Activity of the glycosylating enzyme, core 2 GlcNAc (beta1,6) transferase, is higher in polymorphonuclear leukocytes from diabetic patients compared with age-matched control subjects: relevance to capillary occlusion in diabetic retinopathy

The exact mechanism for capillary occlusion in diabetic retinopathy is still unclear, but increased leukocyte-endothelial cell adhesion has been implicated. We examined the possibility that posttranslational modification of surface O-glycans by increased activity of core 2 transferase (UDP-Glc:Galbeta1-3GalNAcalphaRbeta-N-acetylglucoaminyltr ansferase) is responsible for increased adhesion of leukocytes to vascular endothelium in diabetes. The mean activity of core 2 transferase in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients was higher compared with age-matched control subjects (1,638 +/- 91 [n = 42] vs. 249 +/- 35 pmol x h(-1) x mg(-1) protein [n = 24], P = 0.00013; 1,459 +/- 194 [n = 58] vs. 334 +/- 86 [n = 11], P = 0.01). As a group, diabetic patients with retinopathy had significantly higher mean activity of core 2 transferase compared with individuals with no retinopathy. There was a significant association between enzyme activity and severity of retinopathy in type 1 and type 2 diabetic patients. There was a strong correlation between activity of core 2 transferase and extent of leukocyte adhesion to cultured retinal capillary endothelial cells for diabetic patients but not for age-matched control subjects. Results from transfection experiments using human myelocytic cell line (U937) demonstrated a direct relationship between increased activity of core 2 transferase and increased binding to cultured endothelial cells. There was no relationship between activity of core 2 transferase and HbA(1c) (P = 0.8314), serum advanced glycation end product levels (P = 0.4159), age of the patient (P = 0.7896), and duration of diabetes (P = 0.3307). On the basis that branched O-glycans formed by the action of core 2 transferase participate in leukocyte adhesion, the present data suggest the involvement of this enzyme in increased leukocyte-endothelial cell adhesion and the pathogenesis of capillary occlusion in diabetic retinopathy.

Evaluation of a new commercial enzyme-linked immunosorbent assay for the detection of porcine antibodies against Trichinella spp

Trichinellosis is a zoonotic disease that is caused by the nematode Trichinella spp. Both European Union regulations and guidelines from the World Organization for Animal Health foresee the possibility of conducting serological surveillance for Trichinella spp. A newly developed commercial enzyme-linked immunosorbent assay (ELISA) was evaluated against 2 existing diagnostic techniques: an in-house ELISA and an in-house Western blot. A total of 875 Trichinella larva-negative samples of pigs and 93 Trichinella larva-positive samples of both naturally and experimentally infected pigs were included in the study. Bayesian modeling techniques were used to correct for the absence of a perfect reference test. The sensitivity and specificity of the commercial ELISA was 97.1-97.8% and 99.5-99.8%, respectively. Sensitivity analysis demonstrated high stability in the models. In a serological surveillance system, ELISA-positive samples should be tested by a confirmatory test. The Western blot is a suitable test for this purpose. With the use of the results of the models, the sensitivity and specificity of a test protocol in both ELISA and Western blot were 95.9% and 99.9%, respectively. The high sensitivity and specificity were achieved with a lower limit of detection than that of the routine artificial digestion test, suggesting that serological surveillance is a valuable alternative in surveillance for Trichinella spp. in pig production.