An efficient method for bacterial production and activity assessment of recombinant human insulin like growth factor 1.

Human insulin like growth factor 1 directs physiological roles in cellular proliferation and differentiation process. The protein is considered as an important therapeutic target with clinical significance. In this study, to avoid production of human insulin like growth factor 1 as inclusion body, the thioredoxin was used as a solubilizing fusion tag. The expression of fusion human insulin like growth factor 1 was carried out in E. coli Rosetta-gami by transformation of pET-32b contained functional elements. The evaluation of different conditions involving protein expression including IPTG concentration, temperature and post induction time showed that 0.1 mM IPTG at 34 °C for 4 h was the optimum condition. The isolated fusion protein was purified using nickel affinity purification and digested by entrokinase to produce mature recombinant protein without any additional tag. The accuracy of produced recombinant protein was confirmed by western blot analysis. Biological activity of produced recombinant human insulin like growth factor 1 was determined by its proliferation effects on MCF-7 cells, expansion of bovine granulosa cells and activation of PI3K/Akt signaling pathway in these cells. The present study provides a simple and efficient method for high-level production of soluble, active recombinant human insulin like growth factor 1 in E. coli.

[Assessment of treatment effect for acromegaly with sleep disordered breathing].

Acromegaly is caused by excessive secretion of growth hormone (GH) and presents with a variety of clinical manifestations, including facial disfigurement and abnormally large hands and feet, as well as diabetes mellitus, hypertension, and sleep-disordered breathing (SDB). Although SDB is known to be associated with serious symptoms, there have been few study reports, and no clear consensus has been reached regarding the method of assessment of individual treatments. We report herein on the results of surgical intervention with transsphenoidal surgery (TSS) for acromegaly and assessment of the treatment effect after the intervention. We studied 6 patients who received a diagnosis of acromegaly complicated with SDB and underwent TSS at our hospital. Polysomnography (PSG) was performed before and after TSS, and the polysomnograms were analyzed. We also examined changes in the levels of GH and insulin-like growth factor-1 (IGF-1) on blood biochemistry. In 6 cases of acromegaly with SDB, we were able to confirm endocrinologic improvement of TSS with blood biochemistry. However there was no meaningful improvement in the PSG index for SDB.

Recombinant rat and mouse growth hormones: risk assessment of carcinogenic potential in 2-year bioassays in rats and mice.

Recombinant rat growth hormone (rrGH) and recombinant mouse growth hormone (rmGH) were developed to evaluate the potential carcinogenicity of each biologically active growth hormone (GH) as assessed in the respective species. Biological activities of rrGH and rmGH were demonstrated by showing an increase in body weight gain and serum levels of insulin-like growth factor-1 (IGF-1) in hypophysectomized rats receiving daily sc injections for 6 days. With the exception of pharmacologically mediated weight gain, rrGH and rmGH had no adverse effects in 5-week oral toxicity studies and no production of anti-recombinant GH antibodies. The high doses selected for the carcinogenicity studies provided systemic exposures of GH up to approximately 10-fold over basal levels. In the 105-week mouse carcinogenicity study, daily sc injections of rmGH at 0.1, 0.2, or 0.5 mg/kg/day were well tolerated and had no effects on survival or incidence of tumors. In the 106-week rat carcinogenicity study, daily sc injections of rrGH at 0.2, 0.4, or 0.8 mg/kg/day had a favorable effect on longevity in female rats administered 0.4 or 0.8 mg/kg/day, an increased weight gain in females and males, and no increase in the incidence of tumors. The absence of carcinogenic effects of recombinant GH administered daily for 2 years to rodents was consistent with publications of clinical experience, indicating a lack of convincing evidence for an increased risk of cancer in children receiving human recombinant GH replacement therapy.

Expression of insulin-like growth factor-I (IGF-I) in alveolar macrophages and lymphocytes obtained by bronchoalveolar lavage (BAL) in interstitial lung diseases (ILD). Assessment of IGF-I as a potential local mitogen and antiapoptotic cytokine.

Little is known about IGF-I expression in the alveolar lymphocytes (AL), and about local role of IGF-I in physiological conditions and in interstitial lung diseases. Bronchoalveolar lavage was carried out in patients with silicosis, asbestosis, idiopathic pulmonary fibrosis (IPF) and sarcoidosis, as well as in control subjects (n = 13, 9, 12, 56, 15, resp). Alveolar macrophages (AM) and lymphocytes (AL) were studied for (1) IGF-I, BCL-2, Fas and Fas Ligand expression and (2) cell cycle (incl. sub-G1 peak of late apoptosis) with propidium iodide (PI). Flow cytometry (FC) and immunocytochemistry were used. AL early apoptosis was detected by Annexin V FITC/PI staining. IGF-I was present in AL of all tested groups. The number of IGF-I positive AL was significantly higher in IPF (52 +/- 6.7%) and in later (II and III) stages of sarcoidosis (39 +/- 7.8 vs 16 +/- 4.0% in controls, p < 0.05). Increased BCL-2 expression in AL was detected in IPF and sarcoidosis. In all tested groups, AL were almost exclusively Fas+ T cells. Generally, a low number of AL entered apoptosis; no significant differences were found between patient groups, except decreased apoptosis rate in sarcoidosis (0.60 +/- 0.17 vs 1.15 +/- 0.33% in controls, p < 0.05). Proportion of AL positive for IGF-I was significantly correlated with parameters reflecting AL and AM cell proliferation and BCL-2 expression (e.g. AL IGF-I+ vs AM in S phase of cell cycle: r(S) = +0.50, p = 0.001), but not with apoptosis. The results show that human alveolar lymphocytes express IGF-I in normal conditions, as well as in ILD. The proportion of IGF-I+ lymphocytes was significantly increased in IPF and at later stages of sarcoidosis. In our material there was no evidence for profibrogenic or antiapoptotic activity of IGF-I. We suggest that IGF-I originating from AL may be locally active as a mitogen for alveolar macrophages and lymphocytes in ILD.

[The assessment of plasma levels of insulin-like growth factor (IGF-I) and binding protein (IGFBP-3) during the maintenance therapy and after remissionof acute lymphoblastic leukemia (ALL)]. / Ocena wartosci insulinopodobnego czynnika wzrostu (IGF-I) i bialka wiazacego (IGFBP-3) w czasie leczenia podtrzymujacego remisje i po zakonczeniu leczenia ostrej bialaczki limfoblastycznej (ALL).

INTRODUCTION: Antineoplastic therapy may cause metabolic disturbances, which result in e.g. abnormal growth rate. The aim of the study was the assessment of IGF-1 and IGFBP-3 plasma concentrations in children after intensive therapy of ALL completed according to age, weight, height and therapy protocols (Methotrexate dose, Central Nervous System - CNS prophylaxis). MATERIALS AND METHODS: 63 children (43 boys) were involved in this study with mean age 10.95+/-4.73 years after ALL therapy according to BFM protocols. 35 patients were in maintenance therapy and 28 completely finished the therapy. 20 children had cranial irradiation as CNS prophylaxis (12 Gy). We assessed plasma levels of IGF-1 and IGFBP-3 in radioimmunoassay method and the obtained results were related to mean age values (SDScore). RESULTS: 1. A) IGF-1 mean plasma levels in children (n=30) before puberty (Tanner I) was 151.48+/-75.75 ng/ml and was lower than compared to value in the puberty group (n=16, Tanner II-IV) - 222.06+/-96.61 ng/ml p=0.000008. After puberty (Tanner V) IGF-1 was 332.03+/-75.66 ng/ml. b) IGFBP-3 was highest after puberty (3837.4 ng/ml +/-598.49); before puberty it was 2899.68+/-656.57 ng/ml, and in puberty it was 3593.14+/-598,49 ng/ml. 2. IGF-1 SDS values were lower than mean age values. 3. IGFBP-3 SDS were insignificantly higher than mean age values. 4. There were no differences between IGF-1 SDS and IGFBP-3 SDS according to gender, prior CNS prophylaxis, methotrexate dose and phase of maintenance therapy and completed therapy. 5. IGF-1 and IGFBP-3 correlated to each other and to body mass index, height but IGF-1 SDS and IGFBP-3 SDS was independent on age, CNS prophylaxis, BMI SDS and height SDS. CONCLUSIONS: 1. IGF-1 and IGFBP-3 values are dependent on puberty degree, body mass index and height. 2. IGF-1 SDS are lower and IGFBP-3 values higher, independent on gender, therapy protocol and CNS prophylaxis (12 Gy).

Assessment of growth hormone status in acromegaly: what biochemical markers to measure and how?

Various studies have evaluated the usefulness of measuring insulin-like growth factor I (IGF-I) and IGF-binding proteins, compared with growth hormone (GH), in the diagnosis and follow-up of patients with acromegaly. The clinical use of recently developed, highly sensitive GH assays has improved our understanding of the secretion of GH at very low concentrations. Studies examining the GH nadir after oral glucose administration in healthy adults suggest that GH values should be suppressed to no more than 0.14 microg/l after oral glucose administration. The highly sensitive GH assays are now being used in the diagnosis of patients with acromegaly and in post-treatment follow-up. In addition, the use of IGF-I immunoassays has confirmed that serum IGF-I level is a reliable and useful biochemical marker for determining GH excess. The widespread use of highly sensitive, carefully validated assays offers accurate and reproducible biochemical information that will help in the overall clinical management of patients with acromegaly, including the monitoring of disease remission, disease cure and treatment titration regimen, as well as the early recognition of patients with residual disease.