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Background: Hibiscus Sabdariffa is a plant of the Malvacee family commonly used in Asian and African folk medicine and for Karkadè preparation. In our laboratory, we previously demonstrated that Hibiscus Sabdariffa total extract (HSE) exerted against human multiple myeloma cells a reversible cytostatic effect and reduced cell motility/invasiveness. In order to identify the molecule/s involved in HSE effects, in this study we evaluated the antitumoral activity of a fraction of HSE obtained after ethyl acetate separation (HEF). Material and methods: Human multiple myelomacell s RPMI8226 were treated with different HEF concentrations and cell viability was evaluated by MTT and Trypan blue vital count assays. Apoptosis cell death was evaluated by AnnexinV assay and Caspase3 western blot. Cells migration/invasion was assessed using Boyden Chamber assay. Rat embryo dorsal root ganglia cultures were used to assess HEF neurotoxicity in vitro. HEF and HSE activity was compared. Results: HEF was effective in reducing RPMI8226 cell viability in a dose and time dependent manner. HEF enhanced cell mortality, AnnexinV positive cells and Caspase3 activation. Moreover, HEF was effective in reducing cells migration/invasion and it was not neurotoxic in vitro. Conclusions: In this study we evaluated the in vitro effects of HEF against human multiple myeloma cells RPMI8226. Our results demonstrated that HEF treatment showed an increased effectiveness and Caspase3-dependent apoptosis, but not an increased neurotoxicity, compared to HSE. Considering evident anticancer properties of HEF, it can provide the basis for a further fractionation in order to isolate molecules responsible for Hibiscus sabdariffa effects.

UNLABELLED: Cortical reorganization occurring in multiple sclerosis (MS) patients is thought to play a key role in limiting the effect of structural tissue damage. Conversely, its exhaustion may contribute to the irreversible disability that accumulates with disease progression. Several aspects of MS-related cortical reorganization, including the overall functional effect and likely modulation by therapies, still remain to be elucidated. The aim of this work was to assess the extent of functional cortical reorganization and its brain structural/pathological correlates in Dark Agouti rats with experimental autoimmune encephalomyelitis (EAE), a widely accepted preclinical model of chronic MS. Morphological and functional MRI (fMRI) were performed before disease induction and during the relapsing and chronic phases of EAE. During somatosensory stimulation of the right forepaw, fMRI demonstrated that cortical reorganization occurs in both relapsing and chronic phases of EAE with increased activated volume and decreased laterality index versus baseline values. Voxel-based morphometry demonstrated gray matter (GM) atrophy in the cerebral cortex, and both GM and white matter atrophy were assessed by ex vivo pathology of the sensorimotor cortex and corpus callosum. Neuroinflammation persisted in the relapsing and chronic phases, with dendritic spine density in the layer IV sensory neurons inversely correlating with the number of cluster of differentiation 45-positive inflammatory lesions. Our work provides an innovative experimental platform that may be pivotal for the comprehension of key mechanisms responsible for the accumulation of irreversible brain damage and for the development of innovative therapies to reduce disability in EAE/MS. SIGNIFICANCE STATEMENT: Since the early 2000s, functional MRI (fMRI) has demonstrated profound modifications in the recruitment of cortical areas during motor, cognitive, and sensory tasks in multiple sclerosis (MS) patients. Experimental autoimmune encephalomyelitis (EAE) represents a reliable model of the chronic-progressive variant of MS. fMRI studies in EAE have not been performed extensively up to now. This paper reports fMRI studies in a rat model of MS with somatosensory stimulation of the forepaw. We demonstrated modifications in the recruitment of cortical areas consistent with data from MS patients. To the best of our knowledge, this is the first report of cortical remodeling in a preclinical in vivo model of MS.

Multiple Sclerosis (MS) is a neuroinflammatory and immune-mediated chronic disease of the Central Nervous System which progressively damages the axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. Most MS cases are characterized by a relapsing-remitting course, and current therapies rely only on the use of immunomodulating drugs which are, however, unable to reverse disease progression. Among the newly proposed alternative therapies, Mesenchymal Stem Cells (MSCs) are considered suitable for MS treatment due to their capacity to modulate the immune response and to modify the pattern of the released cytokines. So far, encouraging results have been obtained with the administration of MSCs before disease onset, mainly in animal models of acute Experimental Autoimmune Encephalomyelitis (EAE) in which MSCs were able to reduce inflammation, thus ameliorating also the disease’s clinical symptoms. On the contrary, only a very small number of studies have investigated the effect of MSCs on relapsing-remitting models of the disease. Here, we investigated the therapeutic potential of MSC administration, both before and after the disease’s onset, in an animal model of MS represented by Dark Agouti rats affected by chronic Relapsing-Remitting EAE. Our results demonstrated that in chronic Relapsing-Remitting EAE the administration of MSCs after the clinical disease’s appearance is able to completely abrogate the relapsing phase and to strongly reduce spinal cord demyelination. These encouraging results have demonstrated that MSCs can provide a protective and reparative strategy for MS treatment.

Multiple sclerosis (MS) is an autoimmune disease affecting the CNS and characterized by neuroinflammation. Clinical observations revealed different incidence between males and females, suggesting a possible influence of sex steroids. These molecules, produced by peripheral steroidogenic glands, can exert their actions into the CNS, directly, or after metabolic conversion, and, for this reason, they are also called neuroactive steroids. Observations on experimental autoimmune encephalomyelitis (EAE) rodents, the most used animal model for MS, also suggest an involvement for neuroactive steroids in the pathology. In particular, castration worsen, while testosterone or its metabolite dihydrotestosterone (DHT) improve the pathological course of the disease. Based on these evidences, male Dark Agouti rats were induced with EAE in order to develop a relapsing-remitting pathology, similar to the most common human form of MS. EAE animals were treated with DHT or vehicle (sesame oil) and monitored through the experiment. After 45 days post induction, we evaluated inflammatory parameters in spinal cord tissues. The pathological course was improved in EAE rats treated with DHT compared to vehicle. Moreover, neuroinflammatory parameters such as microglial and astrocytes markers as well as cytokine expression and thiobarbituric acid-reactive substances, that were increased in the EAE-vehicle group, are significantly reduced after treatment with DHT. In conclusion, DHT treatment ameliorates not only the pathological course of the disease but also neuroinflammatory parameters in spinal cord tissues. These observations may suggest DHT as potential therapeutic option for male MS patients.

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 α, 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.

Neurodegenerative diseases are different and many-sided disorders affecting both the Central and the Peripheral Nervous System. Despite the very different peculiar features, all the neurodegenerative diseases are characterized by the neuronal degeneration, which may be the consequence of different processes, such as an altered protein accumulation, an axonal damage, or the exposure to toxic agents. The progressive neuronal death leads to disease progression, which is not effectively counteracted by the current symptomatic therapies. Among the newly proposed therapeutic approaches, encouraging results have been obtained with Mesenchymal Stem Cells (MSCs), adult stem cells initially proposed for their differentiation potential and for their immune-modulatory abilities. Here we first verified in vivo the protective potential of MSCs into an in vivo model of Multiple Sclerosis (MS), represented by Experimental Autoimmune Encephalomyelitis (EAE), demonstrating that intravenous administration of MSCs are able to ameliorate the clinical score and the functional skills, and to reduce demyelinated lesions. We then investigated in vitro the possible molecular mechanisms of MSC protective action, thus demonstrating that, besides immunomodulation, MSCs are able to support neuronal survival after toxic stimuli exposure by reducing the apoptosis and by inhibiting the Metalloprotease pathway, which is supposed to be involved in neurodegenerative disease progression. Moreover, MSCs are able to promote the axonal myelination through the modulation of p75 receptor. For all these abilities, MSCs can represent a promising therapeutic approach for the treatment of neurodegenerative disorders.

Multiple sclerosis (MS) is an autoimmune pathology characterized by inflammatory and demyelinating phases. Clinical observations indicate an involvement for sex hormones in thepathology, so that, recently, a therapy for MS based on hormones has been proposed. A clinicaltrial, involving male MS patients, revealed improvement in cognitive performances after one yeartreatment with testosterone (Sicotte et al., 2007). In the experimental autoimmune encephalomyelitis (EAE) mice, a MS animal model, neuroprotective effects of testosterone, and of its metabolite dihydrotestosterone (DHT), were observed (Dalal et al., 1997; Palaszynski et al.,2004). In the same model, castration has deleterious effects on clinical course, suggesting a protective role for androgens (Bebo et al., 1998). Moreover, in vitro evidences showed that testosterone is able to reduce inflammatory cytokines produced by human macrophages (D’Agostino et al., 1999) and monocytes (Li et al., 1993; Liva and Voskuhl, 2001). These antiinflammatory properties may be mediated by the conversion of testosterone into estradiol, or, alternatively, by some direct effects on androgen receptor (AR), mediated by DHT. This steroid can be subsequently converted into 3 alpha-diol, a GABAA receptor agonist, acting as antiinflammatory agent. Based on these evidences, we were interested in explore the protective effects of androgens treatment in a rat EAE model characterized by acute inflammatory events. We induced male Lewis rats with myelin basic protein in order to develop EAE; animals were treated with 3 alpha-diol or vehicle (sesame oil) every other day for two weeks, whereas control rats (i.e., rats not induced with EAE) received in same days only vehicle. Neurological deficits were assessed throughout the experiment (i.e., until 15 days post induction; dpi), and, at 15dpi, spinal cords were dissected. The clinical course and the weight loss of EAE rats treated with 3 alpha-diol were not different when compared to vehicle treated animals. Significant differences between these two groups were instead observed in the spinal cord after immunostaining analysis for the glial fibrillary acidic protein and the major histocompatibility complex class II, where 3 alpha-diol treatment was able to reduce the inflammatory parameters. In agreement with this observation, gene expression for tumor necrosis factor alpha, a pro-inflammatory cytokine, was decreased after 3 alpha-diol administration compared to oil-treated EAE animals. This effect seems specific for proinflammatory cytokine, because the expression of transforming growth factor beta 1, an antiinflammatory cytokine, was not different between vehicle and 3 alpha-diol treated EAE rats. To asses the metabolic fate of the injected 3 alpha-diol, liquid chromatography tandem mass spectrometry analysis were performed in spinal cord from all groups. 3 alpha-diol levels were significantly increased in steroid treated animals; moreover, a significant raise in DHT levels was observed in the same animals, indicating a retroconversion of 3 alpha-diol into DHT. The data we obtained may suggest that the reduction of inflammatory parameters could be due by both the action of DHT on AR and/or of 3 alpha-diol on GABAA receptor. These observations pointed out, for the first time, the effects of 3 alpha-diol on inflammatory parameters in EAE Lewis rats. Future studies will be aimed to elucidate the involvement of androgen and/or GABAA receptors on the observed effects.

Cholangiocarcinoma (CCA) is the second most frequent primary liver neoplasia. It mainly arises from the malignant transformation of biliary epithelial cells, although it might originate from either hepatic progenitor cells at the Hering canals or transformed hepatocytes. CCA is a highly aggressive tumor with extremely poor prognosis and limited therapeutic approaches. Baicalin (BA) is one of the main bioactive flavonoids identified in the Scutellaria Baicalensis Georgi root dried extract which is extensively used in the Chinese traditional medicine. Together with the anti-inflammatory effect, the anti-neoplastic action is the most relevant BA property demonstrated on cancer cells of different origin. Being aware of the need of new therapeutic weapons for CCA treatment, we investigated whether Baicalin could exert anti-proliferative and anti-migratory effect on EGI-1 cells, a highly metastatic CCA cell line derived from bile duct carcinoma. We first tested different BA concentrations (from 5 to 200μM) in limiting EGI-1 viability using MTT assay. After 24h and 48h treatment, 5 and 10μM BA had no effect while rising from 25μM to 200μM (i.e. 25, 50, 100 and 200μM) BA exerted a significant cell viability reduction already at 24h and increased after 48h BA exposure. This reduction well correlated with the adherent absolute cell number decrease and it cannot be due to BA induced cell cycle impairment after neither 24 nor 48h treatment. We also evaluated the anti-migratory BA potential by a wound healing assay adding different BA concentrations (5, 25, 50,100 and 200μM) to the culture medium immediately after performing a wound on confluent cell cultures. All BA concentrations but 5μM induced a significant reduction in the EGI-1 migration rate after 24h treatment. Moreover 25, 50 and 10μM BA showed similar migration inhibition extent at 24 and 48h whilst 200μM BA exerted a stronger inhibitory effect already after 24h exposure which increased with time in a significant way. Taken together our preliminary results demonstrate that BA impairs CCA cell viability and migration suggesting a promising adjuvant therapeutic use for BA as antitumoral agent., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Experimental Autoimmune Encephalomyelitis (EAE) is a good model of Multiple Sclerosis in rodents. We have applied fMRI to invesigate the alteration in functional response in rats induced with EAE. Cortical activation is altered in EAE rat brain, compared to controls. fMRI showed an increase in the activated volume involving also the cortex ipsilateral and some extra-cortical areas at the observed time points. These results demonstrate brain cortex remodeling in EAE, a remakable feature of MS. The present model seems to be a relevant tool in preclinical MS studies.

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Magnetic Resonance Imaging (MRI), showing the extent of the involvement of CNS, plays a major role in the assessment of patients with MS. Further information can be obtained with functional MRI (fMRI) which may be used in MS patients to investigate the functional reorganization of cortical areas. fMRI observations in MS are already available in humans, but deeper knowledge on its usefulness might be gained using reliable animal models. We investigated by means of fMRI the brain plasticity in a chronic model of MS, i.e. Experimental Autoimmune Encephalomyelitis (EAE) in the Dark Agouti (DA) rat strain. Serial fMRI acquisitions were performed before, 30 and 60 days after EAE induction. fMRI with somatosensory stimulation was performed according to ref [1]. Briefly electrical stimulation (a train of squared pulses with frequency=3Hz, current=2mA, duration=0.5ms) was delivered to the left forepaw during acquisition of MR images sensitive to Blood-Volume. A single stimulation protocol was composed of 30 images under rest condition and 10 images acquired during stimulation. After appropriate image analysis, performed using the FSL software package [2], the brain region activated by the applied stimulus was determined. Prior to EAE induction, electrical stimulation resulted in a localized response in the contralateral sensory motor cortex according to previously reported results [1]. Thirty and 60 days after EAE Induction, the activated area was greatly increased covering large regions of both contra and ipsilateral somatosensory cortex and extending also to extra-cortical regions. Our results show that the experimental model of EAE in DA rats reproduces a remarkable findings observed in MS patients, i.e. the functional reorganization of motor cortex. It remains to be investigated whether this effect could represent an innovative platform for testing new therapeutic approaches for MS., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Multiple sclerosis (MS) is a severe chronic disease characterized by the presence of immuno-mediated demyelinating lesions and impairment of axonal transmission, which cause the reduction of nerve conduction velocity and lead to the development of neurological symptoms. Current therapy for MS is based on immunosuppressant agents, but recently Mesenchymal Stem Cells (MSCs) have been proposed as therapeutic treatment for MS, demonstrating a positive effect when administered before disease onset, mainly due to their immunomodulatory properties. Here, we investigate the therapeutic potential of MSCs into an animal model of multiple sclerosis, represented by Dark Agouti rats affected by chronic Relapsing-Remitting experimental autoimmune encephalomyelitis (EAE). In order to assess their putative effectiveness, 106 MSC were intravenously injected in EAE rats before disease onset (7 days after disease induction), to test the “preventive” schedule, or after disease onset (14 days after MSC induction), to test the “therapeutic” schedule with MSCs. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords were performed to evaluate the demyelinating lesions. Clinical score analysis demonstrated that the “preventive” schedule of treatment had no effect on EAE clinical course, while the therapeutic schedule was able to hamper relapsing phase from day 19 and till the end of the experiment (day 45) with respect to EAE group. At day 45, histological analysis performed on spinal cords of EAE rats demonstrated the presence of demyelinated plaques, assessed by Luxol fast Blue staining and by immunohystochemistry for MBP. The same lesions were present in spinal cords of rats treated with the preventive MSC administration. On the contrary the therapeutic schedule with MSCs was able to significantly reduce the extension of demyelinated areas in the spinal cords, thus confirming clinical score evaluations. These results suggested that MSCs are able to ameliorate the clinical course of EAE animals and to hamper the disease relapsing by reducing the areas of demyelinated lesions. We are now evaluating the possible mechanism of MSCs action by investigating some putative myelinating properties of MSCs.

Multiple Sclerosis (MS) is a crippling chronic disease of the Central Nervous System caused by the presence of self-antibodies which progressively damage axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. MS is characterized by a Relapsing-Remitting course, and current therapies rely only on the use of immunosuppressive drugs, which are however unable to reverse disease progression. Encouraging results have been obtained in preclinical studies with the administration of Mesenchymal Stem Cells (MSCs) before disease onset (Zappia et al., 2005). Here, we investigate the therapeutic potential of MSC administration after disease onset into an animal model of MS, represented by Dark Agouti rats affected by chronic Relapsing-Remitting Experimental Autoimmune Encephalomyelitis (EAE) (Cavaletti et al., 2004). 106 MSC were intravenously injected in EAE rats after disease onset. Clinical score was assessed daily, and after 45 days rats were sacrificed and histological analysis of spinal cords performed to evaluate the demyelinating lesions. After the first peak of disease, no further relapses were observed in EAE rats treated with MSCs, differently from what observed in EAE group. Histological analysis demonstrated the presence of demyelinated plaques in spinal cords of EAE rats, (Luxol fast Blue staining and anti-MBP immunohystochemistry). On the contrary the therapeutic schedule with MSCs significantly reduces the number and the extension of demyelinated areas in the spinal cords, confirming clinical score evaluations. These results demonstrated that MSCs ameliorate the clinical course of EAE and hamper the disease relapsing by reducing the areas of demyelinated lesions. Granted by MIUR – FIRB Futuro in Ricerca 2008 Prot. N° RBFR08VSVI_001., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Besides the immunomodulatory action, Mesenchymal Stem Cells (MSCs) are able to promote neuronal and glial survival both by releasing trophic factors and through cell to cell contact. For these features MSCs are a promising tool for the treatment of inflammatory and demyelinating diseases such as Multiple Sclerosis (MS). Here we reported a pre-clinical study on Dark Agouti rats affected by a Relapsing-Remitting form of Experimental Autoimmune Encephalomyelitis (RR-EAE), one of the most suitable models for the study of RR-MS. In order to assess the possible preventive or therapeutic effect, 106 MSCs were injected i.v. (intra venous) at day 7 or at day 14 post EAE induction (p.i.) and clinical score was evaluated daily. The preventive schedule of treatment (day 7 p.i.) had no effect on EAE clinical course but the therapeutic one (day 14 p.i.) was able to hamper the relapsing phase from day 19 p.i. and till the end of the experiment (day 45 p.i.) with respect to EAE group. At day 45 p.i., histological analysis performed on spinal cord of EAE rats revealed a substantial absence of inflammatory infiltration and the presence of demyelinated plaques, assessed by Luxol fast Blue staining and by immunohistochemistry for MBP (Myelin Basic Protein). Moreover the analysis performed on serial paraffin sections revealed that the therapeutic schedule with MSCs was able to significantly reduce the extension of demyelinated areas in the spinal cord white matter with respect to EAE and EAE+MSCs day 7 p.i. groups, thus confirming clinical score evaluations. These results suggested that MSCs are able to ameliorate the clinical course of EAE animals by reducing the areas of demyelinated lesions. We are now evaluating the possible mechanism of MSCs action by investigating in vitro some putative myelinating and immunomodulating properties of MSCs. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001.

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Magnetic Resonance Imaging (MRI) is used for MS first diagnosis and the evaluation of CNS damage extension during disease course while information about cortical area functional reorganization can be obtained by means of functional MRI (fMRI). The clinical patterns of disease evolution are highly variable and scarcely correlate with structural MRI-detected CNS damage. This phenomenon has been referred to as clinical/MRI paradox. Experimental Autoimmune Encephalomyelitis (EAE) is the animal model for MS and can be induced in Dark Agouti (DA) rat reproducing the condition experienced by the most MS patients, i.e. the remitting-relapsing form. fMRI observations in MS are already available in humans, but deeper knowledge on its usefulness might be gained using reliable animal models. EAE was induced by syngenic spinal cord intrafootpad administration with clinical disease onset around 10 days post EAE induction (dpi) and the worst clinical condition reached on 14dpi without a complete symptom resolution in main animals up to 45dpi. The brain plasticity was investigated by means of serial fMRI acquisitions performed before, 30 and 60 days after EAE induction. A train of squared pulses electrical stimulation (frequency=3Hz, current=2mA, duration=0.5ms) was delivered to the left forepaw during acquisition of MRI sensitive to Blood-Volume. A single stimulation protocol was composed of 30 images under rest condition and 10 images acquired during stimulation. After appropriate image analysis, performed using the FSL software package, the brain region activated by the applied stimulus was determined. The week before EAE induction, electrical stimulation resulted in a localized response only in the contralateral sensory motor cortex according to previously reported results. Thirty and 60dpi, the activated area was greatly increased covering large regions of both contra and ipsilateral somatosensory cortex and extending also to extra-cortical regions. Our results show that the DA rat EAE model is a good model in reproducing the functional reorganization of cortex observed in MS patients. It remains to be investigated whether this effect could represent an innovative platform for testing new therapeutic approaches for MS. AKNOWLEDGMENTS: The present work was partially supported by Fondazione Italiana Sclerosi Multipla (FISM) grant code 10/12/F14. We thank Dr Elisa Ballarini and Dr Virginia Rodriguez-Menendez for the image supply.

Multiple sclerosis (MS) is a chronic immuno-mediated inflammatory and demyelinating disease characterised by both demyelinating lesions and axonal degeneration, leading to the reduction of nerve conduction velocity and the development of neurological deficits. Recently, some in vivo studies have proposed Mesenchymal Stem Cells (MSCs) as promising therapeutic treatment for MS mainly for their capacity to modulate the immune response, although it is not yet known if other mechanisms, different from immune modulation, are involved in MSCs positive. We investigate the therapeutic potential and the clinical effects of MSCs by using an animal model of multiple sclerosis, represented by Lewis rats affected by acute experimental autoimmune encephalomyelitis (EAE). MSCs were intravenously administered immediately after EAE induction (T0) or one week later (T7), in both the cases before disease onset. The clinical course of acute EAE was ameliorated only in EAE animals in which MSCs were injected one week after disease induction, while EAE rats and EAE rats with MSCs injected at T0 showed similar clinical scores. Moreover, the EAE rats which received MSCs at T7 displayed a cytokines pattern expression comparable to untreated control rats, while both EAE group and EAE+MSCs (T0) group showed an increased expression of pro-inflammatory cytokines. These results evidenced that the intravenous administration of MSCs one week after EAE induction (and before disease onset) induces the amelioration of the clinical scores of EAE-rats, thus supporting the potential role for MSCs in cell therapy in multiple sclerosis. We are now investigating the molecular mechanisms of this positive effect, focusing our attention on the Metalloproteinases pathway, involved in multiple sclerosis and modulated by MSCs.

Organic cation transporters (OCTs) play an important role in transporting cationic xeno- and endobiotics across biological membranes. In particular their role in platinum drugs transport, such as cisplatin and oxaliplatin, has recently been studied in many normal tissues and cancer cells. It has become increasingly clear the role of these transporters in key tissues responsible for drug absorption and disposition: kidney, liver and intestine. However, only limited information is available regarding their distribution and activity in nervous system. This study was aimed to investigate (1) the expression of OCT2 in rat DRG and (2) to quantify the developmental changes in the mRNA expression of OCT2 in DRG neurons isolated from embryonic E15 and young adult Wistar rats. OCT2 mRNA expression in rat DRG was localized by in situ hybridization experiments and quantified by TaqMan Real Time PCR. Our results demonstrated the expression of OCT2 mRNA in rat DRG and its neuronal localization. In order to verify possible developmental changes in the OCT2 expression we quantified by TaqMan Real Time PCR the OCT2 mRNA using in vitro models of DRG neurons obtained from embryonic E15 rats and from young adult rats. OCT2 mRNA expression was lower in embryonic neuron cultures with respect to the one obtained from young adult rats, demonstrating a developmental change in the expression of this transporter in rat DRG neurons. The OCT2 mRNA expression in 4 weeks of age rats approached adult expression levels. Furthermore in literature is reported that the OCT2 mRNA expression in the kidney is gender dependent. Our preliminary data obtained in DRG of adult Wistar rats did not demonstrate the gender difference in OCT2 mRNA expression observed in the kidney. Key words: organic cation transporter, dorsal root ganglia neurons, developmental expression Supported in part by an unrestricted research grant from the “Fondazione Banca del Monte di Lombardia”

Since the introduction in clinical practice of vinca alkaloids, tubulin has became a good target for antineoplastic chemotherapy. Microtubules are essential to separate the duplicated chromosome pairs during mitosis but they are also involved in intracellular transportation, cell signaling and cell movement. Microtubules are cytoskeletal polymers built by self-associated alpha and beta-tubulin dimers in a dynamic equilibrium which is the crucial requirement in the mitotic spindle assembly and functionality. Thus microtubules are among the most interesting targets for anticancer drugs. Paclitaxel, a chemotherapeutic drug belonging to the taxanes family, has been for several years the only microtubule depolymerization inhibitors known in literature. Only in 1995 a second class of cytotoxic natural compounds acting with similar mechanism as taxanes was discovered and named epothilones. Binding the beta-tubulin subunit taxanes, natural epothilones and its synthetic analogs are able to induce the microtubule stabilization and to promote the soluble tubulin polymerization causing cell cycle arrest and apoptosis. The peripheral neuropathy associated to tubulin polymerization is a major clinically-relevant side effect of these drugs thus the aim of this project is to study the peripheral neuropathy and the beta-tubulin polymerization in rats treated with paclitaxel and epothilones. Female Wistar rats were treated with different doses of these drugs and morphological and morphometrical changes in sciatic nerve as well as the tubulin polymerization in the protein extract were analyzed. In these models we observed a mild primary axonopathy and an increased beta-tubulin polymerization while we have different responses in morphometrical analysis. In fact we observed a g-ratio value decrease in the animals treated with paclitaxel and an increase of this value in the other models.

L’Encefalite Autoimmune Sperimentale (EAS) è il principale modello animale impiegato nello studio dei meccanismi immunologici responsabili dello sviluppo della sclerosi multipla (SM). L’EAS viene generalmente indotta attivamente in diverse specie animali mediante la somministrazione di peptidi derivati da proteine espresse a livello del sistema nervoso centrale (SNC). L’EAS indotta nel ratto di Lewis rappresenta un eccellente modello per lo studio dei meccanismi immunologici cellulo-mediati responsabili dell’insorgenza della fase acuta della SM nella quale si instaura uno stato di infiammazione a livello del SNC, che compromette la trasmissione efficace del segnale lungo le fibre mieliniche. La EAS ha un andamento monofasico ed è caratterizzata da un decorso ben definito, contraddistinto dall’insorgenza dei primi segni clinici dopo 10 giorni dall’induzione della patologia. L’acme del processo patologico si registra dopo 14 giorni. Istologicamente si osserva la presenza di infiltrati monocellulari perivascolari a livello del SNC, prevalentemente composti da linfoidi CD4. Dopo 21 gironi dall’induzione, la malattia va incontro a remissione completa, e gli animali manifestano resistenza nei confronti di tentativi di reinduzione della malattia. Diversamente dall’uomo, nel modello di Lewis sono stati identificati i principali clonotipi di linfociti T che svolgono un ruolo centrale sviluppo della patologia. Tra questi, il più studiato è il clonotipo di linfociti T CD4 che esprime la subunità beta (β) 8.2 nella porzione variabile (V) del recettore delle cellule T (TCR). La letteratura in merito è basata per lo più su dati di biologia molecolare o immunoistochimica relativi alle cellule presenti nei linfonodi periferici o alle cellule infiltranti il SNC. Non sono attualmente presenti studi che caratterizzino la prevalenza dei linfociti TCR Vβ 8.2+ durante il decorso della EAS, soprattutto applicando metodiche relativamente veloci e accurate. Analogamente vi sono poche informazioni riguardo la cinetica e l’espressione delle molecole di adesione sulla superficie dei linfociti e responsabili dell’interazione con le pareti dei vasi e della migrazione nel parenchima tissutale. Di particolare rilievo per la extravasazione a livello del SNC sembra essere l’integrina alfa-4, riconosciuta da anticorpi diretto contro CD49d, che eterodimerizza con proteine della famiglia delle beta integrine (beta-1) originando molecole responsabili dell’adesione stabile alle pareti dei vasi sanguigni (VLA-4). Anche in questo caso sono poche le informazioni circa la modulazione dell’espressione di alfa-4 sui linfociti durante il decorso della malattia. Solamente Selmaj et al.1 hanno dimostrato un incremento nell’espressione di alpha-4 su splenociti di topo durante lo sviluppo dell’EAS indotta mediante trasferimento di cloni T encefalitogenici in topi sani. Scopo del nostro lavoro è stato quello di colmare, almeno in parte, le lacune concernenti la caratterizzazione del modello di EAS attivamente indotto nel ratto di Lewis. Ci siamo concentrati in particolare sulla frequenza dei linfociti T TCR Vβ8.2+ e sull’espressione della molecola di adesione alpha-4 su splenociti derivati da animali sacrificati all’insorgenza e al picco di EAS, nonché sulle cellule ottenute dal midollo di animali al picco di malattia.

Actively induced Experimental Autoimmune Encephalomyelitis (EAE) is a well reproducible model for portraying the acute phase of multiple sclerosis as well as good model to develop new promising treatments. Both lymphnode and spinal cord (s.c.) have been widely described by means of molecular biology and immunohistochemistry. The aim of this study was to characterize both spleen and s.c. lymphocyte populations and to highlight possible useful markers to be monitored during new therapeutic treatments. Thus at the onset (day 10 after EAE induction) and at the peak of the disease (day 14 after EAE induction) we investigated CD4, CD8 and encephalitogenic TCRVb8.2+ T cells prevalence as well as the expression of adhesion molecules CD49d (VLA-4) and CD11a (LFA-1). EAE was actively induced by subcutaneously inoculation of 50μg guinea pig Myelin Basic Protein (gifted by P. Riccio) in complete Freund’s adjuvant with inactivated Mycobacterium tuberculosis. The spleen and s.c. were dissected from both healthy (controls) and EAE-induced animals, cells were collected, stained with different combinations of conjugated antibodies and acquired using a flow cytometer. In the spleen of EAE animal we reported a significant decrease in the mean absolute cell number on day 14, together with the presence of a CD45+ population other than lymphocytes as soon as day 10. Furthermore we observed an increase in CD4 and decrease in CD8 T cells compared to the controls on day 14, while no changes were reported on day 10. Moreover no alteration in the TCRVb8.2+ lymphocytes percentage as well as in CD49d (VLA-4) and CD11a (LFA-1) expression on CD4 and CD8 T cells were never observed. In the spinal cord no infiltrating lymphocytes were detected until day 14. Therefore the results on s.c. were compared to those obtained in the spleen on day 14. Thus CD4 lymphocytes percentage was higher in the s.c., while CD8 lymphocytes prevalence decreased. We also observed an increase in CD4 TCRVb8.2+ T cells percentage together with a significant increase in the expression of CD49d (VLA-4) which was more accentuated for CD4 than for CD8 T cells. Our results confirm that EAE in Lewis rats is a CD4 T cell mediated disease in which the expression of TCR Vb8.2 and adhesion molecules is probably regulated mainly in the target organ. We show that multiparametric flow cytometry can be an informative method to describe immune system cells during the EAE ongoing. Supported by a MIUR 2006 grant (prot. 2006064219-002)

Extracorporeal photochemotherapy (ECP) is a cell based procedure effective in the treatment of different T-cell mediated diseases such as cutaneous T-cell lymphoma and Graft-versus-Host Disease. During ECP treatment the patient’s blood is processed by means of a cell separator to collect leukocytes which are then added with the photoactive drug 8-methoxypsoralen (8-MOP), exposed to ultraviolet-A light (UV-A) and reinfused into the patient. Even if the mechanisms of action of ECP remain elusive, it has been shown to own in-vivo immunomodulatory effects and to be effective also on experimental allergic encephalomyelitis (EAE) animals and in a small pilot study on multiple sclerosis (MS) patients. It has been suggested that during ECP not only UV-A irradiation but also changes in the environmental condition may be relevant. Therefore, we developed a new peristaltic-based bench device which reliably mimics ex-vivo the complete ECP cycle using 50 ml peripheral blood sample. Peripheral blood mononuclear cells (PBMC) were then collected and treated with 8-MOP and/or UV-A under the same conditions used for the patients’ therapy. Using this strategy we investigated 8-MOP and/or UV-A effect on the production of the inflammatory cytokines IFN-γ, IL-2 and TNF-α in PBMC after polyclonal stimulation. We observed a significant decrease in activated CD4+ and CD8+ T-lymphocytes producing cytokines after UV-A irradiation and a further decrease in the presence of 8-MOP. The decrease in cytokines production seemed to be both cytokine- and cell type-related. In fact TNF-α production was reduced to a lesser extent than IFN-γ and IL-2 ones, while CD4+ T-cells seemed to be more sensitive than CD8+ lymphocytes when IFN-gamma and IL-2 production was considered. Both T-cell population showed similar behaviour when TNF-α production was evaluated. Following this preliminary experience, this ex-vivo protocol will be used to deeply investigate the effect of ECP on EAE rats.

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Experimental Autoimmune Encephalomyelitis (EAE) is an inflammatory T cell mediated disease of the central nervous system (CNS) that serves as model to understand the pathogenesis of multiple sclerosis (MS). An acute form of EAE can be actively induced in Lewis rat by the inoculation of Myelin Basic Protein resuspended in complete Freud’s adjuvant. This model is highly reproducible and it has been commonly used to study the immunopathogenesis as well as to develop different promising treatments for MS. The aim of the present study is to characterize both splenic and CNS infiltrating lymphocytes during the course of the disease mainly by means of flow cytometry. Thus we investigated if there were differences in the prevalence of encephalitogenic T cells (TCR Vbeta8.2+) and in the expression of the adhesion molecule VLA-4 both in the main organ of memory immune system (spleen) and in the target organ of EAE (spinal cord) in both healthy and EAE animals on day 10 and 14. EAE was actively induced in Lewis rats by subcutaneously inoculation of 50 μg guinea pig MBP (gifted by P. Riccio) in 100 μg complete Freund’s adjuvant with 3 mg/ml of inactivated Mycobacterium tuberculosis in both hind limb footpads. Rats were evaluated every 24 hours for neurological signs. The spleen and spinal cord were dissected from both healthy and EAE-induced animals at the onset of clinical signs (day 10) and at the peak of the disease (day 14). Cells from spleen and spinal cord were collected, stained with different combination of conjugated monoclonal antibodies (anti CD3, CD4, CD8, CD45, CD49d, TCR αβ, TCR Vβ8.2) and then acquired using a FACSCanto flow cytometer. We observed a significant decrease in splenic mean absolute cell number in EAE animals on day 14 in comparison with the controls and an inverse correlation between the decrease in mean cell number value and the increase in the clinical score evaluation. Moreover in the spleen we reported the presence of an unexpected leukocyte population as soon as day 10 after treatment while in spinal cord we did not observed infiltrating lymphocytes or leukocytes until day 14; no leukocytes were never present in the spinal cord of healthy animals as expected. Moreover during the course of the disease, in the spleen we did not report an increase in the percentage of the encephalitogenic T cells, both CD4+ and CD8+, bearing TCR Vβ8.2 in the EAE animals compared with the controls. Furthermore we observed an increase in the percentage of CD4+ TCR Vβ8.2+ T cells in the spinal cord of EAE animals at day 14 compared with the spleen values of the same animals. No significant changes in the expression of CD49d (VLA-4) was observed in the spleen on day 10 or at day 14 on both CD4+ and CD8+ T lymphocytes. Furthermore a significant increase in the expression of this protein was evident in infiltrating T cells. This increase was more accentuated for CD4+ than for CD8+ T cells. Flow cytometry is a powerful and very informative method to describe the variation in immune system elements during the ongoing of EAE. It clearly confirms that EAE in Lewis rats is a CD4 T cell mediated disease in which the expression of TCR Vβ8.2 and adhesion molecules is probably regulated mainly in the target organ (CNS). Supported by a MIUR 2006 grant (prot. 2006064219)

Extracorporeal photochemotherapy (ECP) is a procedure effective in the treatment of several human T-cell mediated diseases. During ECP treatment the patient’s blood is processed by means of a cell separator to collect leukocytes (leukapheresis), mostly lymphocytes and monocytes (PBMC), which are then added with the photoactive drug 8-methoxypsoralen (8-MOP), exposed to ultraviolet-A light (UV-A) and reinfused into the patient. Even if the mechanisms of action of ECP remain elusive, it has been shown that it has an in-vivo immunomodulatory effect in experimental ellergic encephalomyelitis (EAE) and in a small pilot study in multiple sclerosis (MS) patients. It has been suggested that during ECP not only UV-A irradiation but also the environmental condition changes may be relevant, an aspect which has never been investigated in detail. Therefore, we developed a new bench device which reliably mimics ex-vivo the complete ECP cycle and we investigated the effect of 8-MOP, UV-A and their combination on the synthesis of IFN-gamma, IL-2 and TNF-alpha on healthy controls and a MS patient. PBMC were collected and treated with 8-MOP and/or UV-A under the same conditions used for the ECP therapy. PBMC were polyclonally stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of Brefeldin A (BFA) to prevent newly synthesized cytokine egression. Intracellular pro-inflammatory cytokines produced by activated T-lymphocytes were evaluated. We observed a significant decrease in activated CD4+ and CD8+ T-lymphocytes producing cytokines after UV-A irradiation and a further decrease in the presence of 8-MOP + UV-A. The decrease in cytokines production seemed to be both cytokine- and cell type-related. In fact TNF-alpha production was reduced to a lesser extent than IFN-gamma and IL-2 ones by both UV-A and the co-treatment, while CD4+ T-cells seemed to be more sensitive than CD8+ lymphocytes when IFN-gamma and IL-2 production was considered. Both T-cell population showed similar behaviour when TNF-alpha production was evaluated. 8-MOP and UV-A co-treatment affected T-lymphocytes activation after polyclonal stimulation and. reduced IFN-gamma, IL-2 and TNF-alpha production with different extent among the cytokines considered. Following this preliminary experience this ex-vivo protocol will be used to extend the determinations in the EAE model and to reproduce these observations in a larger series of healthy controls and MS patients.

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

trans-Resveratrol (3,4',5-trihydroxystilbene) is able to significantly reduce paclitaxel-induced apoptosis in the human neuroblastoma (HN) SH-SY5Y cell line, acting on several cellular signaling pathways that are involved in paclitaxel-induced apoptosis. trans-Resveratrol reverses phosphorylation of Bcl-2 induced by paclitaxel and concomitantly blocks Raf-1 phosphorylation, also observed after paclitaxel exposure, thus suggesting that Bcl-2 inactivation may be dependent on the activation of the Raf/Ras cascade. trans-Resveratrol also reverses the sustained phosphorylation of JNK/SAPK, which specifically occurs after paclitaxel exposure. Overall, our observations demonstrate that (a) the toxic action of paclitaxel on neuronal-like cells is not only related to the effect of the drug on tubulin, but also to its capacity to activate several intracellular pathways leading to inactivation of Bcl-2, thus causing cells to die by apoptosis, (b) trans-resveratrol significantly reduces paclitaxel-induced apoptosis by modulating the cellular signaling pathways which commit the cell to apoptosis. (C) 2002 Elsevier Science Ltd. All rights reserved.

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 situation. The mitotic index [(cells in mitosis/total cell count) x 100] of paclitaxel-treated cultures was 59.4 4 while it was reduced to 11.9 2.5 in co-treated ones. Flow cytometry analysis confirmed that paclitaxel alone arrested cell in G2/M while resveratrol was able to prevent this arrest and cause a slowing down in early S phase. Resveratrol 50µM had effect on cell cycle control proteins such as cyclin E, increasing its level starting from 4/6 hours. The level of cyclin A increased after 14 hours both in cultures exposed to 50µM resveratrol alone or in cultures co-treated with 1µM paclitaxel and 50µM resveratrol. Furthermore, 50µM resveratrol was able to inhibit both 1µM paclitaxel-induced cyclin B1 accumulation as early as at 4-6 hours and cdk1 dephosphorylation, i.e. activation, that began after 14 hours of treatment. Together with the previous results the present ones suggest that the effect of resveratrol on paclitaxel-induced apoptosis was partly due to its effect on apoptotic transduction pathways, and partly to its effects on cell cycle progression, preventing the cells to reach the mitosis phase during which paclitaxel is able to induce apoptosis. (1) Cavaletti G. at al. (2000) Neurotox. 21, 389-93 (2) Nicolini G. et al. (2001) Neurosci Lett. 302, 41-4 (3) Nicolini G. et al. (2003) Neurochem Int. 42,419-29.

Paclitaxel, an anticancer drug, induces apoptosis in human neuroblastoma cell line SH-SY5Y. The addition of trans-resveratrol, a natural antioxidant present in grapes and red wine, to SH-SY5Y cultures exposed to paclitaxel significantly reduces cellular death. The neuroprotective action of trans-resveratrol is due neither to its antioxidant capacity nor to interference with the polymerization of tubulin induced by paclitaxel. However, trans-resveratrol is able to inhibit the activation of caspase 7 and degradation of poly-(ADP-ribose)-polymerase which occur in SH-SY5Y exposed to paclitaxel. Resveratrol, therefore, exerts its anti-apoptotic effect by modulating the signal pathways that commit these neuronal-like cells to apoptosis.