Thirteen monoclonal antibodies (MAbs) were produced against Lol p I (Rye I), the major Lolium perenne (rye grass) pollen allergen. Spleen cells from A/J and SJL mice immunized with highly purified Lol p I (Lol I) were allowed to fuse with cells from the non-secreting Sp2/0-Ag14 myeloma cell line. Each MAb was analyzed for antigenic specificity by radioimmunoassay (RIA) using 125I-Lol I. The epitope specificities of seven of the MAbs were examined by competitive binding against a labelled standard MAb for the Lol I antigen (Ag). The dissociation constant, Kd, of one MAb (No. 3.2) that was studied most extensively was determined by double Ab RIA to be 3.5 X 10(-6) L/M. This MAb recognized the related 27,000-30,000 Group I glycoproteins found in the pollens of nine other species of grass pollens tested, including weak binding to Bermuda grass Group I (Cyn d I), which by conventional analysis using polyclonal anti-Lol I serum shows no detectable binding. Monoclonal antibody No. 3.2 was coupled covalently to Sepharose 4B and used to prepare highly purified Lol I from a partially purified rye pollen extract. Finally, an RIA was developed which permitted the analysis of the Group I components in rye grass and nine other grass pollen species. The latter assay is likely to prove useful in the standardization of grass pollen extracts according to their Group I contents.

Insulin binds to its specific cell surface receptor in cultured human fibroblasts and also stimulates the conversion of glycogen synthase from the glucose-6-phosphate (G-6-P) dependent to the G-6-P independent form. Although these two processes are tightly coupled in most target tissues for insulin action, in the fibroblast a variety of findings question the relationship of these two events to one another. In human fibroblasts the amount of insulin required to displace half of the 125I-insulin bound to the insulin receptor is 4 ng/ml (6.6 X 10(-10)M), but the activation of glycogen synthase is not maximal until 1-10 micrograms/ml with an ED50 of 30 ng/ml insulin. Antibodies directed against the insulin receptor, which activate glycogen synthase in both fat and muscle, do not stimulate the activation of glycogen synthase in the fibroblast. Fab fragments from anti-insulin receptor antibody compete for insulin binding, but do not inhibit the insulin-stimulated rise in independent activity. The insulin-like growth factor, MSA, which is 1% as potent as insulin in stimulating glucose oxidation in rat fat cells and in inhibiting 125I-insulin binding to human fibroblasts, is 25% as potent as insulin in stimulating glycogen synthase. Proinsulin is 2-10% as potent as insulin, but behaves as a "partial agonist" of insulin action in the fibroblast, i.e. proinsulin is able to elicit only 60% of the maximal response of insulin in the glycogen synthase assay, even at high concentrations. Finally, cell lines from patients with clearly defective insulin receptors exhibit normal insulin dose response curves for the activation of glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin resistance is observed in insulin-deficient diabetic states in spite of an increase in insulin binding to its target cells. To characterize this type of insulin resistance, autophosphorylation and kinase activity of the insulin receptor on liver was studied with streptozotocin (STZ)-induced and BB diabetic rats. Insulin binding capacity was increased in proportion to the severity of the diabetic state in the STZ rat. In the diabetic BB rat, the insulin binding capacity was also increased, and this was partially normalized by insulin treatment. By contrast, insulin-stimulated autophosphorylation of the beta-subunit of the insulin receptor was decreased in proportion to the severity of the diabetic state in the STZ rat. Peptide mapping by reverse-phase high-performance liquid chromatography revealed a decrease in labeling at all sites of autophosphorylation. Kinase activity of the insulin receptor to exogenous substrates was also decreased in proportion to the diabetic state. In the BB rat, autophosphorylation and kinase activity of the insulin receptor were both decreased in the diabetic state and partially normalized by insulin treatment. In addition to the beta-subunit of insulin receptor, a 170 kdalton phosphotyrosine-containing protein was also identified in the glycoprotein fraction of liver. Although the phosphorylation of this protein was not insulin dependent, it was decreased markedly in the diabetic state. This protein is immunologically distinct from the insulin receptor, but is rich in phosphotyrosine. Based on its size and phosphotyrosine content, this protein may be the epidermal growth factor receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

To characterize the insulin-like growth factor I (IGF-I) receptor on human erythrocytes, cells were purified from peripheral blood by Ficoll-Hypaque gradient centrifugation and incubated with [125I]IGF-I. Specific binding was maximal at pH 8.0 after 24 h at 4 C and increased linearly with cell number to 3.9 +/- 0.2% (+/- SEM) for 3.0 X 10(9) cells/ml. The Scatchard plot of the binding data was linear, with 7 fmol [125I]IGF-I bound/10(9) cells and an affinity constant (K) of 1.8 X 10(9) M-1. Unlabeled IGF-I inhibited tracer binding half-maximally at 6 ng/ml. Multiplication-stimulating activity (or rat IGF-II) was 40% as potent (ED50, 15 ng/ml), whereas insulin and proinsulin were 30- to 500-fold less potent. A monoclonal antibody to the IGF-I receptor (alpha IR-3) inhibited IGF-I binding by 50% at a 1:1000 dilution and by 80% at a 1:250 dilution. Insulin binding was unaffected by the same dilutions. IGF-I receptor phosphorylation was studied in erythrocyte ghosts prepared by hypotonic lysis and solubilized in 1% Triton. The extract was preincubated with and without 100 ng/ml IGF-I or porcine insulin and incubated with [gamma-32P]ATP in the presence of Mn2+, and the receptor was identified by immunoprecipitation with alpha IR-3 antibody and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IGF-I stimulated 4-fold the incorporation of 32P into a protein of 95,000 mol wt, which was immunoprecipitated by alpha IR-3; insulin produced a 2-fold stimulation of this protein. This protein corresponds to the beta-subunit of the IGF-I receptor. These data demonstrate that human erythrocytes have specific receptors for IGF-I, and that this IGF-I receptor, like the insulin receptor, undergoes ligand-stimulated autophosphorylation. Thus, analysis of erythrocyte IGF-I binding and receptor phosphorylation may be useful tools for the study of patients with a variety of growth disorders.

Deficient production of interleukin-2 has been reported in Type I diabetes, but its cause has not been elucidated. We therefore measured interleukin-2 production in 27 patients with Type I diabetes, 20 patients with Type II diabetes (6 requiring insulin), 5 monozygotic twin pairs discordant for Type I diabetes, and 10 nondiabetic persons with islet-cell antibodies. Interleukin-2 production was decreased in patients with Type I diabetes as compared with controls (35.8 +/- 2.5 vs. 61.6 +/- 4.6 percent, P less than 0.001). Interleukin-2 production did not differ between patients with Type II diabetes and controls, regardless of whether the patients used insulin. Twins with Type I diabetes had decreased interleukin-2 production as compared with normal controls (33.2 +/- 5.4 vs. 61.6 +/- 4.6 percent, P less than 0.001) and with their nondiabetic twins (33.2 +/- 5.4 vs. 54.5 +/- 3.4 percent, P less than 0.005). Interleukin-2 production in nondiabetic twins and in nondiabetic persons with islet-cell antibodies was normal. There was no correlation between glycosylated hemoglobin levels and interleukin-2 production in any diabetic group. We conclude that patients with Type I diabetes have an acquired defect in interleukin-2 production, whereas patients with Type II diabetes do not, and that this defect is not correlated with an ongoing autoimmune process, with hyperglycemia, or with insulin administration or oral hypoglycemic therapy. Thus, the defect appears to be related to marked beta-cell destruction, although not to the metabolic consequences thereof or the responsible autoimmune process.

Various lipids were tested as substrates for the insulin receptor kinase using either receptor partially purified from rat hepatoma cells by wheat-germ-agglutinin-Sepharose chromatography or receptor purified from human placenta by insulin-Sepharose affinity chromatography. Phosphatidylinositol was phosphorylated to phosphatidylinositol 4-phosphate by the partially purified insulin receptor. In contrast, phosphatidylinositol 4-phosphate and diacylglycerol were not phosphorylated. In some, but not all preparations of partially purified insulin receptor, the phosphatidylinositol kinase activity was stimulated by insulin (mean effect 33%). Phosphatidylinositol kinase activity was retained in insulin receptor purified to homogeneity. Insulin regulation of the phosphatidylinositol kinase was lost in the purified receptor; however, dithiothreitol stimulated both autophosphorylation of the purified receptor and phosphatidylinositol kinase activity in parallel about threefold. (Glu80Tyr20)n, a polymeric substrate specific to tyrosine kinases, inhibited the phosphatidylinositol kinase activity of the purified receptor by greater than 90% and inhibited receptor autophosphorylation by 67%. Immunoprecipitation by specific anti-receptor antibodies depleted by greater than 90% the phosphatidylinositol kinase activity in the supernatant of the purified receptor and the phosphatidylinositol kinase activity was recovered in the precipitate in parallel with receptor autophosphorylation activity. These characteristics of the phosphatidylinositol kinase activity of the purified insulin receptor and its metal ion preference paralleled those of the receptor tyrosine kinase activity and differed from bulk phosphatidylinositol kinase activity in cell extracts, which was not significantly inhibited by (Glu80Tyr20)n, stimulated by dithiothreitol or depleted by immunoprecipitation with anti-(insulin receptor) antibody. These results suggest that the insulin receptor is associated with a phosphatidylinositol kinase activity; however, this activity is not well regulated by insulin. This kinase appears to be distinct from the major phosphatidylinositol kinase(s) of cells. Its relationship to insulin action needs further study.

Successful viral infection involves a series of interactions between the virus and the host cell. The outcome of viral infection is, in fact, dependent on intact cellular function; it is required for viral binding, internalization, and uncoating. To determine the potential importance of lysosomal processing on the outcome of infection with a nonenveloped virus, we have studied the effects of NH4Cl on the course of reovirus infection on a beta-cell tumor in culture. Addition of 10 mM NH4C1 to the medium inhibited viral growth by greater than 80% and reduced toxic effects of the virus on cell viability, protein, and DNA synthesis by 30-45%. In addition, synthesis of viral proteins was markedly decreased. Uptake of virus prelabeled with [35S]methionine was not affected by the ammonium; however, cleavage of mu1C, an outer capsid protein of the virus whose cleavage appears to be required for viral replication, was delayed. These results suggest that intracellular processing of reovirus is dependent on a lysosomal pathway and that disruption of this pathway can alter the course of viral infection.

The type A syndrome of insulin resistance and acanthosis nigricans is characterized by severe insulin resistance due to a cellular defect in insulin action. To better understand the molecular nature of this defect, we have investigated insulin binding to circulating monocytes, erythrocytes, and the Triton X-100-solubilized erythrocyte receptor, and insulin-stimulated receptor autophosphorylation using cells and receptor from three type A patients. Insulin binding in both circulating cells and the soluble extract of erythrocytes indicated a heterogeneity of defects. Patients A1 and A2 both presented a major decrease in tracer insulin binding to intact cells and soluble insulin receptor. Determination of stoichiometric binding parameters using a cooperative model indicated that in patient A1 this was due to a reduction in the number of receptors, whereas in patient A2 the affinity constant for binding was decreased. Patient A3 presented near-normal insulin binding to erythrocytes and normal binding in intact monocytes, solubilized erythrocyte receptors, and cultured fibroblasts. Affinity labeling of erythrocyte receptor from this patient revealed a normal alpha-subunit and also a normal relative distribution of the higher-molecular-weight, nonreduced oligomeric forms of the receptor. Receptor autophosphorylation was measured using the solubilized insulin receptor from erythrocytes. The maximal stimulated phosphorylation was reduced by 79%, 76%, and 52% in patients A1, A2, and A3, respectively, relative to the simultaneous control. In all three patients, the autophosphorylation was stimulated only 1.0-3.5 times the basal level compared with controls, in which the stimulation was 5.7-fold +/- 1.2 (mean +/- 1 SD, P less than 0.005). In addition, in patients A1 and A2 a decrease in basal phosphorylation was observed and in patient A2 there was a rightward shift of the dose-response curve for insulin stimulation. These data and the correlation of coupling of receptor phosphorylation with the fractional occupancy of the receptor measured in the same extract suggest that these patients exhibit three types of defects. In patient A1, there is a loss in receptor number manifested by a parallel decrease in insulin binding and receptor phosphorylation. In patient A2, there is an additional decrease in the affinity constant leading to a decrease in both binding and receptor phosphorylation with an almost linear coupling between receptor occupancy and receptor phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

The development of proliferative diabetic retinopathy was studied in three cohorts consisting of 292 patients with recent juvenile-onset, type I (insulin-dependent) diabetes who were followed 20-40 yr beginning in 1939, 1949, and 1959. The risk of this severe eye complication was almost nonexistent during the first 10 yr of diabetes, rose abruptly to its maximum level (approximately 30/100 person-years), and remained at that level for the next 25 yr. This pattern did not vary with sex, age at onset of diabetes, or level of glycemic control during the first 5 yr of diabetes. However, the risk of proliferative retinopathy was strongly related to the level of glycemic control during the several years preceding onset of this complication. This was a dose-dependent relationship, with patients in the highest quartile of the distribution of the index of frequency of hyperglycemia having a 10-fold higher risk than individuals in the lowest quartile. A virtually identical pattern was observed in patients who developed diabetes in 1959 as was observed in those who developed diabetes in 1949 or 1939. In contrast, diabetic nephropathy as evidenced by persistent proteinuria showed a lower incidence in the 1959 than in the 1939 cohort. In conclusion, these incidence data do not support the notion that the risk of proliferative retinopathy is mainly a function of duration of diabetes. Instead, the pattern of occurrence of this severe eye complication in type I diabetes suggests that the process leading to the development of proliferative retinopathy consists of two or more stages and that progression through each stage may be governed by different factors.