Advanced glycation end-products (AGEs) contribute to vascular complications and organ damage in diabetes. The unique AGE epitope (AGE10) has recently been identified in human serum using synthetic melibiose-derived AGE (MAGE). We aimed at developing ELISA for AGE10 quantification, determining whether AGE10 is present in diabetic patients (n = 82), and evaluating its association with diabetic complications. In a competitive ELISA developed, the reaction of synthetic MAGE with anti-MAGE was inhibited by physiological AGE10 present in serum. In this assay, new murine IgE anti-MAGE monoclonal antibodies, which do not recognize conventional AGEs, a synthetic MAGE used to coat the plate, and LMW-MAGE (low molecular mass MAGE) necessary to plot a standard curve were used. AGE10 was significantly higher in patients with microangiopathy, in whom it depended on treatment, being lower in patients treated with aspirin. AGE10 levels were positively correlated with estimated glomerular filtration rate (eGFR) and negatively with creatinine. As a marker of stage ≥3 chronic kidney disease or microangiopathy, AGE10 displayed moderate overall accuracy (respectively, 69% and 71%) and good sensitivity (82.6% and 83.3%) but poor specificity (58.1% and 57.8%). In conclusion, newly developed immunoassay allows for AGE10 quantification. AGE10 elevation is associated with microangiopathy while its decrease accompanies stage ≥3 chronic kidney disease.

Matrix metalloproteinase 2 (MMP-2) is activated in hearts upon ischemia-reperfusion (IR) injury and cleaves sarcomeric proteins. It was shown that carvedilol and nebivolol reduced the activity of different MMPs. Hence, we hypothesized that they could reduce MMPs activation in myocytes, and therefore, protect against cardiac contractile dysfunction related with IR injury. Isolated rat hearts were subjected to either control aerobic perfusion or IR injury: 25 min of aerobic perfusion, followed by 20 min global, no-flow ischemia, and reperfusion for 30 min. The effects of carvedilol, nebivolol, or metoprolol were evaluated in hearts subjected to IR injury. Cardiac mechanical function and MMP-2 activity in the heart homogenates and coronary effluent were assessed along with troponin I content in the former. Only carvedilol improved the recovery of mechanical function at the end of reperfusion compared to IR injury hearts. IR injury induced the activation and release of MMP-2 into the coronary effluent during reperfusion. MMP-2 activity in the coronary effluent increased in the IR injury group and this was prevented by carvedilol. Troponin I levels decreased by 73% in IR hearts and this was abolished by carvedilol. Conclusions: These data suggest that the cardioprotective effect of carvedilol in myocardial IR injury may be mediated by inhibiting MMP-2 activation.

Human alpha- and beta-enolases are highly homologous enzymes, difficult to differentiate immunologically. In this work, we describe production, purification and properties of anti-alpha- and anti-beta-enolase polyclonal antibodies. To raise antibodies, rabbits were injected with enolase isoenzymes that were purified from human kidney (alpha-enolase) and skeletal muscle (beta-enolase). Selective anti-alpha- and anti-beta-enolase antibodies were obtained by affinity chromatography on either alpha- or beta-enolase-Sepharose columns. On Western blots, antibodies directed against human beta-enolase, did not react with human alpha-isoenzyme, but recognized pig and rat beta-enolase. To determine what makes these antibodies selective bioinformatic tools were used to predict conformational epitopes for both enolase isoenzymes. Three predicted epitopes were mapped to the same regions in both alpha- and beta-enolase. Peptides corresponding to predicted epitopes were synthesized and tested against purified antibodies. One of the pin-attached peptides representing alpha-enolase epitope (the C-terminal portion of the epitope 3 - S(262)PDDPSRYISPDQ(273)) reacted with anti-alpha-enolase, while the other also derived from the alpha-enolase sequence (epitope 2 - N(193)VIKEKYGKDATN(205)) was recognized by anti-beta-enolase antibodies. Interestingly, neither anti-alpha-nor anti-beta-antibody reacted with a peptide corresponding to the epitope 2 in beta-enolase (G(194)VIKAKYGKDATN(206)). Further analysis showed that substitution of E-197 with A in alpha-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A(198) with E in peptide representing beta-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E-197 is essential for preserving immunologically active conformation in epitope 2 peptidic homolog, while it is not crucial for this epitope's antigenic activity in native beta-enolase.