Delayed diagnosis and issues with pump usage are the leading causes of diabetic ketoacidosis in children with diabetes living in Newfoundland and Labrador, Canada.

BACKGROUND: Newfoundland and Labrador (NL) has a very high incidence of type 1 diabetes (T1DM) and admission rate for diabetic ketoacidosis (DKA). The purpose of this study was to identify characteristics and precipitating factors associated with pediatric DKA in this population. METHODS: This was a retrospective study on children diagnosed with DKA from 2007-2011 admitted to the province's only tertiary care pediatric hospital. Demographics, biochemical characteristics, and reasons for DKA diagnosis were analyzed. Chi-square and Fisher Exact tests were performed for categorical variables; t- and non-parametric Kruskal-Wallis tests were performed for continuous variables. RESULTS: A total of 90 children were admitted with DKA (39.5% newly diagnosed; 60.5% were previously diagnosed). The rate of DKA on presentation for incident cases was 22.1%. More severe cases of DKA occurred in younger, newly diagnosed patients. Almost half of preexisting diabetes cases were recurrent DKA (49.1%). The most common presenting characteristics of newly diagnosed patients were weight loss, bedwetting, polyuria, polydipsia, and neurologic symptoms. Pre-existing diabetes patients most often presented with abdominal pain and vomiting. Diagnosis of diabetes in new patients and issues related to interrupted insulin delivery in pre-existing patients using insulin pump therapy were the most common factors associated with DKA. Of the newly diagnosed patients presenting in DKA, 64% had seen a physician in the weeks leading up to diagnosis. CONCLUSIONS: Pediatric patients have predictable patterns associated with a diagnosis of DKA. Most cases of DKA could be prevented with earlier diagnosis and improved education and problem-solving by families and health care providers. DKA preventative strategies are recommended and should be aimed at patients, their families, and health care professionals especially those outside of pediatric centers.

Dual immunosuppression enhances vasomotor injury: interactive effect between endothelin-1 and nitric oxide bioavailability.

OBJECTIVE: Cyclosporine A and corticosteroids are associated with many side effects, such as endothelial dysfunction and transplant vasculopathy. We examined the effects of cyclosporine A and hydrocortisone exposure on endothelial function of the rat thoracic aorta. METHODS: Lewis rats were injected with cyclosporine A, hydrocortisone, cyclosporine A + hydrocortisone, or intraperitoneal saline daily for 2 weeks. Endothelial-dependent and independent vascular relaxation were assessed in isolated segments of thoracic aorta, as well as endothelin-1-induced vasoreactivity. Protein expression of endothelial nitric oxide synthase, endothelin(A), and endothelin(B) receptors were also determined in the thoracic aorta. RESULTS: Exposure to cyclosporine A and cyclosporine A + hydrocortisone resulted in a reduction in endothelial-dependent vasorelaxation compared with control and hydrocortisone (P = .001). Cyclosporine A and hydrocortisone-treated rats demonstrated increased vasoreactivity to endothelin-1 compared with control, whereas cyclosporine A + hydrocortisone treatment resulted in a synergistic increase (P = .04). All treatment groups displayed a significant reduction in endothelial nitric oxide synthase expression compared with control (P = .001). Endothelin(A) receptor expression was increased in all treatment groups with a synergistic effect seen after cyclosporine A + hydrocortisone treatment. No differences were seen in endothelin(B) receptor expression. CONCLUSION: Cyclosporine A and hydrocortisone induce vasomotor dysfunction with a synergistic impairment observed after concomitant exposure. Our findings suggest that the resultant vasomotor dysfunction is the result of alterations in both nitric oxide and endothelin-1 regulation.

Endothelin-1 accentuates the proatherosclerotic effects associated with C-reactive protein.

OBJECTIVES: The proinflammatory marker C-reactive protein has been demonstrated to play a role in the development of atherosclerosis. Endothelin-1 and nitric oxide homeostasis is crucial for normal vasomotor function, limiting inflammatory activation and maintaining a nonthrombogenic endothelial surface. In addition to its vasoactive properties, endothelin-1 is also an inflammatory cytokine. We have previously demonstrated that C-reactive protein impairs endothelial cell nitric oxide production. Protein kinase C, an important signal transducer within the cell, is involved in several cellular responses to external stimuli. We therefore sought to determine whether endothelin-1 exposure modulates C-reactive protein's effects on nitric oxide production via protein kinase C. METHODS: Endothelial cells were incubated with C-reactive protein (200 microg), endothelin-1 (100 nM), C-reactive protein + endothelin-1, or phosphate-buffered saline solution (control) for 24 hours. After exposure, endothelial nitric oxide synthase expression was determined in addition to total nitric oxide production and protein kinase C translocation and activity. RESULTS: Endothelial nitric oxide synthase protein expression was reduced following incubation with C-reactive protein and endothelin-1 treatment compared with baseline by 40% and 45%, respectively (P = .04); however, no additive effects were seen with coincubation. C-reactive protein produced a 47% decrease in nitric oxide production compared with control. Coincubation with endothelin-1 resulted in a synergistic 70% reduction in nitric oxide production (P = .001). C-reactive protein exposure inhibited translocation of protein kinase C lambda compared with control (P = .01). Furthermore, coincubation of C-reactive protein with endothelin-1 led to a synergistic inhibition of protein kinase C lambda translocation (P = .01). C-reactive protein exposure reduced protein kinase C activity by 40% compared with control (P = .02), although coincubation with endothelin-1 had a synergistic reduction in activity (P = .02). CONCLUSIONS: Our results indicate that endothelin-1 exposure accentuated C-reactive protein's impairment of endothelial nitric oxide production via synergistic inhibition of protein kinase C lambda translocation and activity. Our investigations suggest that endothelin-1 inhibition and protein kinase C stimulation may provide a novel therapeutic strategy to improve vascular nitric oxide homeostasis and mitigate the proatherosclerotic effects of C-reactive protein.

In vitro studies of antifreeze glycoprotein (AFGP) and a C-linked AFGP analogue.

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes found in deep sea Teleost fish. These compounds have the ability to depress the freezing point of the organism such that it can survive the subzero temperatures encountered in its environment. This physical property is very attractive for the cryopreservation of cells, tissues, and organs. Recently, our laboratory has designed and synthesized a functional carbon-linked (C-linked) AFGP analogue (1) that demonstrates tremendous promise as a novel cryoprotectant. Herein we describe the in vitro effects and interactions of C-linked AFGP analogue 1 and native AFGP 8. Our studies reveal that AFGP 8 is cytotoxic to human embryonic liver and human embryonic kidney cells at concentrations higher than 2 and 0.63 mg/mL, respectively, whereas lower concentrations are not toxic. The mechanism of this cytotoxicity is consistent with apoptosis because caspase-3/7 levels are significantly elevated in cell cultures treated with AFGP 8. In contrast, C-linked AFGP analogue 1 displayed no in vitro cytotoxicity even at high concentrations, and notably, caspase-3/7 activities were suppressed well below background levels in cell lines treated with 1. Although the results from these studies limit the human applications of native AFGP, they illustrate the benefits of developing functional C-linked AFGP analogues for various medical, commercial and industrial applications.

Assessing antifreeze activity of AFGP 8 using domain recognition software.

Domain recognition software was employed to assess recrystallization-inhibition (RI) activity as an index of antifreeze potential. This represents a key step in the development of a high-throughput analysis for RI activity. Analysis of measurement error indicates an average coefficient of variation for individual crystals of about 8%, which is very small in relation to other sources of variation. Our analysis demonstrates an inverse correlation between AFGP 8 concentration and average crystal size with consistently small, but detectable differences in average crystal size at the edge and centre of the ice wafer. Sensitivity analysis using Monte Carlo re-sampling methods indicate that measuring of 12-15 crystals per field of view are sufficient to obtain accurate estimates of the first two moments (mean and variance) of the crystal size distribution, thereby greatly reducing the time required to assess recrystallization activity. These results suggest that this method has considerable potential for high-throughput analysis of RI activity.