Ns were followed and graded daily according to a score method, where 0: no sign, 1: He percentage of wound sealing was observed after 24 h. The invading flaccid tail, 2: hind limbs weakness, 3: hind limbs paralysis, 4: hind paralysis and fore limbs weakness, 5: full paralysis/dead. An intermediate non-toxic concentration (5 mg/kg/day) of chloroquine was used for EAE treatment (five consecutive days, via i.p.). For prophylactic approach, EAE was induced three days after the last dose of CQ (5 mg.kg21), and for therapeutic approach, mice received the CQ treatment after the onset of EAE (day 10th after immunization with neuro-antigens). Fourteen (prophylactic approach) and thirty (therapeutically approach) days after antigen challenge mice were killed spinal cords were removed and snap frozen; 12 um thin slices were made in cryostat and stained with haematoxylin and eosin (H E).Isolation of Treg Cells (CD4+CD25+) and Transfer Experiments?Naive C57BL/6 mice were treated with chloroquine as described above and three days after the last dose spleen cells were collected and CD4+CD25+ cells were isolated by magnetic beads following manufacturers recommendations (CD4+CD25+ Regulatory T Cell Isolation Kit; Miltenyi Biotec., USA). 56105 Treg cells per mouse were adoptively transferred (via i.v.) to EAE mice at the onset of disease (10 days after immunization). As control, EAE mice received equal numbers of CD4+CD252 cells at the same time point. EAE induction and evaluation was performed as described above.Lymphoproliferative Response and Cytokine DosageSplenic cells were aseptically collected from mice after 10 and 30 days of antigen challenge for prophylactic and therapeutic approaches, respectively, and after 16 days for Treg cells transfer experiments. Single cell suspensions were stained with Carboxyfluorescein succinimidyl ester (CFSE, Sigma-Aldrich, USA) following the manufacturers instructions. Cells (56105/well) were diluted in RPMI 1640 media supplemented with Fetal Calf Serum (FCS;10 vol/vol), guaramicine (50 ug/mL), 2-Mercaptoethanol (2 mM) and myelin oligodendrocyte glycoprotein peptide (MOG35?5;20 ug/mL), plated in flat-bottom plates and incubated at 5 CO2 and 37uC for 96 h. After the incubation period, cells were stained with PercPCy5-conjugated anti-CD3 antibodies and fixed in 1 Title Loaded From File paraformaldehyde prior to flow cytometer analysis. CFSElowCD3+ cells were considered proliferating T cells. CultureMaterials and Methods MiceSix-to-eight week-old female C57BL/6 mice from the Multidisciplinary Center for Biological Research, University of Campinas, were used in this study. Mice were kept in specificpathogen free conditions, in a controlled temperature and photoperiod environment, with free access to autoclaved food and water throughout the experiment. All protocols involving laboratory animals were approved and performed in accordance with the guidelines of the State University of Campinas Committee on the ?Use and Care of Animals (Comissao de Etica no Uso de Animais ? CEUA, # 2687-1).Chloroquine Supresses EAEChloroquine Supresses EAEFigure 1. Chloroquine administration alters the frequency of regulatory T (Treg) cells and dendritic cells (DCs), but not the proliferative capability of T cells. Briefly, mice were treated with chloroquine via i.p. for five consecutive days. Three days after the last dose mice were killed and splenic cells were analyzed by flow cytometry. Increased numbers of Treg cells (A) and reduced frequency of DCs (B) was found in mice treated with chloroquine when compared to the cont.Ns were followed and graded daily according to a score method, where 0: no sign, 1: flaccid tail, 2: hind limbs weakness, 3: hind limbs paralysis, 4: hind paralysis and fore limbs weakness, 5: full paralysis/dead. An intermediate non-toxic concentration (5 mg/kg/day) of chloroquine was used for EAE treatment (five consecutive days, via i.p.). For prophylactic approach, EAE was induced three days after the last dose of CQ (5 mg.kg21), and for therapeutic approach, mice received the CQ treatment after the onset of EAE (day 10th after immunization with neuro-antigens). Fourteen (prophylactic approach) and thirty (therapeutically approach) days after antigen challenge mice were killed spinal cords were removed and snap frozen; 12 um thin slices were made in cryostat and stained with haematoxylin and eosin (H E).Isolation of Treg Cells (CD4+CD25+) and Transfer Experiments?Naive C57BL/6 mice were treated with chloroquine as described above and three days after the last dose spleen cells were collected and CD4+CD25+ cells were isolated by magnetic beads following manufacturers recommendations (CD4+CD25+ Regulatory T Cell Isolation Kit; Miltenyi Biotec., USA). 56105 Treg cells per mouse were adoptively transferred (via i.v.) to EAE mice at the onset of disease (10 days after immunization). As control, EAE mice received equal numbers of CD4+CD252 cells at the same time point. EAE induction and evaluation was performed as described above.Lymphoproliferative Response and Cytokine DosageSplenic cells were aseptically collected from mice after 10 and 30 days of antigen challenge for prophylactic and therapeutic approaches, respectively, and after 16 days for Treg cells transfer experiments. Single cell suspensions were stained with Carboxyfluorescein succinimidyl ester (CFSE, Sigma-Aldrich, USA) following the manufacturers instructions. Cells (56105/well) were diluted in RPMI 1640 media supplemented with Fetal Calf Serum (FCS;10 vol/vol), guaramicine (50 ug/mL), 2-Mercaptoethanol (2 mM) and myelin oligodendrocyte glycoprotein peptide (MOG35?5;20 ug/mL), plated in flat-bottom plates and incubated at 5 CO2 and 37uC for 96 h. After the incubation period, cells were stained with PercPCy5-conjugated anti-CD3 antibodies and fixed in 1 paraformaldehyde prior to flow cytometer analysis. CFSElowCD3+ cells were considered proliferating T cells. CultureMaterials and Methods MiceSix-to-eight week-old female C57BL/6 mice from the Multidisciplinary Center for Biological Research, University of Campinas, were used in this study. Mice were kept in specificpathogen free conditions, in a controlled temperature and photoperiod environment, with free access to autoclaved food and water throughout the experiment. All protocols involving laboratory animals were approved and performed in accordance with the guidelines of the State University of Campinas Committee on the ?Use and Care of Animals (Comissao de Etica no Uso de Animais ? CEUA, # 2687-1).Chloroquine Supresses EAEChloroquine Supresses EAEFigure 1. Chloroquine administration alters the frequency of regulatory T (Treg) cells and dendritic cells (DCs), but not the proliferative capability of T cells. Briefly, mice were treated with chloroquine via i.p. for five consecutive days. Three days after the last dose mice were killed and splenic cells were analyzed by flow cytometry. Increased numbers of Treg cells (A) and reduced frequency of DCs (B) was found in mice treated with chloroquine when compared to the cont.
