Hypoglycemia and hyperglycemia due to insulin antibodies against therapeutic human insulin: treatment with double filtration plasmapheresis and prednisolone.

Two diabetic patients, who had been treated with human insulin, suffered from fasting hypoglycemia and postprandial hyperglycemia. The insulin-binding capacities of their sera were high, and a large amount of total insulin and prolonged presence of free insulin in the sera were shown. Scatchard analysis of these insulin antibodies revealed that high-affinity insulin antibodies had larger capacity and stronger affinity compared with commonly insulin-treated patients. Treatment with double filtration plasmapheresis and subsequent administration of prednisolone in the second patient reduced such antibodies and resulted in recovery of glycemic control by insulin. Hypoglycemia and hyperglycemia could be incurred when insulin antibodies with strong affinity and high capacity in high-affinity sites arise. This condition can be treated with double filtration plasmapheresis and subsequent administration of prednisolone.

Effective use of gamma irradiation for pathogen inactivation of monoclonal antibody preparations.

Gamma irradiation has been used for decades as an effective method of pathogen inactivation of relatively inert materials. Until recently, its application to biologicals has resulted in unacceptable losses in functional activity. In this report we demonstrate that the damaging secondary effects of gamma irradiation can be controlled while maintaining the pathogen inactivation properties due to damage by primary effects. Control is achieved by a combination of protection from free radical damage to a monoclonal antibody through the use of the antioxidant ascorbate and by freeze-drying to minimize the potential for generating free radicals. The data demonstrate a synergy of these two approaches that results in quantitative recovery of functional activity while maintaining the ability to inactivate greater than 5 logs of porcine parvovirus infectivity.

Hipoglucemia por hiperinsulinismo endógeno autoinmune. A propósito de un caso y revisión de la bibliografía / Hypoglycemia due to endogenous hyperinsulinism. Case report and review of the literature

La hipoglucemia por hiperinsulinismo endógeno tiene su origen principal en los tumores pancreáticos y extrapancreáticos. En los últimos años asistimos al incremento de las causas autoinmunes, cuya etiología reconoce la presencia de anticuerpos específicos dirigidos contra la insulina, su receptor, e incluso la propia célula beta. Son producidos en el curso de otras enfermedades autoinmunes y están relacionados con las mismas y/o con fármacos empleados en su tratamiento (destacando aquellos que contienen el grupo sulfidrilo), que alterarían de algún modo la molécula de insulina confiriéndole mayor inmunogenicidad y disminuyendo, a la vez, su actividad biológica. El escaso número de publicaciones en la bibliografía mundial, y el hecho de que la gran mayoría se haya descrito en razas orientales, así como su fuerte correlación con determinado sistema HLA, sugieren la existencia de un componente genético predisponente. Describimos un caso de hipoglucemia por hiperinsulinismo endógeno, en el que descartamos origen tumoral y hallamos valores elevados de insulina y péptido C junto con una alta tasa de anticuerpos antiinsulina, coincidiendo con el tratamiento con metimazol para una enfermedad de Graves, cuadro que mejora espontáneamente y desaparece tras suprimir el fármaco. Aunque coincide con el cuadro de síndrome autoinmune de insulina descrito inicialmente por Hirata, existen particularidades que le confieren especial interés: por una parte, la etnia y un sistema HLA diferente del referido por dicho autor; por otra, una tasa de insulina libre muy elevada si la comparamos con la hallada en la mayoría de los casos y, por último, la coexistencia de anticuerpos anti-GAD que permanecen positivos tras la normalización de los parámetros clínicos y biológicos, y que sugieren la posibilidad de aparición futura de una (AU)

A direct two-site peroxidase-based enzyme-linked immunosorbent assay for proinsulin.

A non-competitive sandwich assay using an anti-C-peptide IgG and an anti-insulin IgG was developed to measure fasting levels of proinsulin in human serum. The former antibody provided the lower layer in a sandwich immunoassay, the upper layer being composed of an anti-insulin IgG-horse radish peroxidase conjugate. The assay showed negligible cross reactivity at supraphysiological levels of insulin and C-peptide. The method enabled the estimation of proinsulin in fasting non-diabetic control subjects [13.7 +/- 1.6(4) pM] and in type 2 non-insulin-dependent diabetic patients [23.2 +/- 1.1(8) pM].

Insulin antibodies before and 1 year after the change-over from U40 to U100 insulin preparations in Belgium.

In 1991, Belgium realized, on a national level, a change-over from U40 to U100 insulin. We took advantage of this evolution to investigate the consequences of changing the concentration of insulin. The patients' weight, daily insulin dosis, insulin binding-capacity of plasma and glycated hemoglobin HbA1c were registered before, and after the change of concentration. Overall, none of these parameters underwent an obvious change, except for the percentage of insulin binding that significantly decreased after the adaptation. Especially in the range of 40% or more insulin binding, the decrease becomes very pronounced. In conclusion, changing the insulin concentration from U40 to U100, did not lead to any harmful clinical consequence. On the contrary, a positive influence of this adaptation, in terms of decreased amount of insulin antibodies was suggested. Probably this decrease is not clinically relevant, since neither the glycated hemoglobin, nor the total daily insulin dose underwent a similar change.

An unusual case of recurrent hypoglycaemia: 10-year follow up of a child with insulin auto-immunity.

We report the case of a child with hypoglycaemia due to insulin auto-immunity. Insulin auto-immunity is the third most frequent cause of hypoglycaemia in Japan where the first cases were described. The child has been followed for the past 10 years with recurrence of hypoglycaemic symptoms and high titres of insulin antibodies.

64,000 Mr autoantibodies as predictors of insulin-dependent diabetes.

The occurrence of autoantibodies to an islet-cell protein of 64,000 Mr (64KA) was examined in relation to development of insulin-dependent diabetes. 64KA were absent in 26 normal controls and present in only 1 of 41 first-degree relatives who lacked islet-cell cytoplasmic autoantibodies (ICA) or insulin autoantibodies (IAA). Among first-degree relatives at high risk for IDD, 64KA were identified in 23 of 28 persons positive for ICA and 4 of 5 with IAA but no ICA. Among 31 patients with newly diagnosed IDD, 64KA were found in 26. 64KA were identified in 23 of 28 persons studied up to 75 months before clinical onset of IDD. Of these 23 64KA-positive prediabetic subjects, 5 were ICA-negative and 10 lacked IAA. 64KA were most predictive in those who became diabetic before age 34 (22/24). In several individuals, 64KA were detected before the other autoantibodies appeared. These findings suggest that 64KA may be an early and useful predictive marker for IDD.

Revelation of specificity of 64K autoantibodies in IDDM serums by high-resolution 2-D gel electrophoresis. Unambiguous identification of 64K target antigen.

Antibodies in serums from newly diagnosed insulin-dependent (type I) diabetes mellitus (IDDM) patients and individuals experiencing early phases of beta-cell destruction specifically immunoprecipitate a minor pancreatic islet cell membrane protein of 64,000 Mr (64K). In this report, we demonstrate the use of two-dimensional (2-D) gel electrophoresis to unambiguously identify the 64K antigen. By nonequilibrium pH-gradient gel electrophoresis in the first dimension and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension, the 64K protein separates into two components, designated alpha and beta, that differ in size but display identical charge heterogeneity. The high resolution of the 2-D method efficiently separates the 64K components from background proteins in immunoprecipitates from crude detergent lysates of islets. The background proteins were identified as major cellular proteins carried nonspecifically through the immunoprecipitation procedure. The high affinity and specificity of the 64K autoantibodies were demonstrated by the exclusive and greater than 1000-fold purification of this minor protein by immunoprecipitation with IDDM serums. The 2-D analyses did not reveal additional proteins specifically immunoprecipitated by IDDM serums, suggesting that the 64K protein is the only protein antigen specifically and consistently recognized by IDDM autoantibodies in the relatively stringent conditions of immunoprecipitation. Moreover, the 2-D analyses demonstrate that purification of membrane protein fractions from both human and rat islets before the immunoprecipitation efficiently removes background proteins and substantially increases the specificity of 64K autoantibody measurements by traditional methods.

Antigenicity of the carboxyl terminus of insulin: isolation of human insulin-specific monoclonal antibodies.

Monoclonal technology was used to isolate antibodies binding the B30 (carboxy) terminal residue in the polyclonal-provoked immune response to human insulin. Although both spleen and lymph node cell fusions were carried out, only the latter were successful in isolating monoclonal antibodies that bound the carboxy terminal of human insulin. The binding of such antibodies was abolished or diminished by substitutions of the B30 residue. Studies with insulin species variants showed that the molecular binding between antibody and insulin may be critically determined by a subresidue feature, e.g. presence or absence of a single methyl group, as shown by the binding of the monoclonal antibody D10 to human insulin (threonine at B30) but not to rabbit insulin (serine at B30). Such studies are of interest in the study of the molecular basis of antibody-antigen interaction.

A comparison of the effects of plasma exchange and immunoadsorption on anti-insulin antibody synthesis in rabbits.

Plasma exchange (PE) and ex vivo immunoadsorption (IA) may be applicable to the removal of anti-insulin antibodies (AI-Ab) from diabetic patients. However, the removal of antibodies may prompt an increase in their rate of synthesis and an overshoot of antibody levels which may be deleterious to the patient. The effects of both PE and IA on AI-Ab synthesis were studied in a rabbit model. Rabbits were immunized with insulin and the resulting AI-Abs removed by both plasma exchange and specific immunoadsorption. Following AI-Ab removal by PE no increase in AI-Ab synthesis or antibody overshoot occurred. However a large increase in AI-Ab synthesis and overshoot occurred following specific AI-Ab removal by immunoadsorption. Despite similar reductions in AI-Ab levels by PE and IA, no increase in antibody synthesis occurred due solely to antibody removal. It is likely that antigen released from the immunoadsorbent stimulated the increase in antibody synthesis following immunoadsorption. These findings are relevant to the clinical application of both PE and IA.

Purification of insulin-binding antibody by affinity chromatography using monocomponent insulin as ligand.

Insulin-binding antibody (IBA) was purified by affinity chromatography using porcine monocomponent (MC) insulin as the ligand. The purity of the antibody was compared with that of the antibody extracted using porcine crystalline (Cr) insulin. Comparing the antibody solutions obtained with MC insulin (MC-lig-sol) or Cr insulin (Cr-lig-sol), the content of IBA in Cr-lig-sol was higher than in MC-lig-sol, but the content of proinsulin-binding antibody (PBA) in MC-lig-sol was very small and statistically lower than that in Cr-lig-sol (P less than 0.01). Adding native MC insulin to a competitive radioimmunoassay suppressed the IBA titer obtained with MC insulin more than that obtained with Cr insulin. By adding native proinsulin in a similar assay system, the PBA titer obtained with Cr insulin was suppressed more than that extracted with MC insulin. Scatchard analysis of the 2 solutions showed that the affinity constants of high affinity antibodies were almost identical, but that of low affinity antibody in MC-lig-sol was larger than in Cr-lig-sol. The binding capacity of low affinity antibody in Cr-lig-sol was 15 times as much as that in MC-lig-sol. Using MC insulin, instead of Cr insulin, as the ligand in affinity chromatography increased the purity of recovered IBA. chromatography increased the purity of recovered IBA.

Antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens.

Simple methods for the generation, purification, and assay of antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens have been described. Chicken antibodies against the alpha-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the beta-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.

Failure of ultracentrifugation as a means of separating plasma free insulin from immunoglobulin fraction prior to radioimmunoassay.

It has been reported that ultracentrifugation of plasma will allow direct measurement of free insulin in the serum of diabetic subjects with insulin antibodies. To validate this method, we determined recovery of immunoreactive insulin and immunoglobulin G from the plasma of normal individuals after ultracentrifugation. The upper and middle fractions of plasma after ultracentrifugation were evaluated at several combinations of time and temperature (4 degrees C, 25 degrees C, or 37 degrees C for 3, 4 or 5 hours). None of these conditions effectively removed all immunoglobulin G without causing concomitant loss of insulin. We conclude that ultracentrifugation of plasma prior to radioimmunoassay cannot be used to reliably determine free insulin concentration in the plasma from subjects with circulating insulin antibodies.

Insulin-anti-insulin complexes in diabetic women and their neonates.

It is known that insulin does not cross placenta, whereas maternal anti-insulin antibodies do. We have therefore investigated insulin antibodies and insulin-anti-insulin complexes both in pregnant diabetic women during pregnancy and in umbilical cord blood from their new-born infants. Forty-seven diabetic pregnant women and 23 new-born-infants of these diabetic women were studied. All the pregnant patients were studied at the end of pregnancy and in 27, at least on one other occasion during pregnancy. All the patients were treated with insulin during pregnancy: 26 had Type 1 (insulin-dependent) diabetes, 14 Type 2 (non-insulin-dependent) diabetes and seven had gestational diabetes. Insulin antibodies were found in 62% of the Type 1 diabetic patients, in 71% of the Type 2 diabetic patients and in 43% of the gestational diabetic patients. There were present in 48% of the infants studied. Insulin-anti-insulin complexes were found in 37% of the women with Type 1 diabetes, in 21% of those with Type 2 diabetes and in 14% of those with gestational diabetes. Complexes were found in 38% of the new-born infants. The presence of these complexes in the babies was more strongly correlated with their occurrence in their mothers at the beginning than at the end of pregnancy. Insulin-anti-insulin complexes are thus present in the neonatal circulation. They may differ from those in their mothers and they may have pathophysiological and clinical importance.