Treatment for neurological diseases has been limited by the presence of the protective blood-brain barrier. Recent studies from our laboratory have shown that direct intraventricular implantation of microcapsules containing genetically modified cells can effectively deliver the transgene product to the mouse brain, thereby circumventing the blood-brain barrier. In this thesis, the experiments were aimed at scaling up the murine experiments to determine if direct implantation of alginate-poly-L-lysine-alginate microcapsules to the central nervous system of dogs was a feasible means of treating the large animal brain. In the first two experiments reported here, microcapsules containing cells genetically modified to secrete human growth hormone were injected into the central nervous system of dogs. Two routes of delivery were examined, intraventricular brain surgery and injection into the spinal intrathecal space (cisterna magna). While empty capsules within the central nervous system were benign, microcapsules containing cells induced an acute inflammatory response in the brain and spinal cord tissue, irrespective of the route of delivery. Human growth hormone was detected transiently in four of six dogs, but the data were interpreted with caution due to extraneous variables such as compromised microcapsules in two of the dogs and previous systemic treatment in six of the other dogs. In the last experiment, microcapsules containing cells genetically modified to secrete the lysosomal enzyme a-L-iduronidase were implanted into the lateral ventricles of a dog with Mucopolysaccharidosis type I in an attempt to correct the characteristic neuronal pathology. An immune response ensued and appeared to abolish any possible effect of the microcapsule treatment. The experiments presented here demonstrate the challenges and obstacles that need to be overcome to effectively scale up therapies from rodent experiments to large animals. The data also shed light on the immunological complications that may arise with invasive and repeated treatment in the central nervous system of large animals. / Thesis / Master of Science (MS)

Submitted in fulfillment for the degree of Master of Technology, Clinical Technology: Cardiovascular Perfusion, Durban University of Technology, Durban, South Africa, 2016. / Introduction: A large volume of residual haemodilute blood remains in the cardiopulmonary bypass (CPB) circuit after termination of the bypass. It is common practice in many centres to process residual pump blood with an autologus cell salvage system (ACSS), thereby producing a re-suspended red blood cell (RBC) concentrate and attenuating the need for donor blood RBC concentrate. It has also become standard practice to wash donor pack red blood cells (PRBC) before adding it to neonate cardiopulmonary circuits (Swindell et al., 2007). Manufactures of ACSS recommend 0.9% sodium chloride (NaCl) as a wash solution for processing salvaged blood. Previous studies have demonstrated that washing PRBC with normal saline results in acid-base (Huber et al., 2013) and electrolyte derangements (Varghese et al., 2007). Infusion of normal saline in healthy volunteers also results in significant changes in osmolality (Williams et al., 1999). The use of normal saline as a wash solution in processing residual CPB blood requires investigation. Aims and Objectives: This was a prospective, quantitative in vitro investigation to analyze and compare the quality of residual pump blood post CPB that had been washed with either an unbalanced electrolyte solution (0.9% normal saline) or a balanced electrolyte solution (Balsol®). Both are crystalloid solutions. The primary objective of the present study was to measure and compare the pH, electrolytes, metabolites, osmolality and strong ion difference (SID) of residual pump blood to the pH, electrolytes, metabolites, osmolality and SID of processed cell saver blood, which was washed with either 0.9% normal saline or Balsol® solution. The secondary objective was to measure and compare protein levels (albumin and total protein) in residual pump blood to protein levels in processed cell saver blood, that is washed with either 0.9% normal saline or Balsol® solution. The final objective was to determine the volume, haematocrit and haemoglobin yield post cell saver processing, from the input volume of residual pump blood when washed with either 0.9% normal saline or Balsol® solution. This was the first study of this nature done in the South African population group. Methodology: In this investigation in a series of forty patients (n=40) undergoing elective cardiac surgery with CPB, the first twenty patients were allocated to the NaCl control group (n=20) and the second twenty patients were allocated to the Balsol® interventional group (n=20). The extracorporeal circuit consisted of a standard integral hollow fibre membrane oxygenator and tubing that was primed with 1500-1800 millilitres of balanced crystalloid solution (Balsol®), for both the control group and the interventional group, and addition of 5000 iu heparin. The balanced crystalloid solution (Balsol®) is the approved standard CPB priming solution for all cardiac procedures at Inkosi Albert Luthuli Central Hospital. This setup was used with the Stockert S5 roller pump heart lung machine. The operations were performed as per protocol with standard non-pulsatile CPB and hypothermia was maintained at 28 – 32 ºC (core) and haemodilution (haematocrit 20 % to 30 %). A standard flow rate of 2.4 L/min/m² was used. Cardio protection consisted of either cold Blood Cardioplegia using the Buckberg 4:1 ratio, being four parts blood to one part cardioplegia (with the 35ml of 20 % Dextrose + 1 gram Magnesium Sulphate added per 500ml), or 20ml/kg cold St Thomas II cardioplegia (with addition of 10ml of 8.5% NaHCO3 + 100mg lignocain per litre). Topical cooling was achieved with ice cold 0.9 % saline. Maintenance fluid used during CPB was Balsol® for both the control and the interventional groups. Calcium, potassium and sodium bicarbonate was administered as required during CPB to correct deficits for both groups. Weaning of CPB was performed after re-warming to a rectal temperature of at least 35 ºC for both study groups. Immediately on termination of CPB a blood sample was taken from the sampling manifold of the CPB circuit for pre wash analysis. Residual pump blood was then flushed out with one litre of Balsol® solution for both groups and collected into the Medtronic autolog cell saver reservoir to be processed. In the control study group 0.9% NaCl was used as the wash solution and in the interventional study group Balsol® solution was used as the wash solution. After processing of the salvaged blood is complete, a blood sample was taken for post wash analysis. Clinical data recorded for pre and post wash samples included: pH, pCO2, pO2, [K+], [Na+], [Cl-], [Ca2+], lactate, glucose, [HCO3-], TCO2, haematocrit, haemoglobin (GEM 4000® premier™ blood gas analyser) blood volume (Medtronic autolog) and SID (calculated as per equation). Inorganic phosphate, total magnesium, albumin, total protein (Siemens Advia 1800 blood gas analyser) and osmolality (Gonotech osmometer) were also measured. Results: There was a highly significant decrease (p < 0.05) within the NaCl group after washing with pCO2 (28.3 ± 2.9 vs. <6.0 ± 0.0), [K+] (4.5 ± 0.5 vs. 1.0 ± 0.7), total magnesium (1.7 ± 0.7 vs. 0.29 ± 0), ionized calcium (1.0 ± 0.09 vs. 0.1 ± 0.03), inorganic phosphate (0.9 ± 0.4 vs. 0.09 ± 0.04) and SID (27.1 ± 2.1 vs. 18.4 ± 2.2). There was a highly significant increase (p < 0.05) within the NaCl group after washing with pH (7.5 ± 0.1 vs. 7.7 ± 0.1), [Na+] (132.9 ± 3.2 vs. 146.3 ± 1.9), [Cl-] (107.8 ± 3.1 vs. 127.4 ± 2.1) and osmolaltity (256.9 ± 38.4 vs. 296.2 ± 57.5). There were highly significant decrease (p < 0.05) within the Balsol® group after washing with pCO2 (30.15 ± 6.0 vs. 18.9 ± 4.9), [Na+] (134.7 ± 2.2 vs. 125.6 ± 1), [Cl-] (108.8 ± 2.7 vs. 100.2 ± 1.4), ionized calcium (0.9 ± 0.1 vs. 0.02 ± 0.04), inorganic phosphate (0.8 ± 0.2 vs. 0.1 ± 0.024) and osmolality (288.8 ± 20.6 vs. 272.8 ± 19.9). There were highly significant increase (p < 0.05) within the Balsol® group after washing with pH (7.5 ± 0.1 vs. 7.7 ± 0.1), [K+] (4.2 ± 0.4 vs 4.6 ± 0.3). Total magnesium and SID were similar after washing within the Balsol® group. Albumin and total protein revealed similar significant decreases within both groups after washing. There was a highly significant difference (p < 0.05) in the change between groups after washing in all the variables measured, except for pH, inorganic phosphate, lactate, glucose, albumin, total protein, haematocrit, haemoglobin, and blood volume. Total carbon dioxide and [HCO3-] were not compared because they were incalculable by blood gas analyser in the NaCl group. Conclusion: This investigation concluded that the balanced electrolyte solution Balsol® used for washing residual CPB blood results in a re-suspended RBC concentrate, with an osmolality and electrolyte profile that is superior compared to washing residual CPB blood with 0.9% NaCl solution. / M

Mesenchymal stem cells (MSC) possess a multilineage differentiation potential and have the ability to regulate reactivity of the immune system. They are usually isolated and expanded from the bone marrow, adipose tissue or umbilical cord. MSC represent promising cell population for the treatment of some severe diseases, such as amyotrofic lateral sclerosis (ALS), due to the combination of regenerative and immunomodulatory properties. The aim of this study is to compare MSC from ALS patients and healthy donors in their phenotype, proliferative activity and mainly their immunomodulatory properties. The assessment of impact of the disease on the properties of MSC is important for their autologous use in clinical trials. In this study we used MSC isolated from bone marrow of 14 ALS patients and 15 patients undergoing mostly orthopedic surgery as control group. We also used MSC stimulated for 24 hours by poinflammatory cytokines. Cells were compared in terms of immunophenotype, differentiation in adipocytes and osteoblasts, metabolic activity, expression of selected genes for immunomodulatory molecules and for inhibition of lymphocyte proliferation. Further experiments were focused on evaluation of immunomodulatory properties of MSC. The effect of MSC on peripheral blood mononuclear cells stimulated...

Diabetic foot disease (DFD) is a serious complication of diabetes and, along with critical limb ischemia, significantly exacerbates the prognosis of patients. Peripheral arterial disease in patients with diabetes has an atypical clinical course, its diagnosis is challenging and is one of the most common causes of morbidity and mortality of patients with DFD. The aim of this dissertation focused on the diagnosis and treatment of DFD was to identify a suitable method for evaluating the effect of autologous cell therapy (ACT), to assess options for early diagnosis of Charcot osteoarthropathy (COA) and, possibly, to establish the association between the incidence of cardiovascular disease and DFD. In our studies concerning therapeutic vasculogenesis, we observed a significant increase in the antiangiogenic factor endostatin after ACT in contrast to its unchanged levels after standard percutaneous transluminal angioplasty; the transient increase in endostatin seems to be a marker of therapeutic vasculogenesis after ACT. A benefit of using calf muscle perfusion scintigraphy in the assessment of microcirculation and ACT effect was not clearly demonstrated. By contrast, a promising method for the evaluation of microcirculation and the effect of revascularization after ACT was MR spectroscopy of calf...