Dieter Engelhardt

Overexpression of the insulin-like growth factor I receptor in human colon carcinomas

High concentrations of insulin‐like growth factor (IGF)‐I and IGF‐II have been demonstrated in human colonic adenocarcinomas and exert mitogenic effects through paracrine/autocrine interactions with the IGF‐I receptor (IGF‐IR). However, definitive studies of IGF‐IR expression in these tissues have not been performed.

Therapy of Cushing's syndrome with steroid biosynthesis inhibitors.

Several substances with different inhibitory effects on adrenal steroid biosynthesis were investigated in patients with Cushings syndrome. It has been shown that trilostane, a 3 beta-hydroxysteroid-dehydrogenase inhibitor, is not potent enough to block cortisol biosynthesis in patients with hypercortisolism. Aminoglutethimide inhibits side chain cleavage of cortisol synthesis, but it has been demonstrated that the blocking effect on cortisol secretion is not strong enough to normalize urinary cortisol excretion in patients with Cushings disease. For metyrapone, an inhibitor of adrenal 11 beta-hydroxylase, promising results were reported for the treatment of Cushings syndrome. However, the drug has several side effects and depending on the definition of the desired reduction of cortisol secretion a true remission was only found in a minority of patients. The antifungal drug ketoconazole in vitro predominantly blocks 17,20-desmolase (IC50 1 microM) and to a lesser extent 17 alpha-hydroxylase (IC50 10 microM) and 11 beta-hydroxylase (IC50 15-40 microM). Therefore, ketoconazole in vivo most potently suppresses androgen secretion and only to a lesser extent cortisol biosynthesis. Several therapeutic trials with ketoconazole treatment in patients with pituitary Cushings disease showed various remission rates between 30 and 90%. In contrast, in almost all patients with benign, primary adrenal Cushings syndrome cortisol levels were normalized. In patients with ectopic ACTH syndrome ketoconazole was effective in about 50% of all reported cases, while cortisol hypersecretion due to adrenocortical carcinoma was only rarely inhibited by ketoconazole. The main side effect of ketoconazole treatment was liver toxicity which occurred in 12% of all treated patients. In contrast to ketoconazole, the narcotic drug etomidate shows a strong inhibitory effect on 11 beta-hydroxylase (IC50 0.03-0.15 microM) but only a weak inhibition of 17,20 desmolase (IC50 380 microM). This correlates with in vivo studies where even low, non-hypnotic doses of etomidate induced a pronounced fall in serum cortisol levels in normals and in patients with Cushings syndrome. However, its clinical use is limited by its mandatory intravenous application and its sedative effects. In conclusion, ketoconazole remains the only available steroid-inhibitory drug for a therapeutic trial in patients with Cushings syndrome who cannot be treated definitively by surgery.

Mutations in the VHL tumor suppressor gene and associated lesions in families with von Hippel-Lindau disease from central Europe.

Abstract von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome predisposing to retinal, cerebellar and spinal hemangioblastoma, renal cell carcinoma (RCC), pheochromocytoma and pancreatic tumors. Clinically two types of the disease can be distinguished: VHL type 1 (without pheochromocytoma) and VHL type 2 (with pheochromocytoma). We report VHL germline mutations and trends in phenotypic variation in families from central Europe. We identified 28 mutations in 53/65 (81.5%) families with 18 (64%) mutations being unique to this population. Whereas types and distribution of mutations as well as a strong correlation of missense mutations with the VHL 2 phenotype were similar to those identified in other populations, these families have provided new insights into the molecular basis for variability in the VHL 2 phenotype. Seven different missense mutations in exons 1 and 3 varied in their biological consequences from a minimal VHL 2 phenotype with pheochromocytoma only to a full VHL 2 phenotype with RCC and pancreatic lesion. These findings contribute to a better understanding of the fundamental mechanisms of VHL disease and its phenotypic variability. Further, we have provided rapid VHL screening for the families in central Europe, which has resulted in improved diagnosis and clinical management.

Effect of ketoconazole on human ovarian C17,20-desmolase and aromatase

Ketoconazole, an imidazole antimycotic drug, inhibits steroid biosynthesis in adrenal and testicular tissue by blocking cytochrome P-450 dependent enzymes. To study the effect of ketoconazole on steroid biosynthesis in the human ovary we incubated human ovarian tissue (mainly theca cells) or granulosa cells with radiolabeled precursors and increasing concentrations of ketoconazole. After incubation, steroids were extracted and separated by thin layer chromatography (TLC). Activity of C17,20-desmolase and aromatase was estimated by measuring the amount of their radioactive products with liquid scintillation counting. After incubation of ovarian tissue with [3H]17-hydroxyprogesterone the production of [3H]androstenedione was reduced by increasing concentrations of ketoconazole (0-200 microM) to a minimum of 31% of basal production. This indicates a strong inhibition of ovarian C17,20-desmolase by ketoconazole with a 50% inhibiting concentration (IC50) of 23 microM. After incubation of human granulosa cells with ketoconazole (0-2000 microM) and [3H]androstenedione the production of [3H]estrone and [3H]estradiol was suppressed to minimally 37 and 35% of basal values, indicating a significant inhibition of ovarian aromatase. IC50-values were 105 microM ketoconazole for estradiol and 130 microM for estrone. In conclusion, ketoconazole was shown to inhibit human ovarian C17,20-desmolase and aromatase in vitro. As in human adrenals and testes ovarian C17,20-desmolase seems to be most sensitive to the inhibitory effect of ketoconazole.

Insulin-like growth factors and insulin-like growth factor binding proteins in adult patients with severe liver disease before and after orthotopic liver transplantation.

Introduction: The liver is the main source of serum insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) and the concentration of these proteins might reflect liver function. Methods: In a retrospective longitudinal study we examined serum levels of total and free IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 in 21 adult patients with end-stage liver disease before and after orthotopic liver transplantation (LTX) by sensitive and specific RIAs. In each patient, the mean value of at least three measurements before and after LTX was calculated. Results: Before LTX, serum levels of total and free IGF-I, IGF-II, IGFBP-3 were low and showed a rapid and significant increase in almost all patients after successful LTX (total IGF-I: 30 ± 7 vs. 256 ± 30 ng/ml, p < 0.001; free IGF-I: 1.3 ± 0.3 vs. 3.5 ± 0.6 ng/ml, p < 0.01; IGF-II: 177 ± 28 vs. 618 ± 30 ng/ml, p < 0.001; IGFBP-3: 1,230 ± 136 vs. 3,665 ± 264 ng/ml, p < 0.001). In contrast, IGFBP-1 was found to be high immediately before LTX, and declined to normal levels after LTX (210 ± 40 vs. 90 ± 15 ng/ml, p < 0.01), while IGFBP-2 did not show any significant changes (1,154 ± 296 vs. 1,303 ± 192 ng/ ml). Positive correlations were found between IGF-I, IGF-II or IGFBP-3, and serum pseudocholinesterase (R = 0.50, 0.72 and 0.61 respectively, p < 0.001). Negative correlations were found between IGF-I, IGF-II or IGFBP-3, and prothrombin time (R = 0.50, 0.59 and 0.51 respectively, p < 0.001). Conclusion: Patients with severe liver disease show decreased levels of total and free IGF-I, IGF-II and IGFBP-3, and increased levels of IGFBP-1. These abnormalities are promptly normalized after successful LTX. Thus, serum levels of IGF-I, IGF-II and IGFBP-3 might be useful parameters for the assessment of liver function.

Differential Regulation of Insulin-Like Growth Factor-(IGF) I and IGF-Binding Protein (IGFBP) Secretion by Human Peripheral Blood Mononuclear Cells

Background: Recent studies have shown that immunocompetent cells synthesize and express growth hormone (GH), growth hormone receptors (GH-R), insulin-like growth factor I (IGF-I), IGF-I receptors (IGF-I-R) and different insulin-like growth factor binding proteins (IGFBPs). The aim of the current study was to evaluate the regulation of IGFBP and IGF-I secretion from immunocompetent cells by different mitogens. Methods/Results: We studied the in vitro secretion pattern of IGFBPs and IGF-I from human peripheral blood mononuclear cells (PBMC), derived from 10 normal adults and 8 GH-deficient patients with adult onset. In serum-free conditioned medium of unstimulated PBMC, derived from normal adults, Western ligand blotting (1D-WLB) revealed a 24-kD, a 34-kD and a 39/43-kD doublet band to be most prominent. According to their molecular weight and two-dimensional Western ligand blot analysis (2D-WLB), these bands are deglycosylated IGFBP-4, IGFBP-2 and IGFBP-3, respectively. When the cells were treated with the T-cell mitogen phytohemagglutinin (PHA) (10 µg/ml), a differential stimulation of IGFBPs was found with a 2.57 ± 0.48-fold increase of IGFBP-4 (p < 0.01), a 1.55 ± 0.13-fold increase of IGFBP-2 (p < 0.01), and a 1.35 ± 0.19-fold increase of IGFBP-3 (n.s.). In contrast, treatment with the B-cell mitogen pokeweed mitogen (PWM) (10 µg/ml) caused only a modest 1.40 ± 0.07-fold increase of IGFBP-4 (p < 0.01). Treatment with rhGH (100 ng/ml) or rhIGF-I (200 ng/ml) caused no significant induction of any specific band, respectively. In contrast to the secretion pattern of IGFBPs, IGF-I secretion of the PBMC was not stimulated by either PHA or PWM, but showed a significant increase after GH incubation (p < 0.01). A similar differentiated secretion pattern of IGFBPs and IGF-I was also observed in the conditioned medium of PBMC, derived from GH-deficient patients. Conclusion: In summary, at least three different IGFBPs are secreted by human PBMC. Secretion of IGFBPs by PBMC is differentially regulated by different lymphocyte mitogens. Secretion of IGFBPs by PBMC is independent of GH or IGF-I, whereas the secretion of IGF-I is stimulated by GH. PBMC derived from normal adults and GH-deficient patients show similar patterns of IGF-I and IGFBPs secretion, thus indicating that the paracrine/autocrine IGF-I–IGFBPs interactions of the PBMC are not altered by pituitary GH deficiency.

Comparative Study of gp130 Cytokine Effects on Corticotroph AtT-20 Cells – Redundancy or Specificity of Neuroimmunoendocrine Modulators?

Objective: This comparative in vitro study examined the effects of all known gp130 cytokines on murine corticotroph AtT-20 cell function. Methods: Cytokines were tested at equimolar concentrations from 0.078 to 10 nM. Tyrosine phosphorylation of the signal transducer and activator of transcription (STAT)3 and STAT1, the STAT-dependent suppressor of cytokine signaling (SOCS)-3 promoter activity, SOCS-3 gene expression, STAT-dependent POMC promoter activity and adrenocorticotropic hormone (ACTH) secretion were determined. Results: Leukemia inhibitory factor (LIF), human oncostatin M (OSM) and cardiotrophin (CT)-1 (LIFR/gp130 ligands), as well as ciliary neurotrophic factor (CNTF) and novel neurotrophin-1/B-cell stimulating factor-3 (CNTFRα/LIFR/gp130 ligands) are potent stimuli of corticotroph cells in vitro. In comparison, interleukin (IL)-6 (IL-6R/gp130 ligand) and IL-11 (IL-11R/gp130 ligand) exhibited only modest direct effects on corticotrophs, while murine OSM (OSMR/gp130 ligand) showed no effect. Conclusion: (i) CNTFR complex ligands are potent stimuli of corticotroph function, comparable to LIFR complex ligands; (ii) IL-6 and IL-11 are relatively weak direct stimuli of corticotroph function; (iii) differential effects of human and murine OSM suggest that LIFR/gp130 (OSMR type I) but not OSMR/gp130 (OSMR type II) are involved in corticotroph signaling. (iv) CT-1 has the hitherto unknown ability to stimulate corticotroph function, and (v) despite redundant immuno-neuroendocrine effects of different gp130 cytokines, corticotroph cells are preferably activated through the LIFR and CNTFR complexes.

Alterations of the insulin-like growth factor system in patients with polycythemia vera.

The molecular etiology of Polycythemia vera (PV) is still undetermined. Recently, enhanced tyrosine phosphorylation of the insulin-like growth factor-I receptor (IGF-IR) has been shown in PV bone marrow progenitors and peripheral blood mononuclear cells (PBMNC), and elevated levels of IGF binding protein-1 (IGFBP-1) in the serum of PV patients have been reported. To identify further alterations of circulating IGFBPs, the IGFBP profile in the serum of 12 PV patients was compared with age- and sex-matched controls by Western ligand blot (WLB), two-dimensional WLB, IGFBP-3 immunoblot and specific RIA for IGFBP-1, -2, -3 and IGFBP-4. To elucidate a role for the IGF-IR in the pathogenesis of PV, basal and IGF-I stimulated tyrosine phosphorylation of the IGF-IR beta-subunit in PBMNC of PV patients or controls was determined by WLB. Furthermore, exons 2, 3 and 15-21 of the IGF-IR were screened for mutations by PCR-single strand conformation polymorphism analysis (PCR-SSCP). We found alterations of the IGFBP profile in the serum of eight out of 12 examined patients including elevated levels of IGFBP-1, -2 and -4, decreased levels of IGFBP-3 and an increase in IGFBP-3 fragment. However, no differences in tyrosine phosphorylation of the IGF-IR in PV patients, neither basal nor IGF-I induced, were detected. Furthermore, no mutations within the screened exons of the IGF-IR could be identified by PCR-SSCP. We conclude that there is no direct impairment of IGF-IR structure or function, but an altered IGFBP profile in a significant portion of PV patients which might contribute to the pathogenesis of PV in these patients.

[Primary hyperparathyroidism, adrenal tumors and neuroendocrine tumors of the pancreas--clinical diagnosis and imaging requirements].

ZusammenfassungEinleitung. Die Diagnostik von Erkrankungen der Nebenschilddrüse, der Nebenniere und von neuroendokrinen Tumoren des Pankreas erfolgt primär klinisch-endokrinologisch.Die Anforderungen an die Bildgebung bei der nachfolgenden Lokalisationsdiagnostik sind komplex, und die verschiedenen bildgebenden Verfahren bei den jeweiligen Tumorentitäten von unterschiedlichem Stellenwert. Material und Methodik. Aktuelle Literaturrecherche mittels PubMed. Ergebnisse. Beim primären Hyperparathyreoidismus sind die Bestimmung der Knochendichte mittels DXA und der Nachweis einer Nephrolithiasis mittels Sonographie oder nativem Niedrigdosis-CT indiziert. Eine über die Sonographie von Schilddrüse und Nebenschilddrüse hinausgehende präoperative Lokalisationsdiagnostik (Sestamibiszintigraphie, MRT) ist nur beim geplanten Einsatz von minimalinvasiven Operationstechniken oder bei Zweiteingriffen notwendig. Bei Tumoren der Nebenniere ist die diagnostische Wertigkeit von CT und MRT in etwa gleich.Beim Phäochromozytom ist zusätzlich die MIBG-Szintigraphie indiziert.Jedes Inzidentalom der Nebenniere muss endokrinologisch abgeklärt werden.Eine Feinnadelpunktion oder Stanzbiopsie der Nebenniere ist nur selten indiziert; vorher muss laborchemisch ein Phäochromozytom ausgeschlossen werden.Für die Lokalisationsdiagnostik der neuroendokrinen Tumoren des Pankreas werden v. a.Somatostatinrezeptorszintigraphie, Endosonographie und spezielle Sequenzprotokolle der MRT erfolgreich eingesetzt. Diskussion. Spezifische Lokalisationsstrategien werden für die einzelnen Tumorentitäten vorgeschlagen. Die kontinuierliche technische Weiterentwicklung der einzelnen bildgebenden Verfahren erfordert eine regelmäßige Neubewertung.AbstractIntroduction. Diseases of the parathyroids, the adrenals and of neuroendocrine tumors of the pancreas are primarily diagnosed by clinical and endocrinological evaluation.The requirements concerning various imaging techniques and their relative importance in localization strategies of the different tumors are complex. Materials and methods. Current literature search, using PubMed. Results. Evaluation of primary hyperparathyroidism requires bone densitometry by DXA and search for nephrolithiasis by ultrasound or native CT examination.While ultrasound of the thyroid and parathyroids seems useful before any parathyroid surgery,more extensive preoperative localization strategies (sestamibi scintigraphy, MRI) should be restricted to minimal invasive parathyroid surgery or reoperations.For adrenal tumors CT and MRI are of similar diagnostic value. Imaging of pheochromocytomas should be completed by MIBG scintigraphy. Each adrenal incidentaloma requires an endocrinological work-up.A fine-needle aspiration or core needle biopsy of an adrenal tumor is rarely indicated.Before adrenal biopsy a pheochromocytoma has to be excluded.Successful localization strategies for neuroendocrine tumors of the pancreas include somatostatin receptor scintigraphy, endoscopic ultrasound and MRI. Discussion. Specific localization strategies have been established for the aforementioned tumors.The continuous progress of different imaging techniques requires a regular reevaluation of these localization strategies.

Resection of a branchiomeric paraganglioma at a rare extrapulmonary location

Thoracic paragangliomas are a rare cause of hypertension. We report the occurrence of a sporadic benign norepinephrine-producing branchiomeric paraganglioma in a 32-year-old man with paroxysms of hypertension. After localization by iodine 123-metaiodobenzyl-guanidine scintigraphy and magnetic resonance imaging, the paraganglioma was resected successfully below the right pulmonary artery through a right-sided posterolateral thoracotomy. The particular location was consistent with a branchiomeric paraganglioma in an extremely rare extrapulmonary location.