Transduction of an immortalized olfactory ensheathing glia cell line with the green fluorescent protein (GFP) gene: Evaluation of its neuroregenerative capacity as a proof of concept.

Olfactory ensheathing glia (OEG) cells are known to foster axonal regeneration of central nervous system (CNS) neurons. Several lines of reversibly immortalized human OEG (ihOEG) have been previously established that enabled to develop models for their validation in vitro and in vivo. In this work, a constitutively GFP-expressing ihOEG cell line was obtained, and named Ts14-GFP. Ts14-GFP neuroregenerative ability was similar to that found for the parental line Ts14 and it can be assayed using in vivo transplantation experimental paradigms, after spinal cord or optic nerve damage. Additionally, we have engineered a low-regenerative ihOEG line, hTL2, using lentiviral transduction of the large T antigen from SV40 virus, denominated from now on Ts12. Ts12 can be used as a low regeneration control in these experiments.

[International Association of Dental Traumatology guidelines for the management of traumatic dental injuries: 1. Fractures and luxations of permanent teeth. Hebrew edition].

Traumatic dental injuries (TDIs) of permanent teeth occur frequently in children and young adults. Crown fractures and luxations are the most commonly occurring of all dental injuries. Proper diagnosis, treatment planning and follow up are important for improving a favorable outcome. Guidelines should assist dentists and patients in decision making and for providing the best care effectively and efficiently. The International Association of Dental Traumatology (IADT) has developed a consensus statement after a review of the dental literature and group discussions. Experienced researchers and clinicians from various specialties were included in the group. In cases where the data did not appear conclusive, recommendations were based on the consensus opinion of the IADT board members. The guidelines represent the best current evidence based on literature search and professional opinion. The primary goal of these guidelines is to delineate an approach for the immediate or urgent care of TDIs. In this first article, the IADT Guidelines for management of fractures and luxations of permanent teeth will be presented. The Hebrew Edition is part of the IADT global effort to provide accessibility to these guidelines worldwide.

[International Association of Dental Traumatology guidelines for the management of traumatic dental injuries: 2. Avulsion of permanent teeth. Hebrew edition].

Avulsion of permanent teeth is one of the most serious dental injuries, and a prompt and correct emergency management is very important for the prognosis. The International Association of Dental Traumatology (IADT) has developed a consensus statement after a review of the dental literature and group discussions. Experienced researchers and clinicians from various specialties were included in the task group. The guidelines represent the current best evidence and practice based on literature research and professionals' opinion. In cases where the data did not appear conclusive, recommendations were based on the consensus opinion or majority decision of the task group. Finally, the IADT board members were giving their opinion and approval. The primary goal of these guidelines is to delineate an approach for the immediate or urgent care of avulsed permanent teeth. The Hebrew Edition is part of the IADT global effort to provide a worldwide accessibility to these guidelines. This scond part of the guidelines will focus on avulsion of permanent teeth.

[International Association of Dental Traumatology guidelines for the management of traumatic dental injuries: 3. Injuries in the primary dentition. Hebrew edition].

Traumatic injuries to the primary dentition present special problems and the management is often different as compared with the permanent dentition. The International Association of Dental Traumatology (IADT) has developed a consensus statement after a review of the dental literature and group discussions. Experienced researchers and clinicians from various specialities were included in the task group. In cases where the data did not appear conclusive, recommendations were based on the consensus opinion or majority decision of the task group. Finally, the IADT board members were giving their opinion and approval. The primary goal of these guidelines is to delineate an approach for the immediate or urgent care for management of primary teeth injuries. The IADT cannot and does not guarantee favorable outcomes from strict adherence to the guidelines, but believe that their application can maximize the chances of a positive outcome. The Hebrew Edition is part of the IADT global effort to provide a worldwide accessibility to these guidelines. This third part will discuss injuries in the primary dentition.

Semaphorin 3C preserves survival and induces neuritogenesis of cerebellar granule neurons in culture.

Semaphorins (sema) constitute a family of molecules sharing a common extracellular domain (semaphorin domain). This family includes several types of secreted and membrane-associated molecules that are grouped into eight subclasses (subclasses 1-7 and viral semaphorins). Subclass 3 semaphorins are secreted molecules involved in axonal guidance, mainly through repulsive gradients and induction of growth cone collapse. More recently sema 3 molecules have been identified as positive factors in dependence of the type of neurons. Besides their axonal guidance function, some semaphorins have been implicated in apoptosis and survival. We investigated the effect of sema3C on survival and neurite outgrowth of rat cerebellar granule neurons (CGNs) in culture. 3T3 cells were stably transfected with sema3C. Several clonal lines were established and tested for their neuritogenic activity and one, S3C-8, was selected for the bulk of experiments. S3C-8 was co-cultured with CGNs. Sema3C enhanced CGN viability as assessed in co-cultures of CGNs with monolayers of S3C-8 in comparison with co-cultures of CGNs with control mock-transfected 3T3 cells. Moreover sema3C induced neuritogenesis of cultured CGNs, which express neuropilin-1 and -2. S3C-8 cells, overexpressing sema3C, were significantly more neuritogenic for CGN than poly l-lysine (PLL), a positive substrate for CGNs, as assessed by the measurement of the length of neurites and confirmed by Tau expression along the time of culture. CGNs co-cultured with S3C-8, showed up-regulation of the expression of axonal microtubule-associated proteins (MAPs) such as Tau, phosphorylated MAP2C and mode I-phosphorylated MAP1B compared with neurons cultured on control 3T3 cells. We also found increased expression of a specific marker of neuronal cell bodies and dendrites, high molecular weight MAP2 (HMW-MAP2). Interestingly, there was no accompanying up-regulation of a marker enriched within the neuronal somatodendritic domain, mode II-phosphorylated MAP1B. These data support the idea that secreted sema3C favors survival and neuritogenesis of cultured CGNs.

Fibronectin modulation by A beta amyloid peptide (25-35) in cultured astrocytes of newborn rat cortex.

Fibronectin appears to be present in Senile Plaques of Alzheimer's disease brains. These senile or neuritic plaques are surrounded by dystrophic neurites, activated microglia and reactive astrocytes. The purpose of this work was to establish if a direct correlation exists between the production of Fibronectin (FN) by astrocytes and the presence of amyloid, analysing the modification of this protein produced after the treatment of cultured astrocytes with amyloid peptide (25-35). Our data showed that the addition of previously polymerised A beta-peptide to cultured astrocytes induced a marked increase in FN immunoreactivity that is in part dependent on phosphatases 2A or phosphatase 1, since was partially inhibited by okadaic acid. The increased amount of FN did not appear to be associated to any specific single isoform of which are mainly present in the rat brain. Our data suggest that in vivo FN accumulated in senile plaques may be the result, at least in part, of the response of reactive astrocyte to the presence of amyloid peptide. The importance of FN up-regulation in vivo, as part of a 'positive' response of the astrocytes to produce molecules that favours neurite outgrowth, is discussed.

Increasing neurite outgrowth capacity of beta-amyloid precursor protein proteoglycan in Alzheimer's disease.

Progressive cerebral deposition of beta-amyloid peptide either in blood vessels or around neurites is one of the most important features of Alzheimer's disease (AD). The beta-peptide, known as Abeta or A4, is produced by proteolytic cleavage of the amyloid precursor protein (APP). Two APP processing pathways have been proposed as physiological alternatives; only one of which leads to the production of Abeta or amyloidogenic peptides. However, we have little information regarding these processing pathways in the brain, or on whether posttranslational modifications such as glycosylation affect APP processing in vivo. Furthermore, the physiological function(s) of this protein in nervous tissue remains unclear, although modulatory roles in cell adhesion and neuritic extension have been suggested. It has been reported that APP may be glycosylated as a proteoglycan. We purified this APP population from human brain, and our data indicate that PG-APP supports neurite extension of hippocampal neurons. Neurons grown on this substratum showed an increased capacity to elongate neurites and increased neuritic "branching" compared to culture on laminin. These effects were enhanced with PG-APP samples obtained from AD brains. Our results suggest that this APP population may act as a neurite outgrowth and branching promoter and may thus play a role in some pathological conditions. These findings may have significant implications in understanding normal brain development and pathological situations (such as AD).

The neurite retraction induced by lysophosphatidic acid increases Alzheimer's disease-like Tau phosphorylation.

The bioactive phospholipid lysophosphatidic acid (LPA) causes growth cone collapse and neurite retraction in neuronal cells. These changes are brought about by the action of a cell surface receptor coupled to specific G proteins that control morphology and motility through the action of a group of small GTPases, the Rho family of proteins. Many studies have focused on actin reorganization modulated by Rho-GTPases, but almost no information has been obtained concerning microtubular network reorganization after LPA-induced neurite retraction. In the present study, we demonstrate an increase in site-specific Alzheimer's disease-like Tau phosphorylation during LPA-induced neurite retraction in differentiated SY-SH5Y human neuroblastoma cells. The phosphorylation state of Tau was inferred from its immunoreactivity with antibodies that recognize phosphorylation-sensitive epitopes. The effects of specific kinase inhibitors indicate that this phosphorylation is mediated by glycogen synthase kinase-3 (GSK-3). In support of this idea, we observed an increase of GSK-3 activity upon growth cone collapse. Our results are consistent with the hypothesis that activation of GSK-3 occurs in the Rho pathway and may represent an important link between microtubules and microfilaments dynamics during neuritogenesis and in pathological situations such as Alzheimer's disease.

Expression of presenilin 1 in nervous system during rat development.

Alzheimer's disease (AD) is a polygenic disorder involving at least four different genes. Among them, missense mutations in the presenilins segregate with the vast majority of early onset cases of familial AD. To elucidate possible function(s) of presenilin 1 (PS1), we have studied its expression during the development of the rat nervous system. Analysis by in situ hybridization showed expression of PS1 in a variety of cell types and tissues during development, with prominent expression in the nervous system. During late embryogenesis, the ventricular zone presented the highest levels of expression, paralleling the pattern previously reported for Notch. Later, during postnatal development, we observed a peak of PS1 expression at postnatal day 10, particularly in the cerebellum and hippocampus, a time when proliferation and migration are still ongoing and synapse formation is being completed. We propose that presenilins participate in at least two different developmental processes: (1) one involved in neurogenesis and skeleton formation during embryonic development, probably involving coordinate expression with Notch, and (2) a second one in the postnatal central nervous system, perhaps involved in neuritogenic and/or synaptogenic stages, most likely playing a role in amyloid precursor protein processing and amyloid beta production.

Up-regulation of Eph tyrosine kinase receptors after excitotoxic injury in adult hippocampus.

The molecular mechanisms underlying the response to injury in the central nervous system are incompletely understood. Many cell activation systems may be involved. Tyrosine kinase receptors and their ligands play key roles in cell activation throughout life. The Eph family of tyrosine kinase receptors/ ligands are developmentally regulated and have been implicated in neural pathfinding. However, nothing is known about their role in the adult brain. We have used a model of central nervous system lesion in the rat, in which intraventricular injection of kainate was performed. This produced neuronal death in the CA3-CA4 fields and glial activation in the hippocampus. Highly degenerate primers, corresponding to the catalytic domain of the tyrosine kinase family, were used for reverse transcription-polymerase chain reaction of pooled RNA extracted from injured hippocampi. The amplified products were cloned and 100 clones (arbitrarily named TK1-TK100) were examined and inserts sequenced. We obtained four clones containing inserts which belong to the Eph receptor family. Two of these inserts (TK17 and TK63) were EphA4 and the other were EphB2 (TK25) and EphA5 (TK23). We performed in situ hybridization, and we found our clones to be present in all fields of the hippocampus, their expression being mainly neuronal. Three days after lesion, prominent expression appeared in CA1 as compared to the same field in the non-treated contralateral hippocampus. We performed northern blot analysis for quantification, and found that, three days after injury, the values decreased to 33 +/- 4%, 33 +/- 1% and 46 +/- 1% of control values for TK63 (EphA4), TK25 (EphB2) and TK23 (EphA5), respectively. Neuronal death in CA3-CA4 might account for this fact. Later, five days post-injury, the expression increased to 63 +/- 3%, 71 +/- 1% and 111 +/- 5% of control values, respectively. This increase was due to an up-regulation of these genes in the hippocampal neurons that survive after the injury, as indicated by in situ hybridization.

BetaA amyloid peptide (25-35) induced APP expression in cultured astrocytes.

Alzheimer's disease is characterized by an accumulation of senile or neuritic plaques surrounded by activated microglia and reactive astrocytes, the cell processes of which are frequently in contact with the amyloid core. The major component of this amyloid deposit is the amyloid peptide (betaA or betaA4). These reactive glia are characterized by their hypertrophic phenotype and by the overexpression of some molecules such as glial fibrillary acidic protein and the amyloid precursor protein (APP). The purpose of this work was to analyze whether APP expression was modified in astrocytes by the presence of betaA peptide. To study this, the effects of beta-Amyloid (25-35) on cultured astrocytes were analyzed and compared with those of a scrambled peptide. Our data indicated that the addition of previously polymerized betaA peptide induced a marked morphological change from a flat, polygonal shape to a stellated, process-bearing morphology. This change occurred with an increase in APP immunoreactivity that is dependent of phosphatases PP2A or PP1, since it was inhibited by okadaic acid. Upregulation of APP protein expression appears to be mainly nontranscriptional, because the increase of APP protein precedes the increase of mRNA expression. The analysis of several APP isoforms indicated that this increment is not due to changes of a single isoform. Our data may correlate with some in vivo reports of astrocytic APP induction after brain insult, suggesting an important role for betaA peptide in the initial process and/or maintenance of the reactive phenotype in vivo.

Okadaic acid modulates the cytoskeleton changes induced by amyloid peptide (25-35) in cultured astrocytes.

Amyloid beta-protein (25-35) (betaA) induced a marked morphological change in astrocytes, changing their flat polygonal shape into a stellate process-bearing morphology. The changes induced by betaA were concentration and time-dependent, whereas the addition of a scrambled peptide did not alter astrocyte morphology. We discard the possibility of betaA-astrocytes being type II-like astrocytes. We also analysed the influence of the presence of kinase and phosphate inhibitors on this morphological change. Our data indicate that the betaA-induced phenotype was not affected by the inhibition of protein tyrosine kinase or tyrosine phosphatases. Only the addition of okadaic acid to astrocytes prevented the morphological transformation from flat to stellate shape, induced by betaA (25-35). Inhibition of the stellate phenotype by okadaic acid was initiated at a concentration of 10 nM which suggested that either phosphatase 2A or 1 plays an important role in the betaA astrocytic transformation.

Deletions and rearrangements of cyclin-dependent kinase 4 inhibitor gene p16 are associated with poor prognosis in B cell non-Hodgkin's lymphomas.

In the present study we examined by Southern blot analysis the presence of deletions and rearrangements of the p16 tumor-suppressor gene in B cell non-Hodgkin's lymphomas (NHLs) in order to determine whether or not these changes can be related to a particular histological subtype and the different clinico-biological and prognostic characteristics of the disease. 103 untreated patients were enrolled in the study. Seven cases displayed alterations in the p16 gene: four cases with homozygous deletions and three with gene rearrangement. The presence of these abnormalities did not correlate with any specific histological subtype: three cases were small lymphocytic lymphomas (two of them reclassified as mantle cell lymphoma on the basis of the REAL classification), two diffuse large cell lymphomas and two small non-cleaved cell lymphomas (one of them considered to be a Burkitt-like lymphoma according to the REAL). These seven cases showed a trend towards worse prognostic indicators than the remaining patients, and this was confirmed in the survival analysis, since the presence of p16 gene abnormalities was associated with a shorter survival (10 vs 81 months, P = 0.0006). In the multivariate analysis, p16 abnormalities were selected as an independent prognostic factor together with the LDH and beta2-microglobulin. These findings support a role for the p16 gene in the pathogenesis of B cell NHLs and suggest an association of p16 abnormalities with aggressive forms of the disease that could be useful to predict the prognosis of patients.

Axotomy increases the expression of glucose-regulated protein 78 kDa in rat facial nucleus.

Nerve injuries lead to metabolic and morphological changes in the cell bodies of the neurons of origin. Increases in glucose turnover in axotomized facial and hypoglossal motor nuclei have been described. Glucose-regulated protein 78 kDa (GRP78) is implicated in cellular protein folding and subunit assembly and responds to glucose deficiency. We performed Western blot and immunohistochemistry to determine the effect of axotomy on the expression and regulation of GRP78 in the facial nucleus (FN). Facial nerve axotomy caused a larger and longer increase of GRP78 in the ipsilateral FN than in the contralateral FN. In right ipsilateral FN, axotomy resulted in elevation of GRP78 protein levels, first detected at 12 h and which reached significant, maximal induction at 24 h (75 +/- 27% increase). GRP78 protein levels decreased at later time points, but remained elevated over sham-operated controls. In contrast, no significant increase in GRP78 concentrations was found in contralateral left FN. Immunocytochemically, positive GRP78 staining was found mainly in the cytoplasm of motoneurons; there was no nuclear staining. Prominent GRP78-immunostaining appeared in axotomized motoneurons at 24 h postaxotomy as compared with the contralateral, unoperated controls. This augmentation was also observed at 4 and 7 days postaxotomy. The possibility that glucose metabolism and GRP78 levels are two parallel events in the injured facial nucleus is discussed.