Cytoanalysis of Pancreatic B-cells: Using an Avian Model, Mammalian Tissue Culture and Implications of Antisense Oligonucleotides Transfection

Calbindin-D28k (CaBP28K) is a vitamin D-dependent calcium-binding protein that may alter intracellular calcium ion levels, [Ca2+]i. This dissertation describes experiments done to gain an understanding of the potential role of CaBP28k in pancreatic B-cells in control of insulin secretion. The localization of CaBP28k and insulin in chicken pancreas are shown in Chapter 1. CaBP28k expression was found to be highest in ventral and dorsal lobes and lowest in splenic lobe. Insulin concentrations were distributed similarly among these lobes. Confocal microscopic studies demonstrated colocalization of insulin and CaBP28k in Bcells. These findings suggest a possible role for CaBP28k in chicken B-cells that could contribute to type 2 diabetes-like characteristics of chickens. Experiments done in Chapter 2 tested the effects of changing levels of glucose in pancreatic islets in vitro from transgenically derived CaBP28k-knockout (KO) and wildtype (WT) mice. CaBP28k-KO islets were exposed to increasing glucose concentrations from 2.8 mM to 30 mM, levels that mimic transition from fasting to hyperglycemic states. KO islets showed significantly greater elevations in [Ca2+]i as compared to WT. These experiments provide evidence that levels of CaBP28k could play a role in controlling Ca2+-mediated, glucose-induced insulin secretion in B-cells. In chapter 3 the effects of reduction of CaBP28k levels on genomic and nongenomic factors using CaBP28k-antisense oligonucleotides (AS-ON) transfection in a cultured pancreatic B-cell line (RIN1046-38 cells) are described. Complete inhibition of CaBP28k expression in transfection assays was achieved using 200 nM phosphorothioate-AS-ON (PS-AS-ON) as well as 20 nM propyne-AS-ON (PY-AS-ON). In addition, cDNA microarray analysis showed up-regulation of both vitamin D receptor (VDR) and calbindin-D9k mRNAs in PS-AS-ON-transfected RIN cells as compared to controls. Western blotting indicated VDR overexpression and calbindin-D9k expression in AS-ON-transfected cells. This study is the first demonstration of compensatory expression of calbindin-D9k in response to inhibition of CaBP28k in cultured B-cells. Insulin secretory responses of PS-AS-ON-transfected cells were greater than in controls. These findings suggest that B-cells synthesize an alternative protein, calbindin-D9k, to preserve calcium regulation when expression of CaBP28k is abolished. Additional studies are required to help in understanding possible interactions of calbindin-D9k, [Ca2+]i, and VDR in the AS-ON-transfected B-cells

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially