Corinna Grasemann

Lin28a transgenic mice manifest size and puberty phenotypes identified in human genetic association studies

Recently, genome-wide association studies have implicated the human LIN28B locus in regulating height and the timing of menarche. LIN28B and its homolog LIN28A are functionally redundant RNA-binding proteins that block biogenesis of let-7 microRNAs. lin-28 and let-7 were discovered in Caenorhabditis elegans as heterochronic regulators of larval and vulval development but have recently been implicated in cancer, stem cell aging and pluripotency. The let-7 targets Myc, Kras, Igf2bp1 and Hmga2 are known regulators of mammalian body size and metabolism. To explore the function of the Lin28–Let-7 pathway in vivo, we engineered transgenic mice to express Lin28a and observed in them increased body size, crown-rump length and delayed onset of puberty. Investigation of metabolic and endocrine mechanisms of overgrowth in these transgenic mice revealed increased glucose metabolism and insulin sensitivity. Here we report a mouse that models the human phenotypes associated with genetic variation in the Lin28–Let-7 pathway.

Gains and overexpression identify DEK and E2F3 as targets of chromosome 6p gains in retinoblastoma.

The paediatric eye tumour retinoblastoma is initiated by inactivation of RB1, a tumour suppressor on chromosome 13q. In addition to RB1 loss, many retinoblastomas show other genetic alterations including gains on chromosomes 6p21–pter and 1q31–q32. Recently, the minimal region of gains on chromosome 6 was narrowed to band p22. We examined genomic gains and expression changes in primary retinoblastomas to identify potential target genes in 6p22. Quantitative multiplex PCR detected copy numbers ⩾3 in 25 (33%) tumours and no gains in 31 of 76 (40%) tumours. The remaining 20 (26%) samples showed gains only at some loci, most often including E2F3 and DEK in 6p22.3. Analysis of RNA from 21 primary retinoblastomas showed that expression levels of these and some other genes in 6p22 correspond to DNA gains. However, KIF 13A, a reported candidate oncogene on 6p, was expressed at low levels or absent. Clinical manifestation of tumours with gains at all 6p22 loci was distinct in that distribution of age at diagnosis was markedly shifted to older age compared to tumours with no or partial gains. In summary, our results suggest that DEK and E2F3 are potential targets of 6p gains in retinoblastoma.

Removal of metabolites, cytokines and hepatic growth factors by extracorporeal liver support in children.

Background: Molecular Adsorbents Recirculating System (MARS)-mini has recently been approved and applied in children with hepatic failure. However, its indication, efficacy and capability to induce liver regeneration remain unclear. The aim of our pilot study in children was to analyse the impact of MARS on markers of detoxification and regeneration. Methods: In children with fulminant Wilsons disease and bridged with MARSmini for liver transplantation, we analyzed toxic metabolites (bile acids, bilirubin, lactate, ammonia, tryptophan and copper), regulators of the inflammatory cascade [nitrate, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), methionine, cystine and hyaluronic acid] and hepatic growth factors [hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), cortisol, corticosteroid-binding globulin (CBG), insulin-like growth factor-1 (IGF-1), angiogenin, vascular endothelial growth factor (VEGF), IL-6 and TNF-α] from blood, albumin circuit and haemodialysate from four applications. Results: In all four applications, transfer of toxic metabolites (6/6) and inflammatory mediators (6/6), but also of hepatic growth factors (9/10), into the albumin circuit of MARS was consistently detected. Corresponding blood levels were decreased for 3/6 metabolites, 3/6 inflammatory mediators and 1/10 growth factors and increased for 1/10 growth factors. Bridging for liver transplantation was successful with MARS. Conclusions: In our prospective study, substantial extraction of albumin-bound and water-soluble candidate substances was detected with variable effect on respective blood levels. Notably, essential factors inducing liver regeneration were simultaneously removed. These data provide a basis for evaluation of liver restoration and efficacy of liver support in children with liver failure to devise a collaborative, multicentre trial.

Decreased systemic bioavailability of L-arginine in patients with cystic fibrosis

BackgroundL-arginine is the common substrate for nitric oxide synthases and arginases. Increased arginase levels in the blood of patients with cystic fibrosis may result in L-arginine deficiency and thereby contribute to low airway nitric oxide formation and impaired pulmonary function.MethodsPlasma amino acid and arginase levels were studied in ten patients with cystic fibrosis before and after 14 days of antibiotic treatment for pulmonary exacerbation. Patients were compared to ten healthy non-smoking controls.ResultsSystemic arginase levels measured by ELISA were significantly increased in cystic fibrosis with exacerbation compared to controls (17.3 ± 12.0 vs. 4.3 ± 3.4 ng/ml, p < 0.02). Arginase levels normalized with antibiotic treatment. Plasma L-arginine was significantly reduced before (p < 0.05) but not after treatment. In contrast, L-ornithine, proline, and glutamic acid, all downstream products of arginase activity, were normal before, but significantly increased after antibiotic therapy. Bioavailability of L-arginine was significantly reduced in cystic fibrosis before and after exacerbation (p < 0.05, respectively).ConclusionThese observations provide further evidence for a disturbed balance between the L-arginine metabolic pathways in cystic fibrosis.

Oral L-arginine supplementation in cystic fibrosis patients: a placebo-controlled study

Exhaled nitric oxide (eNO) is decreased in cystic fibrosis (CF). The effect of oral L-arginine, the precursor of enzymatic nitric oxide (NO) formation, on airway NO in patients with CF was studied. In a pilot study, oral L-arginine was given in a single dose of 200 mg·kg−1 body weight to eight healthy controls and eight CF patients. Subsequently, the same L-arginine dose was given to 10 patients with CF (five females) t.i.d. for 6 weeks in a randomised double-blind placebo-controlled crossover study. A single dose of oral L-arginine resulted in a 5.5-fold increase of L-arginine in plasma and a 1.3-fold increase of L-arginine in sputum after 2 h. Maximum eNO, within 3 h of L-arginine intake, increased significantly in both CF patients (5.4±2.1 ppb versus 8.3±3.5 ppb) and controls (18.0±8.1 ppb versus 26.4±12.3 ppb). Supplementation of L-arginine for 6 weeks resulted in a sustained increase in eNO compared to placebo (9.7±5.7 ppb versus 6.3±3.1 ppb). An effect of L-arginine supplementation on forced expiratory volume in one second was not observed. These data indicate that airway nitric oxide formation in cystic fibrosis patients can be augmented with oral L-arginine supplementation.

Effects of RANK-Ligand Antibody (Denosumab) Treatment on Bone Turnover Markers in a Girl With Juvenile Paget's Disease

CONTEXT Juvenile Pagets disease (JPD) is an extremely rare, yet painful and debilitating bone disease with onset occurring during early childhood. JPD can be caused by loss of function of osteoprotegerin, resulting in subsequent stimulation of osteoclasts via the receptor activator of nuclear factor-κB (RANK) pathway. Increased bone turnover and lack of bone modeling lead to severe deformities, frequent fractures, short stature, and loss of hearing. SETTING The treatment for JPD is challenging and has previously been based on administration of either calcitonin or bisphosphonates. However, with the development of denosumab, a receptor activator of nuclear factor-κB-ligand (RANKL) antibody, a treatment targeting the pathophysiology of JPD may be available. We report the effects of denosumab treatment on an 8-year-old girl with a severe form of JPD. PATIENT Before starting the denosumab treatment regimen, the patient had been treated for 3.5 years with iv pamidronate. INTERVENTION AND OUTCOME The administration of denosumab resulted in improved disease control compared with bisphosphonate, as assessed by monitoring markers of bone turnover. Alkaline phosphatase levels dropped within the normal range and remained at normal levels for 5 months after the final dose of denosumab. Additionally, bone pain was more efficiently controlled with denosumab. However, concomitant with the first injection, severe hypocalcemia developed, for which the patient was hospitalized and iv calcium supplementation was required for 13 days. CONCLUSIONS Denosumab appears to be significantly effective for osteoclast inhibition for the treatment of JPD. However, in our patient, denosumab administration was associated with severe hypocalcemia, indicating that close monitoring of calcium levels is required during treatment.

A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial to Evaluate the Metabolic and Respiratory Effects of Growth Hormone in Children With Cystic Fibrosis

OBJECTIVE. Positive effects of growth hormone therapy on growth, nutritional status, and lung function have been observed in patients with cystic fibrosis, but the current evidence is based on unblinded studies that involved a small number of patients. This trial was designed as a multicenter, randomized, placebo-controlled, double-blind study to assess the efficacy and safety of 2 dosages of growth hormone in cystic fibrosis. METHODS. Sixty-three dystrophic patients with cystic fibrosis were randomly assigned for 24 weeks to 1 of 3 treatment arms: growth hormone dosage of 0.11 IU/kg body weight per day, growth hormone dosage of 0.21 IU/kg body weight per day, or placebo. The 24-week double-blind period was followed by an open treatment period of 24 weeks. The primary outcome measure was the change in forced expiratory volume in 1 second in percentage predicted from baseline. Secondary outcome measures were changes in height, weight, and exercise tolerance. RESULTS. Height, growth velocity, and growth factors (insulin-like growth factor 1 and insulin-like growth factor–binding protein 3) increased significantly in both treatment groups, whereas weight gain did not differ between the growth hormone groups and placebo. A trend toward improvement in absolute forced vital capacity was observed in patients who received the higher growth hormone dosage, whereas forced expiratory volume in 1 second did not change significantly with growth hormone treatment. Maximal oxygen uptake during peak exercise increased significantly in treated patients. There were no significant differences in the frequency or severity of adverse effects or in the incidence of abnormalities in glucose metabolism. CONCLUSIONS. These data suggest that in the group investigated, growth hormone therapy was well tolerated and had positive metabolic effects but did not result in short-term improvement of lung function in patients with cystic fibrosis.

Neurotrophin receptor expression in human primary retinoblastomas and retinoblastoma cell lines.

Neurotrophin receptor signaling regulates proliferation, differentiation and death of neuronal cells. Expression of Trk receptors has been implicated in the pathogenesis and prognosis of embryonal tumors, including neuroblastoma, nephroblastoma, and medulloblastoma.

Maternal Vitamin D Status in Preeclampsia: Seasonal Changes Are Not Influenced by Placental Gene Expression of Vitamin D Metabolizing Enzymes

Preeclampsia, a hypertensive disorder in pregnancy develops in 2–8% of pregnancies worldwide. Winter season and vitamin D deficiency have been associated with its onset. Objective To investigate the influence of season on maternal vitamin D status and placental vitamin D metabolism. Methods 25-OH vitamin D and 1,25-(OH)2 vitamin D were measured in maternal serum obtained during the winter or summer months from 63 pregnant women at delivery (43 healthy, 20 preeclampsia). In a subgroup, mRNA expression of CYP24A1 (24-hydroxylase), CYP27B1 (1α-hydroxylase) and VDR (vitamin D receptor) were quantified by real time PCR in placental samples of 14 women with normal pregnancies and 13 with preeclampsia. Results In patients with preeclampsia,25-OH vitamin D levels were lower, but differed significantly from controls only in summer (18.21±17.1 vs 49.2±29.2 ng/mL, P<0.001), whereas 1,25-(OH)2 vitamin D levels were significantly lower only in winter (291±217 vs 612.3±455 pmol/mL, P<0.05). A two-factorial analysis of variance produced a statistically significant model (P<0.0001) with an effect of season (P<0.01) and preeclampsia (P = 0.01) on maternal 25-OH vitamin D levels, as well as a significant interaction between the two variables (P = 0.02). Placental gene expression of CYP24A1, CYP27B1, and VDR did not differ between groups or seasons. A negative correlation between placental gene expression of CYP24A1 and CYP27B1 was observed only in healthy controls (r = −0.81, P<0.0001). Summary Patients with preeclampsia displayed lower vitamin D serum levels in response to seasonal changes.The regulation of placental CYP24A1, but not of the VDR or CYP27B1 might be altered in preeclampsia.

Parental Diabetes: The Akita Mouse as a Model of the Effects of Maternal and Paternal Hyperglycemia in Wildtype Offspring

Aim/Hypothesis Maternal diabetes and high-fat feeding during pregnancy have been linked to later life outcomes in offspring. To investigate the effects of both maternal and paternal hyperglycemia on offspring phenotypes, we utilized an autosomal dominant mouse model of diabetes (hypoinsulinemic hyperglycemia in Akita mice). We determined metabolic and skeletal phenotypes in wildtype offspring of Akita mothers and fathers. Results Both maternal and paternal diabetes resulted in phenotypic changes in wildtype offspring. Phenotypic changes were more pronounced in male offspring than in female offspring. Maternal hyperglycemia resulted in metabolic and skeletal phenotypes in male wildtype offspring. Decreased bodyweight and impaired glucose tolerance were observed as were reduced whole body bone mineral density and reduced trabecular bone mass. Phenotypic changes in offspring of diabetic fathers differed in effect size from changes in offspring of diabetic mothers. Male wildtype offspring developed a milder metabolic phenotype, but a more severe skeletal phenotype. Female wildtype offspring of diabetic fathers were least affected. Conclusions Both maternal and paternal diabetes led to the development of metabolic and skeletal changes in wildtype offspring, with a greater effect of maternal diabetes on metabolic parameters and of paternal diabetes on skeletal development. The observed changes are unlikely to derive from Mendelian inheritance, since the investigated offspring did not inherit the Akita mutation. While fetal programming may explain the phenotypic changes in offspring exposed to maternal diabetes in-utero, the mechanism underlying the effect of paternal diabetes on wildtype offspring is unclear.