Aldana Trabucchi

Detection and characterization of ZnT8 autoantibodies could help to screen latent autoimmune diabetes in adult-onset patients with type 2 phenotype

Autoantibodies to zinc transporter 8 (ZnT8A) constitute an additional marker of autoimmune diabetes, complementing those already used in diagnosis support. ZnT8A could also be found in latent autoimmune diabetes of adults (LADA). The aim of this study was to evaluate the prevalence of ZnT8A in adult-onset diabetic patients in Argentinian population. A total of 271 patients diagnosed for diabetes at mean age 53.4 ± 10.9, body mass index ≤ 30, without insulin treatment for the first year of disease, and initially classified as type 2 diabetic patients were tested for ZnT8A using cDNA plasmids encoding the C-terminal domains (aa 268–369) carrying 325Arg, 325Trp, and a dimeric cDNA construct carrying both 325Arg and 325Trp (ZnT8 Arg–Trp325). We also analyzed proinsulin autoantibodies (PAA), glutamic acid decarboxylase autoantibodies (GADA), and protein tyrosine phosphatase IA-2 autoantibodies (IA-2A). A subset of 101 patients was followed during 6 years in order to analyze insulin requirement. Out of the 271 patients, 22.1% presented at least one humoral marker, 2.6% were PAA+, 12.5% were GADA+, 3.3% were IA-2A+, and 10.7% were ZnT8A+. Among the latter, 7.0% were ZnT8A–Arg325, 51.7% were ZnT8A–Trp325, and 62.1% were ZnT8A–Arg–Trp325. Furthermore, the prevalence of autoantibodies in the group of patients treated with insulin (n = 18) was 55.6%. These results demonstrated that a significant proportion of autoimmune adult-onset diabetic patients presented ZnT8A as the only humoral marker. Between them, the higher prevalence was for ZnT8A–Trp325. We suggest that screening for LADA patients, best performed with a minimal set of marker determination, must include at least the screening of GADA and ZnT8A–Arg–Trp325.

Characterization of Insulin Antibodies by Surface Plasmon Resonance in Two Clinical Cases: Brittle Diabetes and Insulin Autoimmune Syndrome

In this study, the characterization of insulin (auto)antibodies has been described, mainly in terms of concentration (q), affinity (Ka) and Ig (sub)isotypes by Surface Plasmon Resonance (SPR) in two particular clinical cases of individuals with severe episodes of impaired glycemia. Subject 1 suffers from brittle diabetes associated with circulating insulin antibodies (IA) due to insulin treatment. Subject 2 has insulin autoantibodies (IAA) associated with hypoglycemia in spite of not being diabetic and not having ever received exogenous insulin therapy. After conventional screening for IA/IAA by radioligand binding assay (RBA), we further characterized IA/IAA in sera of both patients in terms of concentration (q), affinity (Ka) and Ig (sub)isotypes by means of SPR technology. In both cases, q values were higher and Ka values were lower than those obtained in type 1 diabetic patients, suggesting that IA/IAA:insulin immunocomplexes could be responsible for the uncontrolled glycemia. Moreover, subject 1 had a predominat IgG1 response and subject 2 had an IgG3 response. In conclusion, SPR technology is useful for the complete characterization of IA/IAA which can be used in special cases where the simple positive/negative determination is not enough to achieve a detailed description of the disease fisiopathology.

Surface Plasmon Resonance Reveals a Different Pattern of Proinsulin Autoantibodies Concentration and Affinity in Diabetic Patients

Type 1 diabetes mellitus (DM) is characterized by autoimmune aggression against pancreatic beta cells resulting in absolute deficiency of insulin secretion. The first detectable sign of emerging autoimmunity during the preclinical asymptomatic period is the appearance of diabetes-related autoantibodies. In children at risk for type 1 DM, high-affinity Insulin autoantibodies reactive to proinsulin, are associated with diabetes risk. Autoantibodies are usually measured by radioligand binding assay (RBA) that provides quasi-quantitative values reflecting potency (product between concentration and affinity) of specific autoantibodies. Aiming to improve the characterization of the specific humoral immune response, we selected surface plasmon resonance (SPR) as an alternative method to measure proinsulin autoantibodies (PAA). This novel technology has allowed real time detection of antibodies interaction and kinetic analysis. Herein, we have employed SPR to characterize the PAA present in sera from 28 childhood-onset (mean age 8.31±4.20) and 23 adult-onset diabetic patients (≥65 years old, BMI<30) in terms of concentration and affinity. When evaluating comparatively samples from both groups, childhood-onset diabetic patients presented lower PAA concentrations and higher affinities (median 67.12×10−9 M and 3.50×107 M−1, respectively) than the adults (median 167.4×10−9 M and 0.84×107 M−1, respectively). These results are consistent with those from the reference method RBA (Standard Deviation score median 9.49 for childhood-onset group and 5.04 for adult-onset group) where the binding can be directly related to the intrinsic affinity of the antibody, suggesting that there is a different etiopathogenic pathway between both types of clinical presentation of the disease. This technology has shown to be a useful tool for the characterization of PAAs parameters as an alternative to radioimmunoassay, with high versatility and reproducibility associated to low occupational and environmental risk. However, this technology is not eligible for routine marker screening, but this is a powerful technique for a fine description of the thermodynamic parameters of antigen-antibody interaction.

Characterization of zinc transporter 8 (ZnT8) antibodies in autoimmune diabetic patients from Argentinian population using monomeric, homodimeric, and heterodimeric ZnT8 antigen variants

OBJECTIVE In order to gain further knowledge of the structure of zinc transporter 8 (ZnT8) epitopes, we studied the role of the amino acid at position 325 in the antigen and its dimeric conformation for autoantibodies to ZnT8 (ZnT8A) recognition. METHODS For this purpose, several ZnT8 C-terminal domain variants were designed: monomer carrying Arg325 or Trp325, homo-dimers ZnT8-Arg-Arg325 and ZnT8-Trp-Trp325, and hetero-dimer ZnT8-Arg-Trp325. Two groups of Argentinian diabetic patients were subjected to analysis using [(35)S]-ZnT8 variants by radioligand binding assay (RBA): i) 100 new-onset, insulin-dependent, type 1 diabetic patients and ii) 282 slowly progressing to insulin requirement, non-obese adult-onset diabetic patients. In addition, 50 type 1 diabetic patients and 100 normal control sera provided by the American Diabetes Association (ADA) were evaluated in order to calculate the sensitivity and specificity of ZnT8A assays for each antigenic variant. Other routine β-cell autoantibodies were also tested by RBA. RESULTS Of the 100 Argentinian type 1 diabetic patients, 65 were ZnT8A+. Out of them, 8 patients recognized all recombinant forms of ZnT8 and most patients (56) reacted against the heterodimer. Additionally, out of 282 non-obese adult-onset diabetic patients 46 were ZnT8A+, whereas 29 patients recognized only dimers. Besides, exclusive reactivity against ZnT8A was found in 9.0% for type 1 diabetes mellitus and 10.3% for non-obese adult-onset diabetic patients. CONCLUSIONS Significantly higher signal values in RBA were obtained with the heterodimeric variant. An increased detection of humoral autoimmunity was found in both groups when ZnT8A was employed in combination with the other β-cell autoantibodies. The inclusion of homodimeric immunoreactive peptides revealed the existence of quaternary structure-defined epitopes probably resembling the actual state of the autoantigen in vivo. Finally, the differential profiles of ZnT8A exhibited by type 1 and non-obese adult-onset diabetic patients suggest the different nature of autoimmune processes underlying both pathologies.

The catechin flavonoid reduces proliferation and induces apoptosis of murine lymphoma cells LB02 through modulation of antiapoptotic proteins

Flavonoids are products of secondary metabolism of plants. They are present in herbs and trees and also act as natural chemopreventives and anticancer agents. Ligaria cuneifolia (Ruiz & Pav.) Tiegh., Loranthaceae, is a hemiparasite species that belongs to Argentine flora. Phytochemical studies have disclosed the presence of quercetin, catechin-4β-ol and pro-anthocyanidine as polyphenolic compounds in the active extracts. We previously demonstrated that ethyl acetate extract was capable of reducing cell proliferation and inducing apoptotic death of lymphoid tumor cells. The aim of the current study is to determine whether or not catechin, isolated from L. cuneifolia extracts can induce leukemia cell death and to determine its effect on the cytoplasmatic proteins that modulate cell survival. Our results show that catechin can reduce proliferation of murine lymphoma cell line LB02. The effect is mediated by apoptosis at concentrations upper to 100 µg/mL. Cell death is related to the loss of mitochondrial membrane potential (ΔΨm) and a down regulation of survivin and Bcl-2 together with the increase of pro-apoptotic protein Bax. In summary, the current study indicates that catechin present in the extract of L. cuneifolia is in part, responsible for the anti-proliferative activity of whole extracts by induction of ΔΨm disruption and modulation of the anti-apoptotic proteins over expressed in tumor cells. These results give new findings into the potential anticancer and chemopreventive activities of L. cuneifolia.

Biochemical, biophysical, and functional properties of ICA512/IA-2 RESP18 homology domain

BACKGROUND ICA512 (or IA-2/PTPRN) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Previous studies implied its involvement in generation, cargo storage, traffic, exocytosis and recycling of insulin secretory granules, as well as in β-cell proliferation. While several ICA512 domains have been characterized, the function and structure of a large portion of its N-terminal extracellular (or lumenal) region are unknown. Here, we report a biophysical, biochemical, and functional characterization of ICA512-RESP18HD, a domain comprising residues 35 to 131 and homologous to regulated endocrine-specific protein 18 (RESP18). METHODS Pure recombinant ICA512-RESP18HD was characterized by CD and fluorescence. Its binding to insulin and proinsulin was characterized by ELISA, surface plasmon resonance, and fluorescence anisotropy. Thiol reactivity was measured kinetically. Targeting of ΔRESP18HD ICA512-GFP to the membrane of insulinoma cells was monitored by immunofluorescence. RESULTS ICA512-RESP18HD possesses a strong tendency to aggregate and polymerize via intermolecular disulfide formation, particularly at pH>4.5. Its cysteine residues are highly susceptible to oxidation forming an intramolecular disulfide between cysteine 53 and 62 and intermolecular disulfides via cysteine 40 and cysteine 47. The regulated sorting of ICA512 to secretory granules in INS-1 cells was impaired by deletion of RESP18HD. ICA512-RESP18HD binds with high-affinity to insulin and proinsulin. CONCLUSIONS RESP18HD is required for efficient sorting of ICA512 to secretory granules. GENERAL SIGNIFICANCE RESP18HD is a key determinant for ICA512 granule targeting.