Establishment of a Novel Patient hiPSC-derived in vitro Blood-Brain Barrier Model of Collagen IV Small Vessel Disease

Cerebral small vessel disease (SVD) is a prevalent cause of stroke (25-30%) and dementia (45%). Despite its frequency, little is known about the cause and disease progression of SVD. Matrisome alteration leading to blood-brain barrier (BBB) dysfunction is thought to play a role. Sporadic cases are exacerbated by age and vascular risk factors, predominantly affecting the elderly. However, mutations in Collagen IV α1/α2 (COL4A1/2), a highly abundant matrisome protein, cause monogenic SVD, which shares many clinical features with the sporadic form. In order to better understand the pathomolecular mechanisms leading to SVD, human induced pluripotent stem cells (hiPSC) generated from patients with mutations in COL4A1/2 genes (COL4A1G755R and COL4A2G702D), isogenic controls and multiple wild-type (WT) control lines were used to establish a novel in vitro BBB model of Collagen IV SVD. Encompassing hiPSC-derived brain microvascular endothelial cells (BMEC), mural cells (MC) and astrocytes, the model was used to probe the phenotype of COL4A1/2 SVD. Differentiation of hiPSC into BMEC was extensively optimised to overcome high variability between different hiPSC lines. Once established, hiPSC-BMEC were tested with functional assays including transendothelial electrical resistance (TEER), permeability assays (4kDa FITC-Dextran and Sodium Fluorescein), LDL-uptake and tube formation. hiPSC-BMEC were then combined with hiPSC-MC and hiPSC-astrocytes into a Transwell® co-culture model. WT hiPSC-derived BMEC exhibit high TEER of ~3000-4000Ωxcm2, which is increased and maintained over two weeks in co-culture with astrocytes. Limited, short-term increase in TEER is seen in co-culture with MC. Aspects of the COL4A1/2 SVD disease phenotype were reproduced in vitro. COL4A2G702D BMEC exhibit lower tight junction protein expression; while COL4A1G755R BMEC demonstrate discontinuous tight junctions. COL4A2G702D BMEC also show defective P-glycoprotein (P-gp)-mediated Rhodamine123 efflux compared to the isogenic control, which is suggestive of a transport deficiency across the barrier. COL4A1G755R and COL4A2G702D BMEC and MC have increased levels of MMPs. Notably, these findings replicate what is seen in patients, strengthening the hypothesis that matrisome alterations are important in SVD.Rosetrees Trust, British Heart Foundation, Alzheimer’s Association and Stroke Association

A dermal sludge for targeted genetic auto-inflammatory skin disorders

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Pharmacy. 2016Genetic auto-inflammatory inflammatory skin disorders (GAISDs) are a group of inherited disorders which are characterized by seemingly unprovoked recurrent episodes of fever and severe localised inflammation. GAISDs are associated with abnormal activation of the innate immune system, leading to clinical inflammation and high levels of acute-phase reactants. The most common disorder is Familial Mediterranean Fever (FMF), followed by Tumor Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS). TRAPS episodes generally last longer than FMF and FMF patients tend to respond well with colchicine while TRAPS management seems to be challenging. Hence this work is directed towards improving TRAPS diseases management. A definitive treatment for TRAPS has yet to be identified, and current corticosteroid treatment is mainly limited by the long-term side-effects due to high systemic drug exposure, and the poor availability of drugs at the site of action. A number of measures were taken in order to overcome the limitations of corticosteroids.Herein a novel stimuli responsive nanocolloidal gel system was developed. A nanoliposomal gel was the stimuli responsive gel system of choice due to its advantages of skin penetration enhancement in transdermal drug delivery system. In this research, a phospholipid based system with Eudragit® E100 (EuE100) chemically modified into EuE100-cystamine derivative for dual pH/redox responsive delivery of [Copper-glycylglycine-prednisolone succinate] ([Cu(glygly)(PS)]) was developed. The rationale of using [Cu(glygly)(PS)] complex instead of the pure PS corticosteroid was supported by comparing the biological activities of these two compounds. Results indicated a high inflammatory/oxidant inhibitory activity of [Cu(glygly)(PS)] in comparison to the free PS drug. The [Cu(glygly)(PS)] complex exhibited a significant free radical-scavenging activity (60.1±1.2%) and lipoxygenase (LOX-5) inhibitory activity (36.6±1.3%) in comparison to PS which gave activity of 4.4±1.4% and inhibition of 6.1±2.6% respectively. The [Cu(glygly)(PS)] loaded NLs showed a low level of [Cu(glygly)(PS)] release of 22.9±5.4% in 6h at pH 7.4, in comparison to a significant accelerated release at pH 5 in a reducing environment of 75.9±3.7%in 6h. Thereafter optimized [Cu(glygly)(PS)]-loaded NLs were dispersed in hydroxypropyl methylcellulose (HPMC)/Polyvinyl alcohol(PVA) gel resulting in a [Cu(glygly)(PS)]-loaded nanoliposomal gel termed asdermal sludge.A dermal sludge is defined as a viscous gel suspended with solid particles ([Cu(glygly)(PS)]-loaded nanoliposomes). The sludge was characterized using ex vivo permeation, in vitro release, cytotoxicity and in vivo studies, and compared to the conventional PS formulations. The results indicated that the novel dual redox/pH responsive nanoliposomal dermal sludge holds great potential for targeted bioactive delivery in TRAPS through the transdermal route, hence improving the therapeutic outcome.MT201

Impact of metabolic disorders on the structure, function, and immunological integrity of the blood-brain barrier.

PhD ThesisMetabolic disorders induce a low-grade chronic inflammatory state that is damaging to the peripheral vasculature, leading to cardiovascular diseases. In recent years, there has been mounting evidence linking metabolic disease to neurovascular disorders and cognitive decline. However, the pathophysiological response occurring at the brain microvasculature, the blood-brain barrier (BBB), remains largely unexplored. Using a mouse model of Type II diabetes mellitus, the results of this project demonstrate pro-inflammatory mediators and altered immune responses to impair the protective BBB functionality. Disruption to tight junctions and basal lamina, due to loss of control in activation/production of MMPs and their inhibitors, causes complete BBB breakdown. Metabolic stress triggers endothelial dysfunction resulting in altered metabolism and receptor expression, enhanced ROS production and up-regulated adhesion molecules. Immunologically, there is an increase in the percentage of activated effector and central memory T-cells along with an imbalance in the TH17/Treg subsets. Together these factors augment transmigration of leukocytes across the BBB to trigger activation and shift of microglia to a pro-inflammatory phenotype, initiating neuroinflammation. Loss of the physical, transport and metabolic properties of the BBB will consequently have detrimental effects on neuronal function. Additionally, this thesis explores the use of a pharmacological and dietary intervention to reduce and restore the damage incurred to the BBB. Treatment with human recombinant annexin A1 (a well know anti-inflammatory molecule) and reversion from a high-fat high-sugar diet to a chow diet both attenuate T2DM development, reduce peripheral inflammation and dampen the immune response, resulting in restored BBB integrity. Both interventions confer neuroprotection through stabilisation of cell-cell contacts, consequently limiting leukocyte extravasation and downstream pathological events. This thesis emphasises the growing understanding of long-term impact of metabolic disease and their contribution to the development of neuroinflammatory/ neurodegenerative disorders. Preventive and therapeutic strategies that mediate anti-inflammatory effects are key in limiting the disease progression

Embryonic Stem Cells

Embryonic stem cells are one of the key building blocks of the emerging multidisciplinary field of regenerative medicine, and discoveries and new technology related to embryonic stem cells are being made at an ever increasing rate. This book provides a snapshot of some of the research occurring across a wide range of areas related to embryonic stem cells, including new methods, tools and technologies; new understandings about the molecular biology and pluripotency of these cells; as well as new uses for and sources of embryonic stem cells. The book will serve as a valuable resource for engineers, scientists, and clinicians as well as students in a wide range of disciplines

Drug development progress in duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe, progressive, and incurable X-linked disorder caused by mutations in the dystrophin gene. Patients with DMD have an absence of functional dystrophin protein, which results in chronic damage of muscle fibers during contraction, thus leading to deterioration of muscle quality and loss of muscle mass over time. Although there is currently no cure for DMD, improvements in treatment care and management could delay disease progression and improve quality of life, thereby prolonging life expectancy for these patients. Furthermore, active research efforts are ongoing to develop therapeutic strategies that target dystrophin deficiency, such as gene replacement therapies, exon skipping, and readthrough therapy, as well as strategies that target secondary pathology of DMD, such as novel anti-inflammatory compounds, myostatin inhibitors, and cardioprotective compounds. Furthermore, longitudinal modeling approaches have been used to characterize the progression of MRI and functional endpoints for predictive purposes to inform Go/No Go decisions in drug development. This review showcases approved drugs or drug candidates along their development paths and also provides information on primary endpoints and enrollment size of Ph2/3 and Ph3 trials in the DMD space

Risk factors and Blood borne- biochemical markers in type 2 diabetes mellitus

The burden of Diabetes Mellitus (DM) is increasing worldwide and it is estimated to reach indefinite proportions of about 450 million by year 2030. Patients with type 2 diabetes mellitus (T2DM) have a significantly increased risk of developing cardiovascular diseases (CVD). Moreover, CVD is the major cause of mortality and morbidity (75%) in T2DM patients. DM itself has been long recognised as an independent risk factor for several forms of CVD including coronary heart disease (CHD), peripheral arterial disease, cardiomyopathy and congestive heart failure in both men and women. It is well-known that T2DM is associated with several factors including hyperglycaemia, hypertension, dyslipidemia, obesity all of which contribute to CVD. In order to prevent CVD, early intervention on cardiovascular risk factors is vital during clinical assessment of T2DM patients. A major role of inflammation has been well described in the development of CVD in T2DM patients. Inflammatory process and factors which contribute to CVD in T2DM patients have recently become a focus in diabetic research. Elucidation of common patho-physiological mechanisms among T2DM patients might emphasize the role of inflammation in CVD. The main purpose of this study was to investigate any patho-physiological changes in red blood cells (RBC), white blood cells (neutrophils and lymphocytes) and plasma, measuring RBC membrane fragility and proteins, intracellular free calcium concentrations [Ca2+]i and several cations including Na+, Mg2+, Ca2+, Fe2+, Zn2+ and Cu2+, biochemical parameters and inflammatory mediators which normally serve as independent predisposing risk factors for CVD among T2DM patients compared to age-match healthy controls. The results have shown that fura-2 loaded neutrophils and lymphocytes in blood from T2DM patients contain significantly (p<0.05) less [Ca2+]i than neutrophils and lymphocytes from healthy subjects upon stimulation with physiological doses of either fMLP or thapsigargin indicating a derangement in cellular calcium homeostasis during T2DM. Similarly, RBC membranes from T2DM patients contained significantly (p<0.05) more spectrin, ankyrin, band 3, band 4.1, glycophorin etc compared to RBC membranes from age-matched healthy control subjects. The results also show that the RBCs from T2DM patients were more fragile compared to RBC from healthy controls. Measurement of protein glycation in plasma have revealed significantly (p<0.05) more fluorescence in proteins form T2DM patients compared to control. In relation to plasma cations and intracellular markers and mediators, the results show that plasma from T2DM patients contain significantly (p<0.05) more Na+, Mg2+ , Ca2+, Fe2+, Zn2+ and Cu2+ compared to plasma levels from age-match healthy controls. Similarly, the concentrations of kidney and liver function markers such as urea, creatinine, alkaline phosphatase, ALT, AST, GGT, total protein and albumin increased significantly (p<0.05) compared to healthy controls. The same is also true for glucose, total cholesterol, triglycerides, CRP, HBA1C, WBC where the blood from T2DM patients contained elevated concentrations compared to blood from healthy age-matched control patients. Together, the results of this study have clearly demonstrated marked and significant changes in cellular calcium homeostasis in white blood cells, RBC membrane proteins and fragility, plasma protein glycation and in plasma levels of cations, intracellular markers and mediators of T2DM patients compared to healthy controls. Therefore, it is proposed that an early integrated and multi-factorial intervention of risk factors and inflammatory markers must be done in order to reduce the risk of CVD and possible mortality of T2DM patients

Abstracts from the 50th European Society of Human Genetics Conference: Posters

International audienc

The Gastrointestinal Microbiota and Immunity with Treatment in Paediatric Inflammatory Bowel Disease

Intestinal microbiota is strongly implicated in IBD pathogenesis. These microbes exert their influence directly and indirectly through their metabolites which include short chain fatty acids (SCFA). These interactions can be mutually beneficial but can also be detrimental to the host. Combined with a dysregulated immune system, IBD manifests itself in genetically susceptible individuals. These 3 interrelated factors, intestinal microbiota, SCFA and host immunity, were investigated to explore the extent of their associations. Intestinal luminal (stool and duodenal lavages) and mucosal biopsies were collected prospectively from treatment-naive IBD patients, some before diagnosis. Their clinical histories, results and dietetic assessment were also analysed. Dynamic changes in the intestinal microbial profiles with inflammation and treatment were compared to healthy and non-inflammatory control children. The corresponding alterations in stool SCFA, plasma cytokine and vascular profiles were identified. Dysbiosis was characterised at phyla level, and disease-specific microbial profiles in Crohn’s Disease (CD) and Ulcerative colitis (UC) were recorded. The relative abundances of the 3 main phyla (Bacteroidetes, Firmicutes and Proteobacteria) varied with disease activity. Proteobacteria abundance increased with inflammation. Distinct differences in SCFA profiles were observed between control groups and IBD, with distinguishing features evident between CD, UC and ileostomy samples. Inflammation appears to influence SCFA production, particularly butyrate. In addition, pro-inflammatory cytokines and vascular markers mirrored trends in clinical inflammatory markers, and some correlated with SCFA percentages. In conclusion, alterations in intestinal microbial and SCFA profiles appear to influence the systemic cytokine milieu. Larger cohort studies are needed to confirm and further explore these findings if we are to utilise microbial and immune profiles in our quest for personalised treatment of children with IBD