Characterization of the acid-labile subunit of the growth hormone-dependent insulin-like growth factor binding protein complex

The insulin-like growth factors (IGFs) circulate predominantly in a 150,000-dalton (150K) complex, the IGF-binding component of which appears to be an acid-stable 53K glycoprotein (BP-53). This study tested the hypothesis that an acid-labile subunit (ALS) reacts with the binding subunit to form the 150K complex. ALS activity was quantitated by the conversion of covalent BP-53-[125I]IGF-I tracer from about 60K to about 150K. DEAE-Sephadex chromatography of serum at pH 8.2 yielded separate peaks of 30-60K BP-53 immunoreactivity and 100-110K ALS which, when mixed, converted the BP-53 to 150K. Whole serum also contained 100-110K ALS not complexed with BP-53. ALS was markedly acid labile, irreversibly losing activity below pH 4.5. In an assay involving competition between test substances and BP-53-IGF-I tracer in the reaction with partially purified ALS, serum samples, acidified to inactivate endogenous ALS, reacted with potencies proportional to their immunoreactive BP-53 content. Pure BP-53 alone was inactive, but after preincubation with IGF-I or IGF-II, competed with a potency identical to that of BP-53 in acidified serum. Amniotic fluid IGF-binding protein, with or without IGFs, had no activity. These results confirm that serum contains an acid-labile protein which interacts with the acid-stable IGF-binding protein BP-53, when it is occupied by IGFs, to convert it to the 150K form.     

Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein- 1 alpha

BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU-S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G- CSF) and the combination of G-CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100- fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117-, and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB- 10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation.     

Insulin-like growth factor (IGF) binding protein-3 in pregnancy serum binds native IGF-I but not iodo-IGF-I.

Although serum immunoreactive insulin-like growth factor binding protein-3 (IGFBP-3) increases during pregnancy, radioligand binding methods such as ligand blotting with iodinated IGFs fail to detect the protein in pregnancy serum. Since IGFBP-3 must bind IGF-I or IGF-II to form a complex with the acid-labile subunit (alpha-subunit), we have used ternary complex formation from [125I]alpha-subunit as a measure of IGF binding to serum IGFBP-3. High-pressure liquid chromatography fractions containing IGFBP-3 from pregnancy serum did not bind [125I]IGF-I, although the equivalent fractions from nonpregnancy serum showed dose-dependent binding. In contrast, IGFBP-3 fractions from nonpregnancy and pregnancy sera both bound [125I]alpha-subunit in the presence of either exogenous IGF-I or endogenous serum IGFs, implying that non-iodinated IGFs could bind to the IGFBP-3. Substitution of nonradioactive iodo-IGF-I for native IGF-I in the complex formation assay confirmed that the pregnancy-induced alteration in IGFBP-3, probably resulting from proteolysis, prevents it from binding iodo-IGF-I while having little effect on its binding of the native peptide. This provides an explanation for the failure to detect IGFBP-3 in pregnancy by radioligand binding methods, and raises the question of the significance of proteolysis of IGFBP-3.     

A Reliable Method for Quantification of Tendon Structure Using B‐Mode Ultrasound

Structure is an important clinical marker of tendon health; however, current standards use qualitative scores that are not strongly reliable. Therefore, the purpose of this study was to establish the reliability of an image‐processing technique that quantifies tendon collagen structure using B‐mode ultrasound images. Longitudinal images of the Achilles tendon were collected in 12 healthy young adults, and intra‐ and inter‐rater reliability was assessed over multiple image selections and multiple days. Intraclass correlation coefficients were strong (r ≥ 0.71) for all comparisons. These findings demonstrate that quantitative assessments of tendon structure using B‐mode ultrasound are reliable.     

Imatinib for systemic mast-cell disease

Imatinib has shown to be effective against malignant disease driven by ckit. We prospectively treated 12 adults with symptomatic systemic mast-cell disease at a dose of either 100 mg or 400 mg per day. Of the ten patients who we could assess for response, five (50%) had a measurable response to the drug, four of whom had important mast-cell cytoreduction and two who had complete clinical and histological remission. In the five patients with eosinophilia, three had complete clinical and haematological remission. The other two, who did not respond to treatment, were the only patients with the ckit D816V mutation. Our results suggest that imatinib either inhibits the growth-promoting role of wild type ckit, or targets an oncogenic kinase.     

Pilot study of a screening test for peripheral arterial disease in middle aged men: fibrinogen as a possible risk factor

A simple one minute exercise test was used as a screening test for asymptomatic peripheral arterial disease in a sample of 100 men in their sixth decade with no previous referrals for cardiovascular disease. Other investigations included resting ECG, non-invasive carotid artery assessment, and plasma biochemical analysis. Of these 100 men (mean age 56), 10 had evidence of peripheral disease on exercise testing, four had ischaemic changes on resting ECG, and one showed evidence of carotid artery stenosis. A total therefore of 15 out of 100 (15%) had asymptomatic arterial disease. These 15 men had increased concentrations of plasma fibrinogen (4.3(0.7) g.litre-1) compared with men with no evidence of arterial disease (3.5(0.7) g.litre-1; p less than 0.01). The one minute exercise test is a useful screening test for peripheral arterial disease, and this pilot study suggests that raised plasma fibrinogen concentrations may be an important risk factor.