Proposal for publishing and parallel reporting of case reports on adverse drug reactions to authorities by physicians.

For the safe use of medicinal products, it is important that physicians publish adverse experiences with a medicinal product-particularly regarding side effects-in the scientific literature. However, when searching applicable publications, we determined that adverse drug reactions (ADRs) are often published several months after their occurrence. In the context of patient safety, this is rather questionable as new and important information on ADRs is not available quickly enough to be considered in pharmacovigilance systems. This delay is also not acceptable on the background of the timelines-eg, European Union (EU) legislation requires that marketing authorization holders (MAH) report serious ADRs (SADRs) within 15 calendar days. The legal basis for ADR reporting by physicians and other healthcare professionals is specified in article 102 of the EU Directive 2001/83/ EC as amended (2010/84/EU).

MIA is a potential biomarker for tumour load in neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a frequent genetic disease characterized by multiple benign tumours with increased risk for malignancy. There is currently no biomarker for tumour load in NF1 patients. METHODS: In situ hybridization and quantitative real-time polymerase reaction were applied to investigate expression of cartilage-specific genes in mice bearing conditional inactivation of NF1 in the developing limbs. These mice do not develop tumours but recapitulate aspects of NF1 bone dysplasia, including deregulation of cartilage differentiation. It has been recently shown that NF1 tumours require for their growth the master regulator of cartilage differentiation SOX9. We thus hypothesized that some of the cartilage-specific genes deregulated in an Nf1Prx1 mouse model might prove to be relevant biomarkers of NF1 tumours. We tested this hypothesis by analyzing expression of the SOX9 target gene product melanoma-inhibitory activity/cd-rap (MIA) in tumour and serum samples of NF1 patients. RESULTS: Increased expression of Mia was found in Nf1-deficient cartilage in mice. In humans, MIA was expressed in all NF1-related tumours and its serum levels were significantly higher in NF1 patients than in healthy controls. Among NF1 patients, MIA serum levels were significantly higher in those with plexiform neurofibromas and in those with large number of cutaneous (> 1,000) or subcutaneous (> 100) neurofibromas than in patients without such tumours. Most notably, MIA serum levels correlated significantly with internal tumour burden. CONCLUSIONS: MIA is a potential serum biomarker of tumour load in NF1 patients which could be useful in following the disease course and monitoring the efficacy of therapies.

NOA1 is an essential GTPase required for mitochondrial protein synthesis.

Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated with a severe developmental defect of the embryo and trophoblast. Primary embryonic fibroblasts isolated from NOA1 knockout embryos show deficient mitochondrial protein synthesis and a global defect of oxidative phosphorylation (OXPHOS). Additionally, Noa1⁻/⁻ cells are impaired in staurosporine-induced apoptosis. The analysis of mitochondrial ribosomal subunits from Noa1⁻/⁻ cells by sucrose gradient centrifugation and Western blotting showed anomalous sedimentation, consistent with a defect in mitochondrial ribosome assembly. Furthermore, in vitro experiments revealed that intrinsic NOA1 GTPase activity was stimulated by bacterial ribosomal constituents. Taken together, our data show that NOA1 is required for mitochondrial protein synthesis, likely due to its yet unidentified role in mitoribosomal biogenesis. Thus, NOA1 is required for such basal mitochondrial functions as adenosine triphosphate (ATP) synthesis and apoptosis.

Gata4 and Sp1 regulate expression of the erythropoietin receptor in cardiomyocytes.

Experimental studies indicate significant cardioprotective effects of recombinant erythropoietin (Epo) by binding to the Epo receptor (EpoR) and by inducing various molecular mechanisms, including activation of Gata4, a transcription factor that induces anti-apoptotic genes. However, specific molecular mechanisms of EpoR regulation in cardiomyocytes are unknown. We identified a 774 bp regulatory domain in the EpoR 5' flanking region by reporter gene assays in murine HL-1 cardiomyocytes. The binding sites for Gata and Sp transcription factors both significantly contributed to EpoR promoter activity. DNA-binding studies (EMSA and ChIP assays) identified Gata4 and Sp1 as EpoR promoter-binding proteins in HL1 cardiomyocytes. Although Sp1 alone stimulates EpoR only slightly, forced expression of Gata4 significantly induced EpoR mRNA expression. In addition, knockdown of Gata4 (but also of Sp1) resulted in a significant decrease of EpoR transcript levels in HL-1 cardiomyocytes. Cumulative in vitro data suggest that function of the Sp1 site is essential for the Gata4-mediated transcription. In vivo, analysis of transgenic mice expressing an inducible small-hairpin RNA against Gata4 confirmed suppression of EpoR expression in the heart. Treating mice with high-dose doxorubicin not only resulted in Gata4 protein depletion, but also down-regulated EpoR, followed by up-regulation of EpoR transcripts when Gata4 levels recovered. In conclusion, we identified Gata4 as novel regulator of EpoR transcription in cardiomyocytes. In models of cardiac injury, down-regulation of Gata4 or Sp1 may limit the accessibility of the EpoR for binding of erythropoiesis-stimulating agents (ESA). Thereby our data underline the essential role of Gata4 in mediating cardioprotective effects.

Transgenic mice expressing small interfering RNA against Gata4 point to a crucial role of Gata4 in the heart and gonads.

Homozygous deficiency of the transcription factor Gata4 in mice causes lethality due to defects in ventral morphogenesis and heart tube formation. There is increasing evidence demonstrating that GATA4 function is also relevant for normal developed organ systems, including the heart and endocrinum. To analyze the implication of Gata4 beyond development, we generated transgenic mice expressing inducible small interfering RNA against Gata4. In longitudinal analysis, efficient suppression of Gata4 mRNA (down to 80% of wild-type levels) and protein expression in the heart was detected 38 days after induction of Gata4 short hairpin RNA. Decreased Gata4 expression was associated with reduction in the expression of known cardiac target genes, but the function of the heart remained unperturbed at 20-30% of normal Gata4 levels. Interestingly, Gata4 expression was almost abolished in the ovary and testis. This was accompanied in the testis by a significant reduction of GATA4 downstream target genes, such as the genes encoding Mullerian inhibiting substance (MIS) and steroidogenic acute regulatory (StAR) protein. By contrast, expression levels of Mis and Star were only slightly modified in the ovary, and concentrations of circulating FSH and LH were normal in female transgenic mice after induction of Gata4 short hairpin RNA. However, inhibition of Gata4 expression led to the formation of ovarian teratoma in 10% of females. Histology of the teratomas showed predominantly ectodermal and mesodermal structures. Our data demonstrate that Gata4 is critically involved in the function and integrity of the gonads in vivo.

Modelling neurofibromatosis type 1 tibial dysplasia and its treatment with lovastatin.

BACKGROUND: Bowing and/or pseudarthrosis of the tibia is a known severe complication of neurofibromatosis type 1 (NF1). Mice with conditionally inactivated neurofibromin (Nf1) in the developing limbs and cranium (Nf1Prx1) show bowing of the tibia caused by decreased bone mineralisation and increased bone vascularisation. However, in contrast to NF1 patients, spontaneous fractures do not occur in Nf1Prx1 mice probably due to the relatively low mechanical load. We studied bone healing in a cortical bone injury model in Nf1Prx1 mice as a model for NF1-associated bone disease. Taking advantage of this experimental model we explore effects of systemically applied lovastatin, a cholesterol-lowering drug, on the Nf1 deficient bone repair. METHODS: Cortical injury was induced bilaterally in the tuberositas tibiae in Nf1Prx1 mutant mice and littermate controls according to a method described previously. Paraffin as well as methacrylate sections were analysed from each animal. We divided 24 sex-matched mutant mice into a lovastatin-treated and an untreated group. The lovastatin-treated mice received 0.15 mg activated lovastatin by daily gavage. The bone repair process was analysed at three consecutive time points post injury, using histological methods, micro computed tomography measurements and in situ hybridisation. At each experimental time point, three lovastatin-treated mutant mice, three untreated mutant mice and three untreated control mice were analysed. The animal group humanely killed on day 14 post injury was expanded to six treated and six untreated mutant mice as well as six control mice. RESULTS: Bone injury repair is a complex process, which requires the concerted effort of numerous cell types. It is initiated by an inflammatory response, which stimulates fibroblasts from the surrounding connective tissue to proliferate and fill in the injury site with a provisional extracellular matrix. In parallel, mesenchymal progenitor cells from the periost are recruited into the injury site to become osteoblasts. In Nf1Prx1 mice bone repair is delayed and characterised by the excessive formation and the persistence of fibro-cartilaginous tissue and impaired extracellular matrix mineralisation. Correspondingly, expression of Runx2 is downregulated. High-dose systemic lovastatin treatment restores Runx2 expression and accelerates new bone formation, thus improving cortical bone repair in Nf1Prx1 tibia. The bone anabolic effects correlate with a reduction of the mitogen activated protein kinase pathway hyper-activation in Nf1-deficient cells. CONCLUSION: Our data suggest the potential usefulness of lovastatin, a drug approved by the US Food and Drug Administration in 1987 for the treatment of hypercholesteraemia, in the treatment of Nf1-related fracture healing abnormalities. The experimental model presented here constitutes a valuable tool for the pre-clinical stage testing of candidate drugs, targeting Nf1-associated bone dysplasia.