Production of Recombinant N Protein of Infectious Bronchitis Virus Using the Baculovirus Expression System and Its Assessment as a Diagnostic Antigen.

The avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. Sensitive diagnostics and efficacious vaccines are necessary to combat IBV infections in chickens. The aim of this study was to produce recombinant N protein of IBV in the baculovirus system to use in ELISA diagnostic tests in order to enable the assessment of the sero-prevalence and risk of IBV infections in chickens in Turkey. For this, the gene encoding the N protein of the Beaudette strain of IBV was expressed using a recombinant baculovirus expression system. The recombinant N protein was purified using Ni-NTA affinity chromatography. An estimated 50-kDa recombinant protein corresponding to the expected molecular weight of IBV N including the 6xHis tag was detected using an anti-His monoclonal antibody. Specific immunoreactivity of the recombinant protein was confirmed by Western blot using antiserum obtained from vaccinated and naturally infected chicken from Turkey as well as using a monoclonal antibody raised against the N protein of the IBV Massachusetts strain. The results obtained with the in-house ELISA had high agreement with a commercial ELISA. Immunoreactivity analysis using antisera in Western blotting and the in-house ELISA suggests that the recombinant IBV N protein could be broadly cross-reactive with antisera produced against different IBV strains. We conclude that the recombinant baculovirus expressed IBV N protein could serve as a useful diagnostic antigen for detection of IBV infections in chickens by ELISA.

Postprandial vascular effects of VIAject compared with insulin lispro and regular human insulin in patients with type 2 diabetes.

OBJECTIVE: Recent studies suggested an impact of prandial insulin delivery on postprandial regulation of tissue blood flow. This study compared the effect of VIAject with human regular insulin and insulin lispro on postprandial oxidative stress and endothelial function in patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: Fourteen patients (seven men; aged 61.5 +/- 1.8 years; duration of diabetes 6.6 +/- 4.6 years; A1C 7.2 +/- 0.5% [mean +/- SEM]) received a prandial injection of VIAject, human regular insulin, and insulin lispro. At baseline and after a standardized liquid meal test (Ensure Plus), the postprandial increases in asymmetric dimethylarginine (ADMA) and nitrotyrosine levels were investigated. In addition, the postprandial effects on microvascular blood flow, skin oxygenation, and vascular elasticity were measured. RESULTS: Treatment with VIAject resulted in a significant reduction in the peak postprandial generation of ADMA compared with human insulin and insulin lispro (VIAject -27.3 +/- 22.6, human insulin 97.7 +/- 24.4, and insulin lispro 66.9 +/- 33.9 nmol/l; P < 0.05, respectively). The postprandial increases in nitrotyrosine levels were significantly less after VIAject than after human regular insulin (VIAject -0.22 +/- 0.17 vs. human insulin 0.25 +/- 0.15 microg/ml; P < 0.05), whereas nitrotyrosine after insulin lispro was in between (insulin lispro 0.09 +/- 0.07 microg/ml; NS). In parallel, earlier and more pronounced increases in microvascular blood flow and skin oxygenation were obtained after VIAject compared with those after human insulin or insulin lispro (P < 0.05, respectively). All insulin formulations resulted in comparable improvements in central arterial elasticity. CONCLUSIONS; Treatment with VIAject reduced postprandial oxidative stress and improved endothelial function compared with human regular insulin or insulin lispro.

Reliability of lightguide spectrophotometry (O2C) for the investigation of skin tissue microvascular blood flow and tissue oxygen supply in diabetic and nondiabetic subjects.

BACKGROUND AND AIMS: Skin microvascular assessment has progressed to an important evaluation in patients with diabetes mellitus. This study was done to evaluate a new device using micro-lightguide spectrophotometry in the assessment of skin microvascular function. MATERIAL AND METHODS: Twenty nondiabetic subjects (age 46.6 +/- 14.8 years; mean +/- SD) and 20 diabetic patients (age 59.4 +/- 8.4 years) participated in repeated microvascular measurements using micro-lightguide spectrophotometry. This technique allows simultaneous, noninvasive measurement of microvascular blood flow and hemoglobin oxygenation (SO(2)) at the same anatomical area in different tissue layers. A skin probe was placed on nonhairy skin at the thenar eminence of the left hand for the measurement of SO(2), and the postischemic reactive hyperemia response (PRH) was measured in skin and underlying muscle tissue. RESULTS: Repeated measurements in PRH revealed a good correlation at the superficial skin layer (r = 0.97, p < 0.0001) with a coefficient of variation at 9.2 +/- 1.7% and at the superficial muscle layer (r = 0.80, p < 0.0002) with a coefficient of variation at 9.7 +/- 1.5%. A slightly weaker correlation was observed for the SO(2) measurement at the skin layer (r = 0.69 +/- p < 0.0001) with a coefficient of variation at 17.5 +/- 3.8% and at the muscle layer (r = 0.48; p = 0.0016) with a coefficient of variation at 18.1 +/- 10.5%. CONCLUSIONS: Lightguide spectrophotometry is an easy, noninvasive, and reliable method for simultaneous measurement of superficial microvascular blood flow by laser Doppler fluxmetry and skin oxygenation by spectrophotometry. Further studies are required to clarify the validity of these measures in special patient populations such as diabetes mellitus with specified microvascular complications.