On 2016 Apr 26, Annalisa Forlani commented:

We are thankful to Dr. Cooper for his comment and deep knowledge of the literature. However, we have not included the suggested citation for different reasons, as discussed below.

Images from previous reports of pulmonary hyalinosis may resemble our case. However, there are several histological, histochemical and ultrastructural findings that we believe are not consistent with such diagnosis.

Based on previous reports (Billups et al., 1972; Dagle et al., 1976), hyaline bodies are intracellular globules usually distributed within the cytoplasm of macrophages and multinucleated cells, occasionally calcified and strongly positive for Periodic Acid-Schiff (PAS), crystal violet and Oil Red O. The histological lesions are described as multifocal granulomas effacing smaller bronchi and the subpleural tissue at the margins of lobes.

In our case, the eosinophilic granular material was predominantly extracellular and filled the alveolar spaces without any other pulmonary architectural changes. Moreover, histochemistry revealed a negative Von Kossa and Congo red reaction and only variably positivity for PAS.

Ultrastructurally, the substance in the alveolar lumina was composed by short lamellar thick fascicles rather than whorled intracytoplasmic lamellar membranes as reported for pulmonary hyalinosis. Therefore, our findings were strongly suggestive of accumulation of abnormal surfactant rather than degenerate cells.

Pulmonary alveolar proteinosis and pulmonary hyalinosis are both rare and poorly characterized conditions whose pathogenesis is still not entirely understood. In 1976, Dagle and collaborators hypothesized a similar pathogenetic mechanism, even an overlap between the two . However, no additional efforts have been made toward a better understanding of the two entities.

In conclusion, given the differences between the abovementioned studies on pulmonary hyalinosis and our findings, a comparison with pulmonary hyalinosis seems unnecessary, and no additional comments should be included in our manuscript.

References 1. Billups LH, Liu SK, Kelly DF, Garner FM. Pulmonary granulomas associated with PAS-positive bodies in brachycephalic dogs. Vet Pathol 1972; 9: 294-300. 2. Dagle GE, Filipy RE, Adee RR, Stuart BO. Pulmonary hyalinosis in dogs. Vet Pathol 1976; 13: 138-142.

On 2016 Apr 12, Timothy K Cooper commented:

The authors should consider pulmonary hyalinosis of brachycephalic dogs: Pulmonary Granulomas Associated with PAS-Positive Bodies in Brachycephalic Dogs. Billups LH, et al. Veterinary Pathology 1972: 9(5):294-300.

On 2016 Apr 12, Timothy K Cooper commented:

The authors should consider pulmonary hyalinosis of brachycephalic dogs: Pulmonary Granulomas Associated with PAS-Positive Bodies in Brachycephalic Dogs. Billups LH, et al. Veterinary Pathology 1972: 9(5):294-300.

On 2016 Apr 26, Annalisa Forlani commented:

We are thankful to Dr. Cooper for his comment and deep knowledge of the literature. However, we have not included the suggested citation for different reasons, as discussed below.

Images from previous reports of pulmonary hyalinosis may resemble our case. However, there are several histological, histochemical and ultrastructural findings that we believe are not consistent with such diagnosis.

Based on previous reports (Billups et al., 1972; Dagle et al., 1976), hyaline bodies are intracellular globules usually distributed within the cytoplasm of macrophages and multinucleated cells, occasionally calcified and strongly positive for Periodic Acid-Schiff (PAS), crystal violet and Oil Red O. The histological lesions are described as multifocal granulomas effacing smaller bronchi and the subpleural tissue at the margins of lobes.

In our case, the eosinophilic granular material was predominantly extracellular and filled the alveolar spaces without any other pulmonary architectural changes. Moreover, histochemistry revealed a negative Von Kossa and Congo red reaction and only variably positivity for PAS.

Ultrastructurally, the substance in the alveolar lumina was composed by short lamellar thick fascicles rather than whorled intracytoplasmic lamellar membranes as reported for pulmonary hyalinosis. Therefore, our findings were strongly suggestive of accumulation of abnormal surfactant rather than degenerate cells.

Pulmonary alveolar proteinosis and pulmonary hyalinosis are both rare and poorly characterized conditions whose pathogenesis is still not entirely understood. In 1976, Dagle and collaborators hypothesized a similar pathogenetic mechanism, even an overlap between the two . However, no additional efforts have been made toward a better understanding of the two entities.

In conclusion, given the differences between the abovementioned studies on pulmonary hyalinosis and our findings, a comparison with pulmonary hyalinosis seems unnecessary, and no additional comments should be included in our manuscript.

References 1. Billups LH, Liu SK, Kelly DF, Garner FM. Pulmonary granulomas associated with PAS-positive bodies in brachycephalic dogs. Vet Pathol 1972; 9: 294-300. 2. Dagle GE, Filipy RE, Adee RR, Stuart BO. Pulmonary hyalinosis in dogs. Vet Pathol 1976; 13: 138-142.