Differentiation of neurospheres from the enteric nervous system

The enteric nervous system (ENS) derives from neural crest cells, which migrate from the neural tube into the developing gut. The neuronal and glial precursor cells migrate mainly from the oral towards the anal end of the gastrointestinal tract. So far, knowledge about the multipotent influences upon the ENS development, especially its neurotrophic support, derives mainly from knock-out models. The in vitro technique of isolating enteric neuronal precursor cells allows to study the effects of various factors upon their appropriate development in more detail. We therefore adapted the method of growing neurospheres, which are agglomerates of neuronal precursor cells and differentiated neurones and glial cells, from the central nervous system (CNS) for the ENS. The gut of NMRI mice at E12 were dissected, mildly dissociated and plated in 25-cm² culture flasks. The cultures were maintained in N1 supplemented DMEM/F12 medium with the appropriate neurotrophin cocktails (bFGF, GDNF, Neurturin, CNTF). After several days in culture most of the cells die, while the surviving cells form clusters from which domes, and later spheres arise. The spheres could be harvested and processed for further experiments. First investigations revealed, that the amount of precursor cells was much less in enteric neurospheres as seen in corresponding cultures from the CNS. We found about 43% HNK-1-NCAM+ in enteric and approximately 90% Nestin-+ cells in midbrain neurospheres. Differentiation studies of the enteric neurospheres showed that especially ciliary neurotrophic factor (CNTF) increased the number of enteric neurones (PGP positive), while the amount of HNK-1 precursor cells decreased under the influence of all tested neurotrophins but GDNF. The culture of the freshly dissociated enteric neurospheres in a three-dimensional matrix yielded a secondary network which allows to investigate the pattern formation of the ENS. The generation of enteric neurospheres and the following differentiation and 3D culture in vitro can increase our knowledge of the amount and time point of neurotrophic as well as the ECM-protein influence upon the appropriate development of the ENS.     

One-step detection of c-kit point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes

The prognostic significance of somatic activating codon 816 c-kit mutations in pediatric urticaria pigmentosa has not yet been established in detail. Detection of such mutations in archival paraffin-embedded biopsies is usually hampered by an abundance of surrounding normal cells. Here we describe a method for the selective amplification and specific detection of c-kit mutation Asp816-->Val in complete tissue sections cut from up to 24-year-old paraffin blocks. Peptide nucleic acid-mediated polymerase chain reaction clamping of the wild-type allele was combined with on-line mutation detection using oligonucleotide hybridization probes. In DNA extracted from HMC-1 cells heterozygously carrying the c-kit mutation Asp816-->Val, the one-tube assay allowed specific detection of this mutation in a more than 1000-fold excess of normal background DNA within 1 hour and without the need for additional analytical steps. In a series of 38 cases with pediatric urticaria pigmentosa we detected c-kit codons 815 and 816 mutations in 16 cases. Mutation detection did not correlate with clinical outcome after a mean follow-up of 11.2 years. In conclusion, the procedure described may represent an ideal screening tool for all kinds of clinical applications, using point mutations as markers of, for example, early events in carcinogenesis, circulating metastatic tumor cells, and minimal residual disease.     

Migration and differentiation of myogenic precursors following transplantation into the developing rat brain

There is increasing evidence that muscle-derived precursor cells can, under appropriate conditions, give rise to other than myogenic cell types. Transplantation into the embryonic ventricular zone provides a unique opportunity to study the migration and differentiation of non-neural somatic progenitor cells in response to instructive cues within the developing neuroepithelium. Here, we demonstrate that myogenic cell lines grafted into the ventricles of rat embryos showed widespread migration into several host brain compartments. In contrast to incorporation patterns observed after transplantation of neural cells, grafted myoblasts incorporated virtually exclusively along endogenous blood vessels. Preferential incorporation sites included cortex, olfactory bulb, hippocampus, striatum, thalamus, hypothalamus, and tectum. While the engrafted myoblasts showed no evidence of neural differentiation, a fraction exhibited pronounced coexpression of endothelial marker antigens. These findings support the concept of a close developmental relationship between the myogenic and the endothelial lineages. Used as a delivery system, transfected myoblasts may be exploited for widespread gene transfer to the perivascular compartment of the perinatal central nervous system.     

Possibilities of surgery and their differentiated use in the individual treatment of endometriosis

Numerous medical, surgical, and combined therapies have been proposed in the management of endometriosis. This range of treatment options contrasts remarkably with the evidence regarding their respective proven success rates. An exact preoperative differential diagnosis as well as adherence to operative recommendations that have been established meanwhile are essential for optimal results of surgery.     

Neonatal nucleated red blood cell count and postpartum complications in growth restricted fetuses

AIMS: To study relationships between nucleated red blood cell count (NRBC), persistence of NRBC count elevation and neonatal complications in growth restricted fetuses (IUGR). METHODS: Observational study of IUGR neonates (birthweight < 10th percentile). NRBC's/100 WBC were ascertained in a peripheral blood sample. Subsequent daily samples were analyzed until NRBC's fell < 10/100 WBC. NRBC count and days of NRBC elevation were related to complications (respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH), necrotising enterocolitis (NEC), circulatory insufficiency (CI), day 28 oxygen requirement, mortality). RESULTS: 157/298 IUGR neonates (52.7%) had complications, which were associated with a higher NRBC count and persistence of NRBC elevation (12 vs. 189 NRBC's and 1 vs. 4 days; p < 0.0001 respectively). This relationship applied to each complication. Prematurity was the main determinant of RDS, BPD and mortality, while IVH was related to mechanical ventilation, CI to birthweight percentile and NEC to degree of acidemia. Persistence of NRBC count elevation was a statistical contributor for RDS, CI and mortality and the NRBC count to day 28 oxygen requirement. CONCLUSION: NRBC count elevation and persistent NRBC count elevation are associated with perinatal complications in IUGR. Wide ranges in numbers, complex relationships between triggering factors and impacts of other perinatal variables limit the use of NRBC parameters as predictors of complications.