Histochemical analysis of NPC2 replacement therapy in murine spleen sections.

<p>Hematoxylin<i>-</i>eosin (H&E) staining of spleen from saline treated wild type mice (<i>left panel</i>), saline treated <i>NPC2^−/−</i> mice (<i>middle panel</i>), and NPC2 treated <i>NPC2^−/−</i> mice (<i>right panel</i>). Massive accumulation of lipid droplets was most prominent observed in the spleens of saline treated NPC2<i>^−/−</i> mice. Data are representative of three separate experiments. <i>n</i> = 3 animals in each experimental group. Scale bares represent 100 µm.</p

Effect of NPC2 treatment on animal body weight.

<p>(<b>A</b>) Males (n = 4) and (<b>B</b>) females (n = 6), respectively, of saline-treated wild type mice (•), saline-treated <i>NPC2^−/−</i> mice (○), and NPC2 treated <i>NPC2^−/−</i> mice (▾). The mice were weighed weekly from P21 to P87. Each animal was injected twice weekly with saline or NPC2 (5 mg/kg). Values are means ± SEM.</p

Effect of NPC2 replacement therapy on murine brain cholesterol storage.

<p>Total cholesterol levels in cerebellum, cortex, and hippocampus was measured postmortem in saline treated wild type mice (<i>black bars</i>), saline treated <i>NPC2^−/−</i> mice (<i>light gray bars</i>), and NPC2 treated <i>NPC2^−/−</i> mice (<i>dark gray</i>). Each bar represents the mean ± SEM for 6 animals in each of the three groups. Bars not sharing a letter within a given panel are significantly different (<i>P</i><0.05).</p

Immune response to NPC2 in 129P2 wild type- and <i>NPC2^−/−</i> mice.

<p>NPC2 coated microtiter wells were incubated with serial dilutions of immunized serum as indicated. Bound antibodies were detected by TRIFMA as described in Materials and Methods. An arbitrary concentration of anti-NPC2 antibodies was set to 1000 mU/ml in positive control serum prepared by subcutaneously injections of NPC2 with Freund's complete adjuvant as immune potentiator. Sera from saline treated healthy mice were similarly tested and served as negative controls. (<b>A</b>) Anti-NPC2 antibody concentration in intraperitoneal injected wild type mice, (<b>B</b>) Anti-NPC2 antibody concentration in intravenous injected wild type mice, and (<b>C</b>) Anti-NPC2 antibody concentration in intravenous NPC2 treated <i>NPC2^−/−</i> mice. Data represents mean values of triplicate wells.</p

Effect of purified bovine NPC2 on cholesterol accumulation in wild type and <i>NPC2^−/−</i> fibroblasts.

<p>(<b>A</b>) SDS-PAGE of 5 µg purified NPC2 resolved in a 10–20% gradient gel under nonreducing conditions and stained with Coomassie Brilliant Blue (<i>lane 1</i>). Molecular mass markers are shown on the left (<i>lane M</i>). (<b>B</b>) Upper row human- (Hu) and lower row murine (Mu) fibroblasts cultivated for 48 hours in complete medium (DMEM + 10% FBS). Wild type fibroblasts (<i>left panels</i>), <i>NPC2^-/-</i> fibroblasts (<i>middle panels</i>), <i>NPC2^-/-</i> fibroblasts supplemented with 600 nM NPC2 (<i>right panels</i>). Cells were fixed with 10% phosphate buffered formalin, pH 7.4 and stained with Filipin III and visualized using fluorescence microscope.</p

Histochemical and immunohistochemical analysis of NPC2 replacement therapy in murine liver sections.

<p>Staining of representative tissue sections of 87 days old saline treated wild type mice (<i>left panels</i>), saline treated <i>NPC2^−/−</i> mice (<i>middle panels</i>), and NPC2 treated <i>NPC2^−/−</i> mice (<i>right panels</i>). Hematoxylin<i>-</i>eosin (H&E) staining (<i>first row</i>), immunohistochemical localisation of antigen F4/80 positive macrophages (brownish) (<i>second row</i>), and Masson's trichrome staining to detect collagen (blue) (<i>third row</i>). Lipid laden macrophages (Kupffer cells) are clearly visible and prominent in liver section from saline treated <i>NPC2^−/−</i> mice, whereas only a minority of the macrophages in liver sections from NPC2 treated <i>NPC2^−/−</i> mice are correspondingly loaded. Data are representative of three separate experiments. <i>n</i> = 3 animals in each experimental group. Scale bars represent 80 µm.</p

Hepatic gene expression related to inflammation, fibrogenesis and cholesterol metabolism.

<p>Relative mRNA expressions compared with isocaloric diet fed controls of <i>TNF</i> and <i>COL1A1</i> (Panel A), <i>TGFB1</i> and <i>PPARG</i> (Panel B), <i>SREBF2</i>, <i>LDLR</i>, <i>HMGCR</i> and <i>ABCA1</i> (Panel C) and <i>INSIG1</i>, <i>SRB1</i>, <i>GNMT</i> and <i>NPC2</i> (Panel D) in isocaloric controls, high-fat-high-cholesterol (HFHC) controls, two-week NPC2 (treatment) and four-week NPC2 (prevention) animals. Bars represent median ± interquartile range. *: P < 0.05 compared with Isocaloric Controls. **: P < 0.005 compared with Isocaloric controls. ***: P < 0.0005 compared with Isocaloric controls. #: P< 0.05 compared with HFHC Controls. ¤: P<0.05 compared with two-week NPC2.</p

Relative gene expressions in NAFLD and NASH patients and in fibrotic and non-fibrotic NAFLD (F1/2 versus F0).

<p>Relative gene expressions in NAFLD and NASH patients and in fibrotic and non-fibrotic NAFLD (F1/2 versus F0).</p

Hepatic histology scores.

<p>Hepatic histology scores graded semi-quantitatively according to Kleiner et. al. non-alcoholic fatty liver disease (NAFLD) activity score (NAS), steatosis (0–3), lobular inflammation (0–2), ballooning (0–2) and fibrosis (0–4) in isocaloric controls, high-fat, high-cholesterol (HFHC) controls, two-week NPC2 (treatment) and four-week NPC2 (prevention) animals. No animal had significant histological fibrosis on HE (Panel A) or Masson trichrome staining (Panel B). *: P < 0.05 compared with Isocaloric Controls. #: P < 0.05 compared with HFHC Controls.</p