001 1alpha,25-Dihydroxyvitamin D3 has a direct effect on naive CD4+ T cells to enhance the development of Th2 cells. 1996 1,25-dihydroxyvitamin D3 reversibly blocks the progressions of relapsing encephalomyelitis, a model of multiple sclerosis. 1998 1,25-dihydroxyvitamin D3 prevents and ameliorates symptoms in two experimental models of human arthritis. 1998 1,25-dihydroxy-vitamin D3 restores sensitivity to cyclophosphamide-induced apoptosis in non-obese diabetic (NOD) mice and protects against diabetes. 1998 Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine.
2000 Vitamin D receptor gene polymorphisms influence susceptibility to type 1 diabetes mellitus in the Taiwanese population.
1998 Prevention of type 1 diabetes in nonobese diabetic mice by late intervention with nonhyperclcemic analogs of 1,25-dihydroxyvitamin D3 in combination with a short induction course of cyclosporine A. 1997 The nonobese diabetic mouse as a model of autoimmune diabetes: Immune dysregulation gets the NOD.
1998 Inhibition of IL12 production by 1,25-dihydroxyvitamin D3: involvement of NF-?B down regulation by 1,25-dihydroxy vitamin D3. Protein misfolding and aggregation as the common molecular pathogenesis of neurodegenerative diseases.
2008 The pyridoxamine action on Amadori compounds: A reexamination of its scavenging capacity and chelating effect.
2000 Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of b-sheets in Alzheimer’s b-amyloid fibrils. 2004 Aging-related increase in oxidative stress correlates with developmental pattern of betasecretase activity and beta-amyloid plaque formation in transgenic Tg2576 mice with Alzheimer-like pathology. 2011 Insulin resistance and Alzheimer-like reductions in regional cerebral glucose metabolism for cognitively normal adults with prediabetes or early type 2 diabetes. 2006 Glycogen synthase kinase 3? and Alzheimer’s disease: pathophysiological and therapeutic significance. 2002 Supramolecular structure in full-length Alzheimer’s b-amyloid fibrils: evidence for a parallel b-sheet organizatioin from solid-state nuclear magnetic resonance. DNA Immunotherapies for Type 1 DiabetesAlice Li1 and Alan Escher2[1] Loma Linda University, Loma Linda, California, USA[2] SEKRIS Biomedical Inc., Redlands, California, USA1. This is more likely if blood sugars remain elevated and high blood pressure is not treated aggressively. However, people with type 2 diabetes can sometimes restore their blood sugar levels to normal just by eating a healthy diet, regularly exercising, and losing weight. The medication metformin (Glucophage) offers some additional protection for people with pre-diabetes. Very short-acting insulin is used with meals, to help control the spike in blood sugar levels that occur with a meal. All diabetics should consider taking medication to lower their cholesterol, usually one of the statin medications. Diabetics should use medication to control high blood pressure if it can’t be improved by lifestyle changes. Contact your doctor immediately if you develop vomiting or diarrhea and are not able to drink enough fluids. IntroductionThis chapter will review the role of vitamin D in the pathogenesis and treatment of type 1 diabetes mellitus.
Vitamin D supplement in early childhood and risk for type 1 (insulin-dependent) diabetes mellitus. Some genetic mutations responsible of neurodegenerative diseases render the causative proteins prone to misfold and to form beta-sheet-rich oligomers and amyloid fibrillar aggregates, resulting in their accumulation in the affected neurons and eventually leading to degeneration in the brain. LAN5 untreated (control), treated with rAb42 oligomers oligomers), and rAb42 oligomers and insulin (oligomers+insulin) were incubated with Mito Red and Hoechst 33258. San Biagio2[1] Istituto di Biomedicina e Immunologia Molecolare (IBIM) – CNR, Italy[2] Istituto di Biofisica (Palermo unit) – CNR, Italy1. The table summarizes plasmid DNA based immunotherapies under two categories: Gene therapies and DNA vaccines.
If a person does not eat on a regular schedule, very short-acting insulin can be particularly helpful. And the insulin-producing cells in the pancreas may wear out as the pancreas tries to keep up with the body’s extra insulin needs.
The activation of the T-cell by various stimuli (antigens), is brought by major histocompatibility complex (MHC-HLA II).
At the level of the antigen-presenting cell (such as dendritic cells; DCs), 1?,25(OH)2D3 inhibits the surface expression of MHC class II-complexed antigen and of co-stimulatory molecules, in addition to production of the cytokine IL-12, thereby indirectly shifting the polarization of T cells from a Th1 towards a Th2 phenotype.
This mechanism is retained common to a broad variety of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington disease, Amyothophic Lateral Sclerosis and Frontotemporal Lobar degeneration.
IntroductionProtein aggregation is a very fascinating matter due to its implication in many human neurodegenerative diseases and its relevance in food and pharmaceutical industries. This mechanism is retained common to a broad variety of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington disease, Amyothophic Lateral Sclerosis and Frontotemporal Lobar degeneration.Type 2 diabetes (T2D) is classified as a metabolism disorder and it is often associated with microvascular and macrovascular complications, including retinopathy, nephropathy, neuropathy and cardiovascular disease.
Plasmid DNA immunotherapy for type 1 diabetes can be divided into two categories: DNA vaccines and gene therapies. Furthermore, similar to other autoimmune diseases, allergies and asthma, the incidence of type 1 diabetes is on the increase at an alarming rate in industrialized countries for unknown reasons. People with diabetes who also have high levels of total cholesterol or LDL cholesterol are at greatly increased risk for heart disease and strokes. This figure shows also, inhibitors of T-cell activation: cytotoxic T lymphocyte antigen 4 (CTLA-4) and lymphoid tyrosine phosphatase (LYP). In addition, 1?,25(OH)2D3 has immunomodulatory effects directly at the level of the T cell, by inhibiting the production of the Th1 cytokines IL-2 and IFN-? and stimulating the production of Th2 cytokines. We will summarize the results of in-vitro and animal studies and will conclude with a review of the relevant clinical trials.2.
Mitochondrial activity is indicated by red arrows, fragmentation of nuclei is indcate by blu arrows. In some cases, the aggregation of protein is a natural phenomenon occurring in living organisms. A diabetes affected person has an elevated quantity of glucose in the blood (hyperglycemia) that is caused by the inability of the body either to produce any insulin or enough insulin, or by the inability of the cells to respond properly to the insulin producted in the pancreas.
Mitochondrial activity is indicated by red arrows, fragmentation of nuclei is indcate by blu arrows.Moreover, unpublished results indicate that insulin counteracts oxidative stress induced by amyloid beta by activation of Akt survival pathway.
Plasmid DNA can be delivered using different routes, for example, the intradermal and intramuscular routes.
Information released by the American Diabetes Association shows 23% and 21% increased rates of type 1 and 2 diabetes, respectively, from 2001 to 2009. When levels of glucose in the blood rise (for example, after a meal), the pancreas produces more insulin. Some people with type 2 diabetes become dependent on dialysis treatments because of kidney failure. DefinitionType 1 diabetes mellitus is an autoimmune disease in which the pancreas is unable to respond to secretagogue stimulation with appropriate insulin secretion.
Akt traslocates from the cytoplasm to nucleus where phophorylates FOXO3a that, in turn, moves from the nucleus to the cytoplasm inhibiting, in this way the transcription of the FOXO-dependent genes. The increase in incidence of type 1 diabetes is especially apparent in young children and has generated an urgent need for novel treatments that can safely control diabetes-causing inflammation, and alleviate the need for administration of exogenous insulin. Type 2 diabetes is much more common than type 1 diabetes, and is really a different disease. Both Th2 and Tregs can inhibit Th1 cells through the production of counteracting or inhibitory cytokines.
Hyperglycemia develops when more than 70-90% of the insulin-producing beta cells are destroyed. So, even though the blood has plenty of glucose, the cells are incapable of getting it for their essential energy and growth requirements. Since after Abeta-induced oxidative stress are usually activated pro-apoptotic genes, their trascriptional inhibition helps the survival program (Picone et al., 2011). Indeed, type 1 diabetes has been treated for almost a century in the same fundamental manner using daily insulin injections. DNA vaccine immunotherapiesPlasmid DNA vaccine-based immunotherapy is a promising therapeutic field for treatment of type 1 diabetes. Together, these immunomodulatory effects of 1?,25(OH)2D3 can lead to the protection of target tissues, such as ? cells, in autoimmune diseases and transplantation.
An autoimmune destructive process, which plays a central role in the development of type 1 diabetes mellitus, is facilitated by the subject’s own genetic susceptibility and by non-genetic factors. There are three main types of diabetes: Type 1 diabetes (T1D) (referred to as insulin-dependent diabetes and juvenile diabetes), results from the body's failure to produce insulin, and presently requires the person to inject insulin. Moreover, it has been suggested that since insulin signalling in the brain is known to decline with age, the outcome of the balance of different molecules, as Akt and FOXO, represents a risk factor for AD that is well suited for therapeutic intervention. Although it is a life-saving treatment and one of the most remarkable accomplishments of medicine, administration of exogenous insulin is still not a cure because it does not address the underlying autoimmunity that targets insulin-secreting beta cells.
Type 2 diabetes (referred to as non-insulin-dependent diabetes mellitus, and adult-onset diabetes.) is associated with a reduced ability of insulin to stimulate glucose utilization (insulin resistance) and sometimes it is combined with an absolute insulin deficiency.
By restoring the balance of molecules to favour neuron survival, new drugs, designed to specifically enhance CNS insulin signalling, would provide a new and potentially significant class of AD therapeutics.9.
Because it is currently impossible to mimic regulation of physiological insulin levels faithfully, many type 1 diabetic individuals receiving standard of care are exposed to acute and chronic complications that cause increased morbidity and mortality.
Nevertheless, although beneficial effects were observed, it is clear that efficacy must be significantly improved. Vitamin D deficiency is a non-genetic factor that appears to be associated with an increased risk of developing type 1 diabetes mellitus. The third type, gestational diabetes, is when pregnant women, who have never had diabetes before, have a high blood glucose level during pregnancy and it may precede development of T2D.
ConclusionsThe present chapter highlights the overlap and the many points of intersection existing between T2D and AD. As a result, there have been intense efforts to develop immunotherapies that can eliminate or at least alleviate the need for exogenous insulin. Improving efficacy will be likely dependent on the ability to modulate both the innate immune system, through activation of tolerogenic antigen-presenting cells like dendritic cells, and the adaptive immune system, through activation of various populations of regulatory cells.
Insulin resistence in the CNS results in the dysregulation of multiple extracellular and intracellular signaling cascades and molecular mechanisms, which in turn could lead to decrease in neuronal and synaptic functions up to neurodegeneration.
In this case the goal is to arrest pathological autoimmunity that destroys beta cells so that the cells can regain function, and possibly proliferate and regenerate. DNA vaccines are particularly well positioned to achieve this goal because plasmid DNA is information-based, and can encode molecules that affect the immune system in different manners. The acute complications include life-threatening conditions like severe hypoglycemia or diabetic ketoacidosis (DKA). Diabetes is increasing and the World Health Organization estimates that there will be a doubling of cases by 2025, largely as a result of lack of physical activity and poor dietary habits, both of which are risk factors for the disease. An understanding of how each molecular pathway intersects and affects the others is essential for the development of future drug intervention strategies for these pathologies.10.
The challenge is to identify which combination of functions should be delivered together with a pancreatic autoantigen to treat disease with maximum efficacy and safety.Several beta cell autoantigens have been tested in mice for induction of immune tolerance by DNA vaccines and will be discussed in this section. Chronic diabetic complications can be divided into microvascular complications (retinopathy, neuropathy and nephropathy) and macrovascular complications (cardiovascular, cerebrovascular and peripheral vascular disease).
Obesity, hypertension, hyper-cholesterolemia and hyperlipidemia are all associated with T2D (MedScape).Alzheimer’s disease (AD) is the most common form of dementia in the elderly.
AcknowledgementsThe present work was supported by a Grant from the Italian Ministry of Universty and Scientific Research for Programs of Relevant National Interest (PRIN 2008 - prot. The first path relies on different forms of systemic suppression of inflammation that inhibit effector T lymphocytes in a non-specific manner. Immune mechanisms associated with the therapeutic effects of DNA vaccines can be complex because of the variety of cells that can process the information encoded by plasmid DNA. Severe microvascular and macrovascular complications can lead to renal failure (the most common cause of hemodialysis in the US), blindness or lower extremity amputations.
It is characterized by neuronal cell loss and increasing accumulation of neurofibrillary tangles (NTF) in neurons and amyloid fibers in neuritic plaques and in the walls of blood vessels (Wisniewski et al., 1997). Serious side effects associated with the use of systemic immunotherapies are increased risks of cancer and infection resulting from the decreased activity of effector cells involved in beneficial destructive immune responses against cancer cells and pathogens. Amyloid beta-peptides of varying length (39–43 residues) are produced by cleavage of a transmembrane protein, the amyloid beta-protein precursor (APP) (Wilquet & De Strooper, 2004). These side effects have been observed with broadly acting immunosuppressants used to prevent organ transplant rejection, which have been also investigated for treating type 1 diabetes [1, 2].
Insulin DNA vaccinesThus far, the only DNA vaccine that has been tested in both preclinical and clinical trials is a plasmid DNA construct coding for intracellular proinsulin, which is a partially processed non-functional form of insulin.
EpidemiologyIn 2010, about 215,000 people younger than 20 years of age had diabetes (type 1 or type 2) in the United States. Serious side effects can also be seen, albeit to a lesser degree, with more specific agents like antibodies that target specific molecules involved in inflammation [3, 4].
Insulin is not only the hormone produced by beta-cells that controls carbohydrate and fat metabolism in the body, it is also a main target autoantigen in autoimmune diabetes and the presence of anti-insulin autoantibodies can be an indication of disease initiation [59]. Abeta42 has a much greater neurotoxity than Abeta40 and its aggregation kinetics is faster than other beta-petides (Davis & Van Nostrand, 1996).
DNA vaccines coding for different forms of insulin have been investigated for type 1 diabetes immunotherapy since the late 1990’s. This path is thought to be safer because it aims to induce a regulatory immune response that targets the inflamed islets.
Here the goal is to manipulate endogenous immune mechanisms of homeostasis that can re-establish some form of tolerance to the chosen autoantigen, as well as to other neighboring beta cell autoantigens through a mechanism known as “by-stander suppression” [5]. The DNA vaccine induced insulin B-chain specific CD4+ T regulatory cells that secreted interleukin-4, and locally reduced autoreactive activity of cytotoxic T lymphocytes in the pancreatic draining lymph nodes. About 27% of those with diabetes (approximately 7 million Americans) do not know they have the disease. This incurable, degenerative, and terminal disease was first described by the German psychiatrist and neuropathologist Alois Alzheimer in 1906. Accordingly, it is anticipated that pathological autoimmunity and inflammation of islets can be stopped in an organ-specific manner that does not impair the immune system.
Further work showed that co-delivery of interleukin-4 was required to prevent diabetes onset in male nonobese diabetic mice [61]. Several factors have been considered relevant for the AD pathogenesis and among these the most important is age. In this chapter, we will review how plasmid deoxyribonucleic acid (DNA) has been used as an immunotherapeutic vector platform to treat type 1 diabetes through each immunotherapeutic path.
Two isoforms of insulin are synthesized in rodent animals, insulin I in islets and insulin II in both islets and thymus while humans have only one form of insulin. Type 1 diabetes mellitus continues to be highly prevalent in many countries, with an overall annual increase estimated at 3% (International Diabetes Federation [IDF] 2010). During life small variations occurring in cellular metabolism and structure can modify the functional state of susceptible neurons, leading to dramatic or even lethal changes. For the purpose of this review, we have called the first path “gene-based immunotherapy”, meaning that plasmid DNA does not encode a known autoantigen, and the second path “DNA vaccine immunotherapy” meaning that a beta cell autoantigen is encoded by plasmid DNA (Figure 1). The pancreatic beta cells synthesize proinsulin before converting it to functional insulin.
In other words, gene-based immunotherapy relies on the inherent function of a product encoded by plasmid DNA that can in turn affect cell function.
In that regard, intranasal delivery of plasmid DNA encoding mouse proinsulin II together with injection of an anti-CD154 (also named CD40 ligand) antibody to prevent T cell activation was reported to prevent type 1 diabetes in nonobese diabetic mice [62]. In contrast, DNA vaccine immunotherapy relies on the tolerogenic immune response induced directly by the autoantigen after its processing by immune cells. Delivery of 300 microgram DNA and 50 microgram antibody over a 2-week interval at 4 weeks of age synergistically prevented diabetes, reducing disease incidence from 100% diabetic down to 0% in 40-week old mice. Interestingly, late-onset AD is characterized not only by the neuropathological markers mentioned above, but also by vascular lesions, and hyperglycemia, hyperinsulinemia, insulin resistance, glucose intolerance, adiposity, atherosclerosis and hypertension (Haan, 2006).Diabetes and AD are considered age-related diseases and are both increasing. As we shall see, some DNA vaccine immunotherapies have also a gene-based immunotherapy component that acts as a molecular adjuvant to promote tolerogenic immune responses. Natural historyThe natural history of type 1 diabetes is characterized by an autoimmune destruction of the beta cells in the islands of Langerhans in the pancreas.
Nevertheless, in all cases synthesis of the molecule encoded by plasmid DNA, which is almost always a protein, starts after delivery of the plasmid DNA and its uptake by cells.
However, delivery of the DNA vaccine alone did not reduce diabetes incidence, even though it could induce T regulatory cells and reduce insulitis.
The autoimmune process has cellular and humoral components, leading to the destruction of the beta cells and a decreased insulin secretion. The Centers for Disease Control and Prevention predict that more than 29 million people in the US will be affected by diabetes by 2050, while the Alzheimer’s association forecasts that by this date, 11–16 million Americans will have AD (Han & Li, 2010). Plasmid DNA has several notable advantages compared to other vectors and therapeutic molecules. Another report has shown that co-delivery of 50 microgram plasmid DNA encoding human proinsulin together with 100 microgram insulin peptide twice over a 2-week interval could prevent diabetes until 24 weeks of age in 6 week old nonobese diabetic mice. As beta-cell mass declines, insulin secretion decreases until the available insulin no longer is adequate to maintain normal blood glucose levels.
For example, it consists of relatively low molecular weight circles of double stranded DNA that can be readily isolated from bacteria in a generic and cost-effective manner.

After 70-90% of the beta cells are destroyed, hyperglycemia develops and diabetes may be diagnosed.
In addition, plasmid DNA permits rapid turnaround when developing new candidate products, refrigeration-free storage, and synthesis over time of a chosen antigen in its native conformation. Results also indicated induction of CD4+CD25- islet specific T regulatory cells producing transforming growth factor-beta only in the co-immunization group.In another study, a DNA vaccine encoding proinsulin and pancreatic regenerating (Reg) III protein resulted in a significant reduction of hyperglycemia and diabetes incidence with increased serum insulin in a streptozotocin- induced mice model [64]. In fact, some studies revealed that 80% of patients with AD exhibited either impairments in glucose tolerance or frank diabetes (Schrijvers et al., 2010). Furthermore, plasmid DNA can be given in repeat doses within short periods of time without inducing an immune response to vector and other side effects. In some patients years will go by before the onset of diabetes, while other patients may never develop beta cell insufficiency, perhaps due to the regaining of tolerance. Cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) is a strong negative regulator of T cell activity and another example of an immunomodulator that can be co-delivered with an autoantigen. Most patients with type 1 diabetes mellitus have one or more susceptible human leukocyte antigen (HLA) class II, and over 90% have beta cell autoantibodies present. The inbalance between low and high glucose levels in T2D patients may be responsible for brain vascular damage and neurodegeneration thus facilitating the AD onset.
Although they may sometime have pro-inflammatory properties that can be detrimental to controlling inflammation, these particles have been used to induce immune tolerance with plasmid DNA.
The appearance of circulating islet cell autoantibodies is the first detectable sign of this immune process.4. For example, chitosan-DNA nanoparticles encoding an ovalbumin antigen are tolerogenic when delivered orally [9].
Indeed, route of delivery can play a significant role in the type and strength of immune responses induced by DNA vaccines in animal models [10, 11]. In humans, two microgram of a DNA vaccine for treatment of melanoma delivered with gold particles into skin was found to be as efficacious as 1000 microgram injected intramuscularly [12].
However, extra-genetic components influence the penetrance of diabetes susceptibility genes. The formation of amyloid aggregate occurs both in the intra- and extra-cellular environments; further the proteinaceous aggregates are strictly bound with membranes and calcified. These results illustrate the significant impact that choice of route and method of delivery of a DNA vaccine can have not only on efficacy, but also on cost of treatment. If data are obtained at a single point in time, the risk of type 1 diabetes mellitus between monozygotic twins can be as low as 30%, but if the monozygotic twins are followed long-term, the cumulative incidence of diabetes reaches 65% (Redondo et al., 2008).
Other delivery routes like intravenous, nasal, and sublingual have also been investigated [13].
In the same cohort of monozygotic twins, the rate of persistent autoantibody positivity, type 1 diabetes mellitus, or both, reached 78% (Redondo et al., 2008). Post delivery, expression of coding sequences in plasmid DNA results in significant levels of protein production that may persist for six weeks and longer without serious side effects in human patients [14, 15]. To better understand the genetic susceptibility to diabetes, candidate gene studies were conducted in order to identify genes that are associated with autoimmune type 1 diabetes.Human leukocyte antigen (HLA) associations have been long recognized in many autoimmune diseases. In type 1 diabetes mellitus, the HLA on chromosome 6p21 is well described and is considered to play an important role in more than 50% of the familial cases in Caucasians (Noble et al., 1996). X-ray fiber diffraction showed that amyloid fibrils contain beta-sheet structure lying orthogonally to the major fibril axis (Serpell, 2000). HLA DR4-DQ8 or DR3-DQ2 haplotypes are detected in up to 90% of patients with type 1 diabetes mellitus (Devendra & Eisenbarth, 2003).
They showed that the methyl carbons of Ala-21 and Ala-30 must be placed in groups of at least four with internuclear distances of less than 5.5 A. First-degree relatives of the patients who carry the highest risk haplotype combination also have a higher risk of developing diabetes mellitus as compared to the relatives of diabetes patients who do not have this haplotype and who develop type 1 diabetes mellitus later in life (Gillespie et al., 2002). Although beta-sheets are the main constituent of the amyloid fibrils they are not the only structure present in the fibrils. DNA vaccines and other gene-based vaccines belong to a third generation of vaccines after live and attenuated whole organism vaccine and recombinant protein vaccines.
Another HLA haplotype (DR15-DQ6) might have protective properties, and is found in a much larger percentage in the general population (20%) as compared to less than 1% in patients with type 1 diabetes mellitus (Eisenbarth & Gottlieb, 2004). Liquid state NMR, FTIR and CD measurements in Abeta40 have demonstrated the existence of a turn formed by the amino acids at position 26-29.
These vaccines can be used to either prevent (prophylactic vaccine) or treat (therapeutic vaccine) disease depending on their potency, in which prevention is generally easier to achieve than treatment.
Little information is known about the Abeta42 fibril structure and many mutant peptides have been synthetized to obtain an explanation about its secondary structure.
Recent reports of beneficial results in different clinical trials using delivery of autoantigens indicate that DNA vaccination is reaching a stage where we are likely to see accelerated development of a therapeutic future for vaccines targeting a variety of autoimmune diseases. Glutamic acid decarboxylase (GAD) antibodies are more frequent in patients with HLA DR3-DQ2, whereas insulin auto-antibodies (IAA) and protein tyrosine phosphatase-like protein antibodies (IA-2 antibodies) are more frequent in patients with HLA DR4-DQ8.
In the case of type 1 diabetes, early results using a DNA vaccine encoding insulin have shown promise in humans. In addition, DNA vaccines encoding human heat shock protein 60 and glutamic acid decarboxylase 65 have also shown efficacy in preclinical trials and are reviewed in this chapter. The insulin gene contributes 10% to the genetic susceptibility in developing autoimmune diabetes (Bell et al., 1984). The primary sequence of the peptide is well conserved in organisms and, in particular human and mouse IAPPs differ by only six amino acids but the latter does not form fibrils neither in vitro nor in vivo. In contrast to DNA vaccine immunotherapy, gene-based immunotherapy involves delivery of genetic material by a plasmid vector into a cell, tissue or organ with the aim of improving the clinical status using the function of the encoded product, instead of its properties as an antigen. The risk of developing diabetes depends on the expression of the insulin protein in the thymus which can cause a defective central tolerance to the insulin molecule. Gene-based immunotherapy includes delivery of anti-inflammatory cytokines, chemokines, and other factors to modulate the activity of immune cells [16, 17].2. As in Abeta, IAPP is non-toxic in its monomeric form but it exhibits high toxicity levels when it aggregates into beta-rich amyloid structures.
Gene-based immunotherapiesSeveral pre-clinical trials have used plasmid DNA-based gene therapies in experimental models of autoimmune type 1 diabetes. As in other amyloid peptides, the mechanism of fibrillation occurs through the formation of nuclei with a lag phase whose duration is concentration-dependent and proceeds by addition of monomers or oligomers to both fibril terminals. These strategies involve plasmid DNA designed to weaken pre-existing beta-cell autoimmunity through delivery of anti-inflammatory cytokines, chemokines, and other immune cell manipulating agents. The secondary structure of hIAPP mainly consists of unstructured regions, with small alpha-helical and beta-sheet components (Goldsbury et al., 2000). T cells are recognized to be a major part of the immune process in diabetes mellitus, and several genes involved in T cell regulation are associated with type 1 diabetes mellitus.
Cytokine gene therapiesCytokine gene therapies are strategies that use engineered plasmid DNA to produce therapeutic immune cytokines, which are a group of immune active molecules secreted by different cells of the body. Some of these cytokines are considered beneficial for the suppression of autoimmunity, and thus are applied to disease models to reduce clinical symptoms and improve therapeutic effects.
In fact, recent studies have evidenced the importance of the residues in position 13-18 in the interaction leading to the formation of fibrils (Gilead & Gazit, 2008).
Studies of animals with spontaneous autoimmune diabetes have revealed that an important group of autoreactive T cells that mediates islet beta-cell destruction belongs to the T helper-1 type effector cell subset, and produces cytokines like interleukin-2 and interferon-gamma.
Also the aromatic-aromatic interactions between residues 15, 23 and 37 seem to be important in amyloid formation althought not essential for fiber formation as evidenced using IAPP with a triple mutation (Marek et al., 2007). On the other hand, regulatory T cells that control effector cells can secrete interleukin-4, interleukin-10, and transforming growth factor-beta.
One of the earliest applications of cytokine-engineered plasmid DNA was gene-gun delivery of murine interleukin-4 to prevent spontaneous type 1 diabetes [18].
Autoimmune processOne of the best animal models for type 1 diabetes mellitus is the nonobese diabetic mouse (NOD).
The effect of oxidative stressThe brain has a high energy demand and, although it represents only 2% of body weight, it accounts for 20% of total body oxygen consumption.
The plasmid DNA was delivered as three times two microgram within 4 weeks into 3-week-old nonobese diabetic mice, which is the animal model system closest to human type 1 diabetes.
NOD mouse develops type 1 diabetes mellitus spontaneously, over the course of a few months, allowing the investigators to study this process stage by stage.
This energy requirement is largely driven by neuronal request of energy to maintain the ion gradients across the plasma membrane, which are critical for the generation of action potentials. Type 1 diabetes incidence was reduced from 90% in controls to 20% at 34 weeks of age, and was associated with T helper-2 type immune responses in the periphery and pancreas of mice. Many reports describe in detail the genetics, the immune process, the influence of the environment and most importantly, the potential therapies to prevent, delay or reverse the destructive process that leads to type 1 diabetes mellitus in this model. This intense energy requirement is continuous; even brief periods of oxygen or glucose deprivation result in neuronal death. Two other reports have shown that systemic delivery of plasmid DNA constructs coding for interleukin-4 can prevent insulitis, which is an inflammatory sign of immune cell infiltrating pancreatic islets in nonobese diabetic mice [19, 20]. Delovitch and Singh (Delovitch & Singh, 1997) reviewed the use of NOD mouse in the studies of type 1 diabetes mellitus. Diabetes mellitus leads to functional and structural changes in the brain, which appear to be most pronounced in the elderly.
In NOD mice, the first step is the infiltration of the peri-islet regions of the pancreatic islets by dendritic cells (DC) and macrophages, followed by T cells (CD4+ and CD8+). For example, a report indicates that intramuscular electroporation delivery of 50 µg plasmid DNA encoding interleukin-4 accelerated spontaneous type 1 diabetes in nonobese diabetic mice [21].In addition to interleukin-4, interleukin-10-encoded plasmid DNA alone was also tested for its diabetic suppressive effects. Increasing data support the idea that mitochondrial function declines with aging and in age-related diseases such as diabetes and AD.Normal glucose metabolism is required for the performance of cognitive functions, and impairments in glucose metabolism might contribute to cognitive dysfunction. The plasmid DNA was delivered intramuscularly twice for a total of 200 microgram into 3 and 5 week old female nonobese diabetic mice [22].
It is followed by a slower, progressive T cell destruction of the beta cells (insulitis), by 4-6 months of age (Delovitch & Singh, 1997). Imaging studies have revealed that patients with AD and individuals at risk of developing this disease typically have reductions in glucose metabolism in temporal and parietal brain regions and hippocampus (Garrido et al., 2002).
Although the severity of insulitis at 13 weeks of age was not improved, the incidence of diabetes was markedly reduced to 50% at 35 week old compared to 90% with control mice.
Thus, the T cells and the dendritic cells are key players in the immune process leading to type 1 diabetes mellitus.The dendritic cells (DC) are antigen-presenting cells which originate from the bone marrow. These results show that the progression of autoimmune disease in mice can effectively be suppressed by intramuscular DNA injection coding for anti-inflammatory cytokines alone.
These findings indicates that glucose metabolism and insulin signaling are important in normal brain function. After infiltrating the pancreas and undergoing antigenic maturation, DC secrete IL-12 and present the processed antigen (on their surface and in association with the major histocompatibility complex [MHC] class II) to other cells of the immune system (i.e.
The negative effect of impaired glucose metabolism on cognitive functioning can be caused by an increase in oxidative stress that is associated with mitochondrial dysfunction.
Another report investigating the immune effects of interleukin-10 DNA showed that systemic intramuscular administration of 200 microgram interleukin-10 plasmid DNA could alleviate blood glucose and insulitis in a streptozotocin induced diabetic mouse model up to day 28 post injection [24]. T cells) (see Fig 1).T cells are categorized mainly based on their immune actions, achieved via the different cytokines they secrete. Mitochondria are essential subcellular organelles for generating the energy that fuels normal cellular functioning.
In this model, pancreatic interleukin-1b and tumor necrotic factor-alpha gene expression, serum interferon-gamma concentration, and the numbers of CD4+ and CD8+ lymphocytes were decreased on day 28.
At the same time, the mitochondria have a strategic task because, depending on environmental factors, they can decide whether to continue the healthy life of the cell or to terminate it by apoptosis activation. A similar interleukin-10 construct was modified by introducing nuclear factor kappa-B (NF-kB) binding sites into plasmid DNA to facilitate nuclear transport of the plasmid after delivery into the cell [25].
T helper 2 cells (Th2) are important in humoral immunity (activate B cells and antibody production, down regulating Th 1 cells) and secrete type 2 cytokines: interleukins 4, 5, 6, 9 and 10 (Rabinovitch, 1998) (Fig. Mitochondria are essential for neuronal function because the limited glycolytic capacity of these cells makes them highly dependent on aerobic oxidative phosphorylation for their energetic needs. A single injection of 50 microgram of the plasmid using polyethylenimine as a gene carrier in 5 week old mice reduced the degree of insulitis and serum glucose levels in 100% of mice compared to 40% of the control mice at 32 weeks of age. These results illustrate how plasmid DNA can be easily modified in a generic manner to improve therapeutic efficacy.As mentioned previously, nanoparticle technology has been used to condense plasmid DNA into nanometer-size complexes to improve delivery. A variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in vivo through both enzymatic and non-enzymatic routes.
An interleukin-10 encoding plasmid DNA was assembled into a cationic nanoparticle complex, and a single dose of 50 microgram DNA was delivered intramuscularly into streptozotocin-induced diabetic mice [26]. They have an inhibitory effect on the Th1 cells, which are destructive to the pancreatic beta cells. ROS include hydrogen peroxide, hydroxyl radical, superoxide ion and singlet oxygen, products of normal cellular respiration. Animals showed higher serum levels of interleukin-10, suppression of interferon-gamma level, reduction of islet insulitis, and lower blood glucose levels compared to those treated with interleukin-10 plasmid alone or the nanoparticle alone up to week 6 post injection. In the NOD mouse, it appears that the immunologic self-tolerance to pancreatic beta cells is lost. Histology of muscle showed that nanoparticles were biocompatible and did not cause a chronic inflammatory response. The disruption of the equilibrium between Th1 and Th2 cells in the thymus and in the periphery is believed to play a crucial role in the pathogenesis of autoimmune diabetes mellitus (Delovitch & Singh, 1997). It is abundantly produced in the mitochondria during respiration cycles and reacts with the proteins, lipids, and nucleic acids during their production. In addition to their use alone, delivery of both interleukin-4 and interleukin-10 DNA has also been investigated. Once Th1 cells are produced they will secrete interferon ? (IFN ?) and IL-2, leading to the activation of macrophages and cytotoxic T cells, which are destructive to the pancreatic beta cells (Adorini, 2001). Peroxynitrous acid is one of the major RNS found intracellularly and it is involved in the rapid nitration of aromatic residues of proteins, such as tyrosine, to give 3-nitrotyrosine, which may alter the protein structure. Combined delivery into nonobese diabetic mice of the two plasmid DNA constructs encoding interleukin-4 and interleukin-10 (25 µg each) was done intravenously using a degradable, cationic polymeric carrier, poly (alpha-(4-aminobutyl)-L-glycolic acid)[27].
The same Th1 cells will stimulate the IgG2a autoantibodies against the islet beta cells autoantigens (Delovitch & Singh, 1997).
Overexpression of the two cytokine messenger RNAs was confirmed in the liver of mice 5 days after delivery. Autoimmune diabetes can be transferred from a diabetic NOD mouse to an unaffected mouse via T cells (Bendelac et al., 1987). Six weeks after injection, 75% of observed islets were intact compared with less than 3% in the control group, and development of diabetes was prevented in 75% of treated animals at 30 weeks of age, compared to 20% in control mice receiving plasmid DNA coding for a single cytokine or vector control alone. NOD mice develop a spontaneous loss of T-cell tolerance to glutamic acid decarboxylase antibodies (GAD), leading to autoimmune diabetes (Kaufman et al., 1993). In the absence of an appropriate compensatory response from the endogenous antioxidant network, the system becomes redox imbalanced, leading to the activation of a stress-sensitive intracellular signaling pathway and, in extreme conditions, to apoptosis. The results indicated that the interleukin-4 and interleukin-10 plasmid DNAs had synergistic effects on the prevention of autoimmune diabetes.
A report from the same research group showed that a ‘chimeric’ plasmid expressing both of the interleukin-4 and interleukin-10 under controls of two CMV promoters could also reduce insulitis in the same system [28].More recently, a research group also reported packaging plasmid DNA constructs coding for interleukin-4 and interleukin-10 into cationic nanomicelles to prevent type 1 diabetes [29]. Exposure to glutamic acid decarboxylase (GAD65 and GAD67) led to an increased T cell proliferation as early as 4 weeks of life in NOD mice, coinciding with the onset of insulitis (Tisch 1993).
A single intramuscular dose of 50 microgram of the complex reduced levels of blood glucose and insulitis up to 6-week post delivery in 5-week-old streptozotocin-induced diabetic mouse. Some of the other beta-cell antigens elicited an increased immune response after a few more weeks, but there were other beta-cell antigens that did not trigger an immune reaction (for example, amylin) (Tisch 1993).
Abnormal glucose metabolism can also increase the production of free radicals such as reactive oxygen species (ROS) and reactive nitrogen species (RNS).
Notably, plasmid DNA coding for interleukin-4 and interleukin-10 has also been used as adjuvant to promote the therapeutic effect of DNA vaccines in a murine model for type 1 diabetes, which will describe later in this chapter in the ‘Glutamic Acid Decarboxylase DNA Vaccines’ section.In addition, a number of studies have reported that injection of plasmid DNA coding for cytokines normally considered pro-inflammatory can prevent diabetes.
To further support the central role of GAD antigen in autoimmune diabetes, the beta-cell-specific suppression of GAD expression in antisense GAD transgenic NOD mice was demonstrated to prevent the production of diabetogenic T cells and the onset of diabetes (Yoon et al., 1999) In humans, the pancreas becomes infiltrated with mononuclear cells.
These results reflect the multifaceted role of cytokines on immune response [30], which could be dependent on dosage and time of administration. Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GAD) and insulinoma associated-2 antibody (IA-2) are demonstrated years before the clinical symptoms of diabetes.
For example, administration of interleukin-18, also known as interferon-gamma inducing factor, can prevent diabetes in NOD mice [31, 32]. Furthermore, levels of oxidized proteins are increased in the frontal and parietal lobes and in the hippocampus of patients with mild cognitive impairment compared with healthy controls, indicating that oxidative damage might occur early in the development of AD (Butterfield et al., 2007). However, it was also shown that intramuscular electroporation of 2 x 100 microgram plasmid DNA coding for interleukin-18 into 4-6-week-old nonobese diabetic mice aggravates diabetes [33]. The presence of autoantibodies alone does not explain the development of diabetes, since it is recognized now that children born to type 1 diabetic mother with high antibody titers transferred through the umbilical cord do not develop diabetes more often than expected. Another report showed that intraperitoneal administration of 30 microgram of plasmid DNA encoding interferon-gamma promotes insulitis in reovirus type-2 induced diabetic mice compared to controls [34]. Minor modifications of the nucleic acid bases are repaired through base excision repair involving DNA glycosylase and AP endonuclease, which are located in nuclei and mitochondria. This is in contrast with another report showing that injection of interleukin-12 induces interferon-gamma that prevents diabetes in NOD mice [35].
Environmental component The environment is implicated in the pathogenesis of type 1 diabetes mellitus by many studies.
The progression of AD is associated with the diminished expression of these DNA repair enzymes (Nakabeppu et al., 2004). Together, these results suggest that there is risk associated with direct delivery of cytokines for the treatment of type 1 diabetes. Environmental factors have an important role in initiating an immune process that ultimately leads to pancreatic beta cell destruction and clinically apparent diabetes mellitus. The accumulation of the oxidatively damaged nucleic acids and proteins likely exceeds the limit of cellular repair and detoxification mechanisms and leads to the onset or progression of diabetic and neurological pathologies. This possibility is suggested by a phase I clinical trial where new onset patients with type 1 diabetes received a combination treatment of interleukin-2 and the immunosuppressant rapamycin.
Many environmental factors have been proposed, including viruses (rubella, mumps or coxsackievirus B4), toxic substances and cytotoxins. Further evidence supporting this hypothesis has been obtained from studies of a mouse model of AD in which mutations in the genes encoding APP and presenilin 1 cause an increase in Abeta42 production. Chemokine gene therapiesChemokines are a family of small chemotactic cytokines secreted by cells [36]. Before the eradication of rubella in most countries, congenital rubella was strongly associated with the development of type 1 diabetes mellitus (Menser et al., 1978). In these animals, lipid and protein peroxidation is evident at the disease onset (Matsuoka et al., 2001). Their name is derived from their ability to induce directed chemotaxis, or directed cell migration, in responsive cells.
A recent meta analysis of observational studies has shown an association between type 1 diabetes and enterovirus infection (Yeung 2011). In a triple-transgenic animal model of AD, in which mice develop Abeta plaques, tangles and cognitive defects, a decrease in antioxidant capacity and an increase in lipid peroxidation were noted before the development of AD pathology (Resende et al., 2008). Some chemokines are considered pro-inflammatory and can be induced during an immune response to recruit cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal tissue maintenance and inhibiting abnormal inflammation like pathological autoimmune response. While some theories implicate viral infections in the pathogenesis of type 1 diabetes, a recent hypothesis argues that a decreased exposure to microbes may contribute to the current increase in autoimmune disease.
Oxidative stress seems to affect APP either directly, by increasing APP levels, or indirectly, by modulating APP processing, and both mechanisms could increase levels of Abeta. Chemokines are involved in pathogenesis of autoimmune disease because they can selectively recruit various subsets of immune lymphocytes [37, 38]. This theory is known as “the hygiene hypothesis” (Gale, 2002).It is a known fact that the incidence of autoimmune diabetes follows a geographical pattern, with many studies reporting an association between type 1 diabetes and vitamin D status.
Studies in transgenic mice and postmortem brain tissue from patients with AD suggest also that an increase in Abeta production leads to a rise in the production of ROS and that oxidative stress occurs early in the development of the disease. Based on structural motifs near their N-terminal cysteine residue [C], chemokines are divided into four subfamilies, termed CXC, CX3C, C, and CC.
A few large ecological studies describe a pattern of geographical variation, with an increased incidence of type 1 diabetes in the areas located north of the equator. The function of chemokines is modulated by the type of chemokine receptors they bind to as ligands on the surface of cells, and studies have shown that chemokines and chemokine receptors are involved in the pathogenesis of autoimmune diseases like type 1 diabetes. Furthermore, seasons appear to also influence the incidence of type 1 diabetes, with the highest incidence during winter and the lowest during summer.
Chemokine gene therapies for type 1 diabetes use anti-inflammatory chemokines as well as inhibitors of pro-inflammatory chemokine binding. This suggests that diabetes and aging are risk factors for the neurodegeneration induced by this peptide. Typically, the treatment for type 1 diabetes mellitus involves insulin therapy, but in the last few years new therapies have been approved as well (for example, Symlin).
Mitochondrial dysfunction could be one of the common underlying mechanisms explaining the association between diabetes and AD. It was found that, in type 1 diabetic adult patients, elevated levels of serum CXC ligand-10 are associated with high-risk of disease in latent diabetic subjects [39].
For newly diagnosed patients with autoimmune diabetes, combination therapy has been suggested in an attempt to minimize beta cell destruction and prolong pancreatic function.
Mitochondrial dysfunction and the resulting energy deficit trigger the onset of neuronal degeneration and death.
This finding was translated in animal models where blockade or neutralization of the CXC ligand-10 can prevent type 1 diabetes in nonobese diabetic mice [40]. The new therapeutic options include: immunotherapy, vaccines, drugs that influence T cell action, anti-inflammatory drugs (for example, one time use of anti-IL-1R drug), or long-term treatment with B cell components to induce regulatory T cells (oral or nasal insulin, insulin peptide therapy, GAD-Alum or the proinsulin DNA vaccines).
Mitochondria serve also as high capacity Ca2+ sink, which allows them to follow the changes in cytosolic Ca2+ loads and helps in maintaining cellular Ca2+ homeostasis, required for normal neuronal function (Rizzuto et al., 2000). In virus-induced diabetic mice, virus infection results in rapid and differential expression of CXC receptor-3 and CXC ligand-10, which plays a dominant role in programming the ensuing autoimmune disease [41]. Glucagon-like peptide 1-related drugs (GLP-1) could be also considered as a therapeutic option because they promote peritubular pancreatic cell growth (Von Herrath, 2010).5. Conversely, excessive Ca2+ uptake inside mitochondria has been shown to increase ROS production, inhibit ATP synthesis, release cytochrome C, and induce mitochondrial permeability transition (Brustovetsky et al., 2002). The blockade of CXC ligand-10 by using anti CXC ligand-10 monoclonal antibodies successfully aborts severity of antigen-specific injury of pancreatic beta cells and abrogates type 1 diabetes.

Vitamin DAlthough initially described as a “vitamin”, vitamin D is now recognized to be a hormone, synthesized in the human body and exerting its action on other organs via a nuclear receptor (vitamin D receptor, VDR).
The mitochondrial permeability transition (MPT) is defined as the sudden increase of inner mitochondrial membrane permeability to solutes of molecular mass lower than 1500 Da (Bernardi et al., 1994). Mechanistically, the blockade impedes the expansion of peripheral antigen-specific T effector cells and hinders their migration into the pancreas. Even though vitamin D can be obtained from the diet in small quantities, the main source of vitamin D is the skin. Strong evidence now exists that the MPT is due to the opening of a nonselective megachannel (estimated to be 2–3 nm in diameter).
A similar effect of the antibodies was confirmed in a cyclophosphamide accelerated model of type 1 diabetes [40]. Under the direct influence of ultra violet B light (UVB light), 7-dehydrocholesterol (DHC) (provitamin D3) is converted into pre-vitamin D3, which is then further converted into cholecalciferol (vitamin D3) via thermal isomerization. Because the chemiosmotic theory is based on the impermeability of the inner mitocondrion membrane to solutes that are not specifically transported, MPT would collapse the mitochondrial membrane potential (??m) and uncouple the electron transport system from the production of ATP. Based on these reports, plasmid DNA encoding the CXC ligand-10 was constructed to induce production of anti-CXC ligand-10 antibodies in the host [42]. Interestingly, if pre-vitamin D3 continues to be exposed to UVB, it will be converted into biologically inactive metabolites (tachysterol and lumisterol), preventing a potential UVB- induced vitamin D intoxication (Holick, 1999) The other source of vitamin D is the diet, which contains cholecalciferol (vitamin D3), originating from animal sources, and ergocalciferol (vitamin D2), deriving from plants (Holick, 1999).Regardless of their source, once they enter into the circulation, forms of inactive vitamin D3 or D2 bind to the vitamin D-binding protein (DBP) and are transported to the liver. Additionally, MPT results in mitochondrial swelling and can lead to the release of proapoptotic proteins. The inactive vitamin D is activated through a 2-step hydroxylation process via two hydroxylases that belong to the cytochrome P450- dependent steroid hydroxylases (CYP450). Intramuscular electroporation of 100 microgram of the plasmid DNA at 4 and 6 weeks of age induced synthesis of anti CXC ligand-10 antibodies in vivo, and suppressed the incidence of spontaneous diabetes which went from 75% in control mice down to 25% in treated mice at 30 weeks of age.
In the liver, vitamin D undergoes the first hydroxylation at C-25 via some of the CYP 450 vitamin D 25-hydroxylases, forming calcidiol (25-hydroxyvitamin D) (Prosser & Jones, 2004). Some data show the existence of an age-related impairment of the respiratory chain and an uncoupling of oxidative phosphorylation in brain mitochondria isolated from Goto-Kakizaki (GK) rats, as model of T2D (Moreira et al., 2003). Although this treatment did not inhibit insulitis or alter the immunological response, it enhanced the proliferation of pancreatic beta cells and resulted in an increase of beta-cell mass.A subsequent report from the same research group showed that combining complete Freund’s adjuvant with plasmid DNA encoding the CXC ligand-10 could reverse diabetes [43].
The maintenance of oxidative phosphorylation capacity is extremely important in the brain since a large amount of the energy required for the normal functioning of neurons is provided by mitochondria. Diabetes incidence was reduced from 70% in control mice to 20% in treated mice 10 weeks after plasmid DNA delivery. The activity of 1?-hydroxylase in the immune cells is not under the regulation of parathyroid hormone and 1?-,25(OH)2D3, but rather under immune cytokine regulation. In contrast, mice receiving complete Freund’s adjuvant and control plasmid DNA did not show disease reversal. A defect in the up-regulation of 1?-hydroxylase after immune stimulation is described in NOD mouse (Overbergh et al., 2000). Advanced glycation end products (AGE)Abnormal glucose metabolism and oxidative stress contribute to the formation of advanced glycation end products (AGE).
In mice that were treated successfully, residual beta-cell mass was significantly increased, and some beta-cells were in a proliferative state.
Extrarenal distribution of 1?-hydroxylase becomes important in understanding the extra-skeletal effects of vitamin D.VDR is part of the nuclear receptor super family of ligand-activated transcription factors, which also includes glucocorticoid, thyroid hormone and estrogen receptors. This process occurs through the Maillard reaction or “non-enzymatic browning”, a complex series of reactions between reducing carbohydrates with lysine side chains and N-terminal amino groups of proteins.
Although systemic cytokine profiles were unaffected, the frequency of regulatory T cells expressing CXC receptor-3 was significantly increased in local pancreatic lesions and possibly associated with the regulation of anti-islet autoimmunity. The gene for VDR is located on chromosome 12q12-14, and shows great polymorphism (Haussler et al., 1998).
This process initially leads to rather labile Schiff bases which as a rule rearrange to the more stable Amadori products.
Another research group found that intra-pancreatic CC ligand-4 levels are increased in a model of diabetes protection by interleukin-4 treatment in female nonobese diabetic mice [44]. After 1,25 (OH)2D3 binds to VDR, it induces conformational changes that facilitate heterodimerization with the retinoid X receptor and the recruitment of nuclear receptor coactivator proteins, which then act on the chromatin. The protective effect of CC ligand-4 was confirmed by abrogation of diabetes suppression after injection of anti-CC ligand-4 antibodies [45]. These result led to studies using CC ligand-4-encoded plasmid DNA therapy which showed that gene-gun delivery of 1 microgram of the plasmid DNA protects against type 1 diabetes in NOD mice, with diabetes rates reduced from 75% in control mice to 30% at 35 weeks of age when treated weekly from week 3 to 14, and from 80% in control mice down to 30% when treated weekly from week 9 to 14 [45]. Auto-oxidation of glucose leads to the formation of oxygen radicals, which are intermediates in the AGE pathway and the predominant source of endogenous AGEs. Data also indicated that plasmid DNA delivery could both prevent and treat type 1 diabetes. AGEs may exist as protein cross-links or as modification of the side chains of a single protein, and significantly alter protein conformations leading to protein inactivation.
This protection was associated with a T helper-2-like response in the spleen and pancreas, decreased recruitment of activated CD8 T cells to islets accompanied by diminished CC receptor-5 expression on CD8 T cells, and increased regulatory T cell activity in the draining pancreatic lymph nodes. Numerous AGEs have been isolated and characterized by spectroscopic analysis after cleavage from the protein backbones. To summarize, plasmid DNA encoding CC ligand-4 and CXC ligand-10 have been tested for their type 1 diabetic suppressive effects in spontaneous diabetic mouse models. Diabetes suppression is associated with decreased CD8 T lymphocyte activity and increased CD4 T regulatory cell activity. Examples of AGEs resulting from the single protein modification are pyrraline and N? -(carboxymethyl)lysine (CML), the lysine-residue modified products, and argpyrimidine, an arginine-residue modified protein. Other cell-manipulating gene therapiesSeveral immune cell populations have deficiencies in type 1 diabetes, such as CD4 T lymphocytes, CD8 T lymphocytes, B lymphocytes, dendritic cells, macrophages, and NK cells in both nonobese diabetic mice and human patients [46, 47].
The goal of cell-manipulating gene therapy is to increase the diabetic suppressive function of cells like T regulatory or T helper-2 lymphocytes, which are considered important not only for therapeutic purposes, but also for playing a determining role in the development of type 1 diabetes.As mentioned at the beginning of this chapter, type 1 diabetes is a T helper-1-mediated autoimmune disease and strategies suppressing the function of these cells can be expected to have an impact on disease progression. Furthermore, hippocampal neurons from patients with this neurodegenerative disease contain Abeta-positive, AGE-positive and RAGE positive granules. One of these strategies is the delivery of galectin-9, a carbohydrate-binding protein that regulates T helper-1 cells and induces their apoptosis through the galectin-9 receptor. Apoptosis, or programmed-cell-death, is a constantly ongoing process in steady state in vivo and helps maintain tissue and immune homeostasis.
Whether the modifications of Abeta and tau by AGEs are a primary or secondary event in AD is a controversial topic. Mice treated with plasmid DNA coding for galectin-9 were significantly protected from diabetes: intravenous delivery of 2 x 100 microgram bi-weekly protected 85% of mice from diabetes versus 55% in controls [48].
Nevertheless, AGEs are widely accepted to be active participants in the progression of AD, since AGE-induced glycation of Abeta and tau protein has been shown to cause the Abeta aggregation and the formation of NFTs, respectively (Ledesma et al., 1994).
Moreover, diabetic mice with cognitive impairments exhibit increased RAGE expression in neurons and glia compared with wild-type control mice (Toth et al.
Splenocytes from treated mice were also less responsive to mitogenic stimulation than splenocytes from the control group.
Data indicated that galectin-9 DNA may downregulate T helper-1 immune response in diabetic mice and could be used as a therapeutic agent in autoimmune diabetes.In contrast with galectin-9, decoy receptor 3 inhibits apoptosis.
However, it should be pointed out that glycoxidation and oxidative stress are mutually dependent and reinforce each other.
The membrane protein is a member of the tumor necrosis factor receptor superfamily, and regulates immune responses by neutralizing apoptotic signals transmitted through CD95 (Fas receptor), lymphotoxin beta-receptor, and death receptor 3 on target cells. Thus, while the sources of oxidative stress may widely differ in diabetes and AD, and while a number of AGEs accumulate in both conditions, other AGEs found in diabetes have yet to be characterized in AD.6. As a result, transgenic expression of decoy receptor 3 in pancreatic beta cells protects nonobese diabetic mice from autoimmune diabetes [49].
Antioxidant therapy in Alzheimer’s disease and diabetesGiven the importance of mitochondria as the primary source of oxidative stress in AD and diabetes, the use of antioxidants may also be useful.
When decoy receptor 3 is delivered systemically as plasmid DNA, it inhibits insulitis and diabetes by modulating immune responses. However, the broad occurrence of both diseases, the non-regenerative nature of the CNS and the fact that AD diagnosis often does not occur until late in the disease progression, suggest that the ideal antioxidant should be used as a prophylactic treatment for the aged population.
For example, four weekly intravenous injections of 100 microgram of plasmid DNA coding for decoy receptor 9 into nonobese diabetic mice was reported to reduce diabetes incidence from 90% in controls to 30% when treated at 4 weeks of age, 45% (started at 7 week old), and 70% (as Fc-fusion form, started at 12 week old) in 35-week-old female nonobese diabetic mice [50]. Oxidative stress is one of the earliest events in the neurological and pathological changes of AD, while the effects of oxidative stress are manifested in the slow accumulation of AGEs in diabetes.
Treated mice showed less splenocyte proliferation and adoptive transfer of the cells ameliorated diabetes. Thus, antioxidant therapy in combination with AGE inhibitor therapy may be effective approaches for AD and diabetes-related complications. Oxidative stress leads to irreversible protein aggregation and consequent neuronal degeneration in AD (Liu et al., 2007). Data also indicated that immune modulation by decoy receptor 3 may have been the result of differentiation and maturation of dendritic cells that subsequently regulated T effector differentiation and function.Cell migration is another process that plays a role in pancreatic beta cell destruction. Advanced lipoxidation products, such as HNE, bind to phosphorylated tau protein to form paired helical filaments, accelerating the formation of neurofibrillary tangles.
In this regard, plasmid DNA coding for CD44, which is a protein associated with cell migration and delivery of apoptotic signals by inflammatory cells, was investigated for the suppression of diabetes. Oxidative stress also results in the covalent crosslinking of tau filaments to form large aggregates that are resistant to proteolytic cleavage.
It was found that subcutaneous implants of a silicone tube filled with wound dressing sponge carrying CD44 encoded plasmid DNA could attenuate diabetes in a transfer model [51]. Larbig and coworkers reported a series of inhibitors for tau protein aggregation (Larbig et al., 2007). Diabetes was induced in male nonobese diabetic mice by transfer of diabetogenic splenocytes from female diabetic mice and was reduced from 90% in controls to 20-30% 12 weeks after two implants. Remarkably, thiazolium-based compounds, which are also AGE inhibitors and potentially useful for diabetic therapy, are effective inhibitors of tau aggregation. Here the mechanism of treatment was not thought to be strictly a gene therapy effect, but rather induction of anti-CD44 antibodies that inhibited CD44 function. An increasing body of evidence points to a possible relationship between the central nervous system and diabetes [52].
Common antioxidants include the vitamins A, C, and E, glutathione, and the enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. For example, the pancreatic autoantigen glutamic acid decarboxylase is an enzyme responsible for synthesis of the neurotransmitter gamma-aminobutyric acid (GABA) in the peripheral and central nervous system.
Other antioxidants include lipoic acid, mixed carotenoids, coenzyme Q10, several bioflavonoids, antioxidant minerals (copper, zinc, manganese, and selenium), and the cofactors (folic acid; vitamins B1, B2, B6 and B12). Notably, at least two neurotransmitter-related peptides have been used successfully as plasmid DNA immunotherapies for type 1 diabetes. The first peptide is calcitonin gene-related peptide (CGRP), which has been found to play an important role in the regulation of T lymphocytes and in protecting cells from reactive oxygen species.
It was found that a single injection of 200 microgram plasmid DNA encoding the peptide delivered intramuscularly using electroporation could significantly ameliorate hyperglycemia and insulin deficiency [53]. Vitamins A, C, and E are interesting antioxidant molecules because are diet-derived and directly detoxify free radicals. The treatment decreased diabetes incidence from 73% in controls to 23% at 28 days post delivery in a streptozotocin-induced diabetic model. The gene transfer also significantly inhibited T cell proliferation and secretion of the T helper-1 cytokine interferon-gamma, increased the levels of the T helper-2 cytokine interleukin-10, but had no effect on interleukin-4 and transforming growth factor-beta secretion. Tocopherol is reconstituted when ascorbic acid recycles the tocopherol radical; dihydroascorbic acid, which is generated, is recycled by glutathione.
Therefore, calcitonin gene-related peptide gene transfer appears to ameliorate streptozotocin-induced diabetes through immune deviation.The second peptide is named vasoactive intestinal peptide (VIP) and functions as a neuromodulator and neurotransmitter [54]. The peptide is a potent vasodilator that regulates smooth muscle activity, epithelial cell secretion, and blood flow in the gastrointestinal tract. Vitamin E, a component of the total peroxyl radical-trapping antioxidant system, reacts directly with peroxyl and superoxide radicals and singlet oxygen and protects membranes from lipid peroxidation (Weber et al., 1997). Importantly, a body of evidence points to a significant role of vasoactive intestinal polypeptide in regulating immune responses. A deficiency of vitamin E is concurrent with increased peroxides and aldehydes in many tissues. The peptide acts as a potent endogenous anti-inflammatory molecule and promotes the activity of T regulatory cells, which makes it a promising candidate for the treatments of inflammatory and autoimmune diseases, such as septic shock, arthritis, multiple sclerosis, Crohn disease, and autoimmune diabetes [55, 56]. There have been conflicting reports about vitamin E levels in diabetic animals and human subjects.
For example, a single intramuscular injection of 300 microgram of vasoactive intestinal polypeptide-encoding plasmid DNA significantly reduced the incidence of cyclophosphamide accelerated diabetes in female nonobese diabetic mice, from 70% in control to 30% on day 33 post delivery in 8-10-week-old mice [57]. Some attention to AGE inhibitors was focused on aminoguanidine, which blocks electrophilically activated 1,3-dicarbonyl compounds, the precursors of AGEs (Thomas et al. Summary of Section 2Section 1 covers plasmid DNA encoding small protein molecules like cytokines, chemokines, peptides and other immune cell-manipulating agents with therapeutic effects on preclinical type 1 diabetes (Table 1). These approaches belong to systemic treatments and inevitably bear the risks associated with nonspecific immune suppression and chronic complications resulting from interference with the host immune system. This compound was not approved by the US Food and Drug Administration due to adverse side effects in diabetic patients during Phase III clinical trials, and the search to find alternatives continues. Nonetheless, if used as adjuvants or supplements to pancreatic autoantigen-targeting therapies like DNA vaccines, these approaches could be used selectively in DNA-based combination therapies.
Furthermore, these compounds are also good metal ion chelators and attenuate oxidative stress. N-acetylcysteine and lipoic acid act as inhibitors through attenuation of oxidative stress. While the AGE-inhibitory effect of these compounds is not clearly understood, a similar mechanism may operate in the case of AGEs. Carnosine, homocarnosine, and related compounds are potentially suitable as AGE inhibitors although further studies are needed to prove their efficacy in diabetes and AD (Reddy et al., 2005). Carnosine protects superoxide dismutase, catalase, and ?-crystallin from non-enzymatic glycation and protein crosslinking (Hipkiss, 2007). OPB-9195 inhibits AGE formation, especially pentosidine and N?-(carboxymethyl)lysine (CML), apparently through carbonyl trapping and metal ion chelation (Wada et al., 2001). Thiazolium-based compounds such as alagebrium chloride (ALT-711) and N-phenyacyl-1,3-thiazolium bromide (PTB) are effective AGE crosslink breakers, and are potentially useful drugs for diabetes and AD (Susic, 2007). It should, however, be pointed out that the mechanisms of the action of the latter compounds are not clearly understood. In addition to their action as crosslink breakers of AGEs derived from 1,2-dicarbonyl compounds, they may also act as antioxidants through chelation of transition metal ions. The development of these drugs as therapeutics thus depends on the detailed understanding of their mechanisms of action. An alternative strategy involves removal of AGEs through the soluble receptors for AGEs (sRAGEs). Poor glycemic control in diabetes results in decreased concentrations of sRAGES, and upon insulin treatment, significant improvements in the levels of sRAGEs were observed, with concomitant decrease in AGEs (Devangelio et al., 2007). Treatment of diabetic patients with rosiglitazone, a 2,4-thiazolidine dione derivative, results in increase of plasma sRAGEs, comparable to controls (Tan et al., 2007). However, the protective effect of sRAGEs has been questioned recently as their level are much higher in experimental animal models than those found in vivo, suggesting they may be only markers of inflammation (Humpert et al., 2007). Following the trend in using natural antioxidants, a recent paper has examined the effects of banana (Musa sp. The results indicate that fructosamine and AGEs formed during diabetes were inhibited in treated groups when compared with the diabetic group (Bhaskar et al., 2011). However, the results of clinical trials of antioxidant therapy are not clear because of duration of treatment as well as recruitment of patients at different stages of the diseases. In spite of inconsistency in the conclusions of clinical trials on the beneficial effects of antioxidants on these pathologies, epidemiological studies indicate that antioxidants may reduce the risk of their insurgence. It is suggested that a combination of antioxidants might be of greater potential benefit, especially if these agents work in different cellular compartments or have complementary activity (e.g.
If oxidative stress plays as important a role in AD and diabetes pathologies as the literature suggests, regular intake of antioxidants may be beneficial much before any sign or symptoms of the disease are visible. Insulin resistance, Tau hyperphosphorylation and the amyloid cascadeIn addition to being a modulator of food intake and energy homoeostasis, insulin is also an important neurothrophic factor. It modulates brain activity, particularly for such high glucose demanding functions such as memory. This form of diabetes is characterized by insulin resistance, hyperinsulinemia and impaired insulin signaling. Insulin resistance is the common link of the components of the much invoked metabolic syndrome (a cluster of high adiposity, abnormal glucose level, dyslipidemia, hypertension and high inflammation) and it is known to cause common diseases such as stroke, heart disease, and cancer. Given the aging of the population and the epidemic of elevated insulin resistance, evidenced by the rise in elevated adiposity, prediabetes, and diabetes, it is alarming that insulin resistance could contribute to AD.
Many epidemiologic studies have found an association of elevated adiposity, insulin resistance, and T2D with cognitive impairment and dementia (Baker et al., 2011). However, there are several important questions to be addressed for investigators studying the relation of insulin resistance and AD. Insulin receptors are expressed throughout the CNS, expecially in the hippocampus and cortex, even if their function in the brain is not fully understood. Binding of insulin to its receptor activates the intrinsic tyrosine kinase activity of the cytoplasmic domain of the insulin receptor. This leads to autophosphorylation of tyrosine residues, which initiates several intracellular signaling cascades. In the brain, insulin influences the release and reuptake of neurotransmitters, and also appears to improve learning and memory (Zhao et al., 2004). The initial components of the insulin receptor signaling cascade in the brain are largely similar to those of the periphery. The downstream targets of the cascade are quite different, however, probably involving, among others, neuronal glutamate receptors (Zhao et al., 2004).
Neurodegeneration and cognitive impairment in T2D and AD could be caused, in part, by damage to insulin receptor signaling (de la Monte & Wands, 2005). In fact, decreases in the sensitivity of such receptors are known to affect the expression and metabolism of Abeta and tau and impaired insulin receptor activity and hyperinsulinemia are observed in patients with AD and in animal models of this disease (Frolic et al., 1998). In addition, dysfunction of insulin receptor signaling is associated with impairments in Abeta oligomer clearance (Zhao, 2009) and increases the rate of NFT development (Lesort & Johnson, 2000). In fact, insulin transiently increases tau phosphorylation in primary cortical neurons, and hyperinsulinemia results in tau hyperphosphorylation in rat brains. Furthermore, insulin receptor substrate 2 knockout mice demonstrate typical pathological signs of T2D and have an increased number of NFTs in hippocampal neurons compared with control wild-type mice (Schubert, 2003). Insulin receptor signaling leads to the activation of two major signaling pathways, the mitogen-activated protein kinase (MAPK) pathway and the Akt signaling pathway. Thus, under normal conditions, insulin signaling via the insulin receptor leads to GSK-3? inactivation, whereas insulin resistance leads to GSK-3? dephosphorylation and activation (Balaraman et al., 2006). This enzyme is a metalloprotease responsible for insulin degradation and is also the main enzyme responsible for Abeta degradation (Farris et al., 2003).
IDE is secreted to the extracellular space by microglial cells in the brain, where it degrades Abeta peptide, thus reducing the rate of aggregation and the plaque formation (Qiu et al., 1998). IDE levels have been reported to be decreased in the brains of AD patients (Cook et al., 2003).
It has also been hypothesized that hyperinsulinemia in people with pre-diabetes and T2D effectively sequesters IDE, reducing Abeta peptide degradation. This would increase levels of Abeta, and promote many of the pathological features associated with Alzheimer's disease.
Supporting this model, the affinity for the binding of insulin to IDE is much greater than that for the Abeta (Qiuet al., 1997).
In patients with Alzheimer's disease, IDE expression in the hippocampus is substantially reduced, with regards to controls, in particular among patients with the APOEvar epsilon4 genotype. This latter observation could explain the potential interaction between diabetes and the APOEvar epsilon4 genotype in multiplying the risk of dementia (Cook et al., 2003).
Curiously, although the presence of the APOEvar epsilon4 is associated with an increased incidence of Alzheimer's disease, it seems that insulin resistance is only a significant risk factor for AD in those patients without APOEvar epsilon4 (Craft et al., 1998). This suggests that T2D could affect the pathogenesis of AD through mechanisms other than modulation of Abeta metabolism even if the underlying mechanisms for this association remain largely unknown (Takeda et al., 2011). FOXO: A common biomarker for AD and T2DThere is ongoing interest in defining mechanisms that govern insulin resistance and AD. In particular, in presence of insulin, activated Akt translocates to the nucleus where directly phosphorylates FOXO at distinct sites stimulating interaction with 14-3-3 protein (Greer and Brunet, 2005). This chaperone protein promotes the nuclear export and inhibits the nuclear import of FOXO proteins, driving the cells towards cell survival (van der Heide et al., 2004). In contrast, FOXO proteins, under conditions of oxidative stress, are phosphorylated by other protein kinases, including Mst1 and JNK, able to disrupt its interaction with 14-3-3, promoting FOXO nuclear translocation and thereby inducing cell death in neurons, thus opposing Akt’s action (Sunayama et al., 2005). Thus, it is well established that Akt plays a key role in repressing FOXO transcriptional activity. Immediately upstream from FOXO, the activity of Akt itself is governed by several protein kinases and phosphatases. Akt regulates a variety of key physiological functions, and there is strong evidence suggesting that defective Akt signaling contributes to development of insulin resistance (Zdychova & Komers, 2005). However, although it is clear that FOXO governs multiple events in the insulin signaling cascade, mediating both positive and negative effects, the underlying molecular mechanisms are unknown. Some evidence has been reported that FOXO3 activation is also able to increase basal levels of Akt phosphorylation and kinase activity thus it is capable to activating its own inhibitor, providing a feedback regulation (Ni et al., 2007).
Moreover, FOXO transcription factors are involved in both the insulin action and the cellular response to oxidative stress, thereby providing a potential integrative link between AD and insulin resistence (Manolopoulos et al., 2010). Both insulin resistence and oxidative stress due to Abeta stimulus, may promote the transcriptional activity of FOXO proteins, resulting in hyperglycaemia and a further increased production of ROS.
The consecutive activation of c-Jun N-terminal kinases and inhibition of Wingless (Wnt) signalling may result in the formation of Abeta plaques and tau protein phosphorylation.
Wnt inhibition may also result in a sustained activation of FOXO proteins with induction of apoptosis and neuronal loss, thereby completing a vicious circle from oxidative stress, insulin resistence and hyperglycaemia back to the formation of ROS and consecutive neurodegeneration. Thus, it has been proposed that FOXO proteins may provide a potential molecular target for the treatment of both insulin resistence and AD (Manolopoulos et al., 2010).

Can diabetes cause leg cramps zoloft
R en s zinnen chemische stoffen

Comments to «Type 2 diabetes mellitus statistics in the philippines zip»

  1. writes:
    Vitamin E supplementation on renal non-enzymatic day, whether.

  2. writes:
    Says that while a balanced low-carb diet with weren't a factor in the research because everybody seen excellent.