Effects of PEG minocycline-liposomes on the expression of matrix metalloproteinase (MMP)-9 in splenocytes and CNS-infiltrating leukocytes.

<p>The expression levels of MMP-9 were significantly down-regulated on day 20 after active induction of experimental autoimmune encephalomyelitis (EAE) in splenocytes of mice following treatment with daily intraperitoneal (i.p.) injections of minocycline, and after intravenous (i.v.) injections of PEG minocycline-liposomes every 5 days compared to experimental animals that received no treatment, that were treated with i.v. injections of empty PEG-liposome every five days, or with minocycline i.p. injections every five days, as shown by ELISA (A). The proteolytic activity of MMP-9 measured by zymography was also significantly diminished in mice treated daily with i.p. injections of minocycline and i.v. injections of PEG minocycline-liposomes every five days (B). MMP-9 protein expression in CNS mononuclear cells was not found to be significantly different between experimental groups (C).</p

Effects of long-circulating polyethylene glycol (PEG) minocycline-liposomes on the clinical course of experimental autoimmune encephalomyelitis (EAE).

<p>A single intravenous (i.v.) injection of PEG minocycline-liposomes given after disease onset at day 15 post-immunization resulted in significant amelioration of clinical disease for eight days compared to a single injection of PBS when given shortly after onset of clinical disease (A). This effect was not sustained (A). In subsequent experiments, PEG-liposomes were administered every five days. Treatment with i.v. PEG minocycline-liposomes initiated after disease onset at day 15 post-immunization and administered every five days was as effective in ameliorating clinical EAE as treatment with daily intraperitoneal (i.p.) injections of minocycline (B). In contrast, i.v. injections of empty PEG-liposome every five days, or minocycline i.p. injections every five days had no detectable effect on the clinical course of EAE (B). The time of treatment initiation is indicated by a red arrow.</p

Immunization with human (h)AQP4_281-300 leads to an expansion of antigen-specific CD4⁺ T cells <i>in vivo</i>, and an Ig isotype switch in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice.

<p>(A) Following immunization with human (h)AQP4_281-300, an expansion of antigen-specific CD4⁺ T helper cells was detected by tetramer staining of lymph node cells. The fluorescent signal of <i>HLA-DRB1*03</i>:<i>01</i>-loaded tetramers minus the fluorescent signal of empty <i>HLA-DRB1*03</i>:<i>01</i> tetramers is shown. CD4⁺ T helper cells provide soluble mediators that drive B cell differentiation immunoglobulin (Ig) class switching. To determine whether hAQP4_281-300-reactive CD4⁺ T cells are capable of causing IgM to IgG isotype switching in <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice, the concentration of Ig against hAQP4_281-300, mAQP4284-299, or with whole-length hAQP4 protein in serum of immunized mice was quantified longitudinally. Since the NMO-IgG is a human IgG1 isotype, both, the murine IgG2a and IgG2b isotype were examined as they have similar properties with regard to complement binding and the Fcγ receptor. A switch from IgM to IgG2b was detected in mice immunized with hAQP4_281-300 peptide with regard to (B) antibody responses against hAQP4_281-300 and (C) whole-length AQP4 protein. An Ig isotype switch from IgM to IgG2b was also detectable in mice immunized with whole-length AQP4 protein with regard to (D) antibody responses against hAQP4_281-300 and (E) whole-length AQP4 protein.</p

Effects of long-circulating polyethylene glycol (PEG) minocycline-liposomes on inflammation within the central nervous system (CNS).

<p>A decreased number of inflammatory foci in the brain of mice with experimental autoimmune encephalomyelitis (EAE) treated with PEG minocycline-liposomes at disease onset was detected (A). Specifically, the number of CD11b⁺ macrophages, CD3⁺ T lymphocytes, and the overall expression of matrix metalloproteinase (MMP)-9 were decreased by PEG minocycline-liposomes treatment (A,B). There was no difference with regard to the numbers of immunoreactive cells between mice that were treated daily with intraperitoneal (i.p.) minocycline, and animals that had received intravenous (i.v.) PEG minocycline-liposomes (B). There was no difference with regard to the number of CD11b⁺ macrophages, CD3⁺ T lymphocytes, and the expression of MMP-9 in the CNS between animals that had received no treatment, daily i.v. PBS injection, and empty PEG-liposomes administered i.p every five days (data not shown).</p

Identification of critical residues of human (h)AQP4_281-300 for presentation in the context of <i>HLA-DRB1*03</i>:<i>01</i> and recognition by the B.10 T cell receptor (TCR).

<p>(A) First, the ability of hAQP4_281-300-reactive lymph node cells to recognize the alanine screening peptides was determined by ELISpot. 5.0x10⁵ cells/well lymph node cells taken ten days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with hAQP4_281-300 were restimulated with hAQP4 alanine scanning peptides (2 5 μg/mL) for 48 hours in IFNγ and IL-17 ELISpot plates (* = P-value < 0.05 and ** = P-value < 0.01). (B) Alanine screening peptides that not result in an increased frequency of IFNγ and IL-17 secreting lymph node cells were identified as the key residue peptides, and were subsequently tested in a MHC binding assay. Splenocytes taken from <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice were incubated for 12 hours in the presence of biotinylated hAQP4 alanine scanning peptides. Post incubation, cells were stained utilizing FITC-Avidin, and antigen positive cells were quantified by flow cytometry (* = P-value < 0.05 and ** = P-value < 0.01). (C) There was no Ig isotype class switch in mice immunized with mAQP4_284-299 with regard to antibody responses against whole-length AQP4 protein. (D) Critical <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, and B.10 TCR contact amino acids are specified. E₂₈₈ and L₂₉₄ are required as <i>HLA-DRB1*03</i>:<i>01</i> anchor residues, while T₂₈₉, D₂₉₀, D₂₉₁, and I₂₉₃ are critical B.10 TCR interacting residues.</p

<i>HLA-DRB1*03</i>:<i>01</i> transgenic mice are disease resistant to active immunization with human aquaporin 4 (hAQP4), and adoptive transfer of hAQP4-specific T cells.

<p>(A) <i>HLA-DRB1*03</i>:<i>01</i> mice were actively immunized with proteolipid protein (PLP)_91-110 (100 μg/100 μl/mouse; positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152720#pone.0152720.ref025" target="_blank">25</a>]), or varying AQP4 antigens*(whole-length hAQP4 protein, hAQP4_281-300, murine (m)AQP4_281-300, hAQP4_281-300 with a Quil-A Incomplete Freund Adjuvant (IFA) booster on day 14 post-immunization, mAQP4_281-300 with a Quil-A IFA booster on day 14 post immunization, and hAQP4_281-300 plus mAQP4_281-300) emulsified in Complete Freund Adjuvant (CFA). Immunization with a positive control proteolipid protein (PLP)_91-110, a dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> led to typical EAE. (B) Lymph node cells taken from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 or mAQP4_281-300 were restimulated for three days and passively transferred into <i>HLA-DRB1*03</i>:<i>01</i> mice. None of these experimental approaches resulted in clinical disease. (C) Paraffin sections were stained with haematoxlin eosin (H&E) and luxol fast blue (LFB). Representative sections of the spinal cords from PLP_91-110 and hAQP4_281-300 immunized mice are shown. On histopathological examination there were no visible signs of cellular infiltration, inflammation, or demyelination within the brain and spinal cord in any experimental paradigms other than in active immunization with PLP_91-110, the dominant encephalitogenic determinant in <i>HLA-DRB1*03</i>:<i>01</i> that led to typical EAE (spinal cord shown; inflammatory infiltrates and areas of demyelination are indicated by black arrows). (D) Fifteen days post immunization of <i>HLA-DRB1*03</i>:<i>01</i> transgenic mice with PLP_91-110 or hAQP4_281-300, pupillary reflex was measured via a mouse pupillometry. Mice actively immunized with hAQP4_281-300 and the control antigen PLP_91-110 did not show altered pupillary responses.</p

Human (h)AQP4_284-299 Alanine Scanning Peptides.

<p>The immunogenic region of hAQP4_281-300, hAQP4_284-299, was utilized to generate alanine scanning peptides at which each peptide sequence has a single alanine residue mutation.</p

Effects of long-circulating polyethylene glycol (PEG) minocycline-liposomes on the human peripheral blood mononuclear cells (PBMC) in vitro.

<p>Quantitative analysis of the zones of gelatinolysis detected the reduced the proteolytic activity of matrix metalloproteinase (MMP)-9 in the human PBMC sample treated with minocycline (3.0 mg/ml) compared with findings in controls (PBS). A more significant decrease of MMP-9 activity was detected in cells treated with minocycline for 6 hours compared to cells that were treated for 1 hour (A). At 1 hour, there was no significant MMP-9 activity difference among samples treated by the different PEG liposome preparation either with glucose, CaCl₂ or MgCl₂ (A). Time kinetic studies of PEG minocycline-liposomes <i>in vitro</i> revealed that the MMP-9 gelatinolytic activity is significantly reduced at 6 hours after incubation samples with PEG liposome+minocycline (B). Moreover, PEG minocycline-liposome+CaCl₂ treatment inhibited the MMP-9 activity more strikingly than the PEG minocycline-liposome+MgCl₂ preparation (B). Gelatinolytic activity was detectable by gelatin-zymography at molecular weights of 92 kDa, indicative of MMP-9, in the supernatants from all human PBMC samples studied. Incubation with PEG minocycline-liposomes with CaCl₂ resulted in remarkably decreased sizes of the bands at 92 kDa, pointing to decreased activation of MMP-9 in comparison with PEG minocycline-liposomes with MgCl₂ or glucose, respectively (C).</p

Human (h)AQP4_281-300-specific T cells do not cross-react with murine (m) AQP4_281-300.

<p>(A) There is that a single amino acid substitution from aspartic acid in hAQP4 to glutamic acid in murine (m)AQP4 at position 290. (B) In lymph node cells of <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 there was a significant proliferation of CD4⁺ T cells when hAQP4_281-300 was used as the recall antigen (* = P-value = 0.01). Only a higher recall antigen dose of 25 μg/ml resulted in a significant increase in proliferation, whereas as a dose of 5 μg/ml did not. (C) There was no proliferative response to mAQP4_281-300 at either dose_. (D) There is a significantly increased frequency of IFNγ and IL-17 producing lymph nodes cells from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with hAQP4_281-300 by ELISpot assay when hAQP4_281-300, and hAQP4_281-299 are used as recall antigens. However, we were unable to detect antigen specific IFNγ and IL-17 producing lymph nodes cells when mAQP4_281-300, or the negative control hAQP4_66-79 were used as recall antigens (** = P-value < 0.01). (E) IFNγ and IL-17 producing lymph nodes cells from <i>HLA-DRB1*03</i>:<i>01</i> mice immunized with mAQP4_281-300 were undetectable with any of the recall antigens.</p

B and T cell frequency in therapeutic model of Rituximab administration.

<p>Cells from each organ were pooled from at least three mice per group and counted. Cell counts were then normalized by dividing the total counts by the numbers of mice in each group and then multiplied by the percentage of each cell type as identified by flow cytometry. Total leukocytes were identified by CD45+ events within FSC and SSC gates. B cells were identified using gates for CD19 and B220. T cells were identified by expression of CD3 and either CD4 or CD8. Numbers in parenthesis indicate the percent change in cell counts in the hCD20Tg mice as compared to WTLM controls.</p