Phenotypes and Serum Biomarkers in Sarcoidosis.

Sarcoidosis is a multisystem disease, which is diagnosed on a compatible clinical presentation, non-necrotizing granulomatous inflammation in one or more tissue samples, and exclusion of alternative causes of granulomatous disease. Considering its heterogeneity, numerous aspects of the disease remain to be elucidated. In this context, the identification and integration of biomarkers may hold significance in clinical practice, aiding in appropriate selection of patients for targeted clinical trials. This work aims to discuss and analyze how validated biomarkers are currently integrated in disease category definitions. Future studies are mandatory to unravel the diverse contributions of genetics, socioeconomic status, environmental exposures, and other sociodemographic variables to disease severity and phenotypic presentation. Furthermore, the implementation of transcriptomics, multidisciplinary approaches, and consideration of patients' perspectives, reporting innovative insights, could be pivotal for a better understanding of disease pathogenesis and the optimization of clinical assistance.

Inhaled recombinant GM-CSF reduces the need for whole lung lavage and improves gas exchange in autoimmune pulmonary alveolar proteinosis patients.

RATIONALE: Whole lung lavage (WLL) is a widely accepted palliative treatment for autoimmune pulmonary alveolar proteinosis (aPAP) but does not correct myeloid cell dysfunction or reverse the pathological accumulation of surfactant. In contrast, inhaled recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) is a promising pharmacological approach that restores alveolar macrophage functions including surfactant clearance. Here, we evaluate WLL followed by inhaled rGM-CSF (sargramostim) as therapy of aPAP. METHODS: 18 patients with moderate-to-severe aPAP were enrolled, received baseline WLL, were randomised into either the rGM-CSF group (receiving inhaled sargramostim) or control group (no scheduled therapy) and followed for 30 months after the baseline WLL. Outcome measures included additional unscheduled "rescue" WLL for disease progression, assessment of arterial blood gases, pulmonary function, computed tomography, health status, biomarkers and adverse events. Patients requiring rescue WLL were considered to have failed their assigned intervention group. RESULTS: The primary end-point of time to first rescue WLL was longer in rGM-CSF-treated patients than controls (30 versus 18 months, n=9 per group, p=0.0078). Seven control patients (78%) and only one rGM-CSF-treated patient (11%) required rescue WLL, demonstrating a 7-fold increase in relative risk (p=0.015). Compared to controls, rGM-CSF-treated patients also had greater improvement in peripheral arterial oxygen tension, alveolar-arterial oxygen tension difference, diffusing capacity of the lungs for carbon monoxide and aPAP biomarkers. One patient from each group withdrew for personal reasons. No serious adverse events were reported. CONCLUSIONS: This long-term, prospective, randomised trial demonstrated inhaled sargramostim following WLL reduced the requirement for WLL, improved lung function and was safe in aPAP patients. WLL plus inhaled sargramostim may be useful as combined therapy for aPAP.

ABCA3-related interstitial lung disease beyond infancy.

BACKGROUND: The majority of patients with childhood interstitial lung disease (chILD) caused by pathogenic variants in ATP binding cassette subfamily A member 3 (ABCA3) develop severe respiratory insufficiency within their first year of life and succumb to disease if not lung transplanted. This register-based cohort study reviews patients with ABCA3 lung disease who survived beyond the age of 1 year. METHOD: Over a 21-year period, patients diagnosed as chILD due to ABCA3 deficiency were identified from the Kids Lung Register database. 44 patients survived beyond the first year of life and their long-term clinical course, oxygen supplementation and pulmonary function were reviewed. Chest CT and histopathology were scored blindly. RESULTS: At the end of the observation period, median age was 6.3 years (IQR: 2.8-11.7) and 36/44 (82%) were still alive without transplantation. Patients who had never received supplemental oxygen therapy survived longer than those persistently required oxygen supplementation (9.7 (95% CI 6.7 to 27.7) vs 3.0 years (95% CI 1.5 to 5.0), p=0.0126). Interstitial lung disease was clearly progressive over time based on lung function (forced vital capacity % predicted absolute loss -1.1% /year) and on chest CT (increasing cystic lesions in those with repetitive imaging). Lung histology pattern were variable (chronic pneumonitis of infancy, non-specific interstitial pneumonia, and desquamative interstitial pneumonia). In 37/44 subjects, the ABCA3 sequence variants were missense variants, small insertions or deletions with in-silico tools predicting some residual ABCA3 transporter function. CONCLUSION: The natural history of ABCA3-related interstitial lung disease progresses during childhood and adolescence. Disease-modifying treatments are desirable to delay such disease course.

The Genes-Stemness-Secretome Interplay in Malignant Pleural Mesothelioma: Molecular Dynamics and Clinical Hints.

MPM has a uniquely poor somatic mutational landscape, mainly driven by environmental selective pressure. This feature has dramatically limited the development of effective treatment. However, genomic events are known to be associated with MPM progression, and specific genetic signatures emerge from the exceptional crosstalk between neoplastic cells and matrix components, among which one main area of focus is hypoxia. Here we discuss the novel therapeutic strategies focused on the exploitation of MPM genetic asset and its interconnection with the surrounding hypoxic microenvironment as well as transcript products and microvesicles representing both an insight into the pathogenesis and promising actionable targets.

An exploratory study investigating biomarkers associated with autoimmune pulmonary alveolar proteinosis (aPAP).

Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare lung disorder involving production of autoantibodies against endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF). This study aimed to identify biomarkers that could be used to monitor for aPAP, particularly in patients treated with anti-GM-CSF antibodies. This was an exploratory, prospective, observational, single-center study. Pre-specified biomarkers were evaluated between baseline and Day 120 in serum/plasma, whole blood, sputum and exhaled breath condensate from patients with aPAP, healthy volunteers, and patients with chronic obstructive pulmonary disease (COPD) and asthma (not treated with anti-GM-CSF and with no evidence of aPAP). Pulmonary function tests were also performed. Overall, 144 individuals were enrolled (aPAP: n = 34, healthy volunteers: n = 24, COPD: n = 40 and asthma: n = 46). Plasma GM-CSF levels were lower, and Krebs von den Lungen 6 and GM-CSF autoantibody ranges were higher, in patients with aPAP compared with other populations. Surfactant proteins-A and -D, lactate dehydrogenase and carcinoembryonic antigen ranges partially or completely overlapped across populations. Most plasma biomarkers showed high sensitivity and specificity for detection of aPAP; GM-CSF and GM-CSF autoantibody concentrations demonstrated equivalent sensitivity for differentiating aPAP. In addition to characteristic GM-CSF autoantibodies, assessment of plasma GM-CSF may identify individuals at risk of developing aPAP.Trial registration: EudraCT, 2012-003475-19. Registered 23 July 2012- https://eudract.ema.europa.eu/ .

Potential clinical utility of MUC5B und TOLLIP single nucleotide polymorphisms (SNPs) in the management of patients with IPF.

BACKGROUND: Genetic variants of TOLLIP and MUC5B, both on chromosome 11, have been reported to be associated with the development and/or prognosis of idiopathic pulmonary fibrosis (IPF). This retrospective study was conducted to investigate the association of MUC5B and TOLLIP SNPs with disease outcome in IPF. 62 IPF patients and 50 healthy controls (HC) from our Institution were genotyped for SNPs within MUC5B (rs35705950) and TOLLIP (rs3750920 and rs5743890). Correlation of SNPs genotypes with survival, acute exacerbation (AE) or disease progression (defined as a decline of ≥ 5% in FVC and or ≥ 10% in DLco in one year) was investigated. RESULTS: The MUC5B rs35705950 minor allele (T) was more frequent in IPF subjects than in HC (35% vs 9% p < 0.001). TOLLIP SNPs alleles and genotype distribution did not differ between IPF and HC and did not vary according to gender, age, BMI and lung functional impairment at baseline. The minor allele (C) in TOLLIP rs5743890 was associated with worse survival and with disease progression in all performed analyses. The MUC5B rs35705950 or the TOLLIP rs3750920 minor allele, were not associated with disease progression or AE. CONCLUSION: We confirm that the minor allele of MUC5B rs35705950 is associated with IPF. The minor allele of TOLLIP rs5743890 appears to be a predictor of worse survival and more rapid disease progression, therefore being of potential utility to stratify IPF patients at baseline.

Inhaled Molgramostim Therapy in Autoimmune Pulmonary Alveolar Proteinosis.

BACKGROUND: Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare disease characterized by progressive surfactant accumulation and hypoxemia. It is caused by disruption of granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling, which pulmonary alveolar macrophages require to clear surfactant. Recently, inhaled GM-CSF was shown to improve the partial pressure of arterial oxygen in patients with aPAP. METHODS: In a double-blind, placebo-controlled, three-group trial, we randomly assigned patients with aPAP to receive the recombinant GM-CSF molgramostim (300 µg once daily by inhalation), either continuously or intermittently (every other week), or matching placebo. The 24-week intervention period was followed by an open-label treatment-extension period. The primary end point was the change from baseline in the alveolar-arterial difference in oxygen concentration (A-aDo2) at week 24. RESULTS: In total, 138 patients underwent randomization; 46 were assigned to receive continuous molgramostim, 45 to receive intermittent molgramostim, and 47 to receive placebo. Invalid A-aDo2 data for 4 patients (1 in each molgramostim group and 2 in the placebo group) who received nasal oxygen therapy during arterial blood gas measurement were replaced by means of imputation. For the primary end point - the change from baseline in the A-aDo2 at week 24 - improvement was greater among patients receiving continuous molgramostim than among those receiving placebo (-12.8 mm Hg vs. -6.6 mm Hg; estimated treatment difference, -6.2 mm Hg; P = 0.03 by comparison of least-squares means). Patients receiving continuous molgramostim also had greater improvement than those receiving placebo for secondary end points, including the change from baseline in the St. George's Respiratory Questionnaire total score at week 24 (-12.4 points vs. -5.1 points; estimated treatment difference, -7.4 points; P = 0.01 by comparison of least-squares means). For multiple end points, improvement was greater with continuous molgramostim than with intermittent molgramostim. The percentages of patients with adverse events and serious adverse events were similar in the three groups, except for the percentage of patients with chest pain, which was higher in the continuous-molgramostim group. CONCLUSIONS: In patients with aPAP, daily administration of inhaled molgramostim resulted in greater improvements in pulmonary gas transfer and functional health status than placebo, with similar rates of adverse events. (Funded by Savara Pharmaceuticals; IMPALA ClinicalTrials.gov number, NCT02702180.).

Pulmonary alveolar proteinosis: from classification to therapy.

Pulmonary alveolar proteinosis (PAP) is a rare respiratory syndrome characterised by the accumulation of surfactant lipoproteins within the alveoli. According to various pathogenetic mechanisms and aetiologies, PAP is classified as primary, secondary or congenital. Primary PAP is led by a granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling disruption; the autoimmune form is driven by the presence of anti GM-CSF autoantibodies and represents 90% of all the PAP cases; and the hereditary form is the result of mutations in genes encoding GM-CSF receptor. Secondary PAP is associated with various diseases causing a reduction in function and/or number of alveolar macrophages. Congenital PAP emerges as a consequence of corrupted surfactant production, due to mutations in surfactant proteins or lipid transporter, or mutations affecting lung development. The clinical manifestations are various, ranging from insidious onset to acute or progressive respiratory failure, including premature death within the first days of life in neonates with congenital surfactant production disorders. The diagnostic workup includes clinical and radiological assessment (respiratory function test, high-resolution chest computed tomography), laboratory tests (anti-GM-CSF autoantibodies dosage, GM-CSF serum level and GM-CSF signalling test), and genetic tests. Whole-lung lavage is the current gold standard of care of PAP; however, the therapeutic approach depends on the pathogenic form and disease severity, including GM-CSF augmentation strategies in autoimmune PAP and other promising new treatments. EDUCATIONAL AIMS: To update knowledge about a rare respiratory syndrome, pulmonary alveolar proteinosis, in order to promote early diagnosis and correct management.To highlight recent treatment options based on pathogenesis and disease severity.

Eight novel variants in the SLC34A2 gene in pulmonary alveolar microlithiasis.

BACKGROUND: Pulmonary alveolar microlithiasis (PAM) is caused by genetic variants in the SLC34A2 gene, which encodes the sodium-dependent phosphate transport protein 2B (NaPi-2b). PAM is characterised by deposition of calcium phosphate concretions (microliths) in the alveoli leading to pulmonary dysfunction. The variant spectrum of SLC34A2 has not been well investigated and it is not yet known whether a genotype-phenotype correlation exists. METHODS: We collected DNA from 14 patients with PAM and four relatives, and analysed the coding regions of SLC34A2 by direct DNA sequencing. To determine the phenotype characteristics, clinical data were collected and a severity score was created for each variant, based on type and localisation within the protein. RESULTS: We identified eight novel allelic variants of SLC34A2 in 14 patients with PAM. Four of these were nonsense variants, three were missense and one was a splice site variant. One patient was heterozygous for two different variants and all other patients were homozygous. Four patients were asymptomatic and 10 patients were symptomatic. The severity of the disease was associated with the variant severity. CONCLUSIONS: Our findings support a significant role for SLC34A2 in PAM and expand the variant spectrum of the disease. Thus, SLC34A2 variants were detected in all patients and eight novel allelic variants were discovered. An association between disease severity and the severity of the variants was found; however, this needs to be investigated in larger patient populations.

The influence of genetics on therapeutic developments in pulmonary alveolar proteinosis.

PURPOSE OF REVIEW: Pulmonary alveolar proteinosis (PAP) is characterized by the massive accumulation of lipoproteinaceous material within alveoli, which results in progressive respiratory failure. The abnormalities in surfactant clearance are caused by defective pulmonary macrophages, whose terminal differentiation is GM-CSF-dependent. In hereditary PAP, the rupture of GM-CSF signaling is because of mutations in the GM-CSF receptor genes. This review focus on the innovative technologies of gene-correction proposed for the development of new therapeutic strategies, for hereditary PAP patients. RECENT FINDINGS: Hematopoietic stem cell gene therapy has been successfully experimented in murine models to restore the expression of the GM-CSF receptor, however, a therapeutic approach based on bone marrow transplantation requires a preconditioning, which could be hazardous in PAP patients, who are highly susceptible to pulmonary infections. Gene-corrected pulmonary macrophages, administered directly to the lung, could represent an improved approach. Finally, patient-derived induced pluripotent stem cells seem to be promising to overcome the limited availability of primary patient cells and to generate gene-corrected macrophages, able to recover pulmonary surfactant clearance. SUMMARY: WLL is the gold standard therapy for PAP. However, its use in hereditary PAP is limited by the difficulty of performing this technique in paediatric patients and by its purely symptomatic efficacy. The recent advances in genome engineering could provide efficacious strategies for clinical application.

Pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by the accumulation of alveolar surfactant and dysfunction of alveolar macrophages. PAP results in progressive dyspnoea of insidious onset, hypoxaemic respiratory failure, secondary infections and pulmonary fibrosis. PAP can be classified into different types on the basis of the pathogenetic mechanism: primary PAP is characterized by the disruption of granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling and can be autoimmune (caused by elevated levels of GM-CSF autoantibodies) or hereditary (due to mutations in CSF2RA or CSF2RB, encoding GM-CSF receptor subunits); secondary PAP results from various underlying conditions; and congenital PAP is caused by mutations in genes involved in surfactant production. In most patients, pathogenesis is driven by reduced GM-CSF-dependent cholesterol clearance in alveolar macrophages, which impairs alveolar surfactant clearance. PAP has a prevalence of at least 7 cases per million individuals in large population studies and affects men, women and children of all ages, ethnicities and geographical locations irrespective of socioeconomic status, although it is more-prevalent in smokers. Autoimmune PAP accounts for >90% of all cases. Management aims at improving symptoms and quality of life; whole-lung lavage effectively removes excessive surfactant. Novel pathogenesis-based therapies are in development, targeting GM-CSF signalling, immune modulation and cholesterol homeostasis.

Lung disease caused by ABCA3 mutations.

BACKGROUND: Knowledge about the clinical spectrum of lung disease caused by variations in the ATP binding cassette subfamily A member 3 (ABCA3) gene is limited. Here we describe genotype-phenotype correlations in a European cohort. METHODS: We retrospectively analysed baseline and outcome characteristics of 40 patients with two disease-causing ABCA3 mutations collected between 2001 and 2015. RESULTS: Of 22 homozygous (15 male) and 18 compound heterozygous patients (3 male), 37 presented with neonatal respiratory distress syndrome as term babies. At follow-up, two major phenotypes are documented: patients with (1) early lethal mutations subdivided into (1a) dying within the first 6 months or (1b) before the age of 5 years, and (2) patients with prolonged survival into childhood, adolescence or adulthood. Patients with null/null mutations predicting complete ABCA3 deficiency died within the 1st weeks to months of life, while those with null/other or other/other mutations had a more variable presentation and outcome. Treatment with exogenous surfactant, systemic steroids, hydroxychloroquine and whole lung lavages had apparent but many times transient effects in individual subjects. CONCLUSIONS: Overall long-term (>5 years) survival of subjects with two disease-causing ABCA3 mutations was <20%. Response to therapies needs to be ascertained in randomised controlled trials.

Whole lung lavage therapy for pulmonary alveolar proteinosis: a global survey of current practices and procedures.

BACKGROUND: Whole lung lavage (WLL) is the current standard of care treatment for patients affected by pulmonary alveolar proteinosis (PAP). However, WLL is not standardized and international consensus documents are lacking. Our aim was to obtain a factual portrayal of WLL as currently practiced with respect to the procedure, indications for its use, evaluation of therapeutic benefit and complication rate. METHODS: A clinical practice survey was conducted globally by means of a questionnaire and included 27 centers performing WLL in pediatric and/or adult PAP patients. RESULTS: We collected completed questionnaires from 20 centres in 14 countries, practicing WLL in adults and 10 centers in 6 countries, practicing WLL in pediatric patients. WLL is almost universally performed under general anesthesia, with a double-lumen endobronchial tube in two consecutive sessions, with an interval of 1-2 weeks between sessions in approximately 50 % of centres. The use of saline warmed to 37 °C, drainage of lung lavage fluid by gravity and indications for WLL therapy in PAP were homogenous across centres. There was great variation in the choice of the first lung to be lavaged: 50 % of centres based the choice on imaging, whereas 50 % always started with the left lung. The choice of position was also widely discordant; the supine position was chosen by 50 % of centres. Other aspects varied significantly among centres including contraindications, methods and timing of follow up, use of chest percussion, timing of extubation following WLL and lung isolation and lavage methods for small children. The amount of fluid used to perform the WLL is a critical aspect. Whilst a general consensus exists on the single aliquot of fluid for lavage (around 800 ml of warm saline, in adults) great variability exists in the total volume instilled per lung, ranging from 5 to 40 liters, with an average of 15.4 liters/lung. CONCLUSIONS: This international survey found that WLL is safe and effective as therapy for PAP. However these results also indicate that standardization of the procedure is required; the present survey represents the a first step toward building such a document.

GATA2 deficiency in children and adults with severe pulmonary alveolar proteinosis and hematologic disorders.

BACKGROUND: The majority of cases with severe pulmonary alveolar proteinosis (PAP) are caused by auto-antibodies against GM-CSF. A multitude of genetic and exogenous causes are responsible for few other cases. Goal of this study was to determine the prevalence of GATA2 deficiency in children and adults with PAP and hematologic disorders. METHODS: Of 21 patients with GM-CSF-autoantibody negative PAP, 13 had no other organ involvement and 8 had some form of hematologic disorder. The latter were sequenced for GATA2. RESULTS: Age at start of PAP ranged from 0.3 to 64 years, 4 patients were children. In half of the subjects GATA2-sequence variations were found, two of which were considered disease causing. Those two patients had the typical phenotype of GATA2 deficiency, one of whom additionally showed a previously undescribed feature - a cholesterol pneumonia. Hematologic disorders included chronic myeloic leukemia, juvenile myelo-monocytic leukemia, lymphoblastic leukemia, sideroblastic anemia and two cases of myelodysplastic syndrome (MDS). A 4 year old child with MDS and DiGeorge Syndrome Type 2 was rescued with repetitive whole lung lavages and her PAP was cured with heterologous stem cell transplant. CONCLUSIONS: In children and adults with severe GM-CSF negative PAP a close cooperation between pneumologists and hemato-oncologists is needed to diagnose the underlying diseases, some of which are caused by mutations of transcription factor GATA2. Treatment with whole lung lavages as well as stem cell transplant may be successful.

Facts and promises on lung biomarkers in interstitial lung diseases.

Interstitial lung diseases (ILDs) are a heterogeneous group of >100 pulmonary disorders. ILDs are characterized by an irreversible architectural distortion and impaired gas exchange; however, there is great variability in the clinical course. ILD diagnosis requires a combination of clinical data, radiological imaging and histological findings (when a lung biopsy is required). At the same time, successful management of ILD patients strictly depends on an accurate and confident diagnosis. In this context, the detection of reliable biomarkers able to identify ILD subtypes, avoiding lung biopsy, as well as the capacity to stratify patients and predict over time the disease course, has become a primary aim for all research studies in this field.

Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a severe autoimmune disease caused by autoantibodies that neutralize GM-CSF resulting in impaired function of alveolar macrophages. In this study, we characterize 21 GM-CSF autoantibodies from PAP patients and find that somatic mutations critically determine their specificity for the self-antigen. Individual antibodies only partially neutralize GM-CSF activity using an in vitro bioassay, depending on the experimental conditions, while, when injected in mice together with human GM-CSF, they lead to the accumulation of a large pool of circulating GM-CSF that remains partially bioavailable. In contrast, a combination of three non-cross-competing antibodies completely neutralizes GM-CSF activity in vitro by sequestering the cytokine in high-molecular-weight complexes, and in vivo promotes the rapid degradation of GM-CSF-containing immune complexes in an Fc-dependent manner. Taken together, these findings provide a plausible explanation for the severe phenotype of PAP patients and for the safety of treatments based on single anti-GM-CSF monoclonal antibodies.

Pulmonary alveolar proteinosis.

The term pulmonary alveolar proteinosis (PAP) comprises a heterogeneous group of rare disorders characterized by abundant deposition of surfactant- and lipoproteins in the alveoli. The autoimmune form accounts for 90% of cases and is characterized by the presence of GM-CSF autoantibodies. Secondary PAP is associated with several underlying conditions, mainly hematologic malignancies, infections and inhalation exposure, and is GM-CSF antibody negative. Several conditions can mimic PAP, in particular the radiological findings: the crazy paving pattern on high resolution computed tomography (HRCT) is common also to infections, neoplasms, and other interstitial lung diseases. Bronchoalveolar lavage (BAL) typical findings and the detection of serum GM-CSF antibodies are usually sufficient for the diagnosis of PAP. Whole lung lavage (WLL) is still the gold standard for treatment of PAP and is followed by complete remission in about 50% of cases. Inhalative treatment with GM-CSF alone or in combination with WLL could represent the future approach for patients with autoimmune PAP refractory to WLL alone. The anti CD-20 antibody rituximab represents a further promising approach for autoimmune PAP. The treatment of secondary PAP should be focused on the underlying disease.

Incomplete expression of epithelial-mesenchymal transition markers in idiopathic pulmonary fibrosis.

The hypothesis that epithelial-mesenchymal transition (EMT) contributes to the formation of fibroblast foci (FF), which are the histological hallmark and the site of active disease progression of Idiopathic Pulmonary Fibrosis (IPF), has not yet received a conclusive demonstration. Cells undergoing EMT lose epithelial features and acquire mesenchymal markers and morphology. Cadherin expression switch (from E to N) is one of the first events in EMT. We investigated the immunohistochemical expression of E- and N-cadherin, vimentin, fibronectin, laminin-5-γ2, α-smooth muscle actin, and fibroblast-specific protein-1 involved in EMT in 20 IPF lung biopsies, focusing on metaplastic squamous cells of bronchial basal origin, positive for laminin-5-γ2 and ΔNp63/p40, that cover FF. The results were compared with organizing pneumonia, reactive squamous cell metaplasia of bronchiolar epithelia, and squamous cell carcinoma. Bronchiolar basal metaplastic cells in IPF partially lost E-cadherin and expressed vimentin and fibronectin. Hyperplastic pneumocytes in IPF and controls coexpressed E-cadherin and N-cadherin, and were weakly positive for lam5-γ2. Reactive squamous cell metaplasia did not show any mesenchymal markers. Squamous cell carcinoma only expressed lam5-γ2. In IPF lungs, we observed two epithelial cell populations with a different expression profile of markers involved in EMT. Although neither hyperplastic pneumocytes nor bronchial basal cells showed evidence of complete EMT, only the latter seem to be specific for UIP and might have a role in its development.

Relationship between diffuse pulmonary fibrosis, alveolar proteinosis, and granulocyte-macrophage colony stimulating factor autoantibodies.

Extensive pulmonary fibrosis is a rare occurrence in pulmonary alveolar proteinosis. We report 2 cases that have interesting implications. A female patient was diagnosed with autoimmune pulmonary alveolar proteinosis that evolved over 7 years into diffuse fibrosis. In a male patient with diffuse fibrosis we incidentally detected electron microscopic features of alveolar surfactant accumulation and positive autoantibodies to granulocyte-macrophage colony stimulating factor. In the male patient we speculated that the pulmonary fibrosis might have been preceded by an asymptomatic phase of autoimmune pulmonary alveolar proteinosis, and that we should investigate the involvement of surfactant dysfunction in the pathogenesis of fibrotic lung disease.

Physical properties, lung deposition modeling, and bioactivity of recombinant GM-CSF aerosolised with a highly efficient nebulizer.

Pulmonary alveolar proteinosis (PAP) is a rare condition characterized by the accumulation of lipoproteinaceous material within air spaces. Although whole lung lavage is the current standard of care, recent advances in our understanding of PAP pathophysiology suggest that the disorder may benefit from inhalation of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). The aim of this study was to determine the physical properties and bioactivity of rGM-CSF aerosolised by the highly efficient AKITA² APIXNEB® nebulizer system. The physical properties of aerosolised rGM-CSF were investigated in terms of droplet size, output and output rate by laser diffraction and gravimetrical analysis. Lung deposition was assessed using deposition modeling (ICRP). Molecular mass before and after aerosolisation was determined by SDS-PAGE, while the bioactivity of rGM-CSF was evaluated by measuring the GM-CSF-stimulated increase in pSTAT5 using mAM-hGM-R cells. Ninety-six % of the rGM-CSF filling dose was aerosolised with the Akita² Apixneb® nebulizer system. Particle size was highly reproducible, and the amount deposited within the lung was 80.35% of the delivered dose. The aerosolisation did not alter the molecular structure of rGM-CSF, nor its ability to stimulate the pSTAT5, which increased by 99.5%, similar to values for rGM-CSF prior to aerosolisation. We conclude that the highly efficient AKITA² APIXNEB® nebulizer system is likely to efficaciously deliver rGM-CSF to the airways of patients with autoimmune PAP.