Ruben F. Iacono

Combined measurement of diabetes mellitus immunological markers: an assessment of its benefits in adult-onset patients.

The convenience of combining the measurement of antibodies to glutamic acid decru·boxylase (GADA), protein tyrosine phosphatase (IA-2A), and autoantibodies to insulin (IAA) in diabetic patients was assessed. We analysed 71 type l and 11 5 adult-onset diabetic patient. The latter were grouped into three categories according to the time of evolution to insulin dependence. The main findings were as follows: (i) in type I diabetes, the combined analysis of GADA and IA-2A showed a sensitivity of 87.4% and was not appreciably improved by adding IAA; (ii) out of 31 adults who required insulin immediately or wi thin the first two years of diagnosis, 41.9, 29.0, and 6.5% were positive for at least one, two or all three, and all three markers, respectively; GADA was the most prevalent (35.5%) and IA-2A the least represented (16.1%); (iii) 34 adult patients with slow evolution to insulin dependence bowed a completely different profile: 5.9% were GADA positive and 23.5% were IAA positive and no double or triple positivity was observed as all patients were IA-2A negative; and (iv) 50 type 2 patients who had not required insulin treatment showed a low incidence of GADA (4%) as the only marker present. We conclude that a combined double-antigen test for GADA and TA-2A is a useful strategy for prospective screening of type I diabetes. However, in adults, the profile of individual markers di scloses the course to insulin dependence. There fore, it seems advisable to measure the markers separately, to allow a better classification of these patients, and help define their treatment.

A radioligand-binding assay for detecting antibodies specific for proinsulin and insulin using 35S-proinsulin

A new radioligand-binding assay (RBA) is described for the detection of insulin/proinsulin-specific antibodies using 35S-labeled proinsulin produced by a cell-free reticulocyte extract. Direct use of the crude expression product in the RBA was not feasible because the protein failed to fold properly (or had incorrectly paired disulphide bridges) and purification was hindered by interfering by-products. A refolding protocol and a chromatographic procedure were devised that readily allowed production of purified and immunochemically competent 35S-labeled proinsulin. The new RBA was compared with the reference test, in which the tracer was standard 125I-insulin. The analysis included sera from 41 diabetic patients and 25 healthy controls. Twenty-six (63.4%) and 29 (70.7%) patients scored positive by RBA using 35S-PI and 125I-insulin, respectively. The methods showed a satisfactory correlation with r(2)=0.77 and a slope not significantly different from unity (m=1.16+/-0.10; 95% confidence interval). Since the nuclide used in the assay is 35S, the procedure is compatible with standard assays for GADA and IA-2A, and thus may permit combined assays for the major early markers of autoimmune diabetes.

Glial fibrillary acidic protein (GFAP) immunochemical profile after Junin virus infection of rat cultured astrocytes

Cultured astrocytes derived from newborn rat brain were inoculated with Junin virus (JV) to characterize their response to infection by means of their glial fibrillary acidic protein (GFAP) immunochemical profile. Samples from 1 to 11 days post-inoculation (pi), as well as matched controls, were serially harvested for GFAP labeling by peroxidase-antiperoxidase (PAP) method. It was only at day 3 that significantly greater values of GFAP staining (P < 0.05) were disclosed by three complementary approaches: image analysis, ELISA and immunoblot densitometry. Since such increase was abolished by Triton X-100 treatment, soluble GFAP fraction appeared responsible for the early though transient enhancement of GFAP immunoreactivity that followed viral inoculation.

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.

Long-lasting astrocyte reaction to persistent Junin virus infection of rat cortical neurons.

Summary. Immunoperoxidase labeling was performed in histological sections from rat brain harvested during acute (10–30 days), clinically inapparent (90–270 days) and late (450–540 days) stages of Junin virus-induced neurological disease. In frontoparietal cortex, count of viral antigen (+) neurons peaked during the acute period (27.7±6.8), dropped within the intermediate (4.8±4.0 to 1.4±1.1) and increased (7.6±4.3) at the onset of the late neurological syndrome. In infected vs. control rats, the number of GFAP (+) astrocytes maximized during the acute stage (19±4 vs. 11±5), and from the end of the intermediate (27±5 vs. 21±5) up to the late (37±7 vs. 26±6) periods. In turn, surface density of GFAP (+) material in infected samples peaked at 0.196±0.066, while it failed to exceed 0.090±0.043 in controls. Both astrocyte hypertrophy relapsing into chronicity, as depicted by surface density, and astrocyte hyperplasia preceding the onset of the late neurological syndrome, support their pathogenic contribution to disease expression.

Transplacental infection in guinea pigs inoculated with an attenuated strain of Junin virus.

Transplacental infection by the attenuated XJC13 strain of Junin virus (JV) in the guinea pig model was evaluated. 5 pregnant guinea pigs were infected intramuscularly at 45 +/- 3 days of pregnancy. 4 animals were killed at 14 days postinfection (p.i.), and 1 was sacrificed at 137 days p.i. at the end of its second pregnancy. Evidence of JV was obtained by Vero cell cocultivation in all 14 fetuses harvested (brain and/or spleen) and in 10 of 11 placentas. The results strongly suggest that the attenuated JV strain infected the fetus by the transplacental route, as previously demonstrated for the pathogenic XJ strain. Despite limited sampling, the acute as well as the chronic stage proved viable.

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.

Detection and immunochemical characterization of glutamic acid decarboxylase autoantibodies in patients with autoimmune diabetes mellitus

Since GAD65 undergoes post-translational processing and targeting to subcellular compartments and membranes, it may exhibit different immunochemical properties in the cell context compared with the soluble protein expressed in the cell-free eukaryotic system used in the reference method for GADA assessment (radioligand binding assay (RBA)). In the present work, we detected and characterized GADA in 72 sera from patients with type 1 diabetes mellitus (DM) and 14 sera from adult-onset diabetes patients using analytical systems in which GAD65 is expressed in a cellular context: confocal indirect immunofluorescence (IIF) and electron microscopy after immunogold labeling on monolayers of transfected Chinese hamster ovary (CHO) cells, and immunoprecipitation (IP) of metabolically labeled GAD65. Eighteen serum samples, 16 from type 1 diabetes patients and two from adult-onset diabetes patients, were positive by confocal IIF but scored negative by RBA. All of these 18 sera immunoprecipitated a 65 kDa protein, supporting the existence of the GADA marker in such patients. It may be concluded that GADA negativity by the conventional RBA method using the soluble antigen, as well as negativity for other common markers measured by similar methods, is not enough to rule out the existence of the specific autoimmune component in childhood or adult-onset diabetes. Other analytical methods based in a more physiological presentation of the autoantigen structure, as confocal IIF and IP, bring an extra support to assess the complete repertoire of specific autoantibodies to native-like and membrane-bound, or denatured, β-cell antigens.

Replacement of methionine‐161 with threonine eliminates a major by‐product of human glutamate decarboxylase 65‐kDa variant expression in Escherichia coli

Most insulin‐dependent diabetes mellitus patients gen‐erate conformational autoantibodies to the islet‐cell 65‐kDa variant of human glutamate decarboxylase (GAD65), and several immunochemical tests for the early detection of type‐1 diabetes rely on GAD65 antibody (GADA) assessment using properly folded recombinant GAD65 as the antigen. In addition, preventive therapies based on tolerization by GAD65 administration may be available in the near future. Therefore, there exists a strong interest in a facile and economically sound expression procedure for this antigen. Several attempts to produce, in native form, wild‐type GAD65 in Escherichia coli have failed. However, this difficulty was recently surmounted in our laboratory by expressing GAD65 as a fusion protein with thioredoxin [Papouchado, Valdez, Ghiringhelli, Poskus and Ermácora (1997) Eur. J. Biochem. 246, 350–359]. In this work, a new GAD65 hybrid gene was prepared by joining engineered cDNA obtained from human and rat tissues. The new gene was modified additionally to finally code for human GAD65 with a single amino‐acid substitution: Met‐161→Thr. This change impeded the co‐expression of a 48‐kDa by‐product from an internal translation site. Also, a second 58‐kDa by‐product was identified as a GAD65 C‐terminal proteolytic fragment that co‐purifies with thioredoxin–M161T GAD65. The new GAD65 variant was expressed and easily purified, yielding an antigen that performed equally or better than wild‐type GAD65 in the reference radiobinding assay for GADA. The procedure provides an inexpensive source of large amounts of fully active and immunochemically competent GAD65.