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The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro. This process requires both the overexpression of the nuclear receptor Nurr 1 and factors derived from local type 1 astrocytes. Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons. Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinson's disease.

Histidine-rich glycoprotein (HRG) is implicated in tumor growth and metastasis by regulation of angiogenesis and inflammation. HRG is produced by hepatocytes and carried to tissues via the circulation. We hypothesized that HRG's tissue distribution and turnover may be mediated by inflammatory cells. Biodistribution parameters were analyzed by injection of radiolabeled, bioactive HRG in the circulation of healthy and tumor-bearing mice. 125I-HRG was cleared rapidly from the blood and taken up in tissues of healthy and tumor-bearing mice, followed by degradation, to an increased extent in the tumor-bearing mice. Steady state levels of HRG in the circulation were unaffected by the tumor disease both in murine tumor models and in colorectal cancer (CRC) patients. Importantly, stromal pools of HRG, detected in human CRC microarrays, were associated with inflammatory cells. In agreement, microautoradiography identified 125I-HRG in blood vessels and on CD45-positive leukocytes in mouse tissues. Moreover, radiolabeled HRG bound in a specific, heparan sulfate-independent manner, to differentiated human monocytic U937 cells in vitro. Suppression of monocyte differentiation by systemic treatment of mice with anti-colony stimulating factor-1 neutralizing antibodies led to reduced blood clearance of radiolabeled HRG and to accumulation of endogenous HRG in the blood. Combined, our data show that mononuclear phagocytes have specific binding sites for HRG and that these cells are essential for uptake of HRG from blood and distribution of HRG in tissues. Thereby, we confirm and extend our previous report that inflammatory cells mediate the effect of HRG on tumor growth and metastatic spread.

Background Prediction of preeclampsia is of great interest and the coagulation system as well as the angiogenic pathway is known to be dysfunctional in preeclampsia. Histidine-rich glycoprotein (HRG) is a protein interacting with both these biological systems and the purpose of this prospective, longitudinal cohort study was to analyze whether there is a difference in circulating levels of HRG during pregnancy in women developing preeclampsia compared to normal healthy pregnancies. We furthermore wanted to evaluate whether HRG has the potential of being an early biomarker of preeclampsia. Methods A cohort of healthy pregnant women (n = 469) was enrolled at gestational weeks 8-12. Plasma samples were collected at gestational weeks 10, 25, 28, 33, and 37 and analyzed with an enzyme-linked immunosorbent assay. Results The levels of HRG decreased during pregnancy in all women, but the levels were significantly lower at gestational weeks 10, 25, and 28 in women who later developed preeclampsia than in normal pregnant women (P < 0.05, P < 0.05, and P < 0.05). Conclusion Our data indicates that HRG levels in plasma might be a possible biomarker already in gestational week 10 for prediction of later onset of preeclampsia in a low risk population. American Journal of Hypertension advance online publication 20 January 2011; doi:10.1038/ajh.2010.264

Background The progression of colorectal cancer (CRC) involves recurrent amplifications/mutations in the epidermal growth factor receptor (EGFR) and downstream signal transducers of the Ras pathway, KRAS and BRAF. Whether genetic events predicted to result in increased and constitutive signaling indeed lead to enhanced biological activity is often unclear and, due to technical challenges, unexplored. Here, we investigated proliferative signaling in CRC using a highly sensitive method for protein detection. The aim of the study was to determine whether multiple changes in proliferative signaling in CRC could be combined and exploited as a “complex biomarker” for diagnostic purposes. Methods We used robotized capillary isoelectric focusing as well as conventional immunoblotting for the comprehensive analysis of epidermal growth factor receptor signaling pathways converging on extracellular regulated kinase 1/2 (ERK1/2), AKT, phospholipase Cγ1 (PLCγ1) and c-SRC in normal mucosa compared with CRC stage II and IV. Computational analyses were used to test different activity patterns for the analyzed signal transducers. Results Signaling pathways implicated in cell proliferation were differently dysregulated in CRC and, unexpectedly, several were downregulated in disease. Thus, levels of activated ERK1 (pERK1), but not pERK2, decreased in stage II and IV while total ERK1/2 expression remained unaffected. In addition, c-SRC expression was lower in CRC compared with normal tissues and phosphorylation on the activating residue Y418 was not detected. In contrast, PLCγ1 and AKT expression levels were elevated in disease. Immunoblotting of the different signal transducers, run in parallel to capillary isoelectric focusing, showed higher variability and lower sensitivity and resolution. Computational analyses showed that, while individual signaling changes lacked predictive power, using the combination of changes in three signaling components to create a “complex biomarker” allowed with very high accuracy, the correct diagnosis of tissues as either normal or cancerous. Conclusions We present techniques that allow rapid and sensitive determination of cancer signaling that can be used to differentiate colorectal cancer from normal tissue.

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.

Our previous finding that skin-derived and muscle-derived molecules can be used to sort regenerating rat sciatic nerve axons evoked questions concerning neuron-target interactions at the level of single cells, which prompted the present study. The results show that dorsal root ganglion (DRG) neurons co-cultured with fibroblast-like skin-derived cells emit many neurites. These have a proximal linear segment and a distal network of beaded branches in direct relation to skin-derived cells. Electron microscopic examination of such co-cultures showed bundles of neurites at some distance from the target cells and single profiles closely apposed to subjacent cells. RNase protection assay revealed that cultivated skin-derived cells express nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). In co-cultures of DRG neurons and 3T3 fibroblasts overexpressing either of the neurotrophins produced by skin-derived cells the picture varied. NT-3 transfected 3T3 fibroblasts gave a growth pattern similar to that seen with skin-derived cells. Neurons co-cultured with mock-transfected 3T3 fibroblasts were small and showed weak neurite growth. In co-cultures with a membrane insert between skin-derived cells or 3T3 fibroblasts and DRG neurons few neurons survived and neurite growth was very sparse. We conclude that skin-derived cells stimulate neurite growth from sensory neurons in vitro, that these cells produce NGF, BDNF, NT-3 and NT-4 and that 3T3 fibroblasts producing NT-3 mimic the effect of skin-derived cells on sensory neurons in co-culture. Finally the results suggest that cell surface molecules are important for neuritogenesis.