PMC

Alveolar inflammation was generally more severe in mice challenged with 1918 HA/NA:Tx/91 and A/Vn (a and c, respectively) than in mice challenged with 1918 HA/NA/NS:Tx/91 or Tx/91 (b and d, respectively). Black arrows indicate neutrophils in clusters (especially in a, occasionally in c) and singly (b and d) within alveoli. Mice challenged with 1918 HA/NA:Tx/91 and A/Vn also had more striking necrosis of bronchial and bronchiolar epithelium (green arrows). Bronchioli are indicated by a ‘‘B’’, and vessels are indicated by a ‘‘V’’. All sections were stained with hematoxylin and eosin stain. 40X original magnification

BALB/c mice were challenged on day 0 with 1 × 10⁶ PFU of 1918 HA/NA:Tx/91, 1918 HA/NA/NS:Tx/91 or Tx/91; 1.5 × 10³ TCID50 of A/Vn; or H1N1 or H5N1 vehicle control administered by the intranasal route. A minimum of six animals were included per challenge group. Values represent (a) mean change from baseline body weight (group averages of pre-challenge weights taken on study day 0), and (b) percent survival by group following challenge

BALB/c mice were challenged on day 0 with 1 × 10⁶ PFU of 1918 HA/NA:Tx/91, 1918 HA/NA/NS:Tx/91 or Tx/91; 1.5×10³ TCID50 PFU of A/Vn; or H1N1 or H5N1vehicle control administered by the intranasal route. Mice were euthanized on days 1, 3 and 5 post-challenge, lungs from groups of three mice were homogenized, and viral titers were determined by TCID50 assays. Three pooled samples were assayed per challenge group. Values represent mean TCID50 titers per mL (per lung) and the associated confidence intervals. The limit of detection for the assay was 131 TCID50/mL.

Animals were challenged on study day 0 via the intranasal route with a target dose of 5 × 10⁴ TCID50 of Tx /91, 1.5 × 10³ TCID50 of A/Vn, or H5N1vehicle control. Four days post-challenge, lungs were homogenized, and cytospin preparations of isolated cells were prepared. Slides were fixed in methanol prior to staining with DAPI (blue) and antiarginase (green). Results for (a) Tx/91, (b) A/Vn, (c) vehicle control, and a (d) naïve animal are shown along with (e) cells from a naïve animal stained with secondary antibody alone

BALB/c mice were challenged on day 0 with 1 × 10⁶ PFU of 1918 HA/NA:Tx/91, 1918 HA/NA/NS:Tx/91 or Tx/91; 1.5 × 10³ TCID50 of A/Vn; or H1N1 or H5N1 vehicle control administered by the intranasal route. Mice were euthanized on days 1, 3 and 5 postchallenge, and the lungs and blood were pooled from groups of three mice. Cytokine and chemokine levels were determined using cytometric bead arrays. Values represent concentrations (pg/mL) in (a) lung and (b) plasma as determined for 2–3 pooled samples for each challenge group

Microarray analysis of whole lung tissue collected from animals on days 1, 3 and 5 following challenge with H1N1 or H5N1vehicle control; 1 × 10⁶ PFU of 1918 HA/NA:Tx/91, 1918 HA/NA/ NS:Tx/91 or Tx /91; or 1.5 × 10³ TCID50 of A/Vn. The plots illustrate the number of significant genes as compared to matched vehicle controls within selected significant differentially expressed IPAdefined (a) biological function pathways and (b) canonical pathways as determined by an ANOVA with unequal variance and p<0.01, Tukey HSD posthoc test and Benjamini Hochberg FDR correction and > a twofold change

BALB/c mice were challenged on day 0 with 1 × 10⁶ PFU of 1918 HA/NA:Tx/91, 1918 HA/NA/NS:Tx/91 or Tx/91; 1.5 × 10³ TCID50 of A/Vn; or H1N1 or H5N1vehicle control administered by the intranasal route. Mice were euthanized on days 1, 3 and 5 postchallenge, and the bone marrow was pooled from groups of three mice. Immune-cell populations were identified using a gating strategy that employed a combination of DNA content, forward plus side scatter, and labeling. Immunophenotyping assay results are reported as mean percent values with the associated confidence interval for (a) CD45+ cells (leukocytes) and (b) CD45+CD11b+Gr-1+ cells (granulocytes)

BALB/c mice were challenged on day 0 with 1 × 10⁶ PFU of 1918 HA/NA:Tx/91, 1918 HA/NA/NS:Tx/91 or Tx/91; 1.5 × 10³ TCID50 of A/Vn; or H1N1 or H5N1 vehicle control administered by the intranasal route. Mice were euthanized on days 1, 3 and 5 postchallenge, and the lungs and blood were pooled from groups of three mice. Immune-cell populations were identified using a gating strategy that employed a combination of DNA content, forward plus side scatter, and labeling. (a) For peripheral blood, immunophenotyping assay results were converted to actual cell numbers by multiplying values obtained using an Advia clinical hematology analyzer for total white blood cells by the percentages for CD45+ cells (leukocytes) and CD45+CD11b+Gr-1+ cells (granulocytes). Likewise, T-cell subsets were determined by multiplying Advia clinical hematology analyzer values for lymphocytes with the percentage of CD45+CD3+ cells (T cells), CD45+CD3+CD4+CD8- (T_H cells), and CD45+CD3+CD4-CD8+ (T_C cells) (respectively). (b) For lung, immunophenotyping assay results for the same populations are reported as mean percent values with the associated confidence interval