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The development of 3D printable hydrogels based on the crosslinking between chitosan and gelatin is proposed. Chitosan and gelatin were both functionalized with methyl furan groups. Chemical modification was performed by reductive amination with methyl furfural involving the lysine residues of gelatin and the amino groups of chitosan to generate hydrogels with tailored properties. The methyl furan residues present in both polymers were exploited for efficient crosslinking via Diels-Alder ligation with PEG-Star-maleimide under cell-compatible conditions. The obtained chitosan-gelatin hybrid was employed to formulate hydrogels and 3D printable biopolymers and its processability and biocompatibility were preliminarily investigated.

Oxaliplatin (OHP) Induced Peripheral Neurotoxicity (OIPN) is one of the dose-limiting toxicities of the drug and these adverse effects limit cancer therapy with L-OHP, used for colorectal cancer treatment. Acute neurotoxicity consists of symptoms that are the hallmarks of a transient axonal hyperexcitability; chronic neurotoxicity has a clinical picture compatible with a length-dependent sensory neuropathy. Acute OIPN pathogenesis has been linked to sodium voltage-operated channels (Na + VOC) dysfunction and it has been advocated as a possible predisposing factor to chronic neurotoxicity. We tested if topiramate (TPM), a well-known Na + VOC modulator, was able to modify acute as well as chronic OIPN. The project was divided into two parts. In Experiment 1 we tested by means of Nerve Excitability Testing (NET) a cohort of female Wistar rats to assess TPM effects after a single OHP administration (5 mg/kg, iv). In Experiment 2 we assessed TPM effects after chronic OHP treatment (5 mg/kg, 2qw4ws, iv) using NET, nerve conduction studies (NCS), behavioral tests and neuropathology (caudal nerve morphometry and morphology and Intraepidermal Nerve Fiber [IENF] density). In Experiment 1 TPM was able to prevent OHP effects on Na + VOC: OHP treatment induced a highly significant reduction of the sensory nerve's threshold, during the superexcitability period (p-value = 0.008), whereas TPM co-administration prevented this effect. In Experiment 2 we verified that TPM was able to prevent not only acute phenomena, but also to completely prevent chronic OIPN. This latter observation was supported by a multimodal approach: in fact, only OHP group showed altered findings compared to CTRL group at a neurophysiological (proximal caudal nerve sensory nerve action potential [SNAP] amplitude, p-value = 0.001; distal caudal nerve SNAP amplitude, p-value<0.001, distal caudal nerve sensory conduction velocity, p-value = 0.04), behavioral (mechanical threshold, p-value 0.003) and neuropatho

Exposure to ultrafine particles (UFPs) leads to adverse e_ects on health caused by an unbalanced ratio between UFPs deposition and clearance e_cacy. Since air pollution toxicity is first direct to cardiorespiratory system, we compared the acute and sub-acute e_ects of diesel exhaust particles (DEP) and biomass burning-derived particles (BB) on bronchoalveolar Lavage Fluid (BALf), lung and heart parenchyma. Markers of cytotoxicity, oxidative stress and inflammation were analysed in male BALB/c mice submitted to single and repeated intra-tracheal instillations of 50µg UFPs. This in-vivo study showed the activation of inflammatory response (COX-2 and MPO) after exposure to UFPs, both in respiratory and cardiovascular systems. Exposure to DEP results also in pro- and anti-oxidant (HO-1, iNOS, Cyp1b1, Hsp70) protein levels increase, although, stress persist only in cardiac tissue under repeated instillations. Statistical correlations suggest that stress marker variation was probably due to soluble components and/or mediators translocation of from first deposition site. This mechanism, appears more important after repeated instillations, since inflammation and oxidative stress endure only in heart. In summary, chemical composition of UFPs influenced the activation of di_erent responses mediated by their components or pro-inflammatory and pro-oxidative molecules, indicating DEP as the most damaging pollutant in the comparison.

Background: Chemotherapy-induced peripheral neurotoxicity (CIPN) is a severe adverse effect in patients receiving antitumor agents, and no effective treatment is available. Although the mechanisms responsible for the development of CIPN are poorly understood, recent findings make neuroinflammation an attractive target to be investigated, particularly when neuropathic pain is a prominent feature such as after bortezomib administration. The aim of our study was to evaluate the effect of intravenous immunoglobulins (IVIg) delivery in chronic CIPN. The related neuro-immune aspects were investigated in a well-characterized rat model of bortezomib-induced peripheral neurotoxicity (BIPN). Methods: After determination of a suitable schedule based on a preliminary pharmacokinetic pilot study, female Wistar rats were treated with IVIg 1g/kg every 2weeks. IVIg treatment was started at the beginning of bortezomib administration ("preventive" schedule), or once BIPN was already ensued after 4weeks of treatment ("therapeutic" schedule). Neurophysiological and behavioral studies were performed to assess the extent of painful peripheral neurotoxicity induced by bortezomib, and these functional assessments were completed by pathologic examination of peripheral nerves and intraepidermal nerve fiber quantification (IENF). The role of the innate immune response in BIPN was investigated by immunochemistry characterization of macrophage infiltration in peripheral nerves. Results: Both schedules of IVIg administration were able to significantly reduce bortezomib-induced heat and mechanical allodynia. Although these changes were not evidenced at the neurophysiological examination of peripheral nerves, they behavioral effects were paralleled in the animals treated with the preventive schedule by reduced axonopathy in peripheral nerves and significant protection from loss of IENF. Moreover, IVIg administration was very effective in reducing infiltration in peripheral nerves of macrophages wi

Chemotherapy-induced peripheral neuropathy (CIPN) is a major cause of disability in cancer survivors. CIPN investigations in preclinical model systems have focused on either behaviors or acute changes in nerve conduction velocity (NCV) and amplitude, but greater understanding of the underlying nature of axonal injury and its long-term processes is needed as cancer patients live longer. In this study, we used multiple independent endpoints to systematically characterize CIPN recovery in mice exposed to the antitubulin cancer drugs eribulin, ixabepilone, paclitaxel, or vinorelbine at MTDs. All of the drugs ablated intraepidermal nerve fibers and produced axonopathy, with a secondary disruption in myelin structure within 2 weeks of drug administration. In addition, all of the drugs reduced sensory NCV and amplitude, with greater deficits after paclitaxel and lesser deficits after ixabepilone. These effects correlated with degeneration in dorsal root ganglia (DRG) and sciatic nerve and abundance of Schwann cells. Although most injuries were fully reversible after 3–6 months after administration of eribulin, vinorelbine, and ixabepilone, we observed delayed recovery after paclitaxel that produced a more severe, pervasive, and prolonged neurotoxicity. Compared with other agents, paclitaxel also displayed a unique prolonged exposure in sciatic nerve and DRG. The most sensitive indicator of toxicity was axonopathy and secondary myelin changes accompanied by a reduction in intraepidermal nerve fiber density. Taken together, our findings suggest that intraepidermal nerve fiber density and changes in NCV and amplitude might provide measures of axonal injury to guide clinical practice. Significance: This detailed preclinical study of the long-term effects of widely used antitubulin cancer drugs on the peripheral nervous system may help guide clinical evaluations to improve personalized care in limiting neurotoxicity in cancer survivors.

For the deep geological disposal of high-level radioactive waste in argillaceous rocks, the heat production of the waste is an important driver for thermal–hydraulic-mechanical (THM)-coupled processes. These THM processes influence the properties and conditions of the near field that in many repository designs contains bentonite as a clay buffer. One task in the DECOVALEX-2015 (DEvelopment of COupled models and their VALidation against Experiments) project was the modelling of a heated bentonite column (Villar et al. in Long-term THM tests reports: THM cells for the HE-E test: update of results until February 2014. Deliverable-no: D2.2-7.3. CIEMAT Technical Report IEMAT/DMA/2G210/03/2014, 2014) in preparation for the in situ heater experiment HE-E at the underground rock laboratory Mont Terri. DECOVALEX is an international cooperative project that focuses on the development and validation of mathematical models for simulating such coupled processes associated with disposal in deep geological repositories. Eight modelling teams developed their own THM-coupled models for the bentonite column experiment, using six different simulation codes. Each of the teams individually calibrated the THM parameters for the bentonite material. The eight resulting parameter sets agree well and allow a satisfactory reproduction of the TH measurements by all models. The modelling results for the evolution of temperature and relative humidity over time at three sensors in the bentonite column are in good agreement between the teams and with the measured data. Also, changes of the temperature due to modifications of the insulation and the adjustment of the heating power during the course of the experiment are well reproduced. The models were thus able to reproduce the main physical processes of the experiment, both for vapour-dominated diffusion during the heating phase and combined liquid and vapour transport during a subsequent heating and hydration phase. Based on the parameter sets, t

L’Ambrosiana custodisce un pregevole nucleo tassiano, in gran parte individuabile nella raccolta di Gian Vincenzo Pinelli, la cui collezione libraria alimenta uno dei principali fondi della biblioteca milanese. Tasso e Pinelli si conobbero durante gli anni del primo soggiorno padovano del poeta e il loro legame si mantenne almeno sino ai tempi della prigionia. L’apice di tale sintonia è da collocarsi attorno al 1575, quando Tasso trascorse alcune settimane a Padova revisionando canti della Liberata insieme al dotto bibliofilo, la cui dimora era luogo di discussione delle principali voci del dibattito sulle poetiche. Si spiegano così, oltre le carte sul poema, i manoscritti ambrosiani dei Discorsi dell’arte poetica e quello sugli Estratti dalla poetica di Castelvetro. Ulteriore testimonianza dell’interesse di Pinelli per la circolazione delle opere tassiane sono i manoscritti dell’Aminta e delle Rime, che chiamano in causa anche la figura di Aldo Manuzio il Giovane. Anche l’epistolario tassiano è rappresentato in Ambrosiana: accanto ad alcune lettere in copia si conservano un paio di autografi, per uno dei quali, una lettera a Francesco Polverino, si propone una nuova datazione.

Peripheral neurotoxicity is one of the most distressing side effects of oxaliplatin therapy for cancer. Indeed, most patients that received oxaliplatin experience acute and/or chronic severe sensory peripheral neuropathy. However, despite similar co-morbidities, cancer stage, demographics and treatment schedule, patients develop oxaliplatin-induced peripheral neurotoxicity with remarkably different severity. This suggests individual genetic variability, which might be used to glean the mechanistic insights into oxaliplatin neurotoxicity. We characterized the susceptibility of different mice strains to oxaliplatin neurotoxicity investigating the phenotypic features of neuropathy and gene expression profiles in dorsal root ganglia of six genetically different mice strains (Balb-c, C57BL6, DBA/2J, AJ, FVB and CD1) exposed to the same oxaliplatin schedule. Differential gene expression in dorsal root ganglia from each mice strain were assayed using a genome-wide expression analysis and selected genes were validated by RT-PCR analysis. The demonstration of consistent differences in the phenotypic response to oxaliplatin across different strains is interesting to allow the selection of the appropriate strain based on the pre-defined read-out parameters. Further investigation of the correlation between gene expression changes and oxaliplatin-induced neurotoxicity phenotype in each strain will be useful to deeper investigate the molecular mechanisms of oxaliplatin neurotoxicity

Solid lipid nanoparticles (SLN) are colloidal drug delivery systems characterized by higher entrapment efficiency, good scalability of the preparation process and increased sustained prolonged release of the payload compared to other nanocarriers. The possibility to functionalize the surface of SLN with ligands to achieve a site specific targeting makes them attractive to overcome the limited blood-brain barrier (BBB) penetration of therapeutic compounds. SLN are prepared for brain targeting by exploiting the adaptability of warm microemulsion process for the covalent surface modification with an Apolipoprotein E-derived peptide (SLN-mApoE). Furthermore, the influence of the administration route on SLN-mApoE brain bioavailability is here evaluated. SLN-mApoE are able to cross intact a BBB in vitro model. The pulmonary administration of SLN-mApoE is related to a higher confinement in the brain of Balb/c mice compared to the intravenous and intraperitoneal administration routes, without inducing any acute inflammatory reaction in the lungs. These results promote the pulmonary administration of brain-targeted SLN as a feasible strategy for improving brain delivery of therapeutics.

Type-1 Diabetes is generally treated with exogenous insulin administration. Despite treatment, a very common long term consequence of diabetes is the development of a disabling and painful peripheral neuropathy. The transplantation of pancreatic islets is an advanced alternative therapeutic approach, but its clinical application is still very limited, mainly because of the great number of islets required to complete the procedure and of their short-term survival. An intriguing method to improve the performance of pancreatic islets transplantation is the co-transplantation of Mesenchymal Stem Cells (MSCs), adult stem cells already known to support the survival of different cellular populations. In this proof-of-concept study, we demonstrated using an in vivo model of diabetes, the ability of allogenic MSCs to reduce the number of pancreatic islets necessary to achieve glycemic control in diabetic rats, and overall their positive effect on diabetic neuropathy, with the reduction of all the neuropathic signs showed after disease induction. The cutback of the pancreatic islet number required to control glycemia and the regression of the painful neuropathy make MSC co-transplantation a very promising tool to improve the clinical feasibility of pancreatic islet transplantation for diabetes treatment

Intravenous Immunoglobulin (IVIg) are human IgG derived from plasma pools of healthy donors. Although there are studies in literature evaluating their effectiveness in different pathological animal models, there are no data about their possible role on Bortezomib (BTZ)-induced peripheral neuropathies. Female Wistar rats were treated following a preventive schedule (BTZ and IVIg co-treatment for 3 and 8 weeks) and a therapeutic schedule (4 weeks of BTZ treatment followed by a 4-week IVIg-BTZ cotreatment). Caudal nerve conduction velocity (NCV), plantar and dynamic tests were performed at different time points. Animals were sacrificed after 3ws (acute phase) or 8ws (chronic phase) and tissue samples (Dorsal Root Ganglias -DRG-, sciatic nerve, caudal nerve, skin) were collected for morphological, morphometrical and immunohistological analysis.In the preventive schedule, IVIg was not able to rescue caudal NCV reduction caused by BTZ neither after 3 nor after 8 weeks of co-treatment. Same results were observed in the therapeutic schedule. On the other hand, the evaluation of mechanical allodynia and cold hyperalgesia showed that IVIg injection protected from BTZ effect in both treatment schedules. Morphometric analysis evidenced that, even if not statistically significant only the preventive schedule has a tendency to protect the caudal nerve from BTZ damage. This result is consistent with the morphological evaluation of the nerve. Also, intraepidermal nerve fibers density was preserved in the preventive schedule but not in the therapeutic one. Finally, sciatic nerve and DRG macrophage infiltration levels tended to be reduced in the therapeutic schedule and were brought back to ctrl (rats not treated or injected with IVIg alone) levels in the preventive one. In conclusion, we were able to demonstrate for the first time that IVIg treatment especially used as preventive treatment option may reduce BTZ-induced neuropathic painful pointing out the possible role of inflammati

The difficulties encountered in the treatment of brain diseases with conventional pharmacological tools have created the need for innovative strategies. The combination of nanocarriers and alternative administration routes could represent an efficient approach to reach the brain. Intranasal administration (IN) provides a non-invasive option to deliver drugs to the brain, bypassing the BBB, reducing the first-pass effect and enhancing patient compliance. The objective of the present study was to investigate the biodistribution and bioavailability to the brain of polymeric nanoparticles (PNPs) after IN administration in healthy mice. PNPs were prepared with poly-lactide-co-glycolide polymer using nanoprecipitation method. PNPs had a polymodal distribution around 350 nm. The biodistribution of DiR-loaded PNPs was evaluated by means of 3D fluorescence tomography imaging. Our results show that 3h after a single IN administration, more than 5% of the injected dose was detectable in the brain. PNPs were quickly cleared from the thorax and the abdominal cavity, while the brain fluorescence slowly decreased ranging from 3.7% to 2.3% between 24h and 96h. Repeated IN administrations (2 administrations, 24h apart) provided a significant increment of PNPs-associated fluorescence in the brain, without affecting PNPs accumulation in other organs. These findings support nose-to-brain translocation of PNPs as a noninvasive strategy to enhance the bioavailability of therapeutics to the brain

Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) affecting over 2.000.000 individuals around the world. Although MS etiopathogenesis is still not completely defined environmental factor exposure and genetic background are relevant in disease development. Moreover, MS shows heterogeneous onset and course so that different disease forms can be described which are all characterized by motor and/or sensory and even cognitive impairment. Two steps in the disease progression can be described. First MS lesions are originated by the activated immune system which recognizes CNS myelin as a foreign element thus leading to the formation of demyelinated plaques that evolve into axonal damage and subsequent neurodegeneration over the time. Since the beginning MS therapy has been focused on counteracting immune system action. Nevertheless, besides the immunosuppressive/immunomodulating drugs such as Glatiramer Acetate, Beta-interferons and steroids, the advance in the comprehension of the immune-mediated mechanisms has sustained the development and use of molecular and cellular-focused approaches, e.g. monoclonal antibodies and stem cells. At the same time very few weapons are specifically available for fighting MS neurodegenerative progression. We report an overview on MS and both old and new therapeutic approaches to the disease

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.

The proteasome inhibitor bortezomib is an antineoplastic drug mainly used for the treatment of multiple myeloma (MM). Despite its effectiveness, bortezomib clinical use is often limited by the onset of peripheral neuropathy (BiPN). To better understand the mechanisms of BiPN several rat and mice models have been proposed, but no studies in MM-bearing animals allowing to test the antitumor activity of the selected schedules and the role of MM by itself in peripheral nervous system damage have been reported to date. Here, we carried out a study using immunodeficient C.B-17/Prkdcscid (SCID) mice injected with RPMI8266 human MM cells and treated with bortezomib 1 mg/kg once a week for five weeks. Animals were assessed with neurophysiological, behavioral and pathological methods and tumor volume measurement was performed along the study. At the end of the study BiPN was evident in bortezomib-treated animals, and this neurotoxic effect was evident using a schedule able to effectively prevent tumor growth. However, neurophysiological and pathological evidence of MM induced peripheral nervous system damage was also reported. This model based on MM-bearing animals is more reliable in the reproduction of the clinical setting and it is, therefore, more suitable than the previously reported models of BiPN to study its pathogenesis. Moreover, it represents an optimal model to test the efficacy of neuroprotective agents and at the same time their non-interference with bortezomib antineoplastic activity.

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.

Treatment of type-1diabetes with pancreatic islet transplantation is an intriguing therapeutic option, aimed to replace insulin administration, but very limited in clinical practice, mainly for the great number of islets necessary and for their short survival. Aim of this work is to verify the ability of Mesenchymal Stem Cells (MSCs) co-transplanted with Pancreatic Islets to improve the feasibility of this approach, by acting both on glycaemic control and on long term disease complications, such as the diabetic neuropathy. 5 groups were used (8 rats/group): a) healthy controls; b) Streptozotocin-induced diabetic rats; c) Diabetic rats transplanted with pancreatic islets (3000); d) Diabetic rats co-transplanted with pancreatic islets (2000) and MSCs (106); Diabetic rats treated with MSCs (106). Transplantations were performed after the assessment of neuropathic signs, such as the decrease of Nerve Conduction Velocity (NCV) and the impairment of nociceptive (thermal and mechanical) thresholds. The same parameters were evaluated two months after the transplantation. Diabetic rats transplanted only with pancreatic islets, or co-transplanted with MSCs and a suboptimal number of pancreatic islets, showed a marked and significant glycaemia value reduction, an improvement of thermal and mechanical sensitivity, and a nearly complete restoration of NCV with respect to diabetic-untreated rats. No differences were observed between diabetic rats and diabetic rats treated with only MSCs. Co-transplantation of MSCs with Pancreatic Islets allows to reduce the successful number of pancreatic islets, to obtain a better and more physiologic glycaemic control, and to induce the regression of painful neuropathy signs, thus ameliorating diabetes complications management

The transplantation of pancreatic islets is an innovative and intriguing therapeutic option for the long term treatment of type-1 diabetes (Remuzzi et al., 2009). Unfortunately, their clinical feasibility is limited by the great number of islets necessary to achieve glycaemic control and their short survival. A possible means to improve the performance of this technique can be represented by Mesenchymal Stem Cells (MSCs), adult stem cells alrady known to support the survival of different cellular populations (Scuteri et al., 2014). In this work the ability of Mesenchymal Stem Cells (MSCs) to improve the feasibility of this approach was verified into an in vivo model represented by Streptozotocin-induced diabetic rats. We compared 5 different groups (8 rats/group): a) healthy controls; b) Diabetic rats; c) Diabetic rats transplanted with pancreatic islets (3000); d) Diabetic rats co-transplanted with pancreatic islets (2000) and MSCs (106); Diabetic rats treated with MSCs (106). Transplantations were performed after the assessment of neuropathic signs, represented by a decreased Nerve Conduction Velocity (NCV) and an impairment of nociceptive thermal and mechanical thresholds. The same parameters were evaluated two months after the transplantation. Diabetic rats transplanted only with pancreatic islets, or co-transplanted with MSCs and a suboptimal number of pancreatic islets, showed a significant glycaemia value reduction, an improvement of thermal and mechanical sensitivity, and an improvement of NCV with respect to diabetic-untreated rats. No differences were observed between diabetic rats and diabetic rats treated with only MSCs. In conclusion, we demonstrated that co-transplantation with MSCs reduces the number of pancreatic islets needed to reach glycaemic control, and induces the regression of painful neuropathy signs, thus ameliorating diabetes complications management.

Celiac disease (CD) is an inflammatory disorder of the small Intestine activated in genetically predisposed individuals by the ingestion of gluten from wheat and related cereals (1). The role of adaptative immunity in CD has been widely investigated. However, also innate immunity can have a role in the development of the disease, and it could involve the alteration of proteins expression (determined either at transcriptional and post-transcriptional level). Post-transcriptional regulation could involve also small non coding RNAs (micro RNA, miRNA). In this study we have analyzed miRNAs expression in duodenal biopsies of controls and celiac patients subdivided into 2 groups (Marsh 3A-B and Marsh 3C) according to the severity of the intestinal lesion. Microarray analysis demonstrated that miR-192-5p is significantly down-regulated in biopsies of Marsh 3C CD patients. Several studies suggested a possible role for miR-192-5p in diseases with alteration of the immune system (2, 3). In this study we identified possible target genes by in silico analysis, including CXCL2 and NOD2, molecules involved in innate immunity. qRT-PCR and western blotting demonstrated that both mRNA and protein expression were higher in CD patients compared to controls. qRT-PCR performed after laser microdissection and immunohistochemistry prove that miR-192-5p and CXCL2 and NOD2 mRNA were present principally in villus epithelium (both in controls and in CD patients) suggesting a direct interaction between miR-192-5p and the two target proteins. In conclusion the results of the present study support the role of miRNAs in regulating the innate immunity in celiac disease. References [1] Tonutti et al. (2014) Diagnosis and classification of celiac disease and gluten sensitivity. Autoimmun Rev. 13 (4-5): 472-476. [2] Schee et al. (2013) Deep sequencing the microRNA transcriptome in colorectal cancer. Plos One 18; 8(6): e66165. [3] Wang et al. (2011) Serum and urinary free microRNA level in patients wi

OBJECTIVES: microRNAs (miRNAs) are short RNAs that regulate gene expression in various processes, including immune response. Altered immune response is a pivotal event in the pathogenesis of celiac disease (CD), and miRNAs could have a role in modulating both innate and adaptive response to gluten in celiac patients. METHODS: We compared miRNA profiles in duodenal biopsies of controls and CD patients by miRNA array. Differentially expressed miRNAs were validated in controls, Marsh 3A-B, and Marsh 3C patients by quantitative PCR (qPCR). Target gene expression was assessed by qPCR, western blotting, and immunohistochemistry, and the effect of gliadin was evaluated by in vitro stimulation experiments on duodenal biopsies. RESULTS: Seven miRNAs were identified as significantly downregulated in the duodenum of adult CD patients as compared with controls. qPCR validated the decreased expression of miR-192-5p, miR-31-5p, miR-338-3p, and miR-197, in particular in patients with more severe histological lesions (Marsh 3C). In silico analysis of possible miRNA targets identified several genes involved in innate and adaptive immunity. Among these, chemokine C-X-C motif ligand 2 (CXCL2) and NOD2 showed significantly increased mRNA and protein level in Marsh 3C patients and a significant inverse correlation with the regulatory miR-192-5p. In addition, forkhead box P3 (FOXP3), Run-related transcription factor 1, and interleukin-18 (targets of miR-31-5p, miR-338-3p, and miR-197, respectively) showed upregulation in CD patients. Furthermore, alterations in CXCL2 and NOD2, FOXP3, miR-192-5p, and miR-31-5p expression were triggered by gliadin exposure in CD patients. CONCLUSIONS: miRNA expression is significantly altered in duodenal mucosa of CD patients, and this alteration can increase the expression of molecules involved in immune response.

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 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

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.

Objective: Neutrophils have been shown to own several immunoregulatory properties. Nevertheless few attention has been given to their contribution to both Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis (EAE) etiopathogenesis and progression. We aim to investigate the potential role of neutrophils during EAE sensitization and chronic phase in Dark Agouti (DA) EAE. Methods: Chronic EAE was induced in DA rats by intrafootpad injection of syngenic spinal cord homogenate in incomplete Freund's adjuvant. Neutrophils were depleted by means of intraperitoneal injection of anti-rat neutrophil serum (aNEU) at different disease time course, i.e. either during the sensitization and the chronic phase of the disease. The animals were weighted weekly and aNEU effect was first evaluated on clinical score while deeper analysis were performed on blood formula, spleen morphology and spinal cord cytokine, chemokine and myelin basic protein expression. Results: aNEU had no effect on clinical score when neutrophil depletion was performed during the chronic phase, i.e. from 14 to 35 days post EAE induction (dpi). When aNEU was administered during the EAE sensitization phase up to 8dpi, no difference in mean clinical score could be revealed between EAE and aNEU-treated EAE rats up to the disease peak, i.e. 14 dpi. However a significant improvement in clinical condition could be reported soon after the disease peak so that a chronic course was transformed into an acute/ monophasic one. aNEU treatment induced a faster body weight gain while it did not affect blood formula changes related to EAE course. Moreover while aNEU treatment did not counteract EAE effects on both spleen microscopic morphological changes and Treg (FoxP3) content, it mainly affected the expression of both pro- and anti-inflammatory cytokines in the three main spinal cord traits (cervical, thoracic, lumbar) while relative low effect was observed on both MCP-1 and myelin basic protein expression. Conclus

Studies concerning the role of neuroactive steroids in chronic models of Experimental Autoimmune Encephalomyelitis (EAE) are still scarce. First we considered different pathological targets in Dark Agouti (DA) rats affected by EAE in order to well characterize this chronic model of Multiple Sclerosis (MS) which well reflects the relapsing-remitting form of MS. We analyzed neuroinflammatory profile, assonopathy and neuroactive steroid levels in the spinal cord of DA rats affected by EAE. Data obtained at 14 dpi (i.e. days post induction) showed that acute neurological signs were associated with infiltrating T cells (CD3+) and macrophages (ED1+) and with microglial activation (MHC-II+ cells) which were accompanied by an increased expression of pro- and anti-inflammatory cytokines in the spinal cord. We immunolocalized a few damaged axons (SMI-32+) in the spinal cord white matter, moreover we identified a general decrease in the level of most neuroactive steroids analyzed. At 45 dpi we observed a decrease of inflammatory infiltration, of microglial cell activation and of cytokine gene expression respect to EAE rats at 14dpi. Assonopathy was exacerbated as demonstrated by the increase in SMI-32 immunoreactivity and by the decreased Na+,K+-ATPase activity. Changes in the neuroactive steroid level observed at 14dpi were maintained or reverted at 45dpi. Interestingly, the level of progesterone (PROG) significantly decreased only at 45dpi suggesting a possible role of this neuroactive steroid in the chronicization of EAE. Basing on this evidence, we performed a second study aimed to evaluate the possible neuroprotective properties of PROG treatment in DA rats affected by EAE. Data obtained at 45 dpi showed that PROG was able to improve EAE course by reducing the spinal cord neuroinflammation and microglia activation. Moreover PROG treatment was able to promote a functional recover of axons and to counteract the MBP protein reduction observed in EAE rats. Finally an increase

Observations so far obtained in experimental autoimmune encephalomyelitis (EAE) have revealed the promising neuroprotective effects exerted by progesterone (PROG). The findings suggest that this neuroactive steroid may potentially represent a therapeutic tool for multiple sclerosis (MS). However, up to now, the efficacy of PROG has been only tested in the acute phase of the disease, whereas it is well known that MS expresses different features depending on the phase of the disease. Accordingly, we have evaluated the effect of PROG treatment in EAE induced in Dark Agouti rats (i.e. an experimental model showing a protracted relapsing EAE). Data obtained 45 days after EAE induction show that PROG treatment exerts a beneficial effect on clinical score, confirming surrogate parameters of spinal cord damage in chronic EAE (i.e. reactive microglia, cytokine levels, activity of the Na(+) ,K(+) -ATPase pump and myelin basic protein expression). An increase of the levels of dihydroprogesterone and isopregnanolone (i.e. two PROG metabolites) was also observed in the spinal cord after PROG treatment. Taken together, these results indicate that PROG is effective in reducing the severity of chronic EAE and, consequently, may have potential with respect to MS treatment