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In Vitro Investigations of Antibiotic Influences on Nerve Cell Network Responses to Pharmacological AgentsSawant, Meera 12 1900 (has links)
Neuronal networks, derived from mouse embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate effects of gentamicin pretreatment on pharmacological response to the L-type calcium channel blocker, verapamil. Gentamicin is a broad spectrum antibiotic used to control bacterial contamination in cell culture. The addition of gentamicin directly to medium affects the pharmacological and morphological properties of the cells in culture. A reproducible dose response curve to verapamil from untreated cultures was established and the mean EC50 was calculated to be 1.5 ± 0.5 μM (n=10). 40 μM bicuculline was added to some cell cultures to stabilize activity and verapamil dose response curves were performed in presence of bicuculline, EC50 1.4 ± 0.1 μM (n=9). Statistical analysis showed no significant difference in verapamil EC50s values obtained in presence of bicuculline and hence the data was combined and a standard verapamil EC50 was calculated as 1.4 ± 0.13 μM (n=19). This EC50 was then used to compare verapamil EC50s obtained from neuronal cell cultures with chronic and acute exposures to gentamicin. FC cultures (21- 38 days old) were found to be stable in presence of 2300 μM gentamicin. The recommended concentration of gentamicin for contamination control is 5uL /1 ml medium (108 μM). At this concentration, the verapamil EC50 shifted from 1.4 ± 0.13 μM to 0.9 ± 0.2 μM. Given the limited data points and only two complete CRCs, statistical comparison was not feasible. However, there is a definite trend that shows sensitization of cells to verapamil in presence of gentamicin. The cultures exposed to 108 μM gentamicin for 5 days after seeding showed loss of adhesion and no data could be collected for pharmacological analysis. To conclude, acute gentamicin exposure of neuronal cell cultures causes increased sensitivity to verapamil and chronic or long term exposure to gentamicin may cause loss of adhesion of the cell culture by affecting the glial growth. The effect of chronic exposure to gentamicin on pharmacological responses to verapamil remains inconclusive.
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Inhibition of Colon Cancer in Mice by Microencapsulated ProbioticOdun-Ayo, Frederick Oluwasheyi January 2016 (has links)
Submitted in complete fulfillment for the Degree of Doctor of Philosophy in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Colon cancer is the third most common cancer worldwide with a high morbidity and mortality rate. Therapies are less effective during metastasis, therefore prevention and earlier detection is key to reducing the risk of colon cancer. Increased dietary fibre and probiotic intake is known to lower the risk of colon cancer. Probiotics are defined as “live microorganisms which when administered orally in an adequate amount confer a health benefit on the host”. The International Dairy Federation recommends a viable minimum level of 6–7 log10cfu/g in a probiotic product being consumed. Different biopolymer matrices have been used for encapsulation of probiotics; however, loss of viability is still a major challenge. Citrus pectin is a dietary fibre polysaccharide broken down into smaller fragments to form modified citrus pectin (MCP). The unique bioactivity of MCP against carcinogenesisis is linked to its sugar β-galactose inhibiting the cell signalling protein marker, galectin-3 (gal-3), which is intimately involved in endothelial cell morphogenesis. The vascular endothelial growth factor (VEGF) signalling, which invariably drives angiogenesis can be activated when gal-3 binds to integrins. The bioactivity and uptake of MCP may be improved through a novel approach if conjoined with a supplement for example probiotic. Therefore, the synergistic inhibitory effect of modified citrus pectin alginate (MCPA) probiotic microbeads on gal-3 and VEGF in an azoxymethane (AOM) induced colon carcinogenesis Balb/c mouse model was investigated.
A microencapsulation process was used to produce a MCPA microbead containing probiotic, Lactobacillus acidophilus ATCC 4356. Efficiency of the microbead was evaluated in vitro (simulated conditions) and in vivo (Balb/c mouse model). Genomic identification of faecal lactobacilli samples from the treated mice was analyzed. Optimization of AOM dose-time with 10 and 15 mg/kg AOM intraperitoneal (ip) administered to Balb/c mice for 2 and 4 weeks were performed. The optimal AOM dose was initiated prior to intake of MCPA, AP (alginate calcium) probiotic microbeads and MCP in Balb/c mice for 16 weeks; samples were analyzed for colon histopathology and immunohistochemistry.
The MCPA probiotic microbeads significantly enhanced the viability of L. acidophilus ATCC 4356 compared to the AP microbeads in vitro (p< 0.05). Exposure of the MCPA probiotic microbeads to 3 h of simulated gastric juice (SGJ) resulted in 82.7% survival of L. acidophilus ATCC 4356. Also, the faecal lactobacilli count in the MCPA probiotic treated mice significantly increased after 28 days by 10.2% compared to the AP probiotic, MCP treated and control mice (p< 0.0001). A total of 4DNA encoding 16S rRNA gene closest to the genera namely Lactobacillus, Bacillus, Enterococcus and Bifidobacterium were identified from faecal samples of the colon cancer-induced Balb/c mice. Azoxymethane at 15 mg/kg for 4 weeks induced optimal gal-3 and VEGF immunoexpression. Furthermore, MCPA probiotic treatment significantly reduced gal-3 immunoexpression in the colon of AOM induced cancer Balb/c mice compared to the control mice (p< 0.0001). The immunoexpresion of VEGF in the MCPA and AP probiotic treated groups was weakly positive and significantly reduced when compared to the control group (p<0.05), while the MCP treated group was barely positive (p< 0.001).
Modified citrus pectin alginate is a novel effective means of oral delivery of bacterial cells and bioactive compounds. It has a good biodegradability, inexpensive, non-toxic, proven efficiency, and stability at low temperatures warranting its use as a drug carrier by pharmaceuticals. Modified citrus pectin alginate probiotic microbeads increase bioactivity and chemoprevention against colon pre-cancerous lesions and adenocarcinoma through inhibition of gal-3 and VEGF in the mouse model. Modified citrus pectin alginate can be used in probiotic therapy, which may improve the prevention of colon cancer. / D
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