Autoantibodies and high-risk HLA susceptibility markers in first-degree relatives of Brazilian patients with type 1 diabetes mellitus: a progression to disease based study.

PURPOSE: The objective of this study was to determine the frequencies of autoantibodies to heterogeneous islet-cell cytoplasmic antigens (ICA), glutamic acid decarboxylase(65) (GAD(65)A), insulinoma-associated antigen-2 (IA-2A) and insulin (IAA)-and human leukocyte antigen (HLA) class II markers (HLA-DR and -DQ) in first degree relatives of heterogeneous Brazilian patients with type I diabetes (T1DM). A major focus of this study was to determine the influence of age, gender, proband characteristics and ancestry on the prevalence of autoantibodies and HLA-DR and -DQ alleles on disease progression and genetic predisposition to T1DM among the first-degree relatives. METHODS: IAA, ICA, GAD(65)A, IA-2A and HLA- class II alleles were determined in 546 first-degree-relatives, 244 siblings, 55 offspring and 233 parents of 178 Brazilian patients with T1DM. RESULTS: Overall, 8.9% of the relatives were positive for one or more autoantibodies. IAA was the only antibody detected in parents. GAD(65) was the most prevalent antibody in offspring and siblings as compared to parents and it was the sole antibody detected in offspring. Five siblings were positive for the IA-2 antibody. A significant number (62.1%) of siblings had 1 or 2 high risk HLA haplotypes. During a 4-year follow-up study, 5 siblings (expressing HLA-DR3 or -DR4 alleles) and 1 offspring positive for GAD(65)A progressed to diabetes. CONCLUSIONS: The data indicated that the GAD(65) and IA-2 antibodies were the strongest predictors of T1DM in our study population. The high risk HLA haplotypes alone were not predictive of progression to overt diabetes.

Synthesis and applications of phosphonoacetic derivatives.

In this paper, the synthesis of new phosphonoacetic acid derivatives and their applications in fields of biotechnological interest are discussed. Phosphonoacetic acids are competitive inhibitors of alkaline phosphatase, an enzyme widely used in diagnostics, as colorimetric detection tool. The phosphonoacetic acid's inhibition activity has been exploited by us for the obtainment of an innovative technique for non-radioactive DNA probes detection, the last being based on DNA labeling with the enzyme inhibitor, followed by detection by means of the chromogenic enzyme and substrate. Moreover, we have found a further application of phosphonoacetic acids, by the preparation of an affinity chromatography support that has been revealed to be very effective in the purification of alkaline phosphatase. Finally, phosphonoacetic acid derivatives have been tested also for their antiviral activity. Some of them, examined in preliminary in vitro experiments, have been found very active against Herpes simplex virus.

Alkaline phosphatase inhibitors as labels of DNA probes.

A new approach to nucleic acid labeling was developed by preparing bifunctional reagents containing, in addition to the DNA-linking group, a competitive inhibitor of the chromogenic enzyme alkaline phosphatase. The nucleic acids labeled in such a way were able to bind themselves to the enzyme, whose activity was restored in the presence of a chromogenic substrate. Five phosphonic-acid-containing reagents were synthesized and coupled to linearized pBR322 plasmid DNA by different condensation methods. Eight probes thus obtained were assayed in a modified dot-blot detection procedure obtaining the best nucleic acid detection sensitivity of 25 pg. Finally, five of the above probes were tested in hybridization experiments, reaching sensitivity of 50 pg.