Austin G. Milton

Ultrastructural analysis, zinc transporters, glucose transporters and hormones expression in New world primate (Callithrix jacchus) and human pancreatic islets.

The New world primates (NWP) Callithrix jacchus separated from man approximately 50 million years ago and is a potential alternative small non-human primate model for diabetes research. Ultrastructure, and gene expression of pancreatic islets and the recently described diabetes auto antigenic zinc transporters families in human, NWP and pig pancreas were studied. Morphologically NWP islets were larger than pig islets and similar in size to human islets. NWP islets alpha cells had high dense core surrounded by a limiting membrane, beta cells by the mixed morphology of the granule core, and delta cells by moderate opaque core. Antibody staining for insulin, glucagon, somatostatin and Glucagon-like peptide-1 (GLP-1) showed that the distribution pattern of the different cell types within islets was comparable to pig and human islets. In all three species protein expression of zinc transporter ZnT8 was detected in most of the insulin producing beta cells whereas Zip14 expression was widely expressed in alpha and beta cells. In both human and NWP little or no expression of Glut2 was observed compared to Glut1 and glucokinase at the protein level, however the messenger RNA level of Glut2 was greater than Glut1 and glucokinase. In contrast all three glucose transporters were expressed in pig islets at the protein level. The expression of Zip14 in islets is reported for the first time. In conclusion NWP pancreatic islets express comparable islet cell types and distribution to humans and pigs. Importantly, marmosets have a similar glucose transporter profile to humans, making this non-endangered primate species a useful animal model for pancreatic biology.

Association of the phosphodiesterase 4D (PDE4D) gene and cardioembolic stroke in an Australian cohort

Background Large-scale epidemiological studies support an important role for susceptibility genes in the pathogenesis of ischemic stroke, with phosphodiesterase 4D identified as the first gene predisposing to ischemic stroke. Several single nucleotide polymorphisms within the phosphodiesterase 4D gene have been implicated in the pathogenesis of stroke. Aim Undertake a multivariate analysis of six single nucleotide polymorphisms within the phosphodiesterase 4D gene in a previously defined Australian stroke cohort, to determine whether these single nucleotide polymorphisms have an association with ischemic stroke. Methods This case–control study was performed using an existing genetic database of 180 ischemic stroke patients and 301 community controls, evaluated previously for cerebrovascular risk factors (hypertension, hypercholesterolemia, diabetes, paroxysmal atrial fibrillation, smoking and history of stroke in a first-degree relative). Based on previously reported associations with large vessel disease, ischemic stroke, cardioembolic stroke or a mixture of these, six single nucleotide polymorphisms in the phosphodiesterase 4D gene were selected for study, these being single nucleotide polymorphisms 13, 19, rs152312, 45, 83 and 87, based on previously utilized DeCODE nomenclature. Single nucleotide polymorphisms were genotyped using a sequence-specific polymerase chain reaction method and gel electrophoresis. Logistic regression was undertaken to determine the relevance of each polymorphism to stroke. Further analysis was undertaken to determine the risk of stroke following stratification for stroke sub-type and etiology. Results Significant odds ratios were found to be associated with cardioembolic strokes in two single nucleotide polymorphisms: rs152312 and SNP 45 (P<0·05). Conclusions Our findings demonstrated an association between cardioembolic stroke and phosphodiesterase 4D single nucleotide polymorphisms rs152312 and 45. No significant association was found for the other four single nucleotide polymorphisms investigated within the phosphodiesterase 4D gene. We propose that the results from this Australian population support the concept that a large prospective international study is required to investigate the role of phosphodiesterase 4D in the cardiogenic cause of ischemic stroke.

TOOTH (The Open study Of dental pulp stem cell Therapy in Humans): Study protocol for evaluating safety and feasibility of autologous human adult dental pulp stem cell therapy in patients with chronic disability after stroke

Rationale Stroke represents a significant global disease burden. As of 2015, there is no chemical or biological therapy proven to actively enhance neurological recovery during the chronic phase post-stroke. Globally, cell-based therapy in stroke is at the stage of clinical translation and may improve neurological function through various mechanisms such as neural replacement, neuroprotection, angiogenesis, immuno-modulation, and neuroplasticity. Preclinical evidence in a rodent model of middle cerebral artery ischemic stroke as reported in four independent studies indicates improvement in neurobehavioral function with adult human dental pulp stem cell therapy. Human adult dental pulp stem cells present an exciting potential therapeutic option for improving post-stroke disability. Aims TOOTH (The Open study Of dental pulp stem cell Therapy in Humans) will investigate the use of autologous stem cell therapy for stroke survivors with chronic disability, with the following objectives: (a) determine the maximum tolerable dose of autologous dental pulp stem cell therapy; (b) define that dental pulp stem cell therapy at the maximum tolerable dose is safe and feasible in chronic stroke; and (c) estimate the parameters of efficacy required to design a future Phase 2/3 clinical trial. Methods and design TOOTH is a Phase 1, open-label, single-blinded clinical trial with a pragmatic design that comprises three stages: Stage 1 will involve the selection of 27 participants with middle cerebral artery ischemic stroke and the commencement of autologous dental pulp stem cell isolation, growth, and testing in sequential cohorts (n = 3). Stage 2 will involve the transplantation of dental pulp stem cell in each cohort of participants with an ascending dose and subsequent observation for a 6-month period for any dental pulp stem cell-related adverse events. Stage 3 will investigate the neurosurgical intervention of the maximum tolerable dose of autologous dental pulp stem cell followed by 9 weeks of intensive task-specific rehabilitation. Advanced magnetic resonance and positron emission tomography neuro-imaging, and clinical assessment will be employed to probe any change afforded by stem cell therapy in combination with rehabilitation. Sample size estimates Nine participants will step-wise progress in Stage 2 to a dose of up to 10 million dental pulp stem cell, employing a cumulative 3 + 3 statistical design with low starting stem cell dose and subsequent dose escalation, assuming that an acceptable probability of dose-limiting complications is between 1 in 6 (17%) and 1 in 3 (33%) of patients. In Stage 3, another 18 participants will receive an intracranial injection with the maximum tolerable dose of dental pulp stem cell. Outcomes The primary outcomes to be measured are safety and feasibility of intracranial administration of autologous human adult DPSC in patients with chronic stroke and determination of the maximum tolerable dose in human subjects. Secondary outcomes include estimation of the measures of effectiveness required to design a future Phase 2/3 clinical trial.

Activating Mutation of the Renal Epithelial Chloride Channel ClC-Kb Predisposing to Hypertension

The chloride channel ClC-Kb is expressed in the basolateral cell membrane of the distal nephron and participates in renal NaCl reabsorption. Loss-of-function mutations of ClC-Kb lead to classic Bartter syndrome, a rare salt-wasting disorder. Recently, we identified the ClC-Kb(T481S) polymorphism, which confers a strong gain-of-function effect on the ClC-Kb chloride channel. The present study has been performed to explore the prevalence of the mutation and its functional significance in renal salt handling and blood pressure regulation. As evident from electrophysiological analysis with the 2-electrode voltage-clamp technique, heterologous expression of ClC-Kb(T481S) in Xenopus oocytes gave rise to a current that was 7-fold larger than the current produced by wild-type ClC-Kb. The prevalence of the mutant allele was significantly higher in an African population from Ghana (22%) than in whites (12%). As tested in 1 white population, carriers of ClC-Kb(T481S) were associated with significantly higher systolic (by approximately 6.0 mm Hg) and diastolic (by approximately 4.2 mm Hg) blood pressures and significantly higher prevalence (45% versus 25%) of hypertensive (> or =140/90 mm Hg) blood pressure levels. Individuals carrying ClC-Kb(T481S) had significantly higher plasma Na+ concentrations and significantly decreased glomerular filtration rate. In conclusion, the mutation ClC-Kb(T481S) of the renal epithelial Cl- channel ClC-Kb strongly activates ClC-Kb chloride channel function in vitro and may predispose to the development of essential hypertension in vivo.

Significant increase in thrombolysis therapy rates for stroke in South Australia

Andrew W. O. Moey, Monica A. Hamilton-Bruce, Stuart Howell, James M. Leyden, Woon K. Chong, Lizzie Dodd, Austin G. Milton, Simon A. Koblar, Timothy J. Kleinig, Andrew W. Lee, and Jim Jannes

Endovascular treatment for acute ischemic stroke: Experience in South Australia

Laura I. Rudaks, Idin Ahangar, Elizabeth R. Dodd, Austin G. Milton, Monica A. Hamilton-Bruce, Jim Jannes, Simon A. Koblar

The ClC-KbT481S Chloride Channel Gene Polymorphism, Ischaemic Stroke and Hypertension

Stroke is a polygenic disorder. Previous genetic studies focused on candidate genes influencing pathogenic processes, with little emphasis on genes influencing vascular risk factors. Previous research linked the ClC-Kb T481S poly- morphism to blood pressure (BP). We therefore undertook an association study to determine the relevance of this poly- morphism to stroke, particularly lacunar stroke, given its strong correlation with hypertension. We genotyped DNA from 180 patients with acute ischaemic stroke (44 having lacunar stroke) and 298 age- and gender-matched controls using a se- quence-specific polymerase chain reaction method (SS-PCR). We found no association between the ClC-Kb T481S poly- morphism and ischaemic stroke (Odds Ratio (OR): 0.87, 95% Confidence Interval (CI): 0.57-1.33). Stratification for stroke subtype did not alter this finding. This polymorphism showed a borderline association with history of hypertension (p=0.06) but was not associated with systolic or diastolic BP (p>0.05). To our knowledge there are no other studies pub- lished on this polymorphism and stroke.