Elisa Ballarini

Neuroprotective effects of progesterone in chronic experimental autoimmune encephalomyelitis

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.

miRNAs Affect the Expression of Innate and Adaptive Immunity Proteins in Celiac Disease

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.

Multimodal Analysis in Acute and Chronic Experimental Autoimmune Encephalomyelitis

Different experimental autoimmune encephalomyelitis models (EAE) have been developed. However, due to the different experimental conditions applied, observations simultaneously considering different pathological targets are still scarce. Using EAE induced in Dark Agouti rats with syngenic whole spinal cord homogenate suspended in incomplete Freund’s adjuvant, we here analyze neurosteroidogenic machinery, cytokine levels, microglial cells, infiltration of inflammatory cells, myelin proteins and Na+, K+-ATPase pump activity in the spinal cord. Data obtained in the acute phase of the disease confirmed that neurological signs were accompanied by the presence of perivascular infiltrating T cells (CD3+ cells) and activated monocytic/microglial cells (ED1+ and MHC-II+) in the spinal cord. In particular, the number of MHC-II+ cells was significantly increased in association with increased expression of pro- (i.e., TNF-α, IL-1β) and anti-inflammatory (i.e., TGF-β) cytokines as well as with decreased expression of proteolipid protein and myelin basic protein. During the chronic phase of the disease, the number of MHC-II+ cells was still increased, although less than in the acute phase. Changes in the number of MHC-II+ cells were associated with decreased Na+,K+-ATPase enzymatic activity. A general decrease in the levels of neuroactive steroids, with the exception of an increase in tetrahydroprogesterone and 17β-estradiol, was detected in the acute phase. These changes were maintained or reverted in the chronic phase of EAE. In conclusion, we report that modifications in the neuroimmune response in the acute and chronic phases of EAE are associated with specific changes in myelin proteins, Na+,K+-ATPase pump and in the levels of neuroactive steroids.

ApoE-modified solid lipid nanoparticles: A feasible strategy to cross the blood-brain barrier

&NA; 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. Graphical abstract Solid lipid nanoparticles (SLN), prepared by warm microemulsion, are functionalized with a brain targeting peptide (mApoE). Intratracheally instilled SLN‐mApoE translocate from the lungs to the brain crossing the blood‐brain barrier. Figure. No caption available.

Therapeutic potential of Mesenchymal Stem Cells for the treatment of diabetic peripheral neuropathy.

ABSTRACT 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. HighlightsMSCs reduce the number of Pancreatic Islets necessary to control blood glucose level.MSCs co‐transplanted with Pancreatic Islets ameliorate diabetic neuropathy.MSCs co‐transplanted with Pancreatic Islets reduce nephrotoxicity.

Peripheral neuropathy induced by microtubule-targeted chemotherapies: insights into acute injury and long-term recovery

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. Cancer Res; 78(3); 817-29. ©2017 AACR.

Therapeutic Administration of Mesenchymal Stem Cells Abrogates the Relapse Phase in Chronic Relapsing-Remitting EAE

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.

Susceptibility of different mouse strains to oxaliplatin peripheral neurotoxicity: Phenotypic and genotypic insights

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.

Oxaliplatin-Induced Peripheral Neurotoxicity: Morphological Characterization In Different Mouse Strains

Oxaliplatin is one of the most effective anticancer drug, particularly employed in the treatment of colorectal cancer, but one of the major limitation in its use is peripheral neurotoxicity. Oxaliplatin induced peripheral neurotoxicity (OIPN) has a high incidence and is frequently long lasting or permanent. Neuropathy is characterized by distal sensory impairment initially in the legs, then extending to the arms. A prominent manifestation of sensitive damage is ataxia. Besides chronic neurotoxicity, many patients experience an acute, rapidly developing cold-induced sensory neuropathy, usually resolving within one week. OIPN clinical manifestations reflect the involvement of dorsal root ganglia (DRG) as primary target of the drug toxicity. Although this assumption is largely accepted and some pathogenetic hypothesis have been proposed, mechanisms at the basis of OIPN need to be clearly defined. OIPN may vary in frequency and severity among different cancer patients despite equal treatment schedules. A genetic susceptibility for more severe oxaliplatin-induced peripheral neurotoxicity (OIPN) has been suggested but never confirmed. Therefore we designed a study to assess the phenotypic differences induced by oxaliplatin treatment in six different mice strains (Balb c, AJ, C57Bl6, FVB, DBA, CD1) aiming at identifying the more and less severely affected. Animals were treated with OHP 3.5 mg/Kg/iv twice weekly x 4 weeks and evaluated before and after treatment. In all strains we performed a multimodal characterization of its neurotoxicity through morphological and morphometrical assessment in caudal nerve and DRG at light and electron microscopy, intra-epidermal nerve fibers density quantification, evaluation of mechanical and cold allodynia/hypoaesteshesia, caudal and digital nerve conduction velocity, activity of wide dynamic range (WDR) neurons of the spinal dorsal horn. Our preliminary data suggest that all the strains show signs of OIPN but not the same modifications in the parameters examined. We will show these results with particular attention to morphological data. This study suggests that genetic variability might have a role in the type and severity of OHP-induced peripheral damage.

Therapeutic potential of Mesenchymal Stem Cells for the treatment of type-1 Diabetes.

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 cotransplanted 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. Granted by MIUR – FIRB Futuro in Ricerca 2008 Prot. N° RBFR08VSVI_001.