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1.
Figure 1

Figure 1. From: The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in aspergillus fumigatus sensitized mice.

Lp-PLA2targeting strategy and measurement of PAF-AH activity in mice. (A): Generation of Lp-PLA2-/- mice: Homologous recombination in ES cells was used to generate ES cells heterozygous for the Lp-PLA2 primary targeted allele. Breeding of mice heterozygous for the Lppla2 primary targeted allele with a germline Cre-deleter strain resulted in mice heterozygous for the Lp-PLA2 null allele. Lp-PLA2 exons are indicated as blue boxes; the neor selection cassette as a red box; loxP and FRT sites as yellow and green arrowheads respectively. The positions of the PCR-derived homology arms in relation to the genomic locus structure are indicated by dotted lines. (B): PAF hydrolysis is defective in Lp-PLA2-/- mice. PAF-AH activity in the serum of C57BL/6 wild type (WT) and Lp-PLA2-/- mice was measured as described. Data are expressed as Mean±SEM; n=6-8.

Zhilong Jiang, et al. Respir Res. 2012;13(1):100-100.
2.
Figure 5

Figure 5. From: The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in aspergillus fumigatus sensitized mice.

Lp-PLA2deficiency did not increase inflammatory cell influx and airway hyperresponsiveness after allergen challenge of actively sensitized Lp-PLA2-/- mice. (A): 9-12 week-old male C57/B6 wild-type and Lp-PLA2-/- mice were i.p. injected with 20 μg Af pre-formulated with alum (volume 1:1) on day 1 and 7, then intranasally challenged with 30 μg Af on day 13 and studied on day 14 or 15. Mice received Af challenge alone were negative controls. (B): Macrophages, eosinophils, neutrophils, lymphocytes in BAL were counted after cytospin preparations and Giemsa staining. Data are presented as absolute cell number of BAL (mean± sem, n=10-11). Lung resistance (C) and lung dynamic compliance (D) were measured by FlexiVent in response to increasing doses of MCh.

Zhilong Jiang, et al. Respir Res. 2012;13(1):100-100.
3.
Figure 4

Figure 4. From: The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in aspergillus fumigatus sensitized mice.

Lp-PLA2deficiency did not increase airway inflammation and hyperresponsiveness after allergen challenge of passively sensitized Lp-PLA2-/- mice. (A): 9-12 week-old male C57/B6 wild-type and Lp-PLA2-/- mice were sensitized i.v. by different dilutions of serum (results using 1:200 are shown) containing IgE against Aspergillus fumigatus (Af) on day 1, day 2 and day 3, then intranasally challenged with 30 μg Af on day 5. Lung responses were analyzed on day 6. Control mice received naive serum and/or were challenged with PBS. (B): Lung resistance was measured by FlexiVent in response to increasing doses of MCh. (C): BAL was obtained and cytospin preparations of the cell pellet were assessed under light microscope. Differential cell count was performed using total cell counts and Giemsa-stained cytospin preparations. Cells were identified as macrophages (MP), eosinophils (EP), neutrophils (NP) and lymphocytes (LC). (Mean±SEM n=5-11).

Zhilong Jiang, et al. Respir Res. 2012;13(1):100-100.
4.
Figure 3

Figure 3. From: The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in aspergillus fumigatus sensitized mice.

LPS/PAF-induced inflammatory cell influx and airway hyperresponsiveness was comparable between Lp-PLA2-/- and WT mice. (A-B): C57BL/6 mice were administered nothing (Naïve, white bars), saline (light grey bars), 1 μg LPS (hatched bars), 1 μg PAF (dotted bars) or 1 μg LPS+PAF (dark grey bars) via intra-tracheal instillation under light anesthesia. BAL was obtained 24 hours later. (A): Cytospin preparations of the cell pellet were assessed under light microscope. Giemsa stained panels are showing representative BAL samples from PAF (left panel), LPS (middle panel) and LPS+PAF (right panel) treated mice. (B): Differential cell count was performed using total cell counts and cytospin preparations. Cells were identified as Macrophages (MP), eosinophils (EP), neutrophils (NP) and lymphocytes (LC). Data are presented as absolute cell number of BAL (mean± SEM, n=6). (C-E): C57BL/6 wild type (WT, white circles) and LP-PLA2-/- (black circles) mice were administered 1 μg LPS+PAF via intra-tracheal instillation under light anesthesia. Lung resistance (C) and lung dynamic compliance (D) were measured by FlexiVent in response to increasing doses of MCh 24 hours after treatment. (E): BAL cell pellet was assessed and data are presented as absolute cell number of BAL (individual data points [n=6-7] are shown with the median indicated by a horizontal line).

Zhilong Jiang, et al. Respir Res. 2012;13(1):100-100.
5.
Figure 2

Figure 2. From: The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in aspergillus fumigatus sensitized mice.

Lp-PLA2 deficiency did not enhance the function of bone marrow-derived mast cells. (A): Bone marrow cells harvested from C57BL/6 wild type (WT, white bars) and Lp-PLA2-/- (black bars) mice were cultured for 3-4 weeks in the presence of 20 ng/ml murine IL-3. Mast cells were identified by phase contrast microscope (top panels) and FACS analysis using antibodies against FcεRI and CD117 (bottom panels). (B): Cells were sensitized with pooled WT anti-Af serum (1:100 dilution) and challenged with 3 μg/ml Af in vitro (n=4, left panel). Hexosaminidase release was measured in non-treated cells, cells treated with naïve serum alone or anti-Af serum alone (not shown); naïve serum+Af and anti-Af serum+Af. Anti-DNP IgE (2 μg/ml) and DNP (25 μg/ml) treated cells were used as positive controls (n=4). Data presented as % of the total β-hexosaminidase present in 0.1% Triton X-100 lysed cells. (C): Cells derived from wild-type or LP-PLA2-/- mice were sensitized with individual anti-Af serum samples, derived from WT mice that received active sensitization (n=9-10). Cells then were challenged with 3 μg/ml Af. Median and interquartile ranges. The assay was performed in triplicates (left panel). The right panel shows a correlation between Lp-PLA2 levels (as measured by PAF-AH activity) and mast cell degranulating activity (hexosaminidase release) of 6 serum samples. Data are presented as ratio over naïve serum control. (B-C): Released hexosaminidase in supernatant was measured by reaction with 8 mM p-nitrophenyl-N-acetyl-β-d-glucopyranoside (PNP-NAG). Data calculated from the total β-hexosaminidase present in 0.1% Triton X-100 lysed cells.

Zhilong Jiang, et al. Respir Res. 2012;13(1):100-100.
6.
Figure 6

Figure 6. From: The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in aspergillus fumigatus sensitized mice.

Lp-PLA2-/- mice had similar degree of airway inflammation, circulating and local IgE but attenuated IL-4 and IL-5 release in the BAL fluid when compared with WT mice. (A): Representative photomicrographs of H&E stained sections from formalin fixed and paraffin embedded lung tissue of each group. (B): Inflammation was semi-quantitatively assessed (median shown; n=3-11). The following inflammation score was used: (0-0.5): no infiltrates, intact lung structure. (1-1.5): <10 inflammatory cells/field. (2-2.5): Peri-bronchial/peri-vascular infiltrates. (3-3.5): peribronchial/vascular cuffing with parenchymal extensions. The entire tissue section was scored. (C-D): Total IgE and anti-Af specific IgE was measured by ELISA from serum and BAL samples of either non-sensitized (n=3) or Af-sensitized (n=11-16) mice. All animals received a single Af challenge. Maxisorp 96-well plates were used and IgE was detected by HRP-conjugated anti-mouse IgE. Data presented as ng/ml for total IgE with group median (C) or OD450 nm with group median for anti-Af specific IgE (D). The measurements were performed in duplicates. (D left panel): A strong positive correlation is shown between serum Lp-PLA2/PAF-AH levels (ratio over naïve serum ctr) and Af specific serum IgE levels in WT mice (but not Lp-PLA2-/- mice, not shown), sensitized and challenged with Af. (E-F): Cytokine and chemokine levels in cell-free BAL supernatant were measured by a Luminex assay. (E): Correlation of Th2-related mediators with IL-4 (on the y axis). The points represent log10 transformed individual values. (F): Cytokine profiles shown among 4 groups, n=3-7. Data are presented as mean ± SEM, pg/ml. Student’t test *p<0.05.

Zhilong Jiang, et al. Respir Res. 2012;13(1):100-100.

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