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We used electrophoretic mobility shift assays to investigate the effects of cobalamin (Cbl) deficiency on the levels of activated nuclear factor-kappa B (NF-kappaB) in the spinal cords (SCs) and livers of rats made Cbl-deficient (Cbl-D) by total gastrectomy or a Cbl-D diet. We chose the SC and liver because they are severely or scarcely affected, respectively, by Cbl deficiency in terms of histological damage. We found permanently increased NF-kappaB levels (particularly the p50 and p65 subunits) in the SCs and livers of both types of Cbl-D rats, and Western blot analysis demonstrated increased p65 levels. NF-kappaB and p65 protein levels normalized when the totally gastrectomized (TGX) rats were treated with Cbl replacement. As we have previously demonstrated that Cbl deficiency increases tumor necrosis factor (TNF)-alpha and nerve growth factor (NGF) levels in the SC (each of which is a known NF-kappaB activator), we redetermined NF-kappaB levels in the SCs and livers of TGX rats treated with anti-TNF-alpha or anti-NGF antibodies and found that NF-kappaB levels normalized in both tissues after either treatment. These results demonstrate that: (1) Cbl physiologically and indirectly down-regulates NF-kappaB levels in rat SC and liver, and (2) NF-kappaB is an important signaling molecule after Cbl deficiency injury.

Bortezomib is a new proteasome inhibitor with a high antitumor activity, but also with a potentially severe peripheral neurotoxicity that induces neuropathic pain in the patients treated with this drug. To establish a preclinical model and to characterize the neuropathic pain, bortezomib was administered to Wistar rats (0.20mg/kg three times weekly [3q7d] for a total of 4 weeks). At the end of the treatment period sensory nerve conduction velocity (SNCV) was determined in the tail of each animal. Sciatic nerve, DRG and spinal cord specimens were processed for light and electron microscope observations and morphometry. Plantar test and Dynamic Aesthesiometer Test (Ugo Basile instruments) were used at the end of treatment to evaluate hyperalgesia and allodynia in treated animals. Bortezomib induced a significant reduction in SNCV. Sciatic nerve and DRG examination and morphometric determinations demonstrated mild to moderate pathological changes, while the spinal cord was morphologically normal. Bortezomib-induced changes were also observed in dynamic aesthesiometer test but not in plantar test, indicating the presence of allodynia in treated animals. This model is relevant to the neuropathy induced by bortezomib in the treatment of human malignancies and it could be useful in increasing our knowledge regarding the mechanisms underlying bortezomib neurotoxicity and neuropathic pain. Supported in part by a “Fondazione Banca del Monte” unrestricted research grant

Neurodegenerative diseases do affect glial or neuronal cells in both the peripheral and central nervous systems. Although they are characterized by different features and a different onset, all the neurodegenerative diseases share the final steps that lead to cell death by apoptosis. Apoptosis occurs also during developmental neurogenesis. Neuron survival and differentiation depend on specific neurotrophic factors released by their targets. During degenerative diseases the loss of neuronal or glial cells is responsible for the disease's progression. Current therapies are focused on counteracting the degenerative events by acting on the molecular mechanisms involved in cellular death, or by the exogenous administration of pro-survival factors. The presence in many areas of both the peripheral and central nervous systems of niches of neural progenitors which can differentiate, under specific conditions, into neurons or glial cells opens up new therapeutic perspectives. The Mitogen-Activated Protein Kinase (MAPK) family, that includes ERK1/2, JNK/SAPK, p38 and ERK5, is involved in the survival, proliferation and differentiation of nervous cells. Some of the MAPKs promote the differentiation towards the neuron lineage, others towards the glial one. The MAPKs are also involved in apoptosis and may, therefore, play a role in neurodegeneration. This dual role of MAPKs may make it possible to design alternative and/or synergistic approaches to the treatment of degenerative diseases, either by using specific inhibitors of the MAPKs involved in apoptosis, or by increasing the activation of the MAPKs involved in neuronal survival and differentiation. The increased activation of pro-differentiative MAPKs can lead to the replacement of damaged neurons by undifferentiated progenitors and the slowing down of the disease's progression.

Background Periodontal disease is a degenerative illness that leads to resorption of the alveolar bone. Mesenchymal stromal cells (MSC) represent a novel tool for the production of biologic constructs for the treatment of degenerative bone diseases. The preparation of MSC differentiated into osteogenic lineage for clinical use requires the fulfillment of strict good manufacturing practice (GMP) procedures. Methods MSC were isolated from BM samples and then cultured under GMP conditions. MSC were characterized phenotypically and for their differentiative potential. Cells were seeded onto collagen scaffolds (Gingistat) and induced to differentiate into osteogenic lineages using clinical grade drugs compared with standard osteogenic supplements. Alizarin Red S stain was used to test the deposition of the mineral matrix. Standard microbiologic analysis was performed to verify the product sterility. Results The resulting MSC were negative for CD33, CD34 and HLA-DR but showed high expression of CD90, CD105 and HLA-ABC (average expressions of 94.3%, 75.8% and 94.2%, respectively). Chondrogenic, osteogenic and adipogenic differentiation potential was demonstrated. The MSC retained their ability to differentiate into osteogenic lineage when seeded onto collagen scaffolds after exposure to a clinical grade medium. Cell numbers and cell viability were adequate for clinical use, and microbiologic assays demonstrated the absence of any contamination. Discussion In the specific context of a degenerative bone disease with limited involvement of skeletal tissue, the combined use of MSC, exposed to an osteogenic clinical grade medium, and biomimetic biodegradable scaffolds offers the possibility of producing adequate numbers of biologic tissue-engineered cell-based constructs for use in clinical trials.

Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine kinases that respond to various extracellular signals and are involved in many cellular processes. The MAPK family consists of four major groups extracellular signal regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), p38 and ERK5. Additional MAPKs (ERK3, ERK4, ERK7, ERK8) have been identified on the basis of their homology with the ERK1/2 sequence but their functions and activation have not yet been fully described. MAPKs are activated by a ¿three kinase cascade¿ and after activation they phosphorylate specific cytoplasmic and nuclear substrates. MAPK activity is specifically regulated by phosphatases and by interaction with scaffold and/or anchor proteins. MAPK inhibitors are useful tools for studying MAPK requirements in physiological and pathological processes and it is thought that they may constitute a promising new therapeutic strategy for the treatment of tumors, inflammatory and neurodegenerative diseases. MAPK inhibitors are specific to each member of the MAPK family and can act at different levels of the MAPK cascades. These inhibiting molecules may be ATP-competitive or ATP-noncompetitive depending on their binding sites. Other classes of MAPK inhibitors are represented by peptide inhibitors whose sequences derive from scaffold protein sequences, and by low molecular weight compounds that interact with specific MAPK docking domains. MAPKs play an important role in the nervous system. In vitro studies using cell lines and primary neuronal cultures have demonstrated that MAPKs play a crucial role in neuronal survival and differentiation, apoptotic and non-apoptotic neuronal death, neuronal plasticity, learning and memory. In this review, we summarize the studies in which MAPK involvement in neuronal differentiation and neuritogenesis of different cellular models has been demonstrated by MAPK inhibitors.

Objective: Management of periodontal defects has always been a challenge in clinical periodontics. Recently mesenchymal stem cells (MSC) have been proposed for tissue regeneration in periodontal disease and repair of large bone defects. Bone regeneration has to be supported by a scaffold which has to be biocompatible, biodegradable, and able to support cell growth and differentiation. The aim of this study was to evaluate osteogenic differentiation of MSC seeded on a collagen scaffold. Design: MSC were obtained from adult rat bone marrow, expanded and cultured in plastic dishes or seeded in a collagen scaffold Gingistat(R). MSC were induced towards osteogenic differentiation using osteogenic supplements. Cell differentiation and calcium deposits were evaluated by immunoblotting, immunohistochemistry, histochemical techniques, enzymatic activity assay, and SEM-EDX analysis. Biomaterial in vitro degradation was evaluated by measuring mass reduction after incubation in culture medium. Results: Rat MSC osteogenic differentiation was demonstrated by osteopontin and osteocalcin expression and an increase in alkaline phosphatase activity. MSC were distributed homogeneously in the collagen scaffold. Nodular aggregates and alizarin red stained calcium deposits were observed in MSC induced towards osteogenic differentiation cultured in dishes or seeded in the collagen scaffold. SEM-EDX analysis demonstrated that calcium co-localized with phosphorous. The biomaterial in vitro degraded in 4-5 weeks. Conclusions: MSC from bone marrow differentiate towards osteogenic lineage, representing a suitable cell source for bone formation in periodontal regeneration. Gingistat(R) collagen scaffold supports MSC distribution and differentiation, but its short degradation time may be a limitation for a future application in bone tissue regeneration.

Among the different families of intracellular molecules that can be modulated during cell damage and repair, mitogen-activated protein kinases (MAPKs) are particularly interesting because they are involved in several intracellular pathways activated by injury and regeneration signals. Despite most of the studies have been performed in non-neurological models, recently a causal role for MAPKs has been postulated in central nervous system disorders. However, also in some peripheral neuropathies, MAPK changes can occur and these modifications might be relevant in the pathogenesis of the damage as well as during regeneration and repair. In this review, the current knowledge on the role of MAPKs in peripheral neuropathies will be discussed.

Mesenchymal stem cells (MSCs) can differentiate into multiple cellular lineages including neuronal cells. However, the positive effect of MSCs on repairing the nervous tissue has not yet been completely understood. In order to investigate the influence of MSCs on a neuronal population, we co-cultured MSCs, obtained by flushing the bone diaphisis from adult Sprague-Dawley rats, with DRG post-mitotic sensory neurons obtained from rat embryos at day E15. Co-cultures were maintained for 2 months. The adult rat MSCs, simply harvested in a pure culture of DRG neurons, allow the long-lasting survival and maturation of neurons otherwise committed to die. Neurons, when co-cultured with rat fibroblasts, do not survive as long as with MSCs and do not mature to the same degree. The rescue effect of MSCs on neurons is achieved only by cellular direct contact. These results provide a valid explanation for the functional improvement reported in some in vivo experiments.

Mitogen-activated protein kinase (MAPK) cascades transmit and amplify signals involved in cell proliferation as well as in cell death. In this study, the potential derangement of MAPK pathways has been evaluated in human squamous cell carcinomas (SCC) of the larynx. The expression and activity of the MAPK p38, ERK1/2p44/p42 and JNK/SAPKp46/p54 have been investigated by immunoblot analysis of tissue homogenates in 27 samples of primary laryngeal cancer and in 27 paired non-neoplastic laryngeal mucosa. On the same tissues, the activation of MAPK JNK/SAPKp46/p54 was also analyzed by an ELISA assay. The results obtained showed that both total and phosphorylated levels of JNK/SAPKp46/p54 and p38 were not different between tumor and normal samples. Conversely, while total protein levels for both ERK1p44 and ERK2p42 were not statistically different between tumor and normal samples, the analysis of the level of the activated forms of ERK1/2 showed a statistically significant decreased phosphorylation of both isoforms in the tumor samples compared to the control tissues. The rate of reduction was similar for both isoforms. Immunohistochemical analysis of all the activated MAPK (p38, JNK/SAPKp46/p54 and ERK1/2p44/p42) in both laryngeal SCC and normal mucosa demonstrated no difference of cellular localization. Activated ERK1/2p44/p42 and activated p38 demonstrated a nucleo-cytoplasmic distribution whereas activated JNK/SAPKp46/p54 were localized into the cytoplasmic membrane. The decreased activity of ERK1/2p44/42 in laryngeal SCC might reflect alterations in tumor suppressing activity or might derive from the interplay among various transduction pathways

Paclitaxel is an antineoplastic drug which acts by enhancing tubulin polymerization. The induction of peripheral neuropathy is the main dose-limiting side-effect of paclitaxel treatment. In this study, the neurotoxic effect of this drug in dorsal root ganglion (DRG) explants was analyzed by measuring the neurite length of DRG explants exposed to nerve growth factor (NGF). The neurotoxic effect of paclitaxel is dose- and time-dependent. Moreover, in DRG dissociated post-mitotic neurons, the molecular and morphological features of paclitaxel-induced cellular death were studied and the DRG neurons were observed to die by necrosis. On the contrary, the proliferating human neuroblastoma SH-SY5Y cells exposed to paclitaxel die by apoptosis, as reported for cortical neurons. The different response to the same stimulus of different neuronal populations underlines the importance of the biochemical and molecular phenotype of the neuronal population in determining cellular behavior and vulnerability to the same noxious stimulus.

In previous studies we demonstrated that resveratrol acts in an antiapoptotic manner on the paclitaxel-treated human neuroblastoma (HN) SH-SY5Y cell line inhibiting the apoptotic pathways induced by the antineoplastic drug. In the present study we evaluated the antiapoptotic effect of resveratrol, studying its activity on cell cycle progression. We determined the mitotic index of cultures exposed to resveratrol and paclitaxel alone or in combination, the cell cycle distribution by flow cytometric analysis (FACS), and the modulation of some relevant cell cycle regulatory proteins. Resveratrol is able to induce S-phase cell arrest and this interference with the cell cycle is associated with an increase of cyclin E and cyclin A, a downregulation of cyclin D 1, and no alteration in cyclin B1 and cdk 1 activation. The resveratrol-induced S-phase block prevents SH-SY5Y from entering into mitosis, the phase of the cell cycle in which paclitaxel exerts its activity, explaining the antiapoptotic effect of resveratrol. (C) 2004 Elsevier Ltd. All rights reserved

BACKGROUND: The etiopathogenesis of nasal polyps still is to be clarified. Although hyperplasia is a typical feature of these pathological processes, little attention has been paid to specific aspects of cellular growth in polyps. We have evaluated the expression and localization of some of the regulatory proteins that direct the cell through the specific sequence of events culminating in mitosis or apoptosis in nasal polyps. METHODS: Twenty samples of nasal polyps and 20 samples of normal nasal mucosa have been analyzed for apoptotic index by detecting the DNA 3'OH ends deriving from DNA fragmentation. Moreover, they have been evaluated by immunohistochemical staining for expression of Ki-67, cyclins A and B1, p53, p21, p27, murine double minute clone 2, and Bcl-2. RESULTS: We have identified a greater proportion of proliferating cells in the lining epithelial cells of the polyps when compared with the normal mucosa as stained with anti-Ki-67 antibodies. An overexpression of p53, MDM2, and Bcl-2 and an increased apoptosis were observed in nasal polyps compared with the normal mucosa, whereas no variation of p27 expression was observed. The p21 and cyclins A and B1 were rarely expressed in both pathological and normal tissue. CONCLUSION: The p53-based control system of cell cycle progression appears to be altered in nasal polyps, potentially leading to an abrogation of the DNA damage checkpoint. Evaluation of the expression of the regulatory proteins that direct the cells throughout their cycle in nasal polyps may allow a better understanding of the biological behavior and clinical outcome of these benign pathological entities.

Combined anticancer therapy using platinum compounds and antitubulins has increased the risk of neurotoxicity. However, the combination of low-dose cisplatin (CDDP) with toxic doses of paclitaxel significantly reduces cellular death in a human neuroblastoma SH-SY5Y cell line. To analyze the mechanisms of this protection, we evaluated various signaling molecules possibly involved in apoptosis and some relevant cell cycle regulatory proteins. CDDP does not interfere with the tubulin-stabilizing action of paclitaxel. The evaluation of molecular pathways involved in apoptosis indicates that the Bcl-2 but not the caspases may be involved in the CDDP protection of paclitaxel-induced apoptosis. The increase in p53 protein and its nuclear accumulation suggests a possible involvement of p53 in CDDP protection. The use of the chemical inhibitor of p53, pifithrin alpha, excluded this possibility. The study of cyclins and the flow cytometric analysis (fluorescence-activated cell sorting) suggest that CDDP exerts a protective action by blocking cells early in the cell cycle. The determination of the mitotic index indicates that CDDP prevents cells from reaching the mitosis. We concluded that low doses of CDDP are protective against toxic doses of paclitaxel and that the possible mechanism of this protection is that the CDDP prevents human neuroblastoma SH-SY5Y cells from achieving mitosis

Extra corporeal photochemotherapy (ECP) is an immunomodulating procedure used in several nonneurological diseases which, similarly to multiple sclerosis, are likely to be due to T-cell-mediated autoimmunity and it is probable that ECP can modulate the normal activity of peripheral blood mononuclear cells (PBMC). Using the Lewis rat experimental allergic encephalomyelitis (EAE) model of human multiple sclerosis (MS) we examined the effect of extracorporeal UV-A irradiation on psoralen-activated PBMC. In our experiment the comparison between the two groups of animals (ECP or sham-treatment) evidenced that the ECP treatment reduced the severity of EAE on clinical grounds and this result was confirmed by the pathological examination. The changes in the titers of anti-myelin antigen antibodies typical of EAE were also modulated by the procedure. Ex vivo examination evidenced a significant reduction in tumor-necrosis factor-alpha (TNF-alpha) released by PBMC after lipopolysaccharides (LPS) stimulation in culture. We conclude that ECP modifies the normal activity of PBMC during the course of EAE and it is possible that one of the anti-inflammatory mechanisms of action of ECP is correlated to a down-regulation of T-helper 1 lymphocytes activity.

The main dose-limiting side effect of cancer treatment with platinum compounds is peripheral neurotoxicity. To investigate the intracellular mechanisms of platinum drugs neurotoxicity we have studied the effects of cisplatin and oxaliplatin on the human neuroblastoma cell line SH-SY5Y. Both platinum compounds are toxic causing cellular death by inducing apoptosis but oxaliplatin is less neurotoxic than cisplatin. The study of the proteins involved in the intracellular transduction pathways that may cause apoptotic death, revealed a very similar pattern of changes after exposure to cisplatin or oxaliplatin. In particular, as emonstrated by densitometric analysis, after exposure to both platinum compounds the total amount of the anti-apoptotic protein Bcl-2 was significantly reduced. Conversely, the amount of the pro-apoptotic protein p53 significantly increased. Caspases 3 and 7 were activated, but their activation was a late event, indicating a secondary role in the apoptotic process. Among the mitogen activated protein kinases, only the p38 protein was activated (phosphorylated) early enough to have a possible role in inducing apoptosis, possibly through p53 stabilization. The results of the present study and the data of the literature demonstrate that the ways in which cisplatin and oxaliplatin are neurotoxic are very similar and include not only DNA damage, but also the modulation of specific molecules involved in regulating the cellular equilibrium between apoptotic death and the cell cycle.

Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA-induced neuritogenesis, we used human neuroblastoma SH-SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal-regulated kinase (ERK) 1 and 2 in RA-induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA-induced neuritogenesis (percentage of neurite-bearing cells and neurite length) but significantly reduced cell survival. In addition we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA-induced ERK phosphorylation does not follow the classic Raf kinase-dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI 3-K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3-K inhibitor wortmannin, we observed a significant dose-dependent reduction in ERK phosphorylation. RA-induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration-dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA-induced neuritogenesis

INTRODUCTION: Resveratrol is a polyphenol found in grape and black wine. trans-resveratrol is the biologically active form of the polyphenolic compound. In different models it has been shown to have antioxidant, anti-inflammatory, antiplatelet aggregation activity. It has been also shown to have anticancer activity, to inhibit cell cycle progression and DNA synthesis Paclitaxel is an antineoplastic drug which is active against metastatic tumor of lung and breast but it causes peripheral neuropathy, accumulating in dorsal root ganglia (1). Paclitaxel is able to alter the microtubules polymerization rate and inhibits their depolimeryzation. On tumoral cells, paclitaxel is active during the mitosis phase of the cell cycle because it interferes with mitotic spindle formation. In previous studies we have demonstrated that trans-resveratrol was able to inhibit paclitaxel-induced apoptosis in SH-SY5Y acting on paclitaxel-induced apoptosis pathway, while it did not alter paclitaxel-induced microtubules polymerization (2,3). In the present study we have investigated the hypotesis that the antiapoptotic effect of trans-resveratrol was due to its action on cell cycle progression preventing SH-SY5Y cells enter into mitosis and be killed by the antineoplastic drug. MATERIAL AND METHODS: SH-SY5Y cells, cultured in Dulbecco's Modified Eagle's Medium additioned with 10% Fetal Bovine Serum, were incubated for different times in the presence of paclitaxel 1µM, of resveratrol 50µM or with both the substances simultaneously. SH-SY5Y cell cycle distribution was determined by Propidium Iodide staining and FACS analysis while mitotic index was evaluated through Giemsa stainig. To assess cyclin E, cyclin A, cyclin B1 level and cdk 1 phosphorylation state total cellular protein extracts were prepared at different time points and analyzed by immunoblotting. RESULTS: Paclitaxel 1µM blocked SH-SY5Y treated cells in mitosis while the addition of 50µM resveratrol pratically reverted this situati