Brain gliomas and growth factors: immunohistochemical, immunofluorescence, flow cytometry and RT-PCR profile in pediatric age.

Gliomas represent over 50% of tumors occurring in children. Evidence suggests that glioma stem cells (GSCs), maintained by the transforming growth factor-beta (TGF-ß1) pathway, and vascularization substantially contribute to tumor aggressiveness. The identification of important angiogenic factors such as vascular endothelial growth factor (VEGF) may represent a crucial step in the therapeutic approach against tumor growth and metastatic diffusion. The aim of this study was to identify the expression of TGF-ß1, VEGF and VEGF-receptors in brain gliomas. Specimens of 16 gliomas and 4 controls from children aged 0.2-14 years were used in the study. Immunohistochemical analysis and gene expression study from specimens was performed. Flow cytometry analysis on GSCs was performed to ascertain the expression of VEGF and VEGF-R2 in the tumor stem cell compartment. Newly diagnosed gliomas mainly showed moderate to strong VEGF immunostaining and increased expression of pro-inflammatory molecules in glioma cells. The proportion of TGF-ß1 positive endothelial cells was markedly lower in normal brain vessels compared to tumor vessels. These findings demonstrate that the glioma mass is constituted by a phenotypically immature anoxic central area with a proliferating hypoxic layer; the peripheral area is characterized by cell types with a higher degree of differentiation expressing pro-angiogenic factors. Our data have proven that GSCs play a central role in promoting glioma neovascularization. These findings are useful to understand glioma vascularization, have relevant implications in the therapeutic options and may favor new insights into stem cells biology and suggest therapeutic opportunities for the anti-vascular treatment strategy.

Generation of induced pluripotent stem cells (CSSi017-A)(12862) from an ALS patient carrying a repeat expansion in the C9orf72 gene.

Genetic expansions of the hexanucleotide repeats (GGGGCC) in the C9orf72 gene appear in approximately 40% of patients with familial ALS and 7% of patients with sporadic ALS in the European population, making this mutation one of the most prevalent genetic mutations in ALS. Here, we generated a human induced pluripotent stem cell (hiPSC) line from the dermal fibroblasts of a patient carrying a 56-repeat expansion in an ALS disease-causing allele of C9orf72. These iPSCs showed stable amplification in vitro with normal karyotype and high expression of pluripotent markers and differentiated spontaneously in vivo into three germ layers.

Comparative receptor autoradiography of ex vivo and in vitro [3H]dizocilpine binding in mouse brain after middle cerebral artery occlusion.

In the present study the in vitro and ex vivo distributions of [3H]dizocilpine binding sites in mouse brain after middle cerebral artery occlusion (MCA-O) were compared using receptor autoradiography. The distribution patterns of [3H]dizocilpine binding sites obtained in vitro and ex vivo in normal mouse brain were the same with the highest densities occurring in the hippocampus and cerebral cortex. MCA-O had little or no effect on the in vitro binding density for at least 24 hr post-ischaemia. However after 2-3 days binding densities in the region of infarct were significantly reduced compared to the contralateral cerebral cortex. Further reductions occurred after 5-7 days. By contrast ex vivo [3H]dizocilpine binding was reduced in the infarcted area by 78.7 +/- 4% within 2 hr of the ischaemic insult and at all subsequent times binding was reduced by more than 75%. Ex vivo binding after ischaemia was always less than 30% of in vitro binding and this decrease was apparent within 2 hr of the ischaemic insult whereas in vitro binding was maintained at control levels for at least 24 hr. The neuroprotective activity of the NMDA antagonists dizocilpine and CGP 37849 in this model at different times after MCA-O was assessed. The time scale for receptor access following MCA-O is discussed and it is suggested that although the population of NMDA receptors is maintained in the infarct region for some days access to them in vivo may be sufficiently impaired within 2 or 4 hr of ischaemic insult to reduce the neuroprotective activity of NMDA antagonists after this time.

Stress activated protein kinases, a novel family of mitogen-activated protein kinases, are heterogeneously expressed in the adult rat brain and differentially distributed from extracellular-signal-regulated protein kinases.

Mitogen-activated protein kinases are important mediators of signal transduction from the cell surface to the nucleus and their activation has been implicated in a wide array of physiological processes. The extracellular-signal-regulated kinases are the archetypal and best studied members of the mitogen activated protein kinases. Recently, additional subgroups of mitogen activated protein kinases have been identified which exhibit distinct regulatory elements, substrate specificity and respond to diverse extracellular stimuli. Among these newly identified protein kinases are the rat stress-activated protein kinases. Despite a rapidly expanding literature on the biochemical properties of stress-activated protein kinases no anatomical data are yet available. In the present study, we have investigated the regional distribution of messenger RNA transcripts for stress-activated protein kinases in the adult rat central nervous system and compared this distribution to that observed for extracellular-signal-regulated kinases. Intense labelling for stress-activated protein kinases could be detected in discrete brain areas with high levels in hippocampus, neocortex and some nuclei of the brain stem. The apparent hybridization signal appeared to be selectively neuronal. Stress-activated protein kinases and extracellular-signal-regulated kinases hybridization patterns appeared generally dissimilar although a certain degree of co-expression in some brain areas, such as the hippocampal formation, could be observed. These results reveal an extreme complexity in the mitogen-activated protein kinase signalling pathway and suggest the existence of parallel mitogen-activated protein kinase cascades that can be activated independently or in some cases simultaneously, by extracellular stimuli.

Localization of the messenger RNA for the c-Jun NH2-terminal kinase kinase in the adult and developing rat brain: an in situ hybridization study.

Stress-activated protein kinase/extracellular signal-regulated protein kinase-1/c-Jun NH2-terminal kinase kinase is a dual-specificity kinase which phosphorylates and activates stress-activated protein kinase/c-Jun NH2-terminal kinase, a recently discovered mitogen-activated protein kinase that is stimulated by stressful stimuli and that regulates cellular transcriptional activity. The distribution of the messenger RNA encoding for stress-activated protein kinase/extracellular signal-regulated protein kinase-1 was evaluated in the adult and developing rat central nervous system. In situ hybridization with a 35S-labelled 45mer oligodeoxynucleotide probe was used to map the distribution of the stress-activated protein kinase/extracellular signal-regulated protein kinase-1 messenger RNA in postnatal day 1, 3, 6, 9, 12, 15, 18, 21 and adult rat brains. Specific labelling was generally associated with neuronal profiles. In the adult central nervous system, high hybridization signals were observed in the hippocampus, the granular layer of the cerebellum, the medial habenula, the anterodorsal thalamic nucleus, the red nucleus, the pontine nuclei, the facial nucleus, the motor and mesencephalic nuclei of the trigeminal nerve, the hypoglossal nucleus, the vestibular nucleus and the nucleus ambiguus. Intermediate levels were present in diencephalic and mesencephalic regions and in the neocortex, while basal ganglia displayed a low hybridization signal. In the developing brain, the heterogeneous distribution of the hybridization signal observed in the adult brain was already present, but in the hippocampus and basal ganglia the stress-activated protein kinase/extracellular signal-regulated protein kinase-1 messenger RNA levels were significantly higher at postnatal day 3 and during the second postnatal week than in the adult. The results show that stress-activated protein kinase/extracellular signal-regulated protein kinase-1 is widely expressed in the rat central nervous system and co-localizes with its substrate stress-activated protein kinase. The observed changes in stress-activated protein kinase/extracellular signal-regulated protein kinase-1 messenger RNA levels during postnatal development suggest a role for this protein in the maturation of brain circuits.

Distribution of the messenger RNA for the small conductance calcium-activated potassium channel SK3 in the adult rat brain and correlation with immunoreactivity.

Small conductance calcium-activated potassium channels are voltage independent potassium channels which modulate the firing patterns of neurons by activating the slow component of the afterhyperpolarization. The genes encoding a family of small conductance calcium-activated potassium channels have been cloned and up to now three known members have been described and named small conductance calcium-activated potassium channel type 1, small conductance calcium-activated potassium channel type 2 and small conductance calcium-activated potassium channel type 3; the distribution of their messenger RNA in the rat CNS has already been performed but only in a limited detail. The present study represents the first detailed analysis of small conductance calcium-activated potassium channel type 3 mRNA distribution in the adult rat brain and resulted in a strong to moderate expression of signal in medial habenular nucleus, substantia nigra compact part, suprachiasmatic nucleus, ventral tegmental area, lateral septum, dorsal raphe and locus coeruleus. Immunohistological experiments were also performed and confirmed the presence of small conductance calcium-activated potassium channel type 3 protein in medial habenular nucleus, locus coeruleus and dorsal raphe. Given the importance of dorsal raphe, locus coeruleus and substantia nigra/ventral tegmental area for serotonergic, noradrenergic and dopaminergic transmission respectively, our results pose the morphological basis for further studies on the action of small conductance calcium-activated potassium channel type 3 in serotonergic, noradrenergic and dopaminergic transmission.

Differential expression of SAPK isoforms in the rat brain. An in situ hybridisation study in the adult rat brain and during post-natal development.

MAPK pathways transduce a broad variety of extracellular signals into cellular responses. Despite their pleiotropic effects and their ubiquitous distribution, surprisingly little is known about their involvement in the communication network of nerve cells. As a first step to elucidate the role of MAPK pathways in neuronal signalling, we studied the distribution of SAPK alpha/JNK2, SAPK beta/JNK3, and SAPK gamma/JNK1, three isoforms of SAPK/JNK, a stress-activated MAPK subfamily. We compared the mRNA localisation of the three main isoforms in the adult and developing rat brain using in situ hybridisation. In the adult brain, SAPK alpha and beta were widely but heterogeneously distributed, reproducing the pattern of a probe that does not discriminate the isoforms. Differently, high labelling for the SAPK gamma probe was exclusively localised in the endopiriform nucleus and medial habenula. Intermediate staining was detected in the hippocampus. During post-natal development, SAPK beta showed the same localisation as in the adult. Nevertheless, the semi-quantitative analysis of optical densities showed significantly different mRNA levels. In the adult, SAPK gamma signal was weak, whereas in newborn rats the labelling was intense and widely distributed. SAPK gamma mRNA levels decreased during development, to reach the low signals detected in the adult. These results suggest that in the central nervous system SAPK-type MAP kinases perform significant physiological functions which are particularly relevant during post-natal development. The distinct distribution patterns of SAPK isoforms in the adult rat brain support the hypothesis that separate functions are performed by the products of the three SAPK genes.

Central administration of NPY or an NPY-Y5 selective agonist increase in vivo extracellular monoamine levels in mesocorticolimbic projecting areas.

Selective NPY-Y5 antagonists are known to reduce NPY-evoked increase of food intake under free feeding conditions and drug-reinforced operant responding in rodents suggesting that NPY-Y5 receptors can regulate reinforcers, potentially by modulating the hypothalamic-limbic reward system. However, evidence published to date has revealed a limited expression of NPY-Y5 in the limbic areas. Thus, the first aim of the present study was to investigate the distribution of NPY-Y5 receptor binding sites in rat mesocorticolimbic projection areas such as the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and lateral hypothalamus (LH). Since mesocorticolimbic release of monoamines has been typically associated to the rewarding and motivational significance of reinforcers, we then compared the ability of NPY and an NPY-Y5 selective agonist, [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]hPP, to evoke changes in extracellular monoamines from these brain regions using in vivo microdialysis techniques. Intracerebral doses of each compound were selected on the basis of those previously demonstrated to trigger food intake in a separate set of animals. We found that NPY-Y5 receptors were widely distributed in both the NAc and mPFC but not in the LH nuclei. Central administration of either NPY (4.5 nmol/rat) or the NPY-Y5 agonist (0.6 nmol/rat) induced a significant increase of dopamine (DA) output of up to 150% of basal values in the NAc. In addition, NPY induced a stepped increase of norepinephrine (NE) outflow in the NAc area. Also extracellular levels of NE levels were increased by both treatments in the mPFC (150% vs basal concentration). Hypothalamic monoamine levels were unaffected by both treatments. Extracellular serotonin (5-HT) levels were also unchanged in all regions. Given the NPY-Y5 agonist paralleled the in vivo ability of NPY to increase DA, these data suggest that the release of NPY may modulate behaviours associated to accumbal DA release such reward and reinforcement by, at least in part, acting on mesocorticolimbic NPY-Y5 receptors.

[Relationship between structure and function of a chemotactic factor for human leukocytes]. / Relazione tra struttura e funzione di un fattore chemiotattico per i leucociti umani.

Casein, a phosphoprotein forming aggregates in solution, exerts chemotactic activity for human neutrophils. The protein was filtered on Sephadex G 100 to obtain fractions of homogeneous mol. weight and the column fractions were tested for chemotactic activity. The chemotactic activity was found only in the lighter peak, which was then digested by trypsin. The digested material was purified by filtration through AcA-54 and the 6.000 M.W. peptide containing the most phosphate groups and tyrosines resulted chemotactic for PMN. Most likely the phosphate groups are important in chemotactic recognition.

Defective responsiveness to natural and pharmacological molecules of neutrophil locomotion in rheumatoid arthritis disease.

Neutrophils derived from peripheral blood of patients with rheumatoid arthritis (RA) exhibited a defective responsiveness to natural mediators of inflammation, namely histamine and serotonin, and to the anti-inflammatory drugs ibuprofen and naproxen, in spite of the fact that the basic status of motility was normal. Not even pretreatment of granulocytes with substances restored the capacity to modulate the random and directional locomotion. This neutrophil functional defect was correlated with an anomalous response to rifamycin SV, previously observed in rheumatic states.

The effect of rifamycin SV on neutrophil functions in patients with rheumatoid arthritis.

The chemotaxis, phagocytic capacity and reducing activity of neutrophils derived from peripheral blood of patients with rheumatoid arthritis (RA) did not differ from those of control. However, some significant differences between neutrophils from rheumatic and healthy subjects emerged in the presence of rifamycin SV. The chemotactic response of neutrophils from patients with RA was activated by rifamycin SV, whereas cells from controls did not orient their locomotion towards the drug. Moreover, incubation of RA patient's cells with rifamycin SV in vitro depressed phagocytic and reducing activities; the same treatment on normal cells failed to alter these functions. A correlation between improvement of clinical symptoms after treatment of RA by local infiltration with rifamycin SV, observed by others, and the impairment of phagocytosis and NBT reduction, here described, was suggested.

[Medium molecular weight uremic toxins and endogenous polyamines. Behavior of polymorphonuclear leukocyte chemotaxis with respect to chromatographic peaks of dialysate and standard polyamines]. / Tossine uremiche mediomolecolari e poliamine endogene. Comportamento della chemiotassi dei polimorfonucleati rispetto ai picchi cromatografici di dialisato e poliamine standard.

The aim of our study is to evaluate the eventual activity of the total dialysate of two uremic nephrectomized patients in recirculating dialysis and the chromatographic peak of the dialysate fractionated by Sephadex column G 15 on PMN chemotaxis. Only the total dialysate and the chromatographic peak B showed inhibition of chemotaxis. On the contrary the commercial polyamines in the same concentration range, and the other chromatographic peaks, containing polyamines too, did not revealed inhibition. Our data show, therefore, that the chemotaxis inhibition could be due to the middle-molecules present in the peak B, rather than the polyamines itself. Polyamines were determined by dansylation method, separated by thin layer chromatography and quantified by spectrofluorimeter. Chemotaxis was evaluated using the modified Boyden chamber.

[Effect of a component of human lymphocyte dialysate on the motor activity of polymorphonuclear leukocytes]. / Effetto di un componente del dializzato dei linfociti umani sull'attivita' motoria dei leucociti polimorfonucleati.

Lymphocyte dialysates obtained from healthy volunteers and leukemic subject were chromatographed on Bio-Gel P-4. Partially purified fractions were assayed for neutrophil locomotion: a positive chemokinetic effect and no chemotactic activity was observed in both samples.

[Effect of rifamycin SV on neutrophil functions in patients with rheumatoid arthritis]. / Effetto della rifamicina SV sulle funzioni dei neutrofili in pazienti con artrite reumtoide.

The antibiotic rifamycin SV (RSV) has been successfully used by others on the local treatment of rheumatoid arthritis (R.A.). Since polymorphonuclear leukocytes (PMNL) are involved in the synovial inflammatory process we tested the 'in vitro' effect of RSV on PMNL functions, such as locomotion and phagocytosis. PMNL locomotion was evaluated by using modified Boyden Chamber and phagocytosis was tested by the number of yeast particles ingested and by NBT reduction. The, the functions of PMNL derived from 7 R.A. patients in therapy only with non steroidal anti-inflammatory drugs were compared with those of PMNL from 14 patients with non inflammatory disease (osteoporosis and osteoarthrosis) in the same therapy and with neutrophils from healthy subjects. It was demonstrated that PMNL derived from patients with both R.A. and non inflammatory disease activated their directional locomotion towards RSV, on the contrary, cells from healthy subjects were unresponsive. Moreover, only patients with R.A. showed a defective phagocytic capacity and a depression in NBT reducing activity, when PMNL were treated with RSV. This phenomenon might be correlated with beneficial effect observed after local treatment of R.A. with RSV.