Elena Piras

CAR/FoxP3-engineered T regulatory cells target the CNS and suppress EAE upon intranasal delivery

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the murine experimental autoimmune encephalomyelitis (EAE) model of MS, T regulatory (Treg) cell therapy has proved to be beneficial, but generation of stable CNS-targeting Tregs needs further development. Here, we propose gene engineering to achieve CNS-targeting Tregs from naïve CD4 cells and demonstrate their efficacy in the EAE model.MethodsCD4+ T cells were modified utilizing a lentiviral vector system to express a chimeric antigen receptor (CAR) targeting myelin oligodendrocyte glycoprotein (MOG) in trans with the murine FoxP3 gene that drives Treg differentiation. The cells were evaluated in vitro for suppressive capacity and in C57BL/6 mice to treat EAE. Cells were administered by intranasal (i.n.) cell delivery.ResultsThe engineered Tregs demonstrated suppressive capacity in vitro and could efficiently access various regions in the brain via i.n cell delivery. Clinical score 3 EAE mice were treated and the engineered Tregs suppressed ongoing encephalomyelitis as demonstrated by reduced disease symptoms as well as decreased IL-12 and IFNgamma mRNAs in brain tissue. Immunohistochemical markers for myelination (MBP) and reactive astrogliosis (GFAP) confirmed recovery in mice treated with engineered Tregs compared to controls. Symptom-free mice were rechallenged with a second EAE-inducing inoculum but remained healthy, demonstrating the sustained effect of engineered Tregs.ConclusionCNS-targeting Tregs delivered i.n. localized to the CNS and efficiently suppressed ongoing inflammation leading to diminished disease symptoms.

Low levels of the air pollutant 1-nitropyrene induce DNA damage, increased levels of reactive oxygen species and endoplasmic reticulum stress in human endothelial cells

Both epidemiological and experimental studies suggest that exposure to high levels of air pollution is a risk factor associated with cardiovascular disease. Traffic emission is a major source of exposure to persistent air pollutants such as nitrated polycyclic aromatic hydrocarbons (nitro-PAHs). 1-Nitropyrene (1-NP), one of the most abundant nitro-PAHs in diesel exhausts, was selected as a model nitro-PAH for the present study. The aim of the study was to investigate the effects of 1-NP in human umbilical vein endothelial cells (HUVECs) and the metabolic pathways involved. The nitroreductase inhibitor dicoumarol and the coplanar aryl hydrocarbon receptor (AhR) ligand PCB 126 were used to modulate the metabolism of 1-NP. The results revealed that low levels (< or =10microM) of 1-NP induced DNA damage, increased levels of reactive oxygen species (ROS) and increased protein expression of the endoplasmic reticulum (ER) stress chaperone GRP78. A decrease in cell viability was only observed following exposure to a higher level of 1-NP (15microM). Inhibition of nitroreductive metabolism by dicoumarol attenuated the induction of DNA damage, intracellular ROS levels and GRP78 expression. This suggests that the effects of 1-NP on HUVEC were mediated by metabolites mainly formed at nitroreduction. Our findings suggest that the human blood vessel endothelium is a sensitive target tissue for the major nitro-PAH constituent in diesel exhaust.

Methimazole-Induced Damage in the Olfactory Mucosa: Effects on Ultrastructure and Glutathione Levels

Methimazole is an antithyroid drug that can induce loss of smell and taste in humans. It is also an olfactory toxicant in rodents. The aim of the present study was to examine involvement of glutathione in methimazole-induced damage of the olfactory mucosa (OM) of mice, and to study early onset of this damage using transmission electron microscopy (TEM). We found that an intraperitoneal dose of methimazole induced a dose-dependent decrease of nonprotein sulfhydryl groups (NP-SH; mainly glutathione) in the OM. Hepatic NP-SH was not decreased. One hour after administration (50 mg/kg), TEM demonstrated an extensive damage to acinar and intraepithelial excretory duct cells of Bowmans glands (BG) including dilatation of the endoplasmic reticulum and mitochondrial swelling. Furthermore, large vacuoles were noted in basal intraepithelial duct cells. After 2 hours there were ruptures of secretory granule membranes in BG and mitochondrial swelling and degeneration of sustentacular cells. The basal cells were less damaged. After four hours the neuroepithelium was disorganized although the columnar organization of neurons was largely intact. The acinar organization of the BG was frequently lost. The subsequent detachment of the neuroepithelium is suggested to be secondary to extensive damage of BG excretory ducts and sustentacular cells.

Cell specific expression of CYP2A5 in the mouse respiratory tract: Effects of olfactory toxicants

We performed a detailed analysis of mouse cytochrome P450 2A5 (CYP2A5) expression by in situ hybridization (ISH) and immunohistochemistry (IHC) in the respiratory tissues of mice. The CYP2A5 mRNA and the corresponding protein co-localized at most sites and were predominantly detected in the olfactory region, with an expression in sustentacular cells, Bowmans gland, and duct cells. In the respiratory and transitional epithelium there was no or only weak expression. The nasolacrimal duct and the excretory ducts of nasal and salivary glands displayed expression, whereas no expression occurred in the acini. There was decreasing expression along the epithelial linings of the trachea and lower respiratory tract, whereas no expression occurred in the alveoli. The hepatic CYP2A5 inducers pyrazole and phenobarbital neither changed the CYP2A5 expression pattern nor damaged the olfactory mucosa. In contrast, the olfactory toxicants dichlobenil and methimazole induced characteristic changes. The damaged Bowmans glands displayed no expression, whereas the damaged epithelium expressed the enzyme. The CYP2A5 expression pattern is in accordance with previously reported localization of protein and DNA adducts and the toxicity of some CYP2A5 substrates. This suggests that CYP2A5 is an important determinant for the susceptibility of the nasal and respiratory epithelia to protoxicants and procarcinogens.

Intranasal delivery of central nervous system-retargeted human mesenchymal stromal cells prolongs treatment efficacy of experimental autoimmune encephalomyelitis

Treatment with mesenchymal stromal cells (MSCs) is currently of interest for a number of diseases including multiple sclerosis. MSCs are known to target inflamed tissues, but in a therapeutic setting their systemic administration will lead to few cells reaching the brain. We hypothesized that MSCs may target the brain upon intranasal administration and persist in central nervous system (CNS) tissue if expressing a CNS‐targeting receptor. To demonstrate proof of concept, MSCs were genetically engineered to express a myelin oligodendrocyte glycoprotein‐specific receptor. Engineered MSCs retained their immunosuppressive capacity, infiltrated into the brain upon intranasal cell administration, and were able to significantly reduce disease symptoms of experimental autoimmune encephalomyelitis (EAE). Mice treated with CNS‐targeting MSCs were resistant to further EAE induction whereas non‐targeted MSCs did not give such persistent effects. Histological analysis revealed increased brain restoration in engineered MSC‐treated mice. In conclusion, MSCs can be genetically engineered to target the brain and prolong therapeutic efficacy in an EAE model.

Ret, Abl1 (cAbl) and Trp53 Gene Fragmentations in Comet-FISH Assay Act as In Vivo Biomarkers of Radiation Exposure in C57BL/6 and CBA/J Mice

Abstract Amendola, R., Basso, E., Pacifici, P. G., Piras, E., Giovanetti, A., Volpato, C. and Romeo, G. Ret, Abl1 (cAbl) and Trp53 Gene Fragmentations in Comet-FISH Assay Act as In Vivo Biomarkers of Radiation Exposure in C57BL/6 and CBA/J Mice. Radiat. Res. 165, 553–561 (2006). The International Commission on Radiation Protection (ICRP) has lowered the dose limits for workers and for the general public exposed to ionizing radiation. Consequently, a reliable dosimetric method for monitoring possible radiation-induced damage is of great importance in radioprotection. The counting of dicentric chromosomal aberrations and of micronuclei in peripheral blood lymphocytes is unreliable when it is applied to in vivo biopsies and for low-dose exposures. Single-cell gel electrophoresis (SCGE or comet assay), although sensitive and rapid, shows high variability when applied in vivo, probably due to prompt repair of the DNA breaks and confounding environmental factors. In this paper, we describe specific in situ hybridization of Ret, Abl1 (cAbl), and Trp53 gene fragmentations on SCGE slides (comet-FISH assay) in peripheral blood cells from C57BL/6 and CBA/J mice as an indicator of radiation-induced DNA damage. The results obtained from four mice for each experimental point (0, 1, 2 and 4 Gy of X rays) discriminated in a statistically significant way the effects of all doses when fragmentations were analyzed for the Ret, Ab1 and Trp53 genes. SCGE alone, when applied to the same specimens, produced no significant results because of interindividual and experimental variability.