Biomedical subjects
Publications and source records attributed to K G Petersen.
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A solid-phase immunoenzymatic technique was modified to permit the detection and enumeration of antibody-secreting cells recognizing the amino or carboxy terminal of the insulin B chain. The procedure involves coating well surfaces with avidin and binding insulins specifically labelled with biotin at the B1 or B30 residue. On day 15 after immunization with human insulin, 20 outbred NMRI mice had generated cells secreting anti-insulin antibodies. The recognition of B1- or B30-related epitopes differed between individuals, suggesting that there was genetic determination of the epitopes expressed early in the immune response. The method can be used to find strains of mice with a preferential immune response to defined areas on the surface of small peptide molecules. Such strains could then be used to produce specific monoclonal antibodies.
Anti-insulin immunoglobulin E (IgE) antibodies in sera of insulin-treated diabetic patients were characterized by affinity and concentration, and by binding to proinsulin and insulin A and B chains. The affinities of IgE to insulin scattered between less than 10(7) and 0.6 x 10(9) l/mol, the concentrations between 0.3 and 3.5 ng/ml. All positive sera of type 1 and few of type 2 patients recognized proinsulin, A chain, and B chain with comparable affinities and concentrations. Elevated concentrations of total serum IgE in 12 of the 16 anti-insulin IgE-positive sera from insulin-treated patients indicate that these patients are predisposed to allergies. The incidence of elevated levels of total serum IgE in type 1 (17.7%) and in type 2 (7.8%) diabetic patients did not differ from the general population. Reagins to insulin and related antigens were also observed in sera of non-diabetic allergic persons without previous contact with exogenous insulin. The natural occurrence of insulin-specific reagins makes the use of IgE as a marker of antigenicity of insulin questionable. Discrepancies between the insulin-positive radioallergosorbent test (RAST) or skin test and clinical manifestations of insulin allergy exist, because IgE antibodies with low affinities require high concentrations of insulin for binding. Such amounts of insulin may occur at the injection site, but not in the circulation.
Two patients with insulin-dependent diabetes mellitus (IDDM) for 42 and 46 years respectively were diagnosed to produce factitious hypoglycemia. They had several properties in common: abnormal personality, refusal to stop incipient hypoglycemia, ideal body weight, good hemoglobin A1c (HbA1c) values, non-specific changes in the EEG, and brain atrophy sparing the periventricular areas. The addiction forced the patients to continue their habit during hospital stay. The dose of surreptitiously injected regular insulin always produced serum insulin concentrations large enough to clarify the diagnosis.
The ability of gamma irradiation to substitute for H2O2 in peroxidase-labelled enzyme immunoassays was investigated. Colouring of TMB (3,3',5,5'-tetramethylbenzidine), ABTS [2,2'-azino-di(3-ethylbenzthiazoline-6-sulphonic acid)], and OPD (o-phenylenediamine) was produced with doses of 10 and 20 Gy. Addition of superoxide dismutase enhanced TMB and ABTS, and inhibited OPD. The reaction was terminated when the irradiation was stopped. The use of denaturing compounds to stop the reaction was unnecessary and coated antigens could be re-used for a second immunoassay.
The hypothesis that in hypoglycaemia adrenoceptor-blocking drugs may enhance those metabolic effects of the catecholamines that remain unblocked has been investigated in 12 volunteers. alpha-Adrenoceptor blockade with urapidil increased the heart rate and the plasma noradrenaline level, and increased the beta-adrenoceptor mediated cellular uptake of potassium and phosphate, and the production of lactate. Posthypoglycaemic glucose intolerance and the counterregulatory responses of hGH and cortisol remained unchanged. Plasma adrenaline, the alpha-adrenoceptor mediated responses of cortisol and hGH, and the diastolic blood pressure were increased by propranolol. Adrenoceptor blocking drugs produce an indirect stimulatory effect by eliciting a reflex increase in sympathetic tone, which is manifested as stimulation of receptors of the type that has not been blocked.
Antibodies to GHRH1-44, GHRH1-29, and proinsulin were induced in guinea pigs. GHRH1-44 forms 7 S and 10 S complexes with antibodies. It is a divalent antigen. The sequence 30-44 bound 85% of the antibodies to GHRH1-44 with high affinity (3.8 +/- 0.9 x 10(9) l/mol). The fragment 1-29 bound with low affinity (0.6 +/- 0.3 x 10(9) l/mol) 15% of the antibodies (2p less than 0.001). Antibodies to GHRH1-29 had low affinity towards the native hormone (0.4 +/- 0.2 x 10(9) l/mol) and the region 1-29 (0.3 +/- 0.2 x 10(9) l/mol). Antibodies to proinsulin bound linear C-peptide with lower affinity (0.3 +/- 0.2 x 10(9) l/mol) than the C-peptide loop in proinsulin (3.4 +/- 0.9 x 10(9) l/mol). It is concluded that the conformation of the epitopes on the sequence 1-29, recognized during the immune response, i.e. on the cell membrane, is different from the conformation of GHRH1-29 or GHRH1-44 in aqueous solution.
IgE antibodies to peptide hormones or their fragments were investigated using specific radioallergosorbent tests. Ninety-six sera were obtained from otherwise healthy individuals with a positive RAST to at least one common allergen and without previous contact to exogenous hormones. Total serum IgE was normal (less than 300 ng ml-1) in 41 and elevated in 55 sera. Only one serum with normal total IgE had reagins to proinsulin. In sera with elevated total IgE positive tests (greater than 8 x background) were observed frequently: hGH 9.1%, GHRH1-44 5.5%, GHRH1-29 12.7%, erythropoietin 10.9%, human calcitonin 14.6%, salmon calcitonin 1.8%, ACTH 5.5%, insulin 14.6%, proinsulin 7.3%, insulin A chain 1.8%, insulin B chain 18.2, C-peptide 9.1%, and IGF 9.1%. The data show natural occurrence of reagins to peptide hormones in atopic persons. A risk at exposure to these peptides may exist in about 1% of the general population.
Clinical manifestations of allergy to biosynthetic hGH were not reported in 245 patients treated for one year. A sensitive radioallergosorbent test showed, however, the presence of anti-hGH IgE antibodies in the sera of 13%. All patients with anti-hGH IgE had also elevated concentrations of total serum IgE, similar to atopic persons (2459 +/- 147 micrograms/l, normal 70 +/- 8, 2p less than 0.0001); 83% of all patients who had specific IgE antibodies to hGH had specific antibodies prior to treatment. Administration of hGH did not consistently change concentrations (up to 8 micrograms/l) or affinities (less than 10(7) to 1.3 X 10(9) l/mol) of the specific reagins. Anti-hGH IgE antibodies were related to atopy. Treatment with biosynthetic hGH did not significantly stimulate specific IgE.
The effects of three oral doses of nifedipine 20 mg or nifedipine 20 mg/dihydroergotoxin 2 mg (Pontuc, HN 85) during three subsequent days on an oral glucose load (100 g) were compared to placebo. Neither drug altered the glucose load or inhibited the secretion of insulin or C-peptide. The fall of serum potassium was also identical to controls. Basal plasma norepinephrine concentrations were lower following nifedipine/dihydroergotoxine than after nifedipine alone (2 p less than 0.05). A decrease of the serum sodium concentration by 2 mmol/l was observed with nifedipine but not with the combined drugs.
Posthypoglycemic glucose intolerance is related to counter-regulatory hormones. We have tested the role of the beta 2-sympathomimetic activity of the catecholamines in healthy volunteers. beta 2-stimulation with fenoterol for 25 minutes caused glucose intolerance and insulin resistance for 4 hours. This suggests that the beta 2-sympathomimetic activity induces glucose intolerance after an episode of hypoglycemia.
Proinsulin induced hypoglycemia was characterized in eight healthy male volunteers. Proinsulin cleared slower from the circulation than insulin. The metabolic effects on plasma glucose, free fatty acids, glycerol, 3-hydroxybutyrate, potassium, and phosphate occurred slower. The anti-lipolytic effect of proinsulin was longer than that of insulin (2P less than 0.001). The hormonal responses of epinephrine, norepinephrine, prolactin, hGH, and cortisol were attenuated following proinsulin. The amount of epinephrine secreted during counterregulation and the amount of lactate produced in response to beta-stimulation were both correlated to the fall of plasma glucose (2P less than 0.005). Stress symptoms were milder after proinsulin (2P less than 0.01). The data obtained are consistent with the hypothesis that the slower kinetics of proinsulin cause slower metabolic effects. The assumption of non-insulin-like effects of a differing pattern of insulin-like effects is not necessitated by the results of this study.
To define the specificity and epitope of five monoclonal antibodies (MoAbs) to human insulin, binding studies with artificially modified insulins and a number of native insulins were done. Epitopes on the A-chain (A4, A8-A10) and on the end of the B-chain (B30) could be identified. For two MoAbs, substructures of the amino acid B30 were found, which were essential for binding (hydroxyl and methyl groups of B30). In contrast to most antisera, MoAbs to human insulin show high specificity. However, as the study shows, the specificity is not absolute. With suitable artificial epitope modifications, cross-reaction can be seen. Two of the MoAbs used here show sufficient specificity to discriminate between insulin and proinsulin.
The counterregulatory hormonal response to proinsulin-induced hypoglycemia was investigated in eight volunteers. Proinsulin cleared slower from the circulation than insulin. Hypoglycemia occurred slower (2P less than 0.005) and was prolonged, while the overall hypoglycemic activities were comparable. The antilipolytic effect of proinsulin was also prolonged (2P less than 0.001). The response of epinephrine to hypoglycemia was less pronounced after proinsulin (2P less than 0.05). The amount of epinephrine was correlated to the rate of fall in plasma glucose (P less than 0.005). The production of lactate induced by beta-stimulation was also correlated to the fall of glucose (P less than 0.005). The responses of prolactin (2P less than 0.02), norepinephrine (2P less than 0.02), cortisol, and growth hormone were attenuated following proinsulin. The decreases of serum potassium and serum phosphate (2P less than 0.05) were less pronounced. Symptoms like sweating (2P less than 0.01) and dizziness (2P less than 0.01) were milder after proinsulin. It is concluded that the rate of fall in glucose concentration determines the differing counterregulatory responses. We don't relate the differing counterregulatory responses to special insulin-like properties of proinsulin, but to the slower kinetics which is emphasized by the intravenous bolus injection.
Guinea pigs were immunized with extracted human growth hormone. Human sera were obtained after treatment with biosynthetic methionyl hGH. The size of hGH-anti hGH antibody complexes was determined from the sedimentation velocity at 100,000 g. At an excess of hGH over antibodies 8 S complexes were uniformly observed in human and guinea pig sera. S values between 11.8 and 15.6 were observed at antibody excess in individual guinea pig sera. Antibodies from humans treated with methionyl hGH formed smaller complexes (7.5 S). One patient with GH-deficiency developed resistance to treatment. Complexes of 12.3 S were formed by his antibodies. HGH sustains the formation of antibody complexes containing more than three IgG molecules (15.6 S). It is discussed that human antibodies of higher diversity may form complexes larger than trimers which initiate the complement cascade.
Guinea pigs were immunized with regular insulin in Freund's adjuvant weekly (N = 6) or with NPH insulin daily (N = 10). The concentrations, the affinities, and the sizes of the insulin-immunoglobin G (IgG) antibody complexes were determined by ultracentrifugation. During the immune response to insulin, 7-S complexes were observed after 3 wk, and 10-S complexes were observed after 4 wk (2P less than .05 in each group). "7-S" antibodies were transferred into immunized guinea pigs before the formation of measurable antibody concentrations. The development of 10-S structures was enhanced in outbred Pirbright white guinea pigs (2P less than .05, N = 6) and in inbred strain II l.b.m. guinea pigs (2P less than .05, N = 7). Insulin may act as a monovalent antigen (7 S) after 3 wk and become bivalent after 4 wk (10 S). The change in valency is enhanced by antibody transfer. It reflects the formation of antibodies recognizing new epitopes on insulin.
Congenic male mice with differences in the H-2 complex have been used to investigate insulin secretion in vitro, insulin binding to isolated hepatocytes, plasma glucose, and serum insulin. Plasma glucose and serum insulin did not show consistent differences in the B10.BR, B10.D2, B10.A, B10.G, B10.M, B10.S, C57/10SCSN, and C3H.OH strains. Isolated islets of Langerhans responded to stimulation with 400 mg/dl glucose with a 3-5-fold increase in insulin secretion rates (2P less than 0.01): B10.BR greater than B10.M greater than C57BL/10SCSN greater than B10.G greater than C3H.OH, B10.D2, B10.A, B10.S. The biphasic pattern of insulin secretion was less distinct in B10.M, B10.G, and C3H.OH mice. The high-affinity constants of insulin binding to isolated hepatocytes at 37 degrees C varied between 4.5 X 10(7) L X mol-1 and 4.5 X 10(8) L X mol-1 (2P less than 0.01): B10.A greater than B10.BR greater than C57BL/10SCSN, B10.S, B10.D2 greater than B10.M, B10.G. The glucose-stimulated insulin secretion from isolated islets of Langerhans and the binding of insulin to isolated hepatocytes correlate to the H-2 complex independently.
The clearing of monoclonal and polyclonal and anti-insulin antibodies from homogeneous solutions at 100,000 X g was used to estimate the size of soluble insulin-antibody complexes at physiologic concentrations. Monoclonal antibodies cleared as a uniform population of 6.6 S independent of the insulin concentration. Polyclonal antibodies cleared as 6.6 S monomers at saturation and as 10 S particles when the amount of insulin bound decreased, suggesting that a soluble complex with two antibodies was formed. An increase of the affinity and a decrease of antibody valency can be related to the complex formation. The binding affinity of polyclonal sera depends on the composition of the affinities of the IgG monomers and on their ability to form 10 S complexes. The formation of insulin-antibody dimers precludes cross-linking and precipitation. Both types of insulin-antibody complexes have been found in the sera from patients treated with bovine insulin.