Human Embryonic Stem Cell-Derived Mesenchymal Stromal Cells Decrease the Development of Severe Experimental Autoimmune Uveitis in B10.RIII Mice

<p><i>Purpose</i>: We investigated the effect of exogenously administered human embryonic stem cell-derived mesenchymal stromal cells (hESC-MSCs) in experimental autoimmune uveitis (EAU) in B10.RIII mice, a murine model of severe uveitis.</p> <p><i>Methods</i>: B10.RIII mice were immunized with an uveitogenic peptide, and intraperitoneal injections of 5 million hESC-MSCs per animal were given on the same day. Behavioral light sensitivity assays, histological evaluation, cytokine production, and regulatory T cells were analyzed at the peak of the disease.</p> <p><i>Results</i>: Histological and behavioral evidence demonstrated that early systemic treatment with hESC-MSCs decreases the development of severe EAU in B10.RIII mice. hESC-MSCs suppress Th17 and upregulate Th1 and Th2 responses as well as IL-2 and GM-CSF in splenocytes from hESC-MSC-treated mice.</p> <p><i>Conclusions</i>: MSCs that originate from hESC decrease the development of severe EAU in B10.RIII mice, likely through systemic immune modulation. Further investigation is needed to determine any potential effect on active EAU.</p

MOMP-vault immunization enhances the number and induces the redistribution of Th1 cells in the ILN.

<p>(a) Representative histogram showing CD3+CD4+ cells and IFNγ vs. IL-4 intracellular cytokine staining of ILN cells. The number of (b) Th1 or (c) Th2 cells in the ILNs of individual mice on days 7 and 15 after challenge infection were compared by Students' t-test (*p<0.02). Representative experiment with 4–5 mice per group where at least 50,000 cells were analyzed per mouse. The level of (d) IL-1α, (e) IFNγ, and (f) CXCL10 was measured in triplicate in OD tissue homogenates at two time-points after challenge infection in mice immunized with MOMP-vaults or given a lung infection with <i>C. muridarum</i> using Luminex assays (Milipore Corp). Cytokine levels were compared by Students' t-test (*p<0.05). One representative experiment of 3–5 mice per group.</p

Dendritic cells are efficient at incorporating vaults.

<p>(a) BMDC (1×10⁶) were incubated with media, GL-vaults (500 µg) or FITC-dextran (250 µg) at 37°C for the indicated times. Cells were stained for a marker on mouse BMDC (CD11c-PE) and analyzed using a flow cytometer. (b) BMDC (red) incubated as above for 30 min were viewed on a fluorescent microscope (Carl Zeiss, LSM5 Pascal). The fluorescent particles (GL-vaults or FITC-dextran) appear green and mouse BMDC appear red due to staining with CD11c-PE. Results shown are representative of three experiments.</p

Immunization with MOMP-vaults reduces local bacterial burden following genital infection.

<p>The bacterial burden of chlamydiae following a challenge infection was determined from vaginal swabs to be statistically reduced in the MOMP-vault immunized group and the positive control group immunized i.n. with live <i>C. muridarum</i> (Live CM) compared to the control GL-vault immunized group (two-way RM ANOVA, *p<0.005). Dunn's post-hoc test showed no difference between the Live CM and the MOMP-vault immunized groups.</p

Design of recombinant vault nanoparticles containing immunogenic proteins.

<p>(a) ELISA assay configured using vaults with the z peptide (cp-MVP-z) and without (cp-MVP) reacted with either mouse IgG or IgA. Data points represent duplicates, SD = 0.004–0.034 nm. (b) Schematic diagram of constructs used to prepare baculovirus recombinant vaults containing MOMP indicating their approximate locations within a vault. (c) Western blot of high speed pellet extracts of MOMP-vaults (5 µg/lane). Molecular weight markers (lane 1). The gel was probed with a monoclonal antibody against the VD1 region of MOMP (MoPn-40)(lane 2) or mouse 1023C monoclonal antibody against MVP (lane 3). The size of MOMP fused to mINT is shown in the box and is approximately 58 kDa. (d) Negative stain EM of cp-MVP and cp-MVP-z/MOMP-mINT recombinant vaults. Bar, 100 nm.</p