Analysis of the cellular infiltration of the CNS show reduced IFN-γ and IL-17 producing cells in CQ treated EAE mice.

<p>(A) CQ treated-mice presented reduced infiltration of inflammatory cells. (B) The percentage of IFN-γ- and IL-17-producing cells infiltrating the brain was reduced while the frequency of IL-10- producing cells was found augmented in brain of mice treated with CQ. (C, D and E) Gene expression of IFN-γ, IL-17 and IL-10 in the CNS followed the same pattern, respectively. (F) The expression of FOXP3 was evaluated in the CNS by RT-PCR. (G) The frequency of CD25⁺Foxp3⁺ cells was evaluated in spleens of mice. Results are representative of two independent experiments and are expressed as mean ± SEM for at least five animals. p<0,05 (*) and p<0.01 (**).</p

Chloroquine administration alters the frequency of regulatory T (Treg) cells and dendritic cells (DCs), but not the proliferative capability of T cells.

<p>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 control group. In addition, splenic T cells proliferative response was not altered in the presence of concanavalin-A (C). Subpopulations of leukocytes showed slight changes when compared to control subjects (D). Results are representative of three independent experiments.</p

CQ treatment reduces the severity of EAE.

<p>(A) Briefly, mice were treated with chloroquine for five consecutive days and three days after the last dose of the drug EAE was induced through the administration of MOG_35–55 peptide emulsified in CFA and two injections of Ptx (200 ng/animal) at 0 and 48 h post antigen inoculum. (B and C) The weight change and clinical score were checked routinely. Results are expressed as mean ± SEM for at least five animals. p<0,01 (**) and p<0,005 (***). (D) At 14 days, mice were killed and spinal cords were prepared for histological analysis (H&E stained) to evaluate cellular infiltration in the CNS. The figures are representative of at least three independent experiments performed in different days. Bar: 500 µm.</p

Transfer of CQ-elicited Treg cells reduces the severity of ongoing EAE.

<p>(A) Naïve C57BL/6 mice were treated with chloroquine (5 mg/kg/day) for five consecutive days. Three days after the last dose of the treatment, splenic CD4⁺CD25⁺ cells were isolated using magnetic beads and cells (5×10⁵ cells per mouse) were transferred into mice with ongoing EAE (10 days after immunization). As controls, mice received the same number of CD4⁺CD25⁻ cells. (B) The clinical course of the disease was evaluated routinely. (C) The brains and spinal cords were collected and the enriched infiltrating cells were counted. The frequency of IL-17-, IL-10- and IFN-γ-producing cells was analyzed by flow cytometry as well. (D) The spleens were collected and CFSE-stained cells were cultivated in the presence of MOG_35–55 peptide for 96 h. Dye decay and cytokine production were analyzed by flow cytometry. Results are expressed as mean ± SEM for at least five animals. p<0,05 (*), p<0,01 (**) and p<001 (***).</p

Chloroquine treatment reduces the Ag-specific proliferation of T cells in the spleen and the cytokine production profile.

<p>After 14 days of immunization with MOG_35–55 peptide, mice were killed and CFSE-stained splenocytes were cultivated in the presence of MOG_35–55 for 96 h. (A) The proliferation was calculated in the CFSE^lowCD3⁺ cells. Figures presented are representative of three independent experiments. (B) At the end of the culture period the supernatants were collected and assayed for the detection of cytokines using cytometric beads assay. Results are expressed as mean ± SEM for at least five animals. p<0,05 (*).</p

Cell based assay.

<p>Antibodies to aquaporin-4 (AQP4) as detected by binding of patient IgG to HEK293 cells transfected with human full length AQP4 (left column) but not to non-transfected control HEK293 cells (right column). <b>1A and B:</b> Positive AQP4-Ab test in a patient with NMO according to Wingerchuk’s 2006 criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039372#pone.0039372-WHO1" target="_blank">[29]</a>. <b>2A and B:</b> Negative AQP4-Ab test in a patient with HAM/TSP as defined by World Health Organization criteria <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039372#pone.0039372-Casseb1" target="_blank">[28]</a>.</p

Demographic and baseline clinical characteristics of patients and controls.

*<p>Median (range); **EDSS  =  Expanded disability status scale score;</p>***<p>CSF Oligoclonal bands =  Two or more cerebrospinal fluid restricted IgG oligoclonal bands.</p><p>HRS  =  high risk syndromes (patients at high risk for conversion into NMO); N.a. =  Not applicable; N.d. =  Not done.</p