Epidermal growth factor (EGF) stimulation of cultured brain cells. II. Increased production of extracellular soluble proteins.

The production of extracellular soluble proteins was studied in serum-free aggregating cell cultures of fetal rat telencephalon labeled on culture day 7 with a mixture of radioactive amino acid precursors. Cultures treated continuously with epidermal growth factor (EGF; 20 ng/ml) showed a generally increased protein secretion and a particularly enhanced production of a few distinct extracellular proteins. The time lag of this response after an initial dose of EGF (25 ng/ml) on day 7 was 48 h. The total macromolecular radioactivity that accumulated within 96 h of labeling in the media of EGF-treated cultures was 175% of untreated controls, whereas no difference was found in the proportions of intracellular amino acid incorporation. Cultures which received a single dose of EGF (25 ng/ml) on day 1 showed still a greatly increased protein secretion on day 7. Prevention of extracellular protein accumulation by reducing the initial cell number and increasing the rate of media changes did not affect the EGF-induced stimulation of the two glial enzymes, glutamine synthetase and 2',3'-cyclic nucleotide 3'-phosphohydrolase. The results suggest that both the increased production of extracellular proteins and the enhanced development of glial enzymatic activities reflect the stimulated phenotypic expression of EGF-sensitive brain cells.

Effect of age on intraoperative cerebrovascular autoregulation and near-infrared spectroscopy-derived cerebral oxygenation.

Background. Age is an important risk factor for perioperative cerebral complications such as stroke, postoperative cognitive dysfunction, and delirium. We explored the hypothesis that intraoperative cerebrovascular autoregulation is less efficient and brain tissue oxygenation lower in elderly patients, thus, increasing the vulnerability of elderly brains to systemic insults such as hypotension.Methods. We monitored intraoperative cerebral perfusion in 50 patients aged 18-40 and 77 patients >65 yr at two Swiss university hospitals. Mean arterial pressure (MAP) was measured continuously using a plethysmographic method. An index of cerebrovascular autoregulation (Mx) was calculated based on changes in transcranial Doppler flow velocity due to changes in MAP. Cerebral oxygenation was assessed by the tissue oxygenation index (TOI) using near-infrared spectroscopy. End-tidal CO(2), O(2), and sevoflurane concentrations and peripheral oxygen saturation were recorded continuously. Standardized anaesthesia was administered in all patients (thiopental, sevoflurane, fentanyl, atracurium).Results. Autoregulation was less efficient in patients aged >65 yr [by 0.10 (SE 0.04; P=0.020)] in a multivariable linear regression analysis. This difference was not attributable to differences in MAP, end-tidal CO2, or higher doses of sevoflurane. TOI was not significantly associated with age, sevoflurane dose, or Mx but increased with increasing flow velocity [by 0.09 (SE 0.04; P=0.028)] and increasing MAP [by 0.11 (SE 0.05; P=0.043)].Conclusions. Our results do not support the hypothesis that older patients' brains are more vulnerable to systemic insults. The difference of autoregulation between the two groups was small and most likely clinically insignificant.

Insulin-like growth factor I (IGF I) stimulates DNA synthesis in fetal rat brain cell cultures.

Addition of insulin, IGF I or IGF II to serum-free cultures of fetal rat brain cells (gestation day 15/16) significantly stimulates DNA synthesis. The dose-response curves show that IGF I is more potent than insulin; half maximal stimulation of [3H]thymidine incorporation is obtained at about 0.4 nM IGF I and 14 nM insulin, respectively. Cultures initiated 2 days later (gestation day 17/18) showed a decreased responsiveness to both peptides. No additive effect was observed after combined addition of both peptides at near-maximal doses. Both peptides show a latency of action of about 12-18 h. In the presence of either IGF or insulin, neuronal as well as glial enzymes are increased, suggesting that neuronal and glial precursor cell division is influenced. IGF I and IGF II interact with a specific binding site for which insulin competes very weakly; however IGF I and IGF II bind with relatively high affinity to the insulin specific binding site. The present results support the hypothesis that both insulin and IGF stimulate mitotic activity by interacting with specific somatomedin receptors and suggest a physiological role of IGF in the developing brain.

Granulocyte-macrophage colony-stimulating factor elicits bone marrow-derived cells that promote efficient colonic mucosal healing.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy is effective in treating some Crohn's disease (CD) patients and protects mice from colitis induced by dextran sulfate sodium (DSS) administration. However, its mechanisms of action remain elusive. We hypothesized that GM-CSF affects intestinal mucosal repair. METHODS: DSS colitic mice were treated with daily pegylated GM-CSF or saline and clinical, histological, and inflammatory parameters were kinetically evaluated. Further, the role of bone marrow-derived cells in the impact of GM-CSF therapy on DSS colitis was addressed using cell transfers. RESULTS: GM-CSF therapy reduced clinical signs of colitis and the release of inflammatory mediators. GM-CSF therapy improved mucosal repair, with faster ulcer reepithelialization, accelerated hyperproliferative response of epithelial cells in ulcer-adjacent crypts, and lower colonoscopic ulceration scores in GM-CSF-administered mice relative to untreated mice. We observed that GM-CSF-induced promotion of mucosal repair is timely associated with a reduction in neutrophil numbers and increased accumulation of CD11b(+) monocytic cells in colon tissues. Importantly, transfer of splenic GM-CSF-induced CD11b(+) myeloid cells into DSS-exposed mice improved colitis, and lethally irradiated GM-CSF receptor-deficient mice reconstituted with wildtype bone marrow cells were protected from DSS-induced colitis upon GM-CSF therapy. Lastly, GM-CSF-induced CD11b(+) myeloid cells were shown to promote in vitro wound repair. CONCLUSIONS: Our study shows that GM-CSF-dependent stimulation of bone marrow-derived cells during DSS-induced colitis accelerates colonic tissue repair. These data provide a putative mechanism for the observed beneficial effects of GM-CSF therapy in Crohn's disease.

Genetic vulnerability factor for schizophrenia: association with glutamate cysteine ligase modifier gene and abnormal enzyme activity

Background: We previously reported in schizophrenia patients a decreased level of glutathione ([GSH]), the principal non-protein antioxidant and redox regulator, both in cerebrospinal-fluid and prefrontal cortex. To identify possible genetic causation, we studied genes involved in GSH metabolism. Methods: Genotyping: mass spectrometry analysis of polymerase chain reaction (PCR) amplified DNA fragments purified from peripheral blood. Gene expression: real-time PCR of total RNA isolated from fibroblast cultures derived from skin of patients (DSM-IV) and healthy controls (DIGS). Results: Case-control association study of single nucleotide polymorphisms (SNP) from the GSH key synthesizing enzyme glutamate-cysteine-ligase (GCL) modifier subunit (GCLM) was performed in two populations: Swiss (patients/controls: 40/31) and Danish (349/348). We found a strong association of SNP rs2301022 in GCLM gene (Danish: c2=3.2; P=0.001 after correction for multiple testing). Evidence for GCLM as a risk factor was confirmed in linkage study of NIMH families. Moreover, we observed a decrease in GCLM mRNA levels in patient fibroblasts, consistently with the association study. Interestingly, Dalton and collaborators reported in GCLM knock-out mice an increased feedback inhibition of GCL activity, resulting in 60% decrease of brain [GSH], a situation analogous to patients. These mice also exhibited an increased sensitivity to oxidative stress. Similarly, under oxidative stress conditions, GCL enzymatic activity was also decreased in patient fibroblasts. Conclusions: These results at the genetic and functional levels, combined with observations that GSH deficient models reveal morphological, electrophysiological, and behavioral anomalies analogous to those observed in patients, suggest that GCLM allelic variant is a vulnerability factor for schizophrenia.

Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

PURPOSE: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. METHODS: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. RESULTS: No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. CONCLUSIONS: Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

Up-regulation of macrophage migration inhibitory factor gene and protein expression in glial tumor cells during hypoxic and hypoglycemic stress indicates a critical role for angiogenesis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant variant of human glial tumors. A prominent feature of this tumor is the occurrence of necrosis and vascular proliferation. The regulation of glial neovascularization is still poorly understood and the characterization of factors involved in this process is of major clinical interest. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine released by leukocytes and by a variety of cells outside of the immune system. Recent work has shown that MIF may function to regulate cellular differentiation and proliferation in normal and tumor-derived cell lines, and may also contribute to the neovascularization of tumors. Our immunohistological analysis of MIF distribution in GBM tissues revealed the strong MIF protein accumulation in close association with necrotic areas and in tumor cells surrounding blood vessels. In addition, MIF expression was frequently associated with the presence of the tumor-suppressor gene p53. To substantiate the concept that MIF might be involved in the regulation of angiogenesis in GBM, we analyzed the MIF gene and protein expression under hypoxic and hypoglycemic stress conditions in vitro. Northern blot analysis showed a clear increase of MIF mRNA after hypoxia and hypoglycemia. We could also demonstrate that the increase of MIF transcripts on hypoxic stress can be explained by a profound transcriptional activation of the MIF gene. In parallel to the increase of MIF transcripts, we observed a significant rise in extracellular MIF protein on angiogenic stimulation. The data of our preliminary study suggest that the up-regulation of MIF expression during hypoxic and hypoglycemic stress might play a critical role for the neovascularization of glial tumors.

Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation.

The aim of our work was to show how a chosen normal-isation strategy can affect the outcome of quantitative gene expression studies. As an example, we analysed the expression of three genes known to be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: a straightforward normalisation against a single reference gene, GAPDH, using the 2(-ΔΔCt) algorithm and a more complex normalisation against a normalisation factor calculated from the quantitative raw data from four previously validated reference genes. We found that the two different normalisation strategies revealed contradicting results: normalising against a validated set of reference genes revealed an upregulation of the three genes of interest in three post-mortem tissue samples (cardiac muscle, skeletal muscle and brain) under hypoxic conditions. Interestingly, we found a statistically significant difference in the relative transcript abundance of VEGF in cardiac muscle between donors who died of asphyxia versus donors who died from cardiac death. Normalisation against GAPDH alone revealed no upregulation but, in some instances, a downregulation of the genes of interest. To further analyse this discrepancy, the stability of all reference genes used were reassessed and the very low expression stability of GAPDH was found to originate from the co-regulation of this gene under hypoxic conditions. We concluded that GAPDH is not a suitable reference gene for the quantitative analysis of gene expression in hypoxia and that validation of reference genes is a crucial step for generating biologically meaningful data.

New prognostic factors and calculators for outcome prediction in patients with recurrent glioblastoma: A pooled analysis of EORTC Brain Tumour Group phase I and II clinical trials.

BACKGROUND: Prognostic models have been developed to predict survival of patients with newly diagnosed glioblastoma (GBM). To improve predictions, models should be updated with information at the recurrence. We performed a pooled analysis of European Organization for Research and Treatment of Cancer (EORTC) trials on recurrent glioblastoma to validate existing clinical prognostic factors, identify new markers, and derive new predictions for overall survival (OS) and progression free survival (PFS).¦METHODS: Data from 300 patients with recurrent GBM recruited in eight phase I or II trials conducted by the EORTC Brain Tumour Group were used to evaluate patient's age, sex, World Health Organisation (WHO) performance status (PS), presence of neurological deficits, disease history, use of steroids or anti-epileptics and disease characteristics to predict PFS and OS. Prognostic calculators were developed in patients initially treated by chemoradiation with temozolomide.¦RESULTS: Poor PS and more than one target lesion had a significant negative prognostic impact for both PFS and OS. Patients with large tumours measured by the maximum diameter of the largest lesion (⩾42mm) and treated with steroids at baseline had shorter OS. Tumours with predominant frontal location had better survival. Age and sex did not show independent prognostic values for PFS or OS.¦CONCLUSIONS: This analysis confirms performance status but not age as a major prognostic factor for PFS and OS in recurrent GBM. Patients with multiple and large lesions have an increased risk of death. With these data prognostic calculators with confidence intervals for both medians and fixed time probabilities of survival were derived.

The blood-brain barrier: cellular basis.

Perfusion experiments with horseradish peroxidase have established that the morphological substrate of the blood-brain barrier is represented by microvascular endothelial cells. They are characterized by complexly arranged tight junctions and a very low rate of transcytotic vesicular transport. They express transport enzymes, carrier systems and brain endothelial cell-specific molecules of unknown function not expressed by any other endothelial cell population. These blood-brain barrier properties are not intrinsic to these cells but are inducible by the surrounding brain tissue. Type I astrocytes injected into the anterior eye chamber of the rat or onto the chick chorioallantoic membrane are able to induce a host-derived angiogenesis and some blood-brain barrier properties in endothelial cells of non-neural origin. Recently we have shown that this cellular interaction is due to the secretion of a soluble astrocyte derived factor(s). Astrocytes are also implicated in the maintenance, functional regulation and the repair of the blood-brain barrier. Complex interactions between other constituents of the microenvironment surrounding the endothelial cells, such as the basement membrane, pericytes, nerve endings, microglial cells and the extracellular fluid, take place and are required for the proper functioning of the blood-brain barrier, which in addition is regionally different as reflected by endothelial cell heterogeneity.

Brain metastases in gastro-oesophageal adenocarcinoma: insights into the role of the human epidermal growth factor receptor 2 (HER2).

BACKGROUND: Gastro-oesophageal adenocarcinomas rarely metastasize to the central nervous system (CNS). The role of the human epidermal growth factor receptor 2 (HER2) in patients with these cancers and CNS involvement is presently unknown. PATIENTS AND METHODS: A multicentre registry was established to collect data from patients with gastro-oesophageal adenocarcinomas and CNS involvement both retrospectively and prospectively. Inclusion in the study required a predefined clinical data set, a central neuro-radiological or histopathological confirmation of metastatic CNS involvement and central assessment of HER2 by immunohistochemistry (IHC) and in situ hybridisation (ISH). In addition, expression of E-cadherin and DNA mismatch repair (MMR) proteins were assessed by IHC. RESULTS: One hundred patients fulfilled the inclusion criteria. The population's median age was 59 years (interquartile range: 54-68), of which 85 (85%) were male. Twenty-five patients were of Asian and 75 of Caucasian origin. HER2 status was positive in 36% (95% CI: 26.6-46.2) of cases. Median time from initial diagnosis to the development of brain metastases (BMets) or leptomeningeal carcinomatosis (LC) was 9.9 months (95% CI: 8.5-15.0). Median overall survival from diagnosis was 16.9 months (95% CI: 14.0-20.7) and was not related to the HER2 status. E-cadherin loss was observed in 9% of cases and loss of expression in at least one DNA MMR proteins in 6%. CONCLUSIONS: The proportion of a positive HER2 status in patients with gastro-oesophageal adenocarcinoma and CNS involvement was higher than expected. The impact of anti-HER2 therapies should be studied prospectively.

Neutralization of (NK-cell-derived) B-cell activating factor by Belimumab restores sensitivity of chronic lymphoid leukemia cells to direct and Rituximab-induced NK lysis.

Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

Diffantom: Whole-Brain Diffusion MRI Phantoms Derived from Real Datasets of the Human Connectome Project.

Food allergies are believed to be on the rise and currently management relies on the avoidance of the food. Hen's egg allergy is after cow's milk allergy the most common food allergy; eggs are used in many food products and thus difficult to avoid. A technological process using a combination of enzymatic hydrolysis and heat treatment was designed to produce modified hen's egg with reduced allergenic potential. Biochemical (SDS-PAGE, Size exclusion chromatography and LC-MS/MS) and immunological (ELISA, immunoblot, RBL-assays, animal model) analysis showed a clear decrease in intact proteins as well as a strong decrease of allergenicity. In a clinical study, 22 of the 24 patients with a confirmed egg allergy who underwent a double blind food challenge with the hydrolysed egg remained completely free of symptoms. Hydrolysed egg products may be beneficial as low allergenic foods for egg allergic patients to extent their diet. This article is protected by copyright. All rights reserved.

Analysis of trophic responses in lesioned brain: focus on basic fibroblast growth factor mechanisms

The actions of fibroblast growth factors (FGFs), particularly the basic form (bFGF), have been described in a large number of cells and include mitogenicity, angiogenicity and wound repair. The present review discusses the presence of the bFGF protein and messenger RNA as well as the presence of the FGF receptor messenger RNA in the rodent brain by means of semiquantitative radioactive in situ hybridization in combination with immunohistochemistry. Chemical and mechanical injuries to the brain trigger a reduction in neurotransmitter synthesis and neuronal death which are accompanied by astroglial reaction. The altered synthesis of bFGF following brain lesions or stimulation was analyzed. Lesions of the central nervous system trigger bFGF gene expression by neurons and/or activated astrocytes, depending on the type of lesion and time post-manipulation. The changes in bFGF messenger RNA are frequently accompanied by a subsequent increase of bFGF immunoreactivity in astrocytes in the lesioned pathway. The reactive astrocytes and injured neurons synthesize increased amount of bFGF, which may act as a paracrine/autocrine factor, protecting neurons from death and also stimulating neuronal plasticity and tissue repair

L’implication du Stromal Cell-derived Factor-1 alpha dans le remodelage cardiaque une semaine après un infarctus du myocarde

L’injection de cellules souches provenant de la moelle osseuse est reconnue pour améliorer la fonction ventriculaire ainsi que le remodelage cicatriciel après un infarctus du myocarde (IM). Le Stromal Cell-derived factor-1 alpha (SDF-1 alpha), une chimiokine induite par l’ischémie cardiaque, représente une grande importance en raison de son rôle dans le recrutement de cellules inflammatoires et de cellules souches de la moelle osseuse vers les sites endommagés. Quoique les recherches sur le rôle de la chimiokine SDF-1 alpha dans le remodelage ventriculaire se multiplient, son implication dans la phase aiguë du remodelage reste inexplorée. Le but de la présente étude est de déterminer l’effet du SDF-1 alpha sur la taille de la cicatrice, l’hypertrophie cardiaque ainsi que la fonction ventriculaire chez des rats et des souris une semaine après un IM. La stratégie utilisée implique l’administration de l’AMD3100 (1 mg/kg, 24 heures après l’IM, pendant 6 jours), l’antagoniste sélectif du récepteur du SDF-1 alpha, le CXCR4. Ce récepteur est couplé à une protéine G alpha i et induit la migration et la prolifération cellulaire. Chez les rats du groupe IM, l’expression de la chimiokine a été détectée surtout dans les cellules musculaires lisses et les cellules endothéliales des vaisseaux cicatriciels. Le profil d’expression de la chimiokine dans le cœur infarci indique un gradient de concentration vers la cicatrice. Une semaine après l’IM, le traitement avec l’AMD3100 a diminué la taille de la cicatrice, résultant en une amélioration de la fonction ventriculaire et une diminution de l’élévation de l’expression de l’ARNm de l’ANP dans le ventricule gauche non infarci (VGNI). Chez les souris, le traitement avec l’AMD3100 a engendré les mêmes effets, soit une diminution de la taille de la cicatrice ainsi qu’une amélioration de la fonction ventriculaire. La réduction de la taille de la région infarcie chez les souris traitées avec l’AMD3100 est associée avec une atténuation de l’infiltration des neutrophiles dans la région ischémique. Ces résultats suggèrent que le blocage pharmacologique de l’axe SDF-1 alpha/CXCR4 lors de la phase aiguë du remodelage ventriculaire après un IM diminue la taille de la cicatrice et améliore la fonction ventriculaire, en partie, par la diminution de la réaction inflammatoire.