Levels of HLA protein expression differ between biopsy cases.

<p>In adult human mixed glial cultures, microglia and astrocytes express variable basal levels of HLA (qualitatively assessed by proportion of positive cells and intensity of staining). However, HLA expression is not observed on untreated brain-derived pericytes in culture.</p

IFNy-induced expression of HLA-DP, DQ, DR in meningeal fibroblasts is completely blocked by TGFβ₁.

<p>A) In vehicle-treated leptomeningeal explant cultures only leptomeningeal/perivascular macrophages express HLA-DP, DQ, DR (indicated by arrows). B) IFNy increases intensity of HLA expression on macrophage cells and greatly induces HLA expression in meningeal fibroblasts. C) TGFβ₁ has no effect on basal leptomeningeal/perivascular macrophage or meningeal fibroblast HLA expression. D) However, TGFβ₁ completely inhibits IFNy-induced meningeal fibroblast HLA expression, without affecting leptomeningeal/perivascular macrophage HLA expression. DAB brightfield images have been inverted for image analysis. Scale bar = 100 µm. E) Quantification of HLA expression shows a massive increase in HLA expression in leptomeningeal explant cultures with IFNy but not with TGFβ₁ + IFNy (N = 12).</p

IP-10 is expressed by microglia and astrocytes in primary adult human mixed glial cultures.

<p>A) Following IFNy treatment (1 ng/ml, 96 h) IP-10 expression (green) is co-localised with CD45-immunopositive microglia (red). All nuclei are labelled with Hoechst (blue). Hoechst, IP-10 and CD45 and overlaid in the far right image. B) GFAP-immunopositive astrocytes (red) express IP-10 following IFNy treatment. Hoechst, IP-10 and GFAP are overlaid in the far right image. Scale bar = 100 µm. Arrows indicate high levels of IP-10 expression and insets show close-up examples of cells expressing IP-10.</p

IFNy increases pro-inflammatory cytokine and chemokine release from adult human mixed glial cultures and brain-derived pericytes.

<p>Here each data point indicates an individual case (n = 3). Control and IFNy-treated samples from the same case are indicated by connecting lines. A) IL-6 secretion is slightly, but not significantly, increased by IFNy treatment of mixed glia (microglia, astrocytes and brain-derived pericytes). B) MCP-1 production is significantly increased by IFNy in mixed glia cultures. C) Adult human mixed glia produce a low basal level of IP-10 which is markedly increased by IFNy. D) Relatively low concentrations of IL-6 production by pericytes are not changed by IFNy. E) Pericyte cell cultures produce comparable levels of MCP-1 to mixed glial cultures when stimulated with IFNy. F) Pericytes release IP-10 upon IFNy stimulation only.</p

IFNy-induced expression of HLA-DP, DQ, DR in brain-derived pericytes is inhibited by TGFβ₁ but not by M-CSF.

<p>A) Vehicle-treated pericytes (Hoechst-labelled nuclei) do not express HLA-DP, DQ, DR protein. B) IFNy induces a major up-regulation of HLA-DP, DQ, DR protein (green) in brain pericytes. C) TGFβ₁ treatment alone does not induce expression of HLA-DP, DQ, DR in these cells. However, TGFβ₁ completely inhibits the IFNy-stimulated increase in HLA-DP, DQ, DR (D). M-CSF affects neither basal (E) nor IFNy-induced (F) HLA-DP, DQ, DR expression in brain pericytes. Scale bar = 100 µm. G) HLA-DP, DQ, DR is induced by treatment with IFNy, and inhibited by simultaneous exposure to TGFβ₁ but not M-CSF (N = 12). H) Pericyte cell number (per well) is not influenced by IFNy or M-CSF but is significantly decreased by TGFβ₁ (N = 12).</p

Astrocytic expression of HLA-DP, DQ, DR is increased by IFNy, and not changed by TGFβ₁ or M-CSF.

<p>A) Adult human GFAP +ve astrocytes (red) express variable levels of HLA-DP, DQ, DR (green) in basal conditions without any treatment. B) IFNy (1 ng/ml, 96 h) increased astroglial expression of HLA-DP, DQ, DR. C) TGFβ₁ (10 ng/ml) did not affect astrocyte HLA-DP, DQ, DR expression alone, or when enhanced by IFNy treatment (D). E) M-CSF (25 ng/ml) also did not affect basal HLA-DP, DQ, DR expression in astrocytes or IFNy-enhanced HLA-DP, DQ, DR expression in astrocytes (F). Insets show close-up examples of astrocytes indicated by arrows. Scale bar = 100 µm. G) A significant increase in percentage of HLA-DP, DQ, DR-immunopositive astrocytes is found with IFNy treatment. Neither TGFβ₁ nor M-CSF significantly affect astrocyte HLA-DP, DQ, DR protein expression (N = 12). H) Quantification of GFAP-immunopositive astrocyte cell number (per well) following treatment with IFNy, TGFβ₁ or M-CSF does not result in any significant differences compared to vehicle-treated cells (N = 12).</p

Differential regulation of HLA and IP-10 in adult human microglia, astrocytes, brain pericytes and meningeal fibroblasts by IFNy, TGFβ₁ and M-CSF.

<p>The T cell pro-inflammatory cytokine IFNy upregulates HLA and IP-10 protein expression in adult human brain glial cells, pericytes and meningeal fibroblasts. Microglial HLA was increased by IFNγ (1 ng/ml for 96 h). M-CSF (25 ng/ml), but not TGFβ₁ (10 ng/ml), was found to decrease microglial HLA expression. Astrocytic expression of HLA was also increased by IFNγ, and not modulated by TGFβ₁ or M-CSF. Brain pericytes and meningeal fibroblasts do not basally express HLA but have a marked induction on exposure to IFNγ, which was blocked by TGFβ₁. IFNγ increased adult human microglia, astrocyte and pericyte expression and release of pro-inflammatory cytokines and chemokines, particularly IP-10. IP-10 may be involved in leukocyte trafficking into the CNS.</p

Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles

<div><p>Parkinson's disease (PD) is a progressive and currently incurable neurological disorder characterised by the loss of midbrain dopaminergic neurons and the accumulation of aggregated alpha-synuclein (a-syn). Oligomeric a-syn is proposed to play a central role in spreading protein aggregation in the brain with associated cellular toxicity contributing to a progressive neurological decline. For this reason, a-syn oligomers have attracted interest as therapeutic targets for neurodegenerative conditions such as PD and other alpha-synucleinopathies. In addition to strategies using small molecules, neutralisation of the toxic oligomers by antibodies represents an attractive and highly specific strategy for reducing disease progression. Emerging active immunisation approaches using vaccines are already being trialled to induce such antibodies. Here we propose a novel vaccine based on the RNA bacteriophage (Qbeta) virus-like particle conjugated with short peptides of human a-syn. High titres of antibodies were successfully and safely generated in wild-type and human a-syn over-expressing (<i>SNCA</i>-OVX) transgenic mice following vaccination. Antibodies from vaccine candidates targeting the C-terminal regions of a-syn were able to recognise Lewy bodies, the hallmark aggregates in human PD brains. Furthermore, antibodies specifically targeted oligomeric and aggregated a-syn as they exhibited 100 times greater affinity for oligomeric species over monomer a-syn proteins in solution. In the SNCA-OVX transgenic mice used, vaccination was, however, unable to confer significant changes to oligomeric a-syn bioburden. Similarly, there was no discernible effect of vaccine treatment on behavioural phenotype as compared to control groups. Thus, antibodies specific for oligomeric a-syn induced by vaccination were unable to treat symptoms of PD in this particular mouse model.</p></div

Competition ELISA to estimate relative affinity of vaccine-induced antibodies.

<p>Antigens in the form of peptides or recombinant monomer/oligomer protein preparations as described in the methods were coated on to microtitre plates and ELISA reading measured with fixed concentrations of serum IgG antibodies (at OD50 dilution) purified from pooled sera of vaccinated mice (n = 4), preincubated with serial dilutions of (A) free a-syn peptides (PD1, left; PD3, right); (B) free a-syn protein, either monomeric (left panels) or oligomeric (right panels) species, PD1 (top) and PD3 (bottom); and (C) liberated a-syn from haemolysed RBCs (orange line) or PBS negative control (blue line) for PD1 (top) and PD3 (bottom).</p

Effects of long-term immunisation with Qb-PD vaccines on a-syn aggregates.

<p>Male and female <i>SNCA</i>-OVX mice received 20 μg of Qb, Qb-PD1, Qb-PD3 or PBS every two weeks for a month, followed by monthly injections for 12 months (total duration of immunisation: for 13 months). (A-D) Immunofluorescence analysis was performed to detect a-syn aggregates with PLA in the substantia nigra, striatum, hippocampus and cerebellum. PLA puncta were quantified using ImageJ. (E) PLA puncta in 26–36 TH positive neurons were quantified and expressed as average mean of puncta per positive cell ± SEM (n = 3–4 mice per group), ANOVA followed by Dunn’s post hoc test. Scale bar 50 μm.</p