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Constanze Hantel, Felicitas Lewrick, Martin Reincke, Regine Süss, and Felix Beuschlein

Adrenocortical carcinoma (ACC) is a rare endocrine tumor entity with poor prognosis. Medical treatment is limited to common cytotoxic agents, which are associated with low treatment responses. Thus, lack of therapeutic efficacy demands innovative treatment options for patients with advanced ACC. Recently, we have developed and characterized anti-IGF1 receptor (IGF1-R) immunoliposomes (SSLD-1H7) for the treatment of neuroendocrine tumors of the gastroenteropancreatic system. As previous results indicated putative applicability also for other IGF1-R-overexpressing tumor entities, we initiated testing of liposomal preparations in in vitro and in vivo models of ACC. Adrenocortical NCIh295 cells were used for in vitro association studies with different liposomal formulations. Thereby, flow cytometry revealed high cellular association and internalization of anti-IGF1-R immunoliposomes (soy phosphatidylcholine (SPC)/cholesterol (Chol)–polyethyleneglycol (PEG)-1H7, 50.1±2.2%). Moreover, internalization of pegylated liposomes (SPC/Chol–PEG, 57.1±2.4%) and an even higher uptake of plain liposomes (84.6±0.8%; P<0.0001) were detectable in adrenocortical tumor cells. In vivo, liposomal treatments were investigated on NCIh295 tumor xenografts in pharmacokinetic and therapeutic experiments. A significant reduction in tumor size was detectable in NCIh295 tumor-bearing mice after a single treatment with SSLD-1H7 (0.89±0.15 cm; P=0.006) and a diminished efficacy for SSLD–PEG+ (1.01±0.19 cm; P=0.04) in comparison with untreated controls (1.5±0.0 cm). Thus, anti-IGF1-R immunoliposomes have been successfully tested in vitro and in vivo in a preclinical model for ACCs and could, therefore, represent a promising therapeutic approach for this tumor entity. Moreover, a combination of mitotane plus liposomally encapsulated cytostatic agents instead of free drugs could also be an interesting novel treatment option for ACC in the future.

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Charlotte Steenblock, Nicole Bechmann, Felix Beuschlein, Christian Wolfrum, and Stefan R. Bornstein

Obesity is associated with a higher risk of severe COVID-19 and increased mortality. In the current study, we have investigated the expression of ACE2, NRP1, and HMGB1, known to facilitate SARS-CoV-2 cell entry, in adipose tissue from non-COVID-19 control patients with normal weight, overweight and obesity. All factors were expressed, but no significant differences between the groups were observed. Furthermore, diabetes status and medications did not affect the expression of ACE2. Only in obese men, the expression of ACE2 in adipose tissue was higher than in obese women. In adipose tissue from patients that died from COVID-19, SARS-CoV-2 was detected in the adipocytes even though the patients died more than three weeks after the acute infection. This suggests that adipocytes may act as reservoirs for the virus. In COVID-19 patients, the expression of NRP1 was increased in COVID-19 patients with overweight and obesity. Furthermore, we observed an increased infiltration with macrophages in the COVID-19 adipose tissues compared to control adipose tissue. In addition, crown-like structures of dying adipocytes surrounded by macrophages were observed in the adipose tissue from COVID-19 patients. These data suggest that in obese individuals, in addition to an increased mass of adipose tissue that could potentially be infected, increased macrophage infiltration due to direct infection with SARS-CoV-2 and sustained viral shedding, rather than preinfection ACE2 receptor expression, may be responsible for the increased severity and mortality of COVID-19 in patients with obesity.

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Urs Lichtenauer, Igor Shapiro, Simone Sackmann, Jacques Drouin, Jürgen Scheele, Matthias Maneck, Christoph Klein, and Felix Beuschlein

Somatic adrenal stem cells are believed to reside in the periphery of the adrenal cortex throughout life for organ maintenance. Herein, we used the side population (SP) phenomenon to enrich for these progenitors, which made up to 0.01–0.64% of the total cell count. Microarray analysis revealed an expression profile of SP cells, which clearly differed from that of non-SP cells. However, a promising adrenal specific stem cell marker could not be identified. In vitro, SP cells could be maintained in long-term culture, whereas non-SP cells did not proliferate. After 4 weeks of culturing, immunohistochemistry revealed the expression of steroidogenic enzymes such as 3β-HSD, StAR, and P450SCC, suggesting spontaneous differentiation. Interestingly, the quantity of SP cells was significantly diminished in Pbx1 haploinsufficient mice, suggesting a stem cell deficit. By contrast, the subcapsular zone of ACTH-deficient Tpit −/− mice was significantly wider compared with wild-type adrenals (Tpit −/− 259±10.7 vs Tpit +/− 100±12.3%; P<0.01). Accordingly, the number of SP cells in these mice was significantly higher (Tpit −/− 0.45±0.16 vs Tpit +/− 0.13±0.04%; P<0.004). ACTH treatment of these animals reverted the subcapsular zone width and the SP fraction back to normal (130±10.2%; P=0.33 and 0.09%), providing indirect evidence for a stem cell ‘arrest’ in Tpit −/− mice and the role of ACTH in adrenocortical stem cell modulation and differentiation.

Free access

Inga K Johnsen, Marc Slawik, Igor Shapiro, Michaela F Hartmann, Stefan A Wudy, Brendan D Looyenga, Gary D Hammer, Martin Reincke, and Felix Beuschlein

Mouse models of adrenal tumorigenesis have the potential to give insights in the dysregulation of adrenal growth and differentiation. The inbred mouse strain CE/J has been reported to develop adrenal tumors upon gonadectomy (GDX) similar to mice with targeted deletions of the inhibin alpha subunit (Inh−/−). We performed a detailed morphological and molecular characterization of adrenal glands from CE/J mice 8–50 weeks of age to define the cellular origin of these tumors as well as the spatial and temporal expression of marker genes associated with tumor growth. In contrast to the induction of x-zone growth upon GDX in Inh−/− mice, GDX in CE/J mice induced the appearance of sub-capsular nests of densely packed cells that progress into adrenal tumors. Staining for proliferative cell nuclear antigen confirms a substantial increased in cellular proliferation within this sub-capsular compartment and lack of apoptosis upon GDX. Induction of adrenal tumor growth was accompanied by transcriptional changes that otherwise define gonadal endocrine cells. These regulated genes included transcription factors such as GATA-4, WT-1, FOG-1, and steroidogenic factor-1 (SF-1), peptide hormones such as Mullerian-inhibiting substance (MIS), hormone receptors including luteinizing hormone and MIS receptor, and steroidogenic enzymes such as P450c17 and P450 aromatase. The functional significance of steroid enzyme expression was demonstrated by a gradual increase of adrenal androgens after GDX. Taken together these data suggest that adrenal tumors in gonad-ectomized CE/J mice are direct derivatives from cells of the proposed sub-capsular stem cell zone. The distinct expression pattern of this cell population is consistent with a defect in the differentiation of these cells into a cell population with functional properties that otherwise define a gonadal endocrine phenotype.

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Ariadni Spyroglou, Sibylle Sabrautzki, Birgit Rathkolb, Tarik Bozoglu, Martin Hrabé de Angelis, Martin Reincke, Martin Bidlingmaier, and Felix Beuschlein

Arterial hypertension represents one of the most common diseases in developed countries and the rennin–angiotensin–aldosterone system is among the major factors in the regulation of blood pressure and sodium balance. With the exception of rare monogenetic diseases, however, inheritance of aldosterone secretion is widely unknown. In this study, we investigated the aldosterone levels in male and female mice of two inbred strains, C3HeB/FeJ and C57BL/6J, as well as their offspring of the F1 and F2 generation. In all cases, female animals displayed lower aldosterone levels than males. Furthermore, C57BL/6J animals had significantly higher aldosterone levels than C3HeB/FeJ mice of the same age and gender. Depending on the paternal origin of the animal, the F1 offspring showed a tendency toward higher aldosterone values when the paternal side was from the C57BL/6J strain. This observation was confirmed in the F2 generation and over repeated measurements over three consecutive years. Quantification of the aldosterone to renin ratio in the different mouse groups did not show any significant differences, and, similarly, the determination of plasma potassium and kidney parameters did not provide any differences. On the molecular level, investigation of the expression of the enzymes involved in steroidogenesis displayed the same trend as for the aldosterone values, with animals hosting C57BL/6J background in their paternal origin having also the highest expression levels for StAR, cyp11a1, and cyp11b2 enzymes. Taken together, we could demonstrate that the genetic background of the animals plays a significant role modulating their plasma aldosterone levels without clear interference of other parameters in the renin–angiotensin–aldosterone system.

Free access

Anthony P Coll, Martin Fassnacht, Steffen Klammer, Stephanie Hahner, Dominik M Schulte, Sarah Piper, Y C Loraine Tung, Benjamin G Challis, Yacob Weinstein, Bruno Allolio, Stephen O’Rahilly, and Felix Beuschlein

Pro-opiomelanocortin (POMC) is a polypeptide precursor that undergoes extensive processing to yield a range of peptides with biologically diverse functions. POMC-derived ACTH is vital for normal adrenal function and the melanocortin α-MSH plays a key role in appetite control and energy homeostasis. However, the roles of peptide fragments derived from the highly conserved N-terminal region of POMC are less well characterized. We have used mice with a null mutation in the Pomc gene (Pomc −/−) to determine the in vivo effects of synthetic N-terminal 1–28 POMC, which has been shown previously to possess adrenal mitogenic activity. 1–28 POMC (20 μg) given s.c. for 10 days had no effect on the adrenal cortex of Pomc −/− mice, with resultant cortical morphology and plasma corticosterone levels being indistinguishable from sham treatment. Concurrent administration of 1–28 POMC and 1–24 ACTH (30 μg/day) resulted in changes identical to 1–24 ACTH treatment alone, which consisted of upregulation of steroidogenic enzymes, elevation of corticosterone levels, hypertrophy of the zona fasciculate, and regression of the X-zone. However, treatment of corticosterone-depleted Pomc −/− mice with 1–28 POMC reduced cumulative food intake and total body weight. These anorexigenic effects were ameliorated when the peptide was administered to Pomc −/− mice with circulating corticosterone restored either to a low physiological level by corticosterone-supplemented drinking water (CORT) or to a supraphysiological level by concurrent 1–24 ACTH administration. Further, i.c.v. administration of 1–28 POMC to CORT-treated Pomc −/− mice had no effect on food intake or body weight. In wild-type mice, the effects of 1–28 POMC upon food intake and body weight were identical to sham treatment, but 1–28 POMC was able to ameliorate the hyperphagia induced by concurrent 1–24 ACTH treatment. In a mouse model which lacks all endogenous POMC peptides, s.c. treatment with synthetic 1–28 POMC alone can reduce food intake and body weight, but has no impact upon adrenal growth or steroidogenesis.