Autoimmune responses in T1DM : quantitative methods to understand onset, progression, and prevention of disease

Understanding the physiological processes that underlie autoimmune disorders and identifying biomarkers to predict their onset are two pressing issues that need to be thoroughly sorted out by careful thought when analyzing these diseases. Type 1 diabetes ( T1D ) is a typical example of such diseases. It is mediated by autoreactive cytotoxic CD4 + and CD8 + T‐cells that infiltrate the pancreatic islets of Langerhans and destroy insulin‐secreting β‐cells, leading to abnormal levels of glucose in affected individuals. The disease is also associated with a series of islet‐specific autoantibodies that appear in high‐risk subjects ( HRS ) several years prior to the onset of diabetes‐related symptoms. It has been suggested that T1D is relapsing‐remitting in nature and that islet‐specific autoantibodies released by lymphocytic B‐cells are detectable at different stages of the disease, depending on their binding affinity (the higher, the earlier they appear). The multifaceted nature of this disease and its intrinsic complexity make this disease very difficult to analyze experimentally as a whole. The use of quantitative methods, in the form of mathematical models and computational tools, to examine the disease has been a very powerful tool in providing predictions and insights about the underlying mechanism(s) regulating its onset and development. Furthermore, the models developed may have prognostic implications by aiding in the enrollment of HRS into trials for T1D prevention. In this review, we summarize recent advances made in determining T‐ and B‐cell involvement in T1D using these quantitative approaches and delineate areas where mathematical modeling can make further contributions in unraveling certain aspect of this disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106988/1/pedi12148.pd

Investigating the Role of T-Cell Avidity and Killing Efficacy in Relation to Type 1 Diabetes Prediction

During the progression of the clinical onset of Type 1 Diabetes (T1D), high-risk individuals exhibit multiple islet autoantibodies and high-avidity T cells which progressively destroy beta cells causing overt T1D. In particular, novel autoantibodies, such as those against IA-2 epitopes (aa1-577), had a predictive rate of 100% in a 10-year follow up (rapid progressors), unlike conventional autoantibodies that required 15 years of follow up for a 74% predictive rate (slow progressors). The discrepancy between these two groups is thought to be associated with T-cell avidity, including CD8 and/or CD4 T cells. For this purpose, we build a series of mathematical models incorporating first one clone then multiple clones of islet-specific and pathogenic CD8 and/or CD4 T cells, together with B lymphocytes, to investigate the interaction of T-cell avidity with autoantibodies in predicting disease onset. These models are instrumental in examining several experimental observations associated with T-cell avidity, including the phenomenon of avidity maturation (increased average T-cell avidity over time), based on intra- and cross-clonal competition between T cells in high-risk human subjects. The model shows that the level and persistence of autoantibodies depends not only on the avidity of T cells, but also on the killing efficacy of these cells. Quantification and modeling of autoreactive T-cell avidities can thus determine the level of risk associated with each type of autoantibodies and the timing of T1D disease onset in individuals that have been tested positive for these autoantibodies. Such studies may lead to early diagnosis of the disease in high-risk individuals and thus potentially serve as a means of staging patients for clinical trials of preventive or interventional therapies far before disease onset

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Autoimmune Atrial Fibrillation

BACKGROUND Atrial fibrillation (AF) is by far the most common cardiac arrhythmia. In about 3% of individuals, AF develops as a primary disorder without any identifiable trigger (idiopathic or historically termed lone AF). In line with the emerging field of autoantibody-related cardiac arrhythmias, the objective of this study was to explore whether autoantibodies targeting cardiac ion channels can underlie unexplained AF. METHODS Peptide microarray was used to screen patient samples for autoantibodies. We compared patients with unexplained AF (n=37 pre-existent AF; n=14 incident AF on follow-up) to age- and sex-matched controls (n=37). Electrophysiological properties of the identified autoantibody were then tested in vitro with the patch clamp technique and in vivo with an experimental mouse model of immunization. RESULTS A common autoantibody response against K$_{ir}$3.4 protein was detected in patients with AF and even before the development of clinically apparent AF. K$_{ir}$3.4 protein forms a heterotetramer that underlies the cardiac acetylcholine-activated inwardly rectifying K$^{+}$ current, I$_{KACh}$. Functional studies on human induced pluripotent stem cell-derived atrial cardiomyocytes showed that anti-K$_{ir}$3.4 IgG purified from patients with AF shortened action potentials and enhanced the constitutive form of I$_{KACh}$, both key mediators of AF. To establish a causal relationship, we developed a mouse model of K$_{ir}$3.4 autoimmunity. Electrophysiological study in K$_{ir}$3.4-immunized mice showed that K$_{ir}$3.4 autoantibodies significantly reduced atrial effective refractory period and predisposed animals to a 2.8-fold increased susceptibility to AF. CONCLUSIONS To our knowledge, this is the first report of an autoimmune pathogenesis of AF with direct evidence of K$_{ir}$3.4 autoantibody-mediated AF

The role of intra-islet glucagon signalling in total body glucose homeostasis

Glucagon is best known for opposing the actions of insulin, to increase blood glucose levels through glycogenolysis and gluconeogenesis in response to hypoglycaemia. However, glucagon also increases energy expenditure and suppresses appetite. These pleiotropic effects of glucagon have been harnessed to develop novel agents to treat diabetes and obesity. Drugs which contain a glucagon receptor agonist element have been counterintuitively shown to improve glucose homeostasis. It remains poorly understood how glucagon acts at the level of the pancreatic islet to modulate insulin secretion. This work aims to investigate the weight-loss-independent actions of a long acting glucagon analogue, G778, on islet insulin secretory function. Utilising a novel imaging platform, longitudinally visualising pancreatic islets implanted in the anterior chamber of the murine eye, I have demonstrated that chronic treatment of diet-induced obese and diabetic mice with a glucagon analogue induced superior improvement in islet secretory function compared with the equivalent weight loss achieved through dietary restriction alone. To further investigate the effects of glucagon action in the islet, I developed a mouse model with deletion of the glucagon receptor (GCGR) restricted to pancreatic β-cells. The phenotype of this mouse reveals a defective insulin secretory phenotype, which was confirmed to be a functional deficit and not related to changes in islet development. As part of this thesis I have also developed the novel technique of hyperglycaemic clamps in freely moving mice and the final section of this thesis is dedicated to the method development of this technical challenge. In summary, this thesis provides evidence for the role of glucagon signalling in the enhancement of insulin secretory function and adds to evidence for the use of glucagonergic agents to treat Type 2 diabetes.Open Acces

Neurosteroids as regulators of neuroinflammation

Neuroinflammation is a physiological protective response in the context of infection and injury. However, neuroinflammation, especially if chronic, may also drive neurodegeneration. Neurodegenerative diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD) and traumatic brain injury (TBI), display inflammatory activation of microglia and astrocytes. Intriguingly, the central nervous system (CNS) is a highly steroidogenic environment synthesizing steroids de novo, as well as metabolizing steroids deriving from the circulation. Neurosteroid synthesis can be substantially affected by neuroinflammation, while, in turn, several steroids, such as 17β-estradiol, dehydroepiandrosterone (DHEA) and allopregnanolone, can regulate neuroinflammatory responses. Here, we review the role of neurosteroids in neuroinflammation in the context of MS, AD, PD and TBI and describe underlying molecular mechanisms. Moreover, we introduce the concept that synthetic neurosteroid analogues could be potentially utilized for the treatment of neurodegenerative diseases in the future

Are physiological oscillations 'physiological'?

Despite widespread and striking examples of physiological oscillations, their functional role is often unclear. Even glycolysis, the paradigm example of oscillatory biochemistry, has seen questions about its oscillatory function. Here, we take a systems approach to summarize evidence that oscillations play critical physiological roles. Oscillatory behavior enables systems to avoid desensitization, to avoid chronically high and therefore toxic levels of chemicals, and to become more resistant to noise. Oscillation also enables complex physiological systems to reconcile incompatible conditions such as oxidation and reduction, by cycling between them, and to synchronize the oscillations of many small units into one large effect. In pancreatic beta cells, glycolytic oscillations are in synchrony with calcium and mitochondrial oscillations to drive pulsatile insulin release, which is pivotal for the liver to regulate blood glucose dynamics. In addition, oscillation can keep biological time, essential for embryonic development in promoting cell diversity and pattern formation. The functional importance of oscillatory processes requires a rethinking of the traditional doctrine of homeostasis, holding that physiological quantities are maintained at constant equilibrium values, a view that has largely failed us in the clinic. A more dynamic approach will enable us to view health and disease through a new light and initiate a paradigm shift in treating diseases, including depression and cancer. This modern synthesis also takes a deeper look into the mechanisms that create, sustain and abolish oscillatory processes, which requires the language of nonlinear dynamics, well beyond the linearization techniques of equilibrium control theory

Longitudinal imaging of pancreatic islets transplanted into the anterior chamber of the eye

Diabetes is a growing health problem associated with substantial health and socioeconomic costs. Current medications address hyperglycaemia and related complications, but a definitive cure for diabetes remains elusive. Pharmacotherapy with the potential to restore β-cell function is urgently needed. To this end, it is vital that we achieve a better understanding of islet function in both health and disease states. While major breakthroughs in diabetes research have traditionally resulted from in vitro experimentation, the crucial role of the pancreatic internal milieu is being increasingly recognised. The islet in the eye imaging platform is the first experimental tool which allows for the longitudinal and direct investigation of β-cell function in a non-invasive manner. This thesis describes the application of this imaging platform to three divergent areas of islet research. Firstly the physiology of co-ordinated insulin secretion is examined. Pulsatile insulin secretion is physiologically relevant and is impaired in diabetes. The imaging platform is used to establish, for the first time in vivo, that the calcium waves that underlie insulin secretion arise from the co-ordinated activity of a heterogeneous group of β-cells. Obesity is the greatest risk factor for developing Type 2 diabetes. The effects of high fat diet on islet calcium dynamics are incompletely understood. Hyperglucagonaemia is traditionally thought of as a contributing factor to diabetes disease progression. Emerging evidence however suggests that glucagon signalling has beneficial effects on food intake and energy expenditure. More recently, the insulin potentiating effects of intra-islet glucagon has been suggested. The imaging platform is developed to investigate the longitudinal effects of high fat diet and the subsequent weight-loss independent effects of a synthetic glucagon analogue on islet function. Together, these two studies investigate the utility of the islet in the eye imaging platform to better our understanding of intercellular β-cell calcium dynamics in the acute and in a more chronic setting. Islet transplantation has not provided a reliable cure for patients with Type 1 diabetes, due to a relative lack of suitable donors but, more importantly, because the majority of patients fail to achieve long term insulin independence. The reasons for transplant failure are manifold and poorly 9 understood, although engraftment failure is a major issue. There is a clear need to improve transplant success rates, especially if this can be achieved in line with a more reliable supply of β-cells/islets (for example stem-cell derived therapies). The last experimental chapter aims to investigate the effects of epidermal growth factor receptor (EGFR) overexpression in β-cells and whether this treatment improves islet engraftment. In particular, this chapter focuses on the angiogenesis of newly transplanted islets and whether the islet in eye platform is capable of longitudinally monitoring this process.Open Acces