Differences in Redox Biomarkers in Bronchoalveolar Lavage Fluid of Leisure Horses With and Without Severe Equine Asthma: Preliminary Results

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General assessment
This prospective observational study evaluates redox-related biomarkers (d-ROMs, NPBI, AOPP, BAP) in BALF from leisure horses with and without a history of severe equine asthma (SEA). The topic is clinically relevant and BALF is an appropriate compartment to explore local oxidative imbalance. The methods for BAL and laboratory assays are largely well described. However, the small and unbalanced sample size, a strong age imbalance between groups, possible disease heterogeneity in the non-SEA group, and limited control for confounders currently weaken causal interpretation and biomarker attribution specifically to SEA.

Major comments

Age as a major confounder (must be addressed analytically, not only discussed)
Age differs markedly between groups (median 9 vs 14.5 years; p=0.0002). Age can influence oxidative/antioxidant biomarkers and may partially explain group differences.
Recommendation: Perform adjusted analyses to account for age (e.g., regression models with group as predictor and age as covariate, or stratified/sensitivity analyses). With small n, at least provide effect sizes and discuss robustness (e.g., remove outliers, or compare age-matched subsets if feasible). Presenting only unadjusted Mann–Whitney comparisons is insufficient given the magnitude of age imbalance.

Definition of comparison groups/potential inclusion of MEA within “non-SEA”
The non-SEA group may include both healthy horses and MEA horses (as acknowledged). This could dilute or distort biomarker contrasts.
Recommendation: Provide explicit cytological criteria and summary BALF cytology for each group (median % neutrophils, eosinophils, mast cells, etc.), and, if possible, split non-SEA into “healthy” vs “MEA” or at least report how many non-SEA had abnormal cytology.

SEA case definition and disease phase standardization
SEA is classified by history and BALF cytology. However, severity and current phase (exacerbation vs remission) are not standardized and could affect biomarkers.
Recommendation: Clarify clinical status at sampling (clinical score, respiratory effort, cough, mucus, environmental exposure), and report any current treatment (corticosteroids/bronchodilators) and management. Consider adding these as covariates or at minimum reporting them to interpret variability.

Statistical approach and reporting

Kolmogorov–Smirnov is not ideal with small samples; Shapiro–Wilk is typically more appropriate.

Multiple biomarkers are tested; no correction for multiple comparisons is reported.

Report effect sizes (e.g., Hodges–Lehmann median difference with CI) in addition to p-values.
Recommendation: Consider multiple-comparison control (e.g., Holm) or justify why unadjusted p-values are acceptable (exploratory). Provide confidence intervals.

Interpretation of AOPP decrease
The finding of lower AOPP in SEA is counterintuitive given the typical interpretation as protein oxidation markers, and the manuscript suggests a protective/adaptive mechanism. This interpretation is plausible but currently speculative.
Recommendation: Rephrase to a more cautious interpretation and discuss alternative explanations (e.g., dilutional effects in BALF, protein concentration differences, assay interference, compartment-specific kinetics).

Assay suitability and units in BALF
The d-ROMs and BAP descriptions refer to “plasma” and “blood” in places, yet samples are BALF.
Recommendation: Ensure the text consistently refers to BALF and clarify validation/analytical performance in BALF (matrix effects, LOD/LOQ, intra/inter-assay CV, recovery), or cite studies validating these assays in BALF specifically.

Minor comments/editorial issues

Terminology: In Discussion you define NTBI, but the measured analyte is NPBI—use one term consistently and define it once.

BAL method wording: “ev” for IV dosing should be standardized (e.g., “IV”).

Typos/formatting: “GraphPaD”, “Pharmaco.l Ther.”, “polyunsatured” should be corrected.

Units: Ensure consistent µmol/L formatting and symbols across text/table/figure.

Add a table/statement: clinical characteristics (sex, breed, body weight if available), environmental exposure, and current medications.

Limitations: The limitations section is good; strengthen it by stating that group differences cannot be attributed solely to SEA without age adjustment.

Author Response

Reviewer 1 (RED)

General assessment
This prospective observational study evaluates redox-related biomarkers (d-ROMs, NPBI, AOPP, BAP) in BALF from leisure horses with and without a history of severe equine asthma (SEA). The topic is clinically relevant and BALF is an appropriate compartment to explore local oxidative imbalance. The methods for BAL and laboratory assays are largely well described. However, the small and unbalanced sample size, a strong age imbalance between groups, possible disease heterogeneity in the non-SEA group, and limited control for confounders currently weaken causal interpretation and biomarker attribution specifically to SEA.

Major comments

Age as a major confounder (must be addressed analytically, not only discussed)
Age differs markedly between groups (median 9 vs 14.5 years; p=0.0002). Age can influence oxidative/antioxidant biomarkers and may partially explain group differences.
Recommendation: Perform adjusted analyses to account for age (e.g., regression models with group as predictor and age as covariate, or stratified/sensitivity analyses). With small n, at least provide effect sizes and discuss robustness (e.g., remove outliers, or compare age-matched subsets if feasible). Presenting only unadjusted Mann–Whitney comparisons is insufficient given the magnitude of age imbalance.

Answer: we thank the reviewer for the comment. Given the significant age difference between groups, we performed additional multiple linear regression analyses including group as fixed factor and age as continuous covariate. The interaction term (group × age) was initially tested and was not significant for any biomarker; therefore, it was removed from the final models. After adjustment for age, NBPI remained significantly associated with group (p = 0.0041), whereas d-ROMs, AOPP and BAP were no longer significant. These additional analyses suggest that age partially explained some of the unadjusted differences. The Methods and Results sections have been updated accordingly, and a new table reporting adjusted estimates has been included in the revised manuscript (lines 201-207, 239-246 and 251-261).

 

Definition of comparison groups/potential inclusion of MEA within “non-SEA”
The non-SEA group may include both healthy horses and MEA horses (as acknowledged). This could dilute or distort biomarker contrasts.
Recommendation: Provide explicit cytological criteria and summary BALF cytology for each group (median % neutrophils, eosinophils, mast cells, etc.), and, if possible, split non-SEA into “healthy” vs “MEA” or at least report how many non-SEA had abnormal cytology.

Answer: we agree with the reviewer. We verified the presence of MEA horses enrolled in the non-SEA group. All the horses included were cytologically classified as healthy (Couëtil et al., 2016), and a table summarizing the cytological results has been added (lines 220-222, 232 and 234-238)

 

SEA case definition and disease phase standardization
SEA is classified by history and BALF cytology. However, severity and current phase (exacerbation vs remission) are not standardized and could affect biomarkers.
Recommendation: Clarify clinical status at sampling (clinical score, respiratory effort, cough, mucus, environmental exposure), and report any current treatment (corticosteroids/bronchodilators) and management. Consider adding these as covariates or at minimum reporting them to interpret variability.

Answer: thank for the suggestion. The information required has been added to the text (please see the results section). We did not add clinical scores because not useful to discriminate between SEA horses in remission and healthy ones (lines 227-231).

 

Statistical approach and reporting Kolmogorov–Smirnov is not ideal with small samples; Shapiro–Wilk is typically more appropriate.

Answer: thank you for your recommendation, the Shapiro-Wilk test has been applied as suggested (line 201).

 

Multiple biomarkers are tested; no correction for multiple comparisons is reported.

Answer: To account for multiple comparisons across the four biomarkers, we applied Holm’s step-down procedure, which controls the family-wise error rate while being less conservative than the standard Bonferroni correction. After adjustment, only NBPI remained significantly associated with group (raw p = 0.0041; Holm-adjusted p = 0.0164), whereas no significant associations were observed for the other biomarkers. The main text has been modified (please see the statistical analysis and results sections) (lines 207-210 and 260-261).

 

Report effect sizes (e.g., Hodges–Lehmann median difference with CI) in addition to p-values.
Recommendation: Consider multiple-comparison control (e.g., Holm) or justify why unadjusted p-values are acceptable (exploratory). Provide confidence intervals.

Answer: To address the issue of multiple comparisons across the four biomarkers, we applied Holm’s step-down procedure. After adjustment, only NBPI remained significantly associated with group (raw p = 0.0041; Holm-adjusted p = 0.0164), whereas no significant associations were observed for d-ROMs, AOPP or BAP (please see the statistical analysis and results sections) (lines 207-210 and 260-261).

 

Interpretation of AOPP decrease
The finding of lower AOPP in SEA is counterintuitive given the typical interpretation as protein oxidation markers, and the manuscript suggests a protective/adaptive mechanism. This interpretation is plausible but currently speculative.
Recommendation: Rephrase to a more cautious interpretation and discuss alternative explanations (e.g., dilutional effects in BALF, protein concentration differences, assay interference, compartment-specific kinetics).

Answer: thank you for the suggestion. The sentence has been modified based on the different statistical analysis applied and the results obtained (please see the Discussion section) (lines 311-323).

 

Assay suitability and units in BALF

The d-ROMs and BAP descriptions refer to “plasma” and “blood” in places, yet samples are BALF.

Recommendation: Ensure the text consistently refers to BALF and clarify validation/analytical performance in BALF (matrix effects, LOD/LOQ, intra/inter-assay CV, recovery), or cite studies validating these assays in BALF specifically.

Answer: thank you for your suggestion, the sentence has been corrected, and literature has been added (please see lines 188-190).

 

Minor comments/editorial issues

Terminology: In Discussion you define NTBI, but the measured analyte is NPBI—use one term consistently and define it once.

Answer: thank you for the suggestion. We have revised the manuscript and replaced NTBI with NPBI throughout the text to ensure consistent terminology (lines 276, 277, 284, 346, 349, 401).

 

BAL method wording: “ev” for IV dosing should be standardized (e.g., “IV”).

Answer: Thank you for your comment, the term has been replaced with IV (line 133).

 

Typos/formatting: “GraphPaD”, “Pharmaco.l Ther.”, “polyunsatured” should be corrected.

Answer: The typos have been corrected as suggested (lines 437 and 450).

 

Units: Ensure consistent µmol/L formatting and symbols across text/table/figure.

Answer: thank you for your suggestion, the unit µmol/L has been corrected across the text and the table (lines 194, 198 and 242).

 

Add a table/statement: clinical characteristics (sex, breed, body weight if available), environmental exposure, and current medications.

Answer: thank you for your suggestion, the information requested has been added in the Results section (lines 220-225).

 

Limitations: The limitations section is good; strengthen it by stating that group differences cannot be attributed solely to SEA without age adjustment.

Answer: thank you for your suggestion, the limitations have been improved (lines 394-397 and 400-403).

Reviewer 2 Report

Comments and Suggestions for Authors

This report provides unique findings relevant to the equine pulmonary microenvironment. This topic is well referenced which provides needed justification for the importance of this investigation. There are editorial revisions required throughout the report to improve the quality and clarity of writing. This investigation and report provide novel information pertaining to pulmonary oxidative properties among horses with SEA. Specific comments are provided to improve to overall quality of this report.

Specific comments

Line 94, advanced

Line 106, the authors reference “many papers” – this statement should be revised to accurately reflect the numerous investigations that have been completed and reported in the published veterinary literature on this topic.

Line 121, the authors should more thoroughly describe the assessment that was used for all subjects. Were any historical questions asked regarding clinical evidence of SEA, were any horses showing signs of disease exacerbation?  When examined, did any horses have evidence of hypertrophy of the external abdominal oblique muscle (heave line)? With regard to cytologic evaluation, did any horses have evidence of mild-moderate EA?  Inclusion of this detail will provide the reader with a better understanding of subject selection.

Lines 128, 131, 133 – refrain from excessive use or inappropriate placement of The – the authors are encouraged to review these sentences for the necessity of the at the start of each statement.

Line 138, with the head maintained in an extended position

Line 187, ‘as long as the concentration in bronchoalveolar lavage fluid’ is an unclear statement in this sentence, please clarify this wording.

Line 213, Table 1 would be more clearly reported if the authors add a column with P values, this would clarify statistically significant differences between subject groups.

Line 223, compared to non-affected subjects.

Line 226, Figure 1 would more clearly report statistically different findings if designated in reported data. It would be more clear to the reader to represent data sets for Figure 1 as panel a-d.

Line 238, NBPI was measured

Line 239, Non-transferrin-bound-iron

Line 246, replace specifically with previously

Line 251, BPI was measured

Line 258, antioxidant activity

Line 273, in an SEA study

Line 306-307, neutrophils in tracheal mucous were lower in treated group compared to controls.

Line 316, replace less with reduced

Line 320, only when also combined with

Line 341, relationship with onset of disease

 

Author Response

Reviewer 2 (GREEN)

This report provides unique findings relevant to the equine pulmonary microenvironment. This topic is well referenced which provides needed justification for the importance of this investigation. There are editorial revisions required throughout the report to improve the quality and clarity of writing. This investigation and report provide novel information pertaining to pulmonary oxidative properties among horses with SEA. Specific comments are provided to improve to overall quality of this report.

Specific comments

Line 94, advanced

Answer: corrected as suggested (line 95).

 

Line 106, the authors reference “many papers” – this statement should be revised to accurately reflect the numerous investigations that have been completed and reported in the published veterinary literature on this topic.

Answer: thank you for your suggestion, the exact number of investigations has now been reported (line 107).

 

Line 121, the authors should more thoroughly describe the assessment that was used for all subjects. Were any historical questions asked regarding clinical evidence of SEA, were any horses showing signs of disease exacerbation?  When examined, did any horses have evidence of hypertrophy of the external abdominal oblique muscle (heave line)? With regard to cytologic evaluation, did any horses have evidence of mild-moderate EA?  Inclusion of this detail will provide the reader with a better understanding of subject selection.

Answer: thank you for the suggestions. Information about clinical presentation, management, sex and breed was added to the main text (please see the results section). Moreover, BALF results have been added as a table in the text (lines 220-232 and 234-238).

 

Lines 128, 131, 133 – refrain from excessive use or inappropriate placement of The – the authors are encouraged to review these sentences for the necessity of the at the start of each statement.

Answer: The sentences have been reviewed as suggested (lines 129, 132, 134, 172, 181).

 

Line 138, with the head maintained in an extended position.

Answer: corrected as suggested (lines 139-140)

 

Line 187, ‘as long as the concentration in bronchoalveolar lavage fluid’ is an unclear statement in this sentence, please clarify this wording.

Answer: thank you for your suggestions, the sentence has been rephrased (lines 188-190).

 

Line 213, Table 1 would be more clearly reported if the authors add a column with P values, this would clarify statistically significant differences between subject groups.

Answer: the statistical analysis has been modified accordingly to reviewer 1. So, the materials and methods, results and discussion sections have been modified accordingly with the new results obtained. In Table 2, a column with P values has been added as suggested (lines 242-246)

 

Line 223, compared to non-affected subjects.

Answer: Thank you for your suggestion. However, this sentence has been removed, as the Results section was revised after performing the updated statistical analysis.

 

Line 226, Figure 1 would more clearly report statistically different findings if designated in reported data. It would be more clear to the reader to represent data sets for Figure 1 as panel a-d.

Answer: thank you for your suggestion. Due to the new statistical analysis applied, as suggested by reviewer 1, the figure was not useful to the main text, so it has been erased.

 

Line 238, NBPI was measured

Answer: Thank you, rephrased as suggested (line 276).

 

Line 239, Non-transferrin-bound-iron

Answer: thank you for your suggestion. Non-transferrin-bound iron has been replaced with Non-protein-bound-iron throughout the text to ensure consistent terminology (line 277).

 

Line 246, replace specifically with previously

Answer: corrected as suggested (line 284).

 

Line 251, BPI was measured

Answer: Thank you for your suggestion, the sentence has been improved (line 290).

 

Line 258, antioxidant activity

Answer: Corrected as suggested (line 298).

 

Line 273, in an SEA study

Answer: Thank you for your suggestion. The Discussion section has been updated following the changes in the Results section, and this sentence is no longer present in the revised manuscript.

 

Line 306-307, neutrophils in tracheal mucous were lower in treated group compared to controls.

Answer: The sentence has been corrected as suggested (lines 361-362).

 

Line 316, replace less with reduced

Answer: corrected as suggested (line 371).

 

Line 320, only when also combined with

Answer: thank you for your comment, the sentence has been revised (line 375).

 

Line 341, relationship with onset of disease

Answer: revised as suggested (line 398).

Reviewer 3 Report

Comments and Suggestions for Authors

The occurrence of asthma in horses is a current problem. A growing number of horses are suffering from respiratory problems, and the terms for this group of diseases (asthma, obstructive airway disease, respiratory syndrome) are constantly being redefined. 

On the other hand, the study of redox markers in the fluid that remains in the lungs, in a non-sterile environment, does not explain the pathogenesis of these diseases, because it is not known whether the obtained results are the cause or the effect of health disorders. Nevertheless, the results of the presented studies may have diagnostic significance, indicating the stage of disease progression. I appreciate the idea of ​​such research, and it is therefore worth publishing. However, the biological material used in this study is insufficient (limitations: lines 327-340) to generalize the results to the entire horse population. Therefore, the presented results should be considered preliminary, merely encouraging broader research on this issue. I suggest adding the term "preliminary studies" to the title. Before the manuscript is accepted for publication, additions and corrections are necessary:  - The BALF analysis method is described in detail in Section 2.3, so please provide the results of these studies. I understand that the results were the basis for classifying horses into healthy/diseased groups, but the reader should have insight into the basis of this classification, especially since it is not clear (limitations, lines 331-333).
- Please explain in which research material BAP was determined, as the term "plasma" appears three times in the method description (in lines 180, 181, and 185) and the paper describes the results of the BALF samples study. - In the line 223, please replace the terms "increase" and "decrease" with "higher" and "lower"

Author Response

Reviewer 3 (BLUE)

The occurrence of asthma in horses is a current problem. A growing number of horses are suffering from respiratory problems, and the terms for this group of diseases (asthma, obstructive airway disease, respiratory syndrome) are constantly being redefined. 

On the other hand, the study of redox markers in the fluid that remains in the lungs, in a non-sterile environment, does not explain the pathogenesis of these diseases, because it is not known whether the obtained results are the cause or the effect of health disorders. Nevertheless, the results of the presented studies may have diagnostic significance, indicating the stage of disease progression. I appreciate the idea of such research, and it is therefore worth publishing. However, the biological material used in this study is insufficient (limitations: lines 327-340) to generalize the results to the entire horse population. Therefore, the presented results should be considered preliminary, merely encouraging broader research on this issue. I suggest adding the term "preliminary studies" to the title.

Answer: thank you for your suggestion, the title has now been modified (line 4).

 

Before the manuscript is accepted for publication, additions and corrections are necessary: 

- The BALF analysis method is described in detail in Section 2.3, so please provide the results of these studies. I understand that the results were the basis for classifying horses into healthy/diseased groups, but the reader should have insight into the basis of this classification, especially since it is not clear (limitations, lines 331-333).

Answer: thank you for your suggestion. A table reporting the results on BALF cytology has now been added to the main text. Moreover, the limitations paragraph has been improved (lines 234-238 and 387-391).

 

- Please explain in which research material BAP was determined, as the term "plasma" appears three times in the method description (in lines 180, 181, and 185) and the paper describes the results of the BALF samples study.

Answer: thank you for your suggestion, BAP was determined in BALF. Typos have been corrected (lines 183, 184, 187).

 

- In the line 223, please replace the terms "increase" and "decrease" with "higher" and "lower"

Answer: Thank you for your suggestion. However, the results section has been revised following the updated statistical analysis and this sentence has been removed.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript has significantly improved compared with the previous version. The statistical analysis, presentation of the results, and interpretation of findings are now consistent throughout the manuscript. The conclusions are appropriately supported by the results, and the manuscript is clearly written. I have no further comments and consider the manuscript suitable for publication in its present form.