Isolation, Purification and Characterization of Caprine Pancreatic Islets

Diabetes mellitus is a major late consequence of chronic pancreatitis. Diabetes mellitus type 1 or insulin-dependent diabetes mellitus (IDDM) is characterized by the failure of the pancreatic islets of Langerhans to synthesis or secrete insulin. The longterm complications of IDDM are major health problems, and in these diabetics exogenous insulin may not adequately regulate blood glucose homeostasis and thus fail to avert the late complications. In this case, transplantation of pancreatic islets is arguably the most logical approach to restoring metabolic homeostasis. The limited availability of human donors makes the search for alternative source of islet cells mandatory for future developments in pancreatic transplantation. The present study investigates the potential of goats as an alternative source of pancreatic islets. The objectives of the study were to optimize techniques for goat islet isolation and purification for culture establishment, and to perform functional, morphological and viability assessment of goat islets. Goat pancreatic tissues were collected within 15 min of slaughter, placed in Hanks balance salt solution (HBSS) and maintained at 4oC. Goat islets were obtained successfully using a collagenase-based digestion and isolation technique at an optimized pH of 7.2 to 7.4 and temperature of 37oC. Digested pancreatic islets were purified by Euro-Ficoll density gradients. Islet cell purity and viability were determined by dithizone and trypan blue staining, respectively. Islet clusters of different sizes were positively identified by both staining methods and it was observed that 90% of clusters were viable in the culture system. Following the static incubation, an in vitro insulin secretion assay was carried out by ELISA to determine the islets viability. The islets remained viable for 5 days in the culture system following regular media changes. Pancreatic tissues were fixed in Bouin’s solution stained with hematoxylin-eosin (H&E) and immunohistochemistry (IHC) stains and examined microscopically to estimate the islet mass and determine insulin secretion ability. Under the light microscope, there were minimal connective tissue cells separating the islets from the surrounding exocrine component. The nuclei of islets cellswere uniform in size and surrounded by eosinophilic cytoplasm. Purified islets of Langerhans were fixed in a mixture of paraformaldehyde and glutaraldehyde solution for transmission (TEM) and scanning electron microscopic (SEM) examination. Under TEM, the caprine islet cell exhibited their characteristic secretory granules, which were of various sizes and electron opacity. The cells also showed characteristic abundance of rough endoplasmic reticulum (RER) and mitochondria. In b-cells, the rough endoplasmic reticulum was inconspicuous. The -cells are characterized by their peripheral location in the islet being larger and having electron-dense secretory granules. Based on morphological criterion, intermediate cells are shown to be present in both the endocrine and exocrine tissue of the normal pancreas of goats. Under SEM the size of the islet clusters was shown to range from 50 to 250 μm. Cells with secretory sacs on the surface could possibly be the isolated islet cells. The study had provided an optimized isolation and purification techniques for goat pancreatic islets to be further developed and used for xenotransplantation in diabetic animal models. The findings can lead to further research in identification and sequencing of insulin indicator genes, pancreatic hormones biomarkers and long-term cryopreservation of goat pancreatic islets

The Reality of Project Management Office for Construction Organization in the Oil, Gas and Petrochemical Industry of Iran

Abstract: Over the last decade, the Project Management Office (PMO) has become a prominent feature in many organizations. Despite the proliferation of PMO in practice, our understanding of this phenomenon remains sketchy at best. The objective of this study is describing the reality of PMOs for Construction Organization in the Oil, Gas and Petrochemical Industry of Iran at 2010 year. Nine major Iranian oil, gas and petrochemical organizations were participated in this survey. The study examines the reality of PMOs, the age of PMOs, the reason for their implementation and challenges during their implementation. Research concludes that PMO are a new concept and nearly 60% of PMOs have been in existence for 3 years. Monitoring project performance, supporting project management software tools and preparing project management methodology are the most used functions. Organizations are using PMOs to achieve the golden triangle project objectives (cost/time/quality). The study found that the most significant challenges in PMO implementation are the risk of changing organizational culture and the lack of professional staff

Isolation, density purification, and invitro culture maintenance of functional caprine islets of Langerhans as an alternative islet source for diabetes study

Background : Insufficient availability of human donors makes the search for alternative source of islet cells mandatory for future developments in pancreatic transplantation. The present study investigates the potential of caprine as an alternative source of pancreatic islets. The objectives of the study were to optimize techniques for caprine islet isolation and purification for culture establishment, and to subsequently assess their viable and functional potential. Methods:  Caprine pancreatic tissues were collected from a local slaughterhouse and prior transported to the laboratory by maintaining the cold chain. Islets were obtained by a collagenase-based digestion and optimized isolation technique. Islet cell purity and viability were determined by dithizone and trypan blue staining, respectively. Islet clusters of different sizes were positively identified by staining methods and demonstrated 90% viability in the culture system. Following static incubation, an in vitro insulin secretion assay was carried out and analyzed by ELISA. Results:  The islets remained satisfactorily viable for 5 days in the culture system following regular media changes. The current study has successfully optimized the isolation, purification and culture maintenance of caprine islets. Conclusion:  The successful yield, viability and functionality of islets isolated from the optimized protocol provide promising potential as an alternative source of islets for diabetes and transplantation researches

Caprine pancreatic islet xenotransplantation into diabetic immunosuppressed BALB/c mice

Background: Type 1 diabetes mellitus is a devastating disease for which there is currently no cure, but only lifetime management. Islet xenotransplantation is a promising technique for the restoration of blood glucose control in patients with diabetes mellitus. The purpose of this study was to explore the potential use of caprine (goat) islet cells as xenogeneic grafts in the treatment for diabetes in a mouse model. Methods: Caprine pancreases were harvested and transported to the laboratory under conditions optimized to prevent ischemia. Islets were isolated, purified, and tested for functionality. Caprine islets (2000 islet equivalent) were transplanted beneath the kidney capsules of diabetic BALB/c mice under thalidomide-induced immunosuppression. Blood glucose and insulin levels of grafted mice were evaluated by glucometer and enzyme-linked immunosorbent assay kit, respectively. The functionality and quality of caprine pancreatic islet grafts were assessed by intraperitoneal glucose tolerance tests. Results: The viability of purified islet cells exceeded 90%. Recipient mice exhibited normoglycemia (<11 mm glucose) for 30 days. In addition, weight gain negatively correlated with blood glucose level. The findings verified diabetes reversal in caprine islet recipient mice. A significant drop in non-fasting blood glucose level (from 23.3 ± 5.4 to 8.04 ± 0.44 mm) and simultaneous increase in serum insulin level (from 0.01 ± 0.001 to 0.56 ± 0.17 μg/l) and body weights (from 23.64 ± 0.31 to 25.85 ± 0.34 g) were observed (P < 0.05). Immunohistochemical analysis verified insulin production in the transplanted islets. Conclusions: Purified caprine islets were demonstrated to successfully sustain viability and functionality for controlling blood glucose levels in an immunosuppressed mouse model of diabetes. These results suggest the use of caprine islets as an addition to the supply of xenogeneic islets for diabetes research

An update on type 1 diabetes treatments: insulin treatment, cell therapy and transplantation

Diabetes is one of the major life-threatening health problems worldwide today. It is one of the most fast-growing diseases that cause many health complications and a leading cause of decreasing life expectancy and high mortality rate. Many studies have suggested several different types of intervention to treat Type 1 diabetes such as insulin therapy, islet transplantation, islet xenotransplantation and stem cell therapy. However, issues regarding the efficacy, cost and safety of these treatments are not always well addressed. For decades, diabetes treatments with few side effects and long-lasting insulin independence has remained one of the most challenging tasks facing scientists. Among the treatments mentioned above, application of human islet transplantation in patients with type 1 diabetes has progressed rapidly with significant achievement. Again, the lack of appropriate donors for islet transplantation and its high cost have led researchers to look for other alternatives. In this review, we discuss very pertinent issues that are related to diabetes treatments, their availability, advantages, disadvantages and also cost

Effect of semen collection methods on the quality of pre- and post-thawed Bali cattle (Bos javanicus) spermatozoa.

This study was conducted to evaluate the response of Bali bulls (Bos javanicus) to different semen collection methods and their effects on fresh and post-thawed semen quality. The collection methods employed were electro-ejaculation (EE), transrectal massage (RM) and RM followed by EE (RM + EE). A total of 25 untrained Bali bulls (age between 2 and 4 years old) were subjected to the different semen collection methods. Fresh semen samples from all the 25 bulls were evaluated for volume, pH, general motility, live/dead ratio and abnormality using the conventional method. For fresh and frozen samples collected by EE and RM from 10 bulls, computer-assisted semen analysis system was used for precise quantitative measurement of motility, velocity and forward progression. Accucell photometer was used to measure sperm concentration in all samples, regardless fresh and frozen. Semen samples were obtained 100% of the attempts using EE, 84% using RM and 96% using RM + EE. There were no differences among the collection methods for fresh semen quality characteristics, including motility, morphology and viability, but pH and volume were higher for EE than RM and RM + EE. Higher sperm concentration was observed in semen collected by RM than the other two methods. Different age groups (2–3 and >3–4 years old) of the bulls did not show significant differences in volume, pH, sperm concentration, percentages in motility, live/dead ratio and normal sperm morphology. The quality of semen for general and progressive motility, VAP, VSL and VCL and acrosomal integrity after thawing was higher for RM than EE. In conclusion, Bali bulls appeared to respond best to EE and the combination of RM + EE than RM, as a method of semen collection, with a shorter time of stimulation required. Differences in age of the Bali bulls did not affect the semen quality

Gene expression level of toll-like receptor 4 and insulin receptor substrate 1 in type II diabetic Malay patients and their first-degree relatives

Background: Type II diabetes mellitus (T2DM) is a polygenic disorder that can be prevented or delayed in case of the adoption of proper interventions. The identification of susceptible genes and novel biomarkers of T2DM could be of great help in the early detection of high-risk individuals. First-degree relatives of T2DM patients have a high risk of this disease, even when they have no major abnormalities in glucose metabolism. The present study was conducted to examine the status of the expression of two genes, namely toll-like receptor 4 (TLR4) and insulin receptor substrate (IRS1), involved in glucose metabolism in peripheral blood, in individuals genetically predisposed to T2DM development. Methods: Blood samples were collected from 54 participants in three research groups, including Malay subjects with T2DM, first-degree relatives of T2DM patients, and healthy controls. The measurement of gene expression was accomplished using a quantitative real-time polymerase chain reaction. Result: The results were indicative of the significant upregulation and downregulation of TLR4 in patients with T2DM and their first-degree relatives, respectively (P<0.05). With regard to IRS1, the data revealed a decreased expression in T2DM patients as compared to that in the healthy controls (p<0.05). Conclusion: The results indicated that TLR4 and IRS1 might be involved in the pathogenesis of T2DM. Moreover,the altered expression of TLR4 in the first-degree relatives of diabetic patients is an important marker showing a genetic predisposition to T2DM. Therefore, the two investigated genes could be used as a diagnostic tool for the prediction of T2DM in this population

Establishment of rat brain endothelial cells susceptible to rat cytomegalovirus ALL-03 infection

Endothelial cells have been implicated as key cells in promoting the pathogenesis and spread of cytomegalovirus (CMV) infection. This study describes the isolation and culture of rat brain endothelial cells (RBEC) and further evaluates the infectious potential of a Malaysian rat CMV (RCMV ALL-03) in these cultured cells. Brain tissues were mechanically fragmented, exposed to enzymatic digestion, purified by gradient density centrifugation, and cultured in vitro. Morphological characteristics and expression of von Willebrand factor (factor VIII-related antigen) verified the cells were of endothelial origin. RBEC were found to be permissive to the virus by cytopathic effects with detectable plaques formed within 7 d of infection. This was confirmed by electron microscopy examination which proved the existence of the viral particles in the infected cells. The susceptibility of the virus to these target cells under the experimental conditions described in this report provides a platform for developing a cell-culture-based experimental model for studies of RCMV pathogenesis and allows stimulation of further studies on host cell responses imposed by congenital viral infections

Wide dynamic range of surface-plasmon-resonance-based assay for hepatitis B surface antigen antibody optimal detection in comparison with ELISA

Limit of detection (LOD), limit of quantification, and the dynamic range of detection of hepatitis B surface antigen antibody (anti-HBs) using a surface plasmon resonance (SPR) chip-based approach with Pichia pastoris-derived recombinant hepatitis B surface antigen (HBsAg) as recognition element were established through the scouting for optimal conditions for the improvement of immobilization efficiency and in the use of optimal regeneration buffer. Recombinant HBsAg was immobilized onto the sensor surface of a CM5 chip at a concentration of 150 mg/L in sodium acetate buffer at pH 4 with added 0.6% Triton X-100. A regeneration solution of 20 mM HCl was optimally found to effectively unbind analytes from the ligand, thus allowing for multiple screening cycles. A dynamic range of detection of ∼0.00098–0.25 mg/L was obtained, and a sevenfold higher LOD, as well as a twofold increase in coefficient of variance of the replicated results, was shown as compared with enzyme-linked immunosorbent assay (ELISA). Evaluation of the assay for specificity showed no cross-reactivity with other antibodies tested. The ability of SPR chip-based assay and ELISA to detect anti-HBs in human serum was comparable, indicating that the SPR chip-based assay with its multiple screening capacity has greater advantage over ELISA