Effects of ultrafine particles-induced oxidative stress on Clara cells in allergic lung inflammation.
Study subjects
Balb/c mouse bronchoalveolar lavage fluid
Five to seven-week-old Balb/c mice (Charles River, Sulzfeld, Germany); OVA/alum sensitized groups had OVA-specific IgE 24.2 +/- 1.8 vs 0.1 +/- 0.01 ug/ml and OVA-specific IgG1 1392.6 +/- 182.6 vs <0.1 ug/ml compared to non-sensitized controls; data as mean +/- SD. Group sizes differ per endpoint and are stated in each assay description - BALF and serum CC16 n = 4-7 per time point (Fig. 2), BALF total protein n = 4-8 per time point (Fig. 3), lung mRNA n = 3-6 per group (Fig. 4) - so no single n is carried on this shared subject record
Balb/c mouse serum
Five to seven-week-old Balb/c mice (Charles River, Sulzfeld, Germany); OVA/alum sensitized groups had OVA-specific IgE 24.2 +/- 1.8 vs 0.1 +/- 0.01 ug/ml and OVA-specific IgG1 1392.6 +/- 182.6 vs <0.1 ug/ml compared to non-sensitized controls; data as mean +/- SD. Group sizes differ per endpoint and are stated in each assay description - BALF and serum CC16 n = 4-7 per time point (Fig. 2), BALF total protein n = 4-8 per time point (Fig. 3), lung mRNA n = 3-6 per group (Fig. 4) - so no single n is carried on this shared subject record
Balb/c mouse whole lung homogenate
Five to seven-week-old Balb/c mice (Charles River, Sulzfeld, Germany); OVA/alum sensitized groups had OVA-specific IgE 24.2 +/- 1.8 vs 0.1 +/- 0.01 ug/ml and OVA-specific IgG1 1392.6 +/- 182.6 vs <0.1 ug/ml compared to non-sensitized controls; data as mean +/- SD. Group sizes differ per endpoint and are stated in each assay description - BALF and serum CC16 n = 4-7 per time point (Fig. 2), BALF total protein n = 4-8 per time point (Fig. 3), lung mRNA n = 3-6 per group (Fig. 4) - so no single n is carried on this shared subject record
Balb/c mouse lung histological sections
Five to seven-week-old Balb/c mice (Charles River, Sulzfeld, Germany); lungs retrieved 7 days after OVA challenge / EC-UFP inhalation, n = 6-12 per group, data as mean +/- SD
Balb/c mouse bronchiolar epithelium for electron microscopy
Five to seven-week-old Balb/c mice (Charles River, Sulzfeld, Germany); n = 2 per time point per group for electron microscopy; analysis focussed on the small airways (circumference <= 800 um) at the one week time point
Unrestrained Balb/c mouse in whole body plethysmograph
Five to seven-week-old Balb/c mice (Charles River, Sulzfeld, Germany); unrestrained, measured 24 h after OVA challenge, n = 5-7 per group
BALF sputum assays
BALF total protein concentration - EC-UFP inhalation before allergen challenge, with and without NAC
Figure 3, Coomassie protein assay on cell-free BALF, mean +/- SD, n = 4-8 per time point, one-way ANOVA with post-hoc Scheffe test, at 0, 1 and 7 days. OVA challenge alone raised BALF total protein above the untreated baseline and NAC significantly reduced that rise. EC-UFP inhalation before OVA challenge produced a further increase at the 7 day time point (S/UFP/OVA), which NAC significantly reduced. The non- challenged groups (NS/UFP, S/UFP, with and without NAC) showed no induction of protein levels at all, so the particle effect on barrier leakage required the allergic challenge. Values are plotted only, so no numeric measurements are recorded here.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Untreated baseline control - no sensitization, no EC-UFP inhalation, no OVA challenge (n = 8) EXPOSURE:alessandrini-untreated |
— | — | — |
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| Non sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (NS/NAC/UFP) EXPOSURE:alessandrini-ns-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice exposed to EC-UFP, 507 ug/m3 for 24 h (S/UFP) EXPOSURE:alessandrini-s-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (S/NAC/UFP) EXPOSURE:alessandrini-s-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized and OVA-challenged mice treated with NAC, exposed to filtered air (S/NAC/OVA) EXPOSURE:alessandrini-s-nac-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| baseline | BALF total protein - untreated baseline BALF:alessandrini-total-protein-output-untreated · under EXPOSURE:alessandrini-untreated |
|
| treated | BALF total protein - NS/UFP BALF:alessandrini-total-protein-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| treated | BALF total protein - NS/NAC/UFP BALF:alessandrini-total-protein-output-ns-nac-ufp · under EXPOSURE:alessandrini-ns-nac-ufp |
|
| treated | BALF total protein - S/UFP BALF:alessandrini-total-protein-output-s-ufp · under EXPOSURE:alessandrini-s-ufp |
|
| treated | BALF total protein - S/NAC/UFP BALF:alessandrini-total-protein-output-s-nac-ufp · under EXPOSURE:alessandrini-s-nac-ufp |
|
| vehicle_control | BALF total protein - S/OVA BALF:alessandrini-total-protein-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | BALF total protein - S/NAC/OVA BALF:alessandrini-total-protein-output-s-nac-ova · under EXPOSURE:alessandrini-s-nac-ova |
|
| treated | BALF total protein - S/UFP/OVA BALF:alessandrini-total-protein-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | BALF total protein - S/NAC/UFP/OVA BALF:alessandrini-total-protein-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
In sensitized mice, OVA challenge alone caused an increase in BALF total protein concentration compared to control, which was significantly reduced by NAC treatment (Fig. 3b).
Supports the S/OVA vs untreated difference and the NAC effect in S/NAC/OVA that this assay's output records stand for.
A further increase of BALF total protein was detected in sensitized mice exposed to EC-UFP inhalation prior to OVA challenge (S/UFP/OVA) at the 7 days time point (Fig. 3b).
The particle-plus-allergen increase in BALF total protein recorded by the S/UFP/OVA output record.
NAC significantly reduced BALF total protein concentrations (S/NAC/UFP/OVA) at the 7 days time point.
Supports the reversal recorded by the S/NAC/UFP/OVA output record and the corresponding response comparison.
No induction of protein levels were observed in any of the non-challenged groups (NS/UFP, S/UFP, with and without NAC treatment, Fig. 3a).
The explicit null result carried by the four non-challenged output records of this assay.
Untreated mice served as baseline controls (dotted line). Data presented as mean ± SD (n = 4-8/time point).
Fixes the baseline group, the central tendency and the group sizes stated for this figure.
BAL inflammatory infiltrate and BALF cytokines IL-5, IL-13 and IFN-gamma - EC-UFP inhalation before allergen challenge, with and without NAC
Differential BAL cell count and two site ELISAs for IL-5, IL-13 and IFN- gamma, reported in the paper's additional file 1 (functional characterization of the mouse model) rather than in a main figure, mean +/- SD, one-way ANOVA with post-hoc Scheffe test. EC-UFP inhalation before OVA challenge significantly increased the BAL inflammatory infiltrate and BALF IL-5 and IL-13 at 7 days compared with the filtered- air allergic control, and NAC significantly reduced that augmentation; NAC reduced inflammation in S/OVA only non-significantly. Non sensitized mice exposed to EC-UFP developed no inflammatory infiltrate and no cytokine release at all, and IFN-gamma was unchanged in every group. IL-5 and IL-13 have no named measurement slot in this schema and no values are tabulated in the fetched text, so no numeric measurements are recorded here.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Untreated baseline control - no sensitization, no EC-UFP inhalation, no OVA challenge (n = 8) EXPOSURE:alessandrini-untreated |
— | — | — |
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice exposed to EC-UFP, 507 ug/m3 for 24 h (S/UFP) EXPOSURE:alessandrini-s-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized and OVA-challenged mice treated with NAC, exposed to filtered air (S/NAC/OVA) EXPOSURE:alessandrini-s-nac-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| baseline | BAL inflammatory infiltrate and cytokines - untreated baseline BALF:alessandrini-inflammation-output-untreated · under EXPOSURE:alessandrini-untreated |
|
| treated | BAL inflammatory infiltrate and cytokines - NS/UFP BALF:alessandrini-inflammation-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| treated | BAL inflammatory infiltrate and cytokines - S/UFP BALF:alessandrini-inflammation-output-s-ufp · under EXPOSURE:alessandrini-s-ufp |
|
| vehicle_control | BAL inflammatory infiltrate and cytokines - S/OVA BALF:alessandrini-inflammation-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | BAL inflammatory infiltrate and cytokines - S/NAC/OVA BALF:alessandrini-inflammation-output-s-nac-ova · under EXPOSURE:alessandrini-s-nac-ova |
|
| treated | BAL inflammatory infiltrate and cytokines - S/UFP/OVA BALF:alessandrini-inflammation-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | BAL inflammatory infiltrate and cytokines - S/NAC/UFP/OVA BALF:alessandrini-inflammation-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
In sensitized mice, EC-UFP inhalation prior to OVA challenge (S/UFP/OVA) caused a significant increase of BAL inflammatory infiltrate and BALF cytokines IL-5 and IL-13 7 days after OVA challenge, compared with sensitized and challenged mice exposed to filtered air (S/OVA); NAC treatment significantly reduced EC-UFP-induced augmentation of lung allergic inflammation (S/NAC/UFP/OVA)
Carries both the S/UFP/OVA increase and the S/NAC/UFP/OVA reversal that the treated output records of this assay stand for.
Non sensitized mice exposed to EC-UFP did not develop an inflammatory infiltrate and showed no cytokine release (NS/UFP, see additional file 1: functional characterization of the mouse model).
The null result recorded by the NS/UFP output record: particles alone did not inflame a non-allergic lung.
No significant alterations in the IFN-γ concentration were observed in any of the groups
Supports the unchanged IFN-gamma noted in this assay's description across every output record.
Differential BAL cell count (400 cells/sample) was performed on cytospins (600 rpm for 10 min) fixed and stained with Diff-Quick
Documents how the inflammatory infiltrate behind these output records was counted.
Gene expression assays
CC16 protein concentration in BALF - EC-UFP inhalation before allergen challenge, with and without NAC
Figure 2a and 2b, upper panels: CC16 (Clara cell protein, the main secretory product of bronchiolar Clara cells) measured by automated latex immunoassay in cell-free BALF at 0, 1 and 7 days, mean +/- SD, n = 4-7 per time point, one-way ANOVA with Least Significant Difference test. EC-UFP for 24 h significantly DECREASED BALF CC16 at the 1-day time point in both non sensitized and sensitized mice, and NAC abrogated that decrease; by one week BALF CC16 had almost returned to the control level. In sensitized mice EC-UFP before allergen challenge gave a biphasic response - an initial significant reduction followed by a significant INCREASE at 7 days above untreated, NS/UFP, S/UFP and S/OVA - which NAC significantly reduced. OVA challenge alone caused a transitional reduction at 1 day that NAC only minimally reduced. Values are plotted only, so no numeric measurements are recorded here; the direction of each effect is carried in response_comparisons.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Untreated baseline control - no sensitization, no EC-UFP inhalation, no OVA challenge (n = 8) EXPOSURE:alessandrini-untreated |
— | — | — |
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| Non sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (NS/NAC/UFP) EXPOSURE:alessandrini-ns-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice exposed to EC-UFP, 507 ug/m3 for 24 h (S/UFP) EXPOSURE:alessandrini-s-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (S/NAC/UFP) EXPOSURE:alessandrini-s-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized and OVA-challenged mice treated with NAC, exposed to filtered air (S/NAC/OVA) EXPOSURE:alessandrini-s-nac-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| baseline | BALF CC16 - untreated baseline GE:alessandrini-cc16-protein-balf-output-untreated · under EXPOSURE:alessandrini-untreated |
|
| treated | BALF CC16 - NS/UFP GE:alessandrini-cc16-protein-balf-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| treated | BALF CC16 - NS/NAC/UFP GE:alessandrini-cc16-protein-balf-output-ns-nac-ufp · under EXPOSURE:alessandrini-ns-nac-ufp |
|
| treated | BALF CC16 - S/UFP GE:alessandrini-cc16-protein-balf-output-s-ufp · under EXPOSURE:alessandrini-s-ufp |
|
| treated | BALF CC16 - S/NAC/UFP GE:alessandrini-cc16-protein-balf-output-s-nac-ufp · under EXPOSURE:alessandrini-s-nac-ufp |
|
| vehicle_control | BALF CC16 - S/OVA GE:alessandrini-cc16-protein-balf-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | BALF CC16 - S/NAC/OVA GE:alessandrini-cc16-protein-balf-output-s-nac-ova · under EXPOSURE:alessandrini-s-nac-ova |
|
| treated | BALF CC16 - S/UFP/OVA GE:alessandrini-cc16-protein-balf-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | BALF CC16 - S/NAC/UFP/OVA GE:alessandrini-cc16-protein-balf-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
Non sensitized and sensitized mice exposed to EC-UFP for 24 h (NS/UFP, S/UFP) showed a significant decrease in CC16 in BALF at 1-day time point (Fig. 2a, upper panel).
The EC-UFP-alone decrease in BALF CC16 recorded by the NS/UFP and S/UFP output records.
EC-UFP-induced decrease in BALF CC16 concentrations were abrogated by NAC treatment (NS/NAC/UFP; S/NAC/UFP).
The NAC reversal recorded by the NS/NAC/UFP and S/NAC/UFP output records - the paper's basis for an oxidative-stress mechanism.
One week after EC-UFP inhalation, BALF CC16 concentrations almost reached the control level.
Establishes that the particle-only BALF CC16 decrease was transient, which the assay description records.
Sensitized mice exposed to EC-UFP inhalation prior to allergen challenge showed an initial significant reduction in BALF CC16, followed by a significant increase in CC16 BALF content at the 7-day time point compared to untreated animals
The biphasic S/UFP/OVA response carried by that output record and by the two corresponding response comparisons.
NAC minimally reduced OVA-induced CC16 alterations (S/NAC/OVA).
Supports the weak NAC effect recorded by the S/NAC/OVA output record, in contrast to its strong effect in the particle-exposed groups.
Untreated mice served as baseline controls (dotted line). Data presented as mean ± SD (n = 4-7/time point). *p < 0.05, **p < 0.01 vs untreated;
Fixes the baseline group, central tendency, group sizes and significance thresholds for this figure.
CC16 protein concentration in serum - EC-UFP inhalation before allergen challenge, with and without NAC
Figure 2a and 2b, bottom panels: serum CC16 by automated latex immunoassay on blood taken from the retro orbital plexus before BAL, mean +/- SD, n = 4-7 per time point, one-way ANOVA with Least Significant Difference test. In contrast to the BALF response, serum CC16 INCREASED significantly after EC-UFP in both non sensitized and sensitized mice and was still raised one week after inhalation; in sensitized mice EC-UFP before allergen challenge increased serum CC16 further, and NAC significantly reduced that increase. NAC reduced serum CC16 in sensitized EC-UFP-exposed mice at the 7 day time point but had no effect in S/OVA. Because CC16 is a marker of alveolo-capillary barrier permeability, the opposite directions in BALF and serum are read by the authors as increased leakage across the lung/blood barrier, which is why this assay informs on the epithelial barrier key event. Values are plotted only, so no numeric measurements are recorded here.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Untreated baseline control - no sensitization, no EC-UFP inhalation, no OVA challenge (n = 8) EXPOSURE:alessandrini-untreated |
— | — | — |
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| Non sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (NS/NAC/UFP) EXPOSURE:alessandrini-ns-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice exposed to EC-UFP, 507 ug/m3 for 24 h (S/UFP) EXPOSURE:alessandrini-s-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (S/NAC/UFP) EXPOSURE:alessandrini-s-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized and OVA-challenged mice treated with NAC, exposed to filtered air (S/NAC/OVA) EXPOSURE:alessandrini-s-nac-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| baseline | Serum CC16 - untreated baseline GE:alessandrini-cc16-protein-serum-output-untreated · under EXPOSURE:alessandrini-untreated |
|
| treated | Serum CC16 - NS/UFP GE:alessandrini-cc16-protein-serum-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| treated | Serum CC16 - NS/NAC/UFP GE:alessandrini-cc16-protein-serum-output-ns-nac-ufp · under EXPOSURE:alessandrini-ns-nac-ufp |
|
| treated | Serum CC16 - S/UFP GE:alessandrini-cc16-protein-serum-output-s-ufp · under EXPOSURE:alessandrini-s-ufp |
|
| treated | Serum CC16 - S/NAC/UFP GE:alessandrini-cc16-protein-serum-output-s-nac-ufp · under EXPOSURE:alessandrini-s-nac-ufp |
|
| vehicle_control | Serum CC16 - S/OVA GE:alessandrini-cc16-protein-serum-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | Serum CC16 - S/NAC/OVA GE:alessandrini-cc16-protein-serum-output-s-nac-ova · under EXPOSURE:alessandrini-s-nac-ova |
|
| treated | Serum CC16 - S/UFP/OVA GE:alessandrini-cc16-protein-serum-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | Serum CC16 - S/NAC/UFP/OVA GE:alessandrini-cc16-protein-serum-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
In contrast to BALF CC16 concentrations, serum CC16 concentrations increased significantly following exposure to EC-UFP in both non sensitized and sensitized mice (Fig. 2a, bottom panel).
The serum CC16 increase after EC-UFP recorded by the NS/UFP and S/UFP output records, and the BALF/serum divergence stated in the description.
In sensitized mice, inhalation of EC-UFP prior to OVA challenge caused most significant alterations of BALF and serum CC16 concentration, BALF total protein and TNF-α relative expression compared to relevant controls
Establishes S/UFP/OVA as the group with the largest serum CC16 alteration, which is what that output record stands for.
Our results showed that sensitized and challenged mice exposed to EC-UFP prior to allergen challenge showed a transient decrease, followed by a late increase of CC16 in BALF
The authors' own summary of the biphasic BALF course that the divergent serum course is contrasted with here.
BALF total protein concentration was also most increased in this group, confirming that the exudative response caused by the increased airway permeability plays an important role.
Ties the serum CC16 rise to increased airway permeability, the key event this assay is mapped to.
CC16 relative mRNA expression in whole lung homogenate - EC-UFP inhalation before allergen challenge, with and without NAC
Figure 4a and 4b, upper panels: TaqMan real-time RT-PCR for CC16 in lung homogenates retrieved 24 h after EC-UFP / filtered air exposure, normalised to 18S and to the untreated group by the delta-delta-CT method, mean +/- SD, n = 3-6 per group, one-way ANOVA with Least Significant Difference test. EC-UFP exposure alone INCREASED CC16 mRNA in both non sensitized and sensitized mice, which the authors read as increased intracellular storage of CC16 protein. Allergen challenge alone had no significant effect on CC16 mRNA. EC-UFP before allergen challenge raised CC16 mRNA similarly to NS/UFP and S/UFP. NAC moderately reduced CC16 mRNA in non challenged animals but FAILED to reduce the EC- UFP-induced increase in sensitized and challenged animals - the authors' reason for proposing post-transcriptional regulation of CC16 by NAC. Note that this assay is mapped to the club cell marker key event, whose canonical wording is a decrease; the direction actually observed here is an increase and is recorded in the response_comparisons. Values are plotted only, so no numeric measurements are recorded here.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Untreated baseline control - no sensitization, no EC-UFP inhalation, no OVA challenge (n = 8) EXPOSURE:alessandrini-untreated |
— | — | — |
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| Non sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (NS/NAC/UFP) EXPOSURE:alessandrini-ns-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice exposed to EC-UFP, 507 ug/m3 for 24 h (S/UFP) EXPOSURE:alessandrini-s-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized and OVA-challenged mice treated with NAC, exposed to filtered air (S/NAC/OVA) EXPOSURE:alessandrini-s-nac-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| baseline | Lung CC16 mRNA - untreated baseline GE:alessandrini-cc16-mrna-lung-output-untreated · under EXPOSURE:alessandrini-untreated |
|
| treated | Lung CC16 mRNA - NS/UFP GE:alessandrini-cc16-mrna-lung-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| treated | Lung CC16 mRNA - NS/NAC/UFP GE:alessandrini-cc16-mrna-lung-output-ns-nac-ufp · under EXPOSURE:alessandrini-ns-nac-ufp |
|
| treated | Lung CC16 mRNA - S/UFP GE:alessandrini-cc16-mrna-lung-output-s-ufp · under EXPOSURE:alessandrini-s-ufp |
|
| vehicle_control | Lung CC16 mRNA - S/OVA GE:alessandrini-cc16-mrna-lung-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | Lung CC16 mRNA - S/NAC/OVA GE:alessandrini-cc16-mrna-lung-output-s-nac-ova · under EXPOSURE:alessandrini-s-nac-ova |
|
| treated | Lung CC16 mRNA - S/UFP/OVA GE:alessandrini-cc16-mrna-lung-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | Lung CC16 mRNA - S/NAC/UFP/OVA GE:alessandrini-cc16-mrna-lung-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
In sensitized and non sensitized mice EC-UFP exposure alone induced an increase in CC16 and TNF-α relative mRNA expression compared to untreated mice (Fig. 4a, upper and bottom panel respectively).
The particle-only CC16 mRNA increase recorded by the NS/UFP and S/UFP output records.
alone had no significant effects on relative CC16 mRNA expression, but significantly increased TNF-α expression
The null CC16 mRNA result for allergen challenge alone, carried by the S/OVA output record.
prior to allergen challenge increased CC16 mRNA expression similarly to NS/UFP and S/UFP (S/UFP/OVA, Fig. 4b, upper panel), whereas it significantly increased TNF-α expression compared to all other groups
Records that CC16 mRNA in S/UFP/OVA rose no further than with particles alone, unlike TNF-alpha - the dissociation this assay pair documents.
animals NAC failed to reduce EC-UFP-induced increase in CC16 expression, but strongly reduced EC-UFP-induced increase in TNF-α mRNA expression
The NAC-resistant CC16 mRNA increase recorded by the S/NAC/UFP/OVA output record.
NAC moderately reduced CC16 and TNF-α mRNA expression in non challenged animals (NS/NAC/UFP, Fig. 4a, striped bars).
The partial NAC effect recorded by the NS/NAC/UFP output record.
implying that the regulation of CC16 by NAC may function at the post-transcriptional level.
The authors' interpretation of the NAC-resistant mRNA response, noted in this assay's description.
The lungs were retrieved 24 h after EC-UFP/filtered air exposure. Data presented as mean ± SD (n = 3-6/group).
Fixes the sampling time, central tendency and group sizes for the mRNA figure.
TNF-alpha relative mRNA expression in whole lung homogenate - EC-UFP inhalation before allergen challenge, with and without NAC
Figure 4a and 4b, bottom panels: TaqMan real-time RT-PCR for TNF-alpha in lung homogenates retrieved 24 h after EC-UFP / filtered air exposure, normalised to 18S and to the untreated group by the delta-delta-CT method, mean +/- SD, n = 3-6 per group, one-way ANOVA with Least Significant Difference test. EC-UFP alone increased TNF-alpha mRNA; allergen challenge alone also increased it significantly; and EC-UFP before allergen challenge increased it significantly compared with ALL other groups, which the authors describe as a synergistic effect of allergic inflammation and particle inhalation. Unlike CC16 mRNA, the TNF-alpha increase was strongly reduced by NAC - the clearest evidence in the paper that this arm is oxidative-stress dependent. TNF-alpha is relevant here because it is both pro-inflammatory and a known stimulator of CC16 production. Values are plotted only, so no numeric measurements are recorded here.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Untreated baseline control - no sensitization, no EC-UFP inhalation, no OVA challenge (n = 8) EXPOSURE:alessandrini-untreated |
— | — | — |
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| Non sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (NS/NAC/UFP) EXPOSURE:alessandrini-ns-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice exposed to EC-UFP, 507 ug/m3 for 24 h (S/UFP) EXPOSURE:alessandrini-s-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized and OVA-challenged mice treated with NAC, exposed to filtered air (S/NAC/OVA) EXPOSURE:alessandrini-s-nac-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| baseline | Lung TNF-alpha mRNA - untreated baseline GE:alessandrini-tnfa-mrna-lung-output-untreated · under EXPOSURE:alessandrini-untreated |
|
| treated | Lung TNF-alpha mRNA - NS/UFP GE:alessandrini-tnfa-mrna-lung-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| treated | Lung TNF-alpha mRNA - NS/NAC/UFP GE:alessandrini-tnfa-mrna-lung-output-ns-nac-ufp · under EXPOSURE:alessandrini-ns-nac-ufp |
|
| treated | Lung TNF-alpha mRNA - S/UFP GE:alessandrini-tnfa-mrna-lung-output-s-ufp · under EXPOSURE:alessandrini-s-ufp |
|
| vehicle_control | Lung TNF-alpha mRNA - S/OVA GE:alessandrini-tnfa-mrna-lung-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | Lung TNF-alpha mRNA - S/NAC/OVA GE:alessandrini-tnfa-mrna-lung-output-s-nac-ova · under EXPOSURE:alessandrini-s-nac-ova |
|
| treated | Lung TNF-alpha mRNA - S/UFP/OVA GE:alessandrini-tnfa-mrna-lung-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | Lung TNF-alpha mRNA - S/NAC/UFP/OVA GE:alessandrini-tnfa-mrna-lung-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
In sensitized and non sensitized mice EC-UFP exposure alone induced an increase in CC16 and TNF-α relative mRNA expression compared to untreated mice (Fig. 4a, upper and bottom panel respectively).
The particle-only TNF-alpha mRNA increase recorded by the NS/UFP and S/UFP output records.
prior to allergen challenge increased CC16 mRNA expression similarly to NS/UFP and S/UFP (S/UFP/OVA, Fig. 4b, upper panel), whereas it significantly increased TNF-α expression compared to all other groups
The S/UFP/OVA TNF-alpha increase above every other group, which that output record and its response comparison carry.
animals NAC failed to reduce EC-UFP-induced increase in CC16 expression, but strongly reduced EC-UFP-induced increase in TNF-α mRNA expression
The strong NAC reversal recorded by the S/NAC/UFP/OVA output record of this assay.
At the mRNA level, NAC failed to reduce EC-UFP-induced alterations of CC16, but reduced EC-UFP-induced TNF-α relative expression
The paper's own section heading stating the CC16 / TNF-alpha dissociation under NAC.
the antioxidant strategy reduced TNF-α mRNA expression in whole lung homogenate.
Confirms in the discussion that the antioxidant lowered lung TNF-alpha mRNA, the basis for the oxidative stress key event relationship.
The lungs were retrieved 24 h after EC-UFP/filtered air exposure. Data presented as mean ± SD (n = 3-6/group).
Fixes the sampling time, central tendency and group sizes for the mRNA figure.
Goblet cell assays
Volume density of PAS positive material in small airways (circumference <= 800 um) - EC-UFP inhalation before allergen challenge, with and without NAC
Figure 5a: morphometrical analysis of the volume density of PAS positive material in cross sections of small airways (circumference <= 800 um), 7 days after OVA challenge / EC-UFP inhalation, expressed as percentage of PAS positive material per total epithelial cells, n = 6-12 per group, one-way ANOVA with post-hoc Scheffe test. NS/UFP showed no PAS positive material at all. Allergen challenge alone (S/OVA) produced significant goblet cell metaplasia and mucus hypersecretion. EC-UFP before allergen challenge INCREASED goblet cell metaplasia and mucus hypersecretion in the small airways - the airway-size-specific finding of this paper, which the authors attribute to predominant ultrafine particle deposition in smaller airways. NAC significantly decreased it. Values are plotted only, so no numeric measurements are recorded here. NOTE a tension inside the paper itself: the results text calls the allergen-only (S/OVA) effect significant while citing both the small- and large-airway panels, whereas the discussion narrows it to significant only in the large airways. The S/OVA output record is kept here because the results text covers the small airways, but the discussion's narrower reading is quoted on the companion large-airway assay and should be read alongside this one.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| treated | PAS positive volume density, small airways - NS/UFP GCM:alessandrini-pas-small-airways-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| vehicle_control | PAS positive volume density, small airways - S/OVA GCM:alessandrini-pas-small-airways-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | PAS positive volume density, small airways - S/UFP/OVA GCM:alessandrini-pas-small-airways-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | PAS positive volume density, small airways - S/NAC/UFP/OVA GCM:alessandrini-pas-small-airways-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
Interestingly, sensitized and challenged mice exposed to EC-UFP prior to challenge showed increased goblet cell metaplasia induced by EC-UFP exposure only in the small airways, in contrast to large airways.
States the small-airway-specific increase that this assay's S/UFP/OVA output record carries.
In sensitized animals allergen challenge alone had a significant effect on goblet cell metaplasia and mucus hypersecretion (Fig. 5a, b, d).
Supports the S/OVA output record, the allergic control against which the particle effect is read.
NAC strongly reduced EC-UFP-induced mucus hypersecretion in allergic animals
The paper's section heading for the NAC reversal recorded by the S/NAC/UFP/OVA output record.
The data are expressed as percentage of PAS positive material/total epithelial cells.
Defines the quantity the output records of this assay refer to.
n = 6-12/group. **p < 0.01 vs NS/UFP; ##p < 0.01 vs S/UFP/OVA.
Fixes the group sizes and the two reference groups the figure's significance markers are against.
Volume density of PAS positive material in large airways (circumference > 800 um) - EC-UFP inhalation before allergen challenge, with and without NAC
Figure 5b: the same morphometrical analysis in cross sections of large airways (circumference > 800 um), 7 days after OVA challenge / EC-UFP inhalation, expressed as percentage of PAS positive material per total epithelial cells, n = 6-12 per group, one-way ANOVA with post-hoc Scheffe test. The allergen-driven metaplasia of Clara cells into mucous cells was significant in the large airways, but EC-UFP exposure before allergen challenge did NOT increase it further here - the mirror of the small-airway result. NAC significantly decreased goblet cell metaplasia and mucus hypersecretion in the large bronchi as well. Values are plotted only, so no numeric measurements are recorded here.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| treated | PAS positive volume density, large airways - NS/UFP GCM:alessandrini-pas-large-airways-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| vehicle_control | PAS positive volume density, large airways - S/OVA GCM:alessandrini-pas-large-airways-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | PAS positive volume density, large airways - S/UFP/OVA GCM:alessandrini-pas-large-airways-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | PAS positive volume density, large airways - S/NAC/UFP/OVA GCM:alessandrini-pas-large-airways-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
In the morphometrical analysis of PAS positive material we showed that this phenomenon was significant only in the large airways.
States that the allergen-driven mucous cell metaplasia reached significance in the large airways, which the S/OVA output record carries.
Interestingly, sensitized and challenged mice exposed to EC-UFP prior to challenge showed increased goblet cell metaplasia induced by EC-UFP exposure only in the small airways, in contrast to large airways.
The explicit null result for the large airways recorded by this assay's S/UFP/OVA output record.
NAC strongly reduced EC-UFP-induced mucus hypersecretion in allergic animals
The paper's section heading for the NAC reversal recorded by the S/NAC/UFP/OVA output record.
The data are expressed as percentage of PAS positive material/total epithelial cells.
Defines the quantity the output records of this assay refer to.
n = 6-12/group. **p < 0.01 vs NS/UFP; ##p < 0.01 vs S/UFP/OVA.
Fixes the group sizes and the two reference groups the figure's significance markers are against.
TEM/SEM goblet cell metaplasia and Clara cell ultrastructural damage - EC-UFP inhalation before allergen challenge, with and without NAC
Figures 6 and 7: transmission and scanning electron microscopy of the small airways at the one week time point, n = 2 per time point per group. This is a QUALITATIVE ultrastructural assessment, so the output records carry no numeric values at all. In untreated mice Clara cells were cuboidal with dense homogenous secretory granules near the apex and no goblet cells were detectable. In S/OVA the bronchioli featured goblet cell metaplasia: the dense secretory granules converted to mucus containing vesicles, with signs of partial necrosis. In S/UFP/OVA the damage was most profound - more pronounced goblet cell metaplasia than S/OVA, areas with no rudiments of secretory granules left, smooth endoplasmic reticulum and dimorphic mitochondria entirely displaced by mucous vesicles, widening of intercellular spaces, detachment of the epithelium and necrosis. NAC treatment extensively reduced these alterations: over broad ranges of bronchioli Clara cells displayed normal features and necrosis appeared to a strikingly lesser degree. No particles were ever detected inside Clara cells, so the authors read the damage as an indirect particle effect mediated by inflammatory mediators in the lung lining fluid.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Untreated baseline control - no sensitization, no EC-UFP inhalation, no OVA challenge (n = 8) EXPOSURE:alessandrini-untreated |
— | — | — |
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| Non sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (NS/NAC/UFP) EXPOSURE:alessandrini-ns-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice exposed to EC-UFP, 507 ug/m3 for 24 h (S/UFP) EXPOSURE:alessandrini-s-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (S/NAC/UFP) EXPOSURE:alessandrini-s-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| baseline | Bronchiolar ultrastructure - untreated baseline GCM:alessandrini-em-goblet-metaplasia-output-untreated · under EXPOSURE:alessandrini-untreated |
|
| treated | Bronchiolar ultrastructure - NS/UFP GCM:alessandrini-em-goblet-metaplasia-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| treated | Bronchiolar ultrastructure - NS/NAC/UFP GCM:alessandrini-em-goblet-metaplasia-output-ns-nac-ufp · under EXPOSURE:alessandrini-ns-nac-ufp |
|
| treated | Bronchiolar ultrastructure - S/UFP GCM:alessandrini-em-goblet-metaplasia-output-s-ufp · under EXPOSURE:alessandrini-s-ufp |
|
| treated | Bronchiolar ultrastructure - S/NAC/UFP GCM:alessandrini-em-goblet-metaplasia-output-s-nac-ufp · under EXPOSURE:alessandrini-s-nac-ufp |
|
| vehicle_control | Bronchiolar ultrastructure - S/OVA GCM:alessandrini-em-goblet-metaplasia-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | Bronchiolar ultrastructure - S/UFP/OVA GCM:alessandrini-em-goblet-metaplasia-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | Bronchiolar ultrastructure - S/NAC/UFP/OVA GCM:alessandrini-em-goblet-metaplasia-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
In S/OVA mice, the bronchioli featured goblet cell metaplasia: the dense homogenous secretory granules of the Clara cells converted to mucus containing vesicles, some of them containing dark residues of the secret
The allergen-driven metaplasia recorded by the S/OVA output record of this assay.
Electron miscroscopy shows most dramatic alterations of Clara cell ultrastructure in this group, characterized by extreme hyperplasia, metaplasia with complete loss of secretory granules and necrosis.
The authors' summary of the S/UFP/OVA ultrastructure, which that output record stands for (the typo 'miscroscopy' is as printed).
In some areas no rudiments of the secretory granules could be detected
The complete secretory granule loss in S/UFP/OVA that distinguishes it from the allergen-only group.
Widening of the intercellular spaces and detachment of the epithelium were frequently observed (Fig. 6, S/UFP/OVA, arrows).
The epithelial detachment in S/UFP/OVA, the ultrastructural counterpart of the raised BALF total protein in the same group.
Over broad ranges of bronchioli Clara cells displayed normal features. Necrosis appeared to a striking less degree.
The NAC rescue recorded by the S/NAC/UFP/OVA output record.
Following EC-UFP exposure, no particles could be detected within Clara cells. EC-UFP inhalation alone induced alterations in the morphology of Clara cells.
Establishes that the Clara cell changes were not caused by particles inside the cells, as noted in this assay's description.
In the analysis of the samples performed by SEM and TEM, we focussed on the small airways (circumference ≤ 800 μm), one week time point.
Fixes the airway segment and time point these output records refer to.
TEM/SEM Clara cell hyperplasia after EC-UFP inhalation alone, with and without NAC
Figures 6 and 7, the non-challenged arm: EC-UFP inhalation alone induced hyperplasia and proliferation of the Clara cells of the bronchioli, so that ciliated cells seemed reduced in number and appeared constricted, and in sensitized non-challenged mice clusters of 3 or 4 Clara cells formed. Crucially this happened WITHOUT goblet cell metaplasia, which the authors call surprising for such relatively inert particles - it is the finding that separates the particle-only response from the particle- plus-allergen response recorded in the companion EM assay. NAC reduced these morphological alterations to a great extent: no clusters were found and the Clara cell surface displayed the typical dome-shaped apical projections as in untreated mice. Qualitative ultrastructural assessment, n = 2 per time point per group, so no numeric values are recorded.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Untreated baseline control - no sensitization, no EC-UFP inhalation, no OVA challenge (n = 8) EXPOSURE:alessandrini-untreated |
— | — | — |
| Non sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h (NS/UFP) EXPOSURE:alessandrini-ns-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| Non sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (NS/NAC/UFP) EXPOSURE:alessandrini-ns-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice exposed to EC-UFP, 507 ug/m3 for 24 h (S/UFP) EXPOSURE:alessandrini-s-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized, non challenged mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h (S/NAC/UFP) EXPOSURE:alessandrini-s-nac-ufp |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| baseline | Clara cell hyperplasia - untreated baseline GCM:alessandrini-em-clara-hyperplasia-output-untreated · under EXPOSURE:alessandrini-untreated |
|
| treated | Clara cell hyperplasia - NS/UFP GCM:alessandrini-em-clara-hyperplasia-output-ns-ufp · under EXPOSURE:alessandrini-ns-ufp |
|
| treated | Clara cell hyperplasia - NS/NAC/UFP GCM:alessandrini-em-clara-hyperplasia-output-ns-nac-ufp · under EXPOSURE:alessandrini-ns-nac-ufp |
|
| treated | Clara cell hyperplasia - S/UFP GCM:alessandrini-em-clara-hyperplasia-output-s-ufp · under EXPOSURE:alessandrini-s-ufp |
|
| treated | Clara cell hyperplasia - S/NAC/UFP GCM:alessandrini-em-clara-hyperplasia-output-s-nac-ufp · under EXPOSURE:alessandrini-s-nac-ufp |
Evidence
In TEM as well as in SEM the Clara cells of the bronchioli exhibited hyperplasia and proliferation
The hyperplasia and proliferation after EC-UFP alone that the NS/UFP and S/UFP output records carry.
Surprisingly, we showed hyperplasia of Clara cells (but no goblet cell metaplasia) following inhalation of relatively inert particles, such as EC-UFP
States that the particle-only response was hyperplasia WITHOUT metaplasia, the distinction this assay exists to record.
In non sensitized mice EC-UFP inhalation caused alterations in CC16 concentration, both at protein and mRNA level, and induced Clara cell hyperplasia.
The abstract's statement of Clara cell hyperplasia in non sensitized mice, matching the NS/UFP output record.
NAC strongly reduced EC-UFP-induced morphological alterations in Clara Cells
The paper's section heading for the NAC reversal recorded by the NS/NAC/UFP and S/NAC/UFP output records.
Lung function assays
Airway hyperresponsiveness to methacholine (enhanced pause, PenH) - EC-UFP inhalation before allergen challenge, with and without NAC
Unrestrained whole body plethysmography 24 h after allergen challenge, n = 5-7 per group, enhanced pause (PenH) before and after five methacholine levels (10 mg/ml for 1, 2 and 4 min, then 40 mg/ml for 2 and 4 min) as the index of airway hyperresponsiveness, one-way ANOVA with Least Significant Difference test. PenH rose significantly with increasing methacholine concentration in sensitized mice exposed to EC- UFP before OVA challenge compared with untreated mice, and the increase over the filtered-air allergic control was close to significant. NAC reduced the EC-UFP-dependent increase in airway hyperresponsiveness. The PenH traces live in the paper's additional file 1 rather than a main figure and no values are tabulated in the fetched text, so no numeric measurements are recorded here; PenH also has no named measurement slot in this schema.
Exposure conditions
| Condition | Agent | Concentration | Duration |
|---|---|---|---|
| Untreated baseline control - no sensitization, no EC-UFP inhalation, no OVA challenge (n = 8) EXPOSURE:alessandrini-untreated |
— | — | — |
| OVA-sensitized and OVA-challenged mice exposed to filtered air (S/OVA) EXPOSURE:alessandrini-s-ova |
— | — | — |
| OVA-sensitized mice exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/UFP/OVA) EXPOSURE:alessandrini-s-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
| OVA-sensitized mice treated with NAC and exposed to EC-UFP, 507 ug/m3 for 24 h, 24 h prior to OVA challenge (S/NAC/UFP/OVA) EXPOSURE:alessandrini-s-nac-ufp-ova |
ultrafine respirable suspended particulate matter ENVO:01000416 |
507 microgram per cubic meter | 24 hour |
Outputs
| Group | Output | Description |
|---|---|---|
| baseline | PenH airway hyperresponsiveness - untreated baseline LF:alessandrini-penh-methacholine-output-untreated · under EXPOSURE:alessandrini-untreated |
|
| vehicle_control | PenH airway hyperresponsiveness - S/OVA LF:alessandrini-penh-methacholine-output-s-ova · under EXPOSURE:alessandrini-s-ova |
|
| treated | PenH airway hyperresponsiveness - S/UFP/OVA LF:alessandrini-penh-methacholine-output-s-ufp-ova · under EXPOSURE:alessandrini-s-ufp-ova |
|
| treated | PenH airway hyperresponsiveness - S/NAC/UFP/OVA LF:alessandrini-penh-methacholine-output-s-nac-ufp-ova · under EXPOSURE:alessandrini-s-nac-ufp-ova |
Evidence
NAC reduced EC-UFP-induced airway hyperresponsiveness
The paper's own results heading for this endpoint: particles raised airway hyperresponsiveness and the antioxidant lowered it, which is what the S/UFP/OVA and S/NAC/UFP/OVA output records carry.
For whole body plethysmography, the 'enhanced pause' (PenH) determined before and after methacholine exposure was applied as an index of airway hyperresponsiveness (AHR), as previously described
Defines the quantity these output records refer to and why the assay is mapped to the decreased-lung-function adverse outcome.
Lung function tests were performed 24 h after allergen challenge in unrestrained animals using whole body plethysmography
Fixes the timing and the unrestrained plethysmography method behind these output records.
NAC had minor effects on lung CC16 concentrations of sensitized and challenged mice maybe because our model reproduces a mild allergic inflammation of the lung, as shown by the data on inflammatory cells, cytokines and airway hyperresponsiveness
The authors' caveat that this model is a mild allergic inflammation, which bounds how much the airway hyperresponsiveness result can carry.
Response comparisons
BALF CC16, NS/UFP vs untreated baseline - decreased
Figure 2a upper panel. EC-UFP inhalation alone for 24 h significantly decreased BALF CC16 at the 1-day time point in non sensitized mice. Marked p < 0.05 or p < 0.01 vs untreated off the plot; no magnitude is stated, so none is recorded.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
BALF CC16, S/UFP vs untreated baseline - decreased
Figure 2a upper panel. The same significant 1-day decrease in BALF CC16 occurred in sensitized but non-challenged mice, so allergic sensitization was not required for it.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
BALF CC16, NS/NAC/UFP vs untreated baseline - no change
Figure 2a upper panel. NAC abrogated the EC-UFP-induced BALF CC16 decrease, so the NAC-treated particle-exposed group no longer differed from the untreated baseline. This NAC reversibility is the paper's evidence that the CC16 response is oxidative-stress driven.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
BALF CC16 at 7 days, S/UFP/OVA vs untreated baseline - increased
Figure 2b upper panel. In sensitized mice, EC-UFP before allergen challenge gave a biphasic course: an initial significant reduction and then a significant INCREASE in BALF CC16 at the 7-day time point above untreated animals, NS/UFP, S/UFP and S/OVA. The record here is for the late increase; the early reduction is stated in the same sentence and in the assay description.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
Serum CC16, NS/UFP vs untreated baseline - increased
Figure 2a bottom panel. Serum CC16 increased significantly after EC-UFP inhalation in non sensitized mice and was still detectable one week later - the opposite direction to BALF CC16 in the same animals, which the authors read as leakage across the air/blood barrier.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
Serum CC16, S/UFP/OVA vs untreated baseline - increased
Figure 2b bottom panel. S/UFP/OVA showed the most significant serum CC16 alteration of any group; NAC significantly reduced it (S/NAC/UFP/OVA).
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
BALF total protein, S/OVA vs untreated baseline - increased
Figure 3b. Allergen challenge alone raised BALF total protein above the untreated baseline; NAC significantly reduced that rise (S/NAC/OVA).
Statistical test: One-way ANOVA with post-hoc Scheffe test
BALF total protein at 7 days, S/UFP/OVA vs S/OVA - increased
Figure 3b. EC-UFP inhalation before OVA challenge produced a further increase of BALF total protein over the filtered-air allergic control at the 7 days time point - the exudative response the authors attribute to increased airway permeability.
Statistical test: One-way ANOVA with post-hoc Scheffe test
BALF total protein at 7 days, S/NAC/UFP/OVA vs S/UFP/OVA - decreased
Figure 3b. NAC significantly reduced BALF total protein concentrations at the 7 days time point, so the particle-driven barrier leakage is oxidative-stress dependent.
Statistical test: One-way ANOVA with post-hoc Scheffe test
BALF total protein, NS/UFP vs untreated baseline - no change
Figure 3a. NULL RESULT: no induction of protein levels was observed in any of the non-challenged groups, with or without NAC. EC-UFP alone did not open the lung/blood barrier - the allergic challenge was required.
Statistical test: One-way ANOVA with post-hoc Scheffe test
Lung CC16 mRNA, NS/UFP vs untreated baseline - increased
Figure 4a upper panel. EC-UFP exposure alone increased CC16 relative mRNA expression in non sensitized mice. Note the direction: the BALF CC16 protein fell at the same time point while its mRNA rose, which the authors interpret as increased intracellular storage of CC16 protein.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
Lung CC16 mRNA, S/OVA vs untreated baseline - no change
Figure 4b upper panel. NULL RESULT: allergen challenge alone had no significant effect on relative CC16 mRNA expression, although it did significantly increase TNF-alpha in the same animals.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
Lung CC16 mRNA, S/NAC/UFP/OVA vs S/UFP/OVA - no change
Figure 4b upper panel. NULL RESULT for the antioxidant: in sensitized and challenged animals NAC FAILED to reduce the EC-UFP-induced increase in CC16 mRNA, in contrast to its strong effect on TNF-alpha mRNA in the very same samples. This dissociation is why the authors propose post- transcriptional regulation of CC16 by NAC.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
Lung TNF-alpha mRNA, NS/UFP vs untreated baseline - increased
Figure 4a bottom panel. EC-UFP exposure alone increased TNF-alpha relative mRNA expression in non sensitized mice, i.e. the particles engaged a pro-inflammatory transcriptional response even without allergen.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
Lung TNF-alpha mRNA, S/UFP/OVA vs S/OVA - increased
Figure 4b bottom panel. EC-UFP before allergen challenge increased TNF- alpha mRNA significantly compared with ALL other groups, including the filtered-air allergic control - the synergistic effect of allergic inflammation and particle inhalation on lung TNF-alpha that the discussion describes.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
Lung TNF-alpha mRNA, S/NAC/UFP/OVA vs S/UFP/OVA - decreased
Figure 4b bottom panel. NAC strongly reduced the EC-UFP-induced increase in TNF-alpha mRNA expression. Paired with the unchanged CC16 mRNA under NAC, this is the cleanest oxidative-stress-dependent endpoint in the paper.
Statistical test: One-way ANOVA with Least Significant Difference (LSD) test
PAS positive volume density in small airways, S/OVA vs NS/UFP - increased
Figure 5a. Allergen challenge alone had a significant effect on goblet cell metaplasia and mucus hypersecretion, against an NS/UFP group that showed no PAS positive material at all. NS/UFP is the reference group the figure's significance markers are against, which is why it is the control output here.
Statistical test: One-way ANOVA with post-hoc Scheffe test
PAS positive volume density in small airways, S/UFP/OVA vs S/OVA - increased
Figure 5a. EC-UFP exposure before allergen challenge increased goblet cell metaplasia and mucus hypersecretion in the SMALL airways over the filtered-air allergic control. The authors explain the airway-size specificity by predominant ultrafine particle deposition in smaller airways.
Statistical test: One-way ANOVA with post-hoc Scheffe test
PAS positive volume density in small airways, S/NAC/UFP/OVA vs S/UFP/OVA - decreased
Figure 5a. NAC treatment prior to and during EC-UFP inhalation significantly decreased goblet cell metaplasia and mucus hypersecretion in the small airways, marked ## p < 0.01 vs S/UFP/OVA.
Statistical test: One-way ANOVA with post-hoc Scheffe test
PAS positive volume density in large airways, S/UFP/OVA vs S/OVA - no change
Figure 5b. NULL RESULT and the mirror of the small-airway finding: the EC-UFP-induced increase in goblet cell metaplasia occurred only in the small airways, in contrast to the large airways, where the allergen- driven metaplasia was already significant on its own.
Statistical test: One-way ANOVA with post-hoc Scheffe test
BAL inflammatory infiltrate, S/UFP/OVA vs S/OVA - increased
Additional file 1. EC-UFP inhalation before OVA challenge significantly increased the BAL inflammatory infiltrate (and BALF IL-5 and IL-13) 7 days after challenge, compared with sensitized and challenged mice exposed to filtered air.
Statistical test: One-way ANOVA with post-hoc Scheffe test
BAL inflammatory infiltrate, S/NAC/UFP/OVA vs S/UFP/OVA - decreased
Additional file 1. NAC treatment significantly reduced the EC-UFP- induced augmentation of lung allergic inflammation; by contrast NAC reduced inflammation in S/OVA only non-significantly, so the antioxidant acted on the particle-driven component.
Statistical test: One-way ANOVA with post-hoc Scheffe test
BAL inflammatory infiltrate, NS/UFP vs untreated baseline - no change
Additional file 1. NULL RESULT: non sensitized mice exposed to EC-UFP did not develop an inflammatory infiltrate and showed no cytokine release. The particles only aggravated inflammation in an already allergic lung.
Statistical test: One-way ANOVA with post-hoc Scheffe test
Key-event relationships
EC-UFP-induced oxidative stress leads to mucus secretory program activation in the small airways
The antioxidant NAC, administered only before and during the EC-UFP exposure, limited goblet cell metaplasia and protected Clara cell ultrastructure, and the PAS morphometry showed the same reversal in the small airways. Support is moderate rather than strong because no oxidative stress marker was measured in this study - the upstream event is inferred from NAC reversibility - and quantitative understanding is low because the endpoints are plotted volume densities and qualitative ultrastructure with no dose-response.
We show that oxidative stress plays an important role, since NAC treatment is able to limit goblet cell metaplasia and protect Clara cell ultrastructure.
The authors' own statement of this upstream-to-downstream link, resting on the NAC reversal of both metaplasia and ultrastructural damage.
NAC strongly reduced EC-UFP-induced mucus hypersecretion in allergic animals
The results heading for the morphometric half of the same reversal, which the PAS assays record group by group.
EC-UFP-induced oxidative stress leads to loss of the club (Clara) cell marker CC16
The paper's central claim. EC-UFP inhalation altered CC16 at both protein and mRNA level and damaged Clara cell morphology, and NAC strongly reduced both the functional and the morphological alterations. Support is moderate, not strong, for two reasons: no oxidative stress marker was measured here, and the antioxidant did NOT reverse every arm - NAC failed to reduce the EC-UFP-induced CC16 mRNA increase in sensitized and challenged animals, which the authors read as post- transcriptional regulation. Quantitative understanding is low: all CC16 values are plotted.
Our findings demonstrate that oxidative stress plays an important role in EC-UFP-induced augmentation of functional and morphological alterations of Clara cells in allergic lung inflammation.
The paper's conclusion, stating exactly this upstream-to-downstream relationship.
NAC strongly reduced both functional and morphological alterations of Clara cells.
The experimental basis for the link: the antioxidant reversed the club cell changes, which is what makes the upstream event inferable.
EC-UFP-induced decrease in BALF CC16 concentrations were abrogated by NAC treatment (NS/NAC/UFP; S/NAC/UFP).
The specific NAC-reversible CC16 endpoint behind this relationship.
EC-UFP-induced oxidative stress leads to augmented airway inflammation in the allergic lung
EC-UFP inhalation before allergen challenge increased the BAL inflammatory infiltrate, BALF IL-5 and IL-13, and lung TNF-alpha mRNA above the filtered-air allergic control, and NAC significantly reduced that augmentation at both the cellular and the TNF-alpha mRNA level. The TNF-alpha arm is the best-supported one in the paper because NAC reversed it strongly, in contrast to CC16 mRNA. Support is moderate because the upstream oxidative stress event is inferred from NAC reversibility and the inflammatory endpoints are reported in an additional file rather than quantified in the text.
In sensitized mice, EC-UFP inhalation prior to OVA challenge (S/UFP/OVA) caused a significant increase of BAL inflammatory infiltrate and BALF cytokines IL-5 and IL-13 7 days after OVA challenge, compared with sensitized and challenged mice exposed to filtered air (S/OVA); NAC treatment significantly reduced EC-UFP-induced augmentation of lung allergic inflammation (S/NAC/UFP/OVA)
Both halves of the link in one sentence: the particle-driven increase in airway inflammation and its reversal by the antioxidant.
the antioxidant strategy reduced TNF-α mRNA expression in whole lung homogenate.
The molecular arm of the same reversal, measured by the TNF-alpha mRNA assay.
Augmented airway inflammation leads to increased lung/blood barrier permeability
BALF total protein was highest in the group with the strongest inflammation (S/UFP/OVA) and the authors read this as an exudative response caused by increased airway permeability; serum CC16 rose while BALF CC16 fell in the same animals, the classic signature of leakage across the alveolo-capillary barrier. Recorded as indirect with weak support: no barrier measurement independent of the leaked proteins was made - no transepithelial resistance, no tracer flux - so inflammation and permeability are correlated here rather than shown to be sequential. Importantly, the non-challenged particle-exposed groups showed no protein induction at all, which is consistent with inflammation being required.
BALF total protein concentration was also most increased in this group, confirming that the exudative response caused by the increased airway permeability plays an important role.
The authors' statement tying the inflamed, particle-exposed group to increased airway permeability, which is this relationship.
No induction of protein levels were observed in any of the non-challenged groups (NS/UFP, S/UFP, with and without NAC treatment, Fig. 3a).
The control that makes the link directional: without the allergic challenge, and therefore without the inflammation, there was no barrier leakage.
Protocols
Bronchoalveolar lavage with BALF total protein and cytokine ELISA
Airways lavaged five times with 0.8 ml PBS. Aliquots of cell-free BAL fluid assayed in duplicate for total protein by Coomassie Protein Assay (Pierce Chemical Co.) and for IL-5, IFN-gamma and IL-13 by two site ELISAs using antibodies from BD Biosciences (IL-5, IFN-gamma) and BioSource (IL-13). BAL performed at 0, 1 or 7 days after OVA challenge or after termination of EC-UFP exposure, n = 4 per time point; BALF stored at -80 C after centrifugation.
Equipment: Coomassie Protein Assay (Pierce Chemical Co.); Two site ELISA kits (BD Biosciences; BioSource)
Differential BAL cell count on Diff-Quick stained cytospins
Viability and yield of BAL cells quantified via trypan blue exclusion in a hemocytometer. Differential BAL cell count of 400 cells per sample performed on cytospins (600 rpm for 10 min) fixed and stained with Diff- Quick (Dade Behring).
Equipment: Hemocytometer; Cytospin centrifuge; Diff-Quick stain (Dade Behring, Marburg, Germany)
Automated latex immunoassay for CC16 in BALF and serum
Cell-free BALF and serum aliquots assayed in duplicate for CC16 by an automated latex immunoassay using a polyclonal rabbit antibody raised against rat CC16 and applicable to both rat and mouse CC16 analysis. Before BAL, blood samples were taken from the retro orbital plexus and centrifuged; serum aliquots stored at -80 C.
Equipment: Automated latex immunoassay analyser; Polyclonal rabbit anti-rat CC16 antibody
TaqMan real-time RT-PCR for CC16 in whole lung homogenate
Lungs retrieved 1 day after EC-UFP exposure / allergen challenge, homogenized, total RNA isolated with the RNeasy Mini kit (Qiagen) and first strand cDNA synthesised with the iScript cDNA synthesis kit (BioRad). Quantitative PCR with TaqMan Gene Expression Assay Mm01230908_m1 for CC16 and TaqMan Universal PCR Master Mix on an ABIprism7000 Sequence Detection System (Applied Biosystems). Expression normalised to the housekeeping gene 18S (Hs99999901_s1) and to the mean of the untreated group by the delta-delta-CT method. One protocol id per target gene because each target uses its own TaqMan assay.
Equipment: ABIprism7000 Sequence Detection System (Applied Biosystems, Darmstadt, Germany); TaqMan Gene Expression Assay Mm01230908_m1; RNeasy Mini kit (Qiagen GmbH); iScript cDNA synthesis kit (BioRad)
TaqMan real-time RT-PCR for TNF-alpha in whole lung homogenate
Lungs retrieved 1 day after EC-UFP exposure / allergen challenge, homogenized, total RNA isolated with the RNeasy Mini kit (Qiagen) and first strand cDNA synthesised with the iScript cDNA synthesis kit (BioRad). Quantitative PCR with TaqMan Gene Expression Assay Mm00443258_m1 for TNF-alpha and TaqMan Universal PCR Master Mix on an ABIprism7000 Sequence Detection System (Applied Biosystems). Expression normalised to the housekeeping gene 18S (Hs99999901_s1) and to the mean of the untreated group by the delta-delta-CT method. One protocol id per target gene because each target uses its own TaqMan assay.
Equipment: ABIprism7000 Sequence Detection System (Applied Biosystems, Darmstadt, Germany); TaqMan Gene Expression Assay Mm00443258_m1; RNeasy Mini kit (Qiagen GmbH); iScript cDNA synthesis kit (BioRad)
PAS staining and morphometrical analysis of volume density of PAS positive material
Lungs removed after BAL and fixed in 10% buffered formalin; left lobe used for histopathology. After paraffin embedding, 3.5 um sections stained with hematoxylin-eosin and periodic acid Schiff. Volume density of PAS positive material determined with the double square lattice test system E80 and the Optimas software package in cross sections of small (circumference <= 800 um) and large (circumference > 800 um) airways. Data expressed as percentage of PAS positive material per total epithelial cells; lungs retrieved 7 days after OVA challenge / EC-UFP inhalation, n = 6-12 per group.
Equipment: Double square lattice test system E80; Optimas software package (Washington, USA); Periodic acid Schiff and hematoxylin-eosin stains
Transmission and scanning electron microscopy of bronchiolar epithelium
Lungs fixed by intratracheal perfusion with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), n = 2 per time point per group. For TEM peripheral and central sections of the right lung were minced, for SEM the left lung was cut longitudinally; all samples fixed a further 4-7 days in glutaraldehyde and postfixed in 1% osmiumtetroxide. TEM: EMbed 812 embedding, semithin 500 nm sections stained with Richardson solution to select the region, then ultrathin sections on formvar-coated 75-mesh nickel grids double-stained with uranyl acetate and lead citrate, observed at 80 kV (JEOL 1210). SEM: cacodylate rinses, ascending ethanol dehydration, critical point drying, sputter-coating with 10 nm gold, examined at 15 kV (JEOL 6300). Analysis focussed on the small airways at the one week time point.
Equipment: JEOL 1210 transmission electron microscope (Tokyo, Japan), 80 kV; JEOL 6300 scanning electron microscope (Tokyo, Japan), 15 kV; EMbed 812 (Science Services); uranyl acetate and lead citrate
Unrestrained whole body plethysmography for enhanced pause (PenH)
Lung function tests performed 24 h after allergen challenge in unrestrained animals using whole body plethysmography (Buxco Electronics). The enhanced pause (PenH) determined before and after methacholine exposure was applied as an index of airway hyperresponsiveness. Methacholine doses M1-M5: 10 mg/ml nebulized for 1, 2 and 4 minutes, then 40 mg/ml for 2 and 4 minutes, with a 3 min recording interval after each level; the mean of the PenH values of the 2nd and 3rd min was used to quantify airway hyperresponsiveness. n = 5-7 per group.
Equipment: Buxco Electronics whole body plethysmograph (Sharon, Connecticut, USA); Methacholine aerosol, 10 mg/ml and 40 mg/ml