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Adult PAP patients experience similar #COVID19 rates to the general population, and high rates of hospitalisation and deaths, underscoring their vulnerability and the need for measures to prevent infection. The impact of iGM-CSF must be considered. https://bit.ly/3M0wKnZ.

BackgroundThe 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.MethodOver 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.ResultsAt 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, theABCA3sequence variants were missense variants, small insertions or deletions with in-silico tools predicting some residual ABCA3 transporter function.ConclusionThe natural history of ABCA3-related interstitial lung disease progresses during childhood and adolescence. Disease-modifying treatments are desirable to delay such disease course.

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/.

Introduction: Pulmonary alveolar proteinosis (PAP) is an ultra-rare syndrome the peculiarity of which is the accumulation of surfactant within the alveolar spaces, which can lead to respiratory fai...

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 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.

BackgroundPulmonary 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.MethodsWe 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.ResultsWe 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.ConclusionsOur 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.

International audience; 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.). Copyright © 2020 Massachusetts Medical Society.

A autoimmune Pulmonary Alveolar Proteinosis (PAP) patient with persistent disease underwent 3 Whole Lung Lavages (WLLs), 10 plasmapheresis sessions and further 3 WLL, from October 2004 to May 2007.

Background/Objectives: Increasing evidence suggests the importance of neuroinflammation in the pathogenesis of Alzheimer’s disease (AD), which is a complex neurodegenerative disorder. Complement activation occurs in the brain of patients with AD and seems to contribute to an important local inflammatory state. Increased expression of the fourth serum complement component 4 (C4) has been observed in AD patients in many studies. This protein has two isoforms, encoded by two genes: C4A and C4B localized to the HLA class III region. These genes exhibit copy number variations (CNVs) and this different gene copy number can influence C4 protein levels. We focalized our attention on these two genes, determining the distribution of CNVs in AD patients, compared with healthy controls, in order to analyse their possible involvement in AD pathogenesis. Methods: We investigated 191 AD patients and 300 healthy controls. The C4A and C4B copy numbers were assessed by quantitative PCR (qPCR). Results: The results obtained showed a statistically significant increase in the number of copies for both C4A and C4B in AD patients, compared with healthy controls (p Conclusion: The presence of high C4A and C4B copy numbers in AD patients could explain the increased C4 protein expression observed in AD patients, thus highlighting a possible role for C4A and C4B CNVs in the risk of developing AD.

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

Background: PAP is a ultra-rare syndrome characterized by the accumulation of surfactant material within the alveolar spaces. The current therapy with whole lung lavage (WLL) is still the gold standard even if clinical trials with inhaled rGM-CSF supplementation are ongoing. Aims: To prospectively study the Italian PAP population. Methods: We carried out a descriptive study of the baseline characteristics of the PAP patients enrolled in the Italian registry from 1989 until 2019. Results: We enrolled 126 PAP, among these 92.8% were affected by autoimmune (aPAP), while the remaining patients were divided in: hereditary (0.8%), secondary (3.2%) and PAP-like (3.2%). Considering the aPAP patients, the mean age at diagnosis was 43±14, with a male/female ratio = 2. Smoking history was reported in 66.7% of cases; 37.2% of them are currently smokers. 49 patients did not necessitate WLL treatment in the long-term follow up. The baseline pulmonary function test (PFT) (% of predicted value) of aPAP patients is shown in tab. 1, as mean±SD. Differences are significant also in a multivariate regression analysis, taking into account age, sex and smoking habit. Conclusions: The establishment of a national registry for PAP has enabled a detailed characterization of the natural course of the disease, moreover our results indicate that also PFT could recommend on when to treat, along with persistent or progressive respiratory failure and exercise desaturation.

Pulmonary alveolar microlithiasis (PAM) is caused by genetic variants in theWe collected DNA from 14 patients with PAM and four relatives, and analysed the coding regions ofWe identified eight novel allelic variants ofOur findings support a significant role for

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.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.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 (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.

Background: Genetic variants of TOLLIP and MUC5B have been reported to be associated with development and/or prognosis of idiopathic pulmonary fibrosis (IPF). Real-life experiences on the application of SNPs in the clinical management of IPF are lacking. This study was conducted to investigate the association of MUC5B and TOLLIP SNPs with disease course and outcome in a single-centre cohort. Patients and Methods: 50 IPF patients (M 43 and F 8, age 66±10 y) and 50 healthy controls (HC) (M 37, F 13, age42±2 y) were genotyped for SNPs within TOLLIP (rs3750920 and rs5743890) and MUC5B (rs35705950). Diagnosis of IPF was made according to the ATS/ERS guideline 2011. Genotype frequency was compared between IPF and HC subjects. Correlation of SNPs genotypes with survival, acute exacerbation or disease progression (a decline of ≥ 10% in FVC) was investigated. Results: TOLLIP SNPs genotype distribution did not differ between IPF and HC (p=0.6). MUC 5B T minor allele was more frequent in IPF subjects than in HC (67% vs 16% p Conclusion: While the minor allele T in MUC5B is associated with IPF, TOLLIP gene variants appear to correlate with disease progression, therefore being of potential utility to stratify IPF patients.

Rationale: PAP is a an ultra-rare disease where a perturbation in surfactant homeostasis results in its accumulation within airspaces, thus impairing gas transfer. WLL currently remains the therapeutic standard, even if it may induce complete resolution of the disorder only in 30% of patients. Aim: To characterize the gene network able to predict the outcome of the WLL, i.e. total resolution/persistent improvement vs transient resolution/progressive deterioration. Methods: We conducted a gene interaction network analysis, with web-based software (esyN), to calculate hub and bottleneck genes previously investigated by a microarray analysis (1), performed on the PBMCs of 16 PAP patients treated with WLL and followed for 24 months. Hub genes were identified by the number of interactions within the network. Bottleneck genes were identified by calculating the betweenness centrality index (BCI) for each gene in the network. BCI reflects the amount of control that a gene exerts over the interactions of other genes in the network. Results: We found that SMAD3, which acts as a mediator of the profibrotic signals initiated by TGF-β and SYNCRIP, which plays a role in multiple aspects of mRNA maturation, are top ranked hubs genes. Interestingly, SMAD3 is also the top ranked bottleneck gene. Conclusion: The SMAD3 transcription factor seems to be the central mediator and conductor of the gene network involved in the response to the WLL treatment in PAP patients. 1- M Zorzetto, et al. A gene network to predict the clinical response to whole lung lavage (WLL), in pulmonary alveolar proteinosis (PAP). ERS Annual Congress, Milano, Italy, September 9-13, 2017.

Rationale: PAP is a ultra-rare disease, where surfactant accumulation within alveolar spaces could lead to respiratory failure and death. WLL is the current standard treatment for PAP, nevertheless disease persistence after treatment is reported in 30% of cases. Aim: This work is aimed to identify a common gene co-expression network able to predict the outcome of the WLL: total resolution/persistent improvement vs transient resolution/progressive deterioration. Methods: We applied a weighted genes co-expression network analysis to transcriptional profiles of total RNA, collected from peripheral blood mononuclear cells (PBMC) of 16 PAP patients who underwent WLL. After a 24-month follow up, they were dichotomized in positive vs negative outcome and a microarray analysis, with around 20000 genes screened (SurePrint G3 Human Gene, Agilent) was performed. Finally Gene Ontology (GO), Reactome, KEGG, and Disease Ontology enrichment analysis was performed for differentially expressed genes (DEGs). Results: Four biologically meaningful modules of genes highly connected were identified. In particular, one of these (comprehending 111 genes with a p value Conclusion: For the first time we provide a modular profile of PAP which could explain the regulatory mechanism underlying the clinical outcome.

A autoimmune Pulmonary Alveolar Proteinosis (PAP) patient with persistent disease underwent 3 Whole Lung Lavages (WLLs), 10 plasmapheresis sessions and further 3 WLL, from October 2004 to May 2007. Nevertheless HRTC and pulmonary function test (PFT) showed a persistent residual disease of mild degree. At the beginning of 2010, the patient was admitted to inhaled rGM-CSF (Sargramostim) therapy as compassionate treatment. GM-CSF was administered by Akita 2 nebulizer (Vectura), as follow: 250 mcg/day every other week for 12 weeks, then 250 mcg/day on 2 consecutive days every 2 weeks for 6 months. Follow up visits were scheduled at 3, 10, 18, 30 months and after that once a year. Functional and HRCT data and PaO2 were collected. Since the start of the inhaled GM-CSF therapy, the patient no more required WLL. Furthermore we found a significant increase in DLCO% (p=0.013) and FVC% (p=0.023) while %FEV1 show a positive trend. No substantial differences in blood gas analysis. The pulmonary involvement at HRCT shows a significant decrease of lung infiltrates (p=0.039) in terms of pathological segments. These data underscore the utility of inhaled GM-CSF not only in case of progressing disease but also in case of refractory patients with persistent lung infiltrates, in order to increase the response rate.

Pulmonary alveolar proteinosis (PAP) is a diffuse pulmonary disease, characterised by the accumulation of lipoproteinaceous material in the distal air spaces, which results in impaired gas transfer. Autoimmune PAP accounts for the vast majority of cases in humans and is caused by autoantibodies directed towards granulocyte-macrophage colony-stimulating factor (GM-CSF), which causes a defect in the function of alveolar macrophages linked to the disruption of surfactant homeostasis. Whole lung lavage (WLL) is the current standard of care for PAP patients and although it is effective in the majority of cases, disease persistence is not an unusual outcome, even if airspace accumulation is well controlled by WLL. Even though WLL remains the current standard therapy for PAP, in this review we focus on novel treatment approaches for autoimmune PAP.

Background: A patient with autoimmune PAP with persistent disease underwent 3 WLL, 10 plasmapheresis cycles and further 3 WLL, from October 2004 to May 2007. Despite a substantial clinical and functional benefit, the radiographic appearance showed a partial resolution of lung infiltrates(1). At the beginning of 2010, after a worsening of respiratory conditions, the patient was admitted to inhaled GM-CSF (Sagramostin) therapy as compassionate treatment. Methods: GM-CSF, dispensed byAkita 2 nebulizer (Vectura),was administered following:250 mcg/day every other week for 12 weeks, then 250 mcg/day on 2 consecutive days every 2 weeks for 6 months. Follow up visits were scheduled at 3, 10, 18, 30 months and after that once a year. Functional and HRCT data and PaO2 were collected. Results: From the start of the inhalatory therapy the patient no more required WLL. Furthermore we found a significant increase in DLCO%(p=0.013) and FVC%(p=0.023) while FEV1% show a positive trend(Fig.1). No substantial differences in blood gas analysis. The pulmonary involvement at HRCT shows a significant decrease of lung infiltrates(p=0.039) in terms of pathological segments. Conclusion: These data underscore the utility of inhaled GM-CSF not only in case of progressing disease but also in case of persistence/stabilization, in order to increase response rate. 1.Luisetti et al. Eur Respir J 2009; 33: 1220–1222.

Background: aPAP is a rare disease characterized by neutralizing GM-CSF autoantibodies and alveolar surfactant accumulation. WLL is the standard therapy but results in complete resolution only in 30% of patients. We hypothesized that the combination WLL and inhaled hGM-CSF (Sargramostim) is more effective than WLL alone. Methods: We conducted a phase II clinical trial:aPAP patients were randomized to receive either WLL alone (group A) or WLL followed by inhaled sargramostim (250 mcg/day every other week for 12 weeks followed by 250 mcg/day on 2 consecutive days every 2 weeks for 6 months) (group B). GM-CSF was administered by the Akita 2 nebulizer (Vectura). Here, we report interim results obtained 30 months after WLL. Results: 18 aPAP patients (7 females/11 males) were randomized (equally) into each group. 7 in the initial group A required re-randomization because of recurrence while only 1 in the initial group B required re-randomization. Compared to the group A, the group B had significant improvement in pulmonary function including increases in mean (95% CI) DLCO%(15.7, 10.0-21.4), FVC%(11.8, 7.4-16.3), TLC%(10, 6.4-13.8), FEV1%(9.6, 5.5-13.7) (P Conclusion: Interim results demonstrate that inhaled sargramostim was well tolerated and effective in aPAP and that combination therapy is more effective than WLL alone. Funding: AIFA(FARM7MCPK4).

Background: WLL, the current standard treatment for patients affected by PAP, is not a standardized procedure. Aim: To describe WLL as currently practiced with respect to the procedure, indications for its use, evaluation of therapeutic benefit, and complication rate. Methods: We developed a questionnaire on several aspects of WLL and performed a global survey among centers performing WLL in either pediatric and/or adulthood setting. Results: We have collected expert opinions from 20 centers in 14 countries performing WLL in adults and 10 centers in 6 countries performing WLL in pediatric patients.In about half of centres, WLL is performed under general anesthesia with a double-lumen endobronchial tube in two consecutive sessions (one lung per session), with an interval of 1-2 weeks between procedures. Other common aspects are indications in PAP, use of saline warmed to 37 o C and drainage of Instilled fluid by gravity. Differences consisted of contraindications, methods and timing of follow up evaluation, choice of first lung to be lavaged, patient position, total volume of lavage per lung, use of chest percussion, timing of extubation following WLL, and lung isolation and lavage methods for small children. Conclusions: This international survey found that WLL is safe and effective as therapy of PAP. Results also indicate that standardization of the procedure is required.

PAP is a rare disorder characterized by the accumulation of lipoproteinaceous material within alveolar spaces. WLL is the current standard treatment for PAP patients and although it is effective in the majority of cases, disease persistence is not unusual. To identify blood biomarkers able to predict the outcome of the WLL: total resolution/persistent improvement (positive outcome) or transient resolution/progressive deterioration (negative outcome). We collected total RNA from peripheral mononuclear cells of 8 PAP patients who underwent WLL, at the baseline and 24 months from the lavage. After the 24-month follow up, they were dichotomized in positive vs negative outcome and a microarray analysis was performed. Co-expression eigengenes module search and Gene Ontology, Reactome, KEGG, and Disease Ontology enrichment analysis was performed for differentially expressed genes (DEGs). Our preliminary data show 86 significantly DEGs, between PAP patients with a positive vs negative outcome; whereas WLL seems not to induce difference in gene expression, in the same subject, at least after two years. The gene co-expression networks analysis, selected a module comprehending 764 genes, which showed a gene significance mean p value=0.04 and a strong relationship between pairwise gene connections and gene significance. The enrichment analysis showed a common predominance of the GO biological process and Reactome pathways. This preliminary study allows to identify key pathways whose different expression could discriminate between PAP patients with a positive outcome vs negative outcome. The validation of our results will lead us to select biomarkers useful to follow up the clinical response to WLL.

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.We retrospectively analysed baseline and outcome characteristics of 40 patients with two disease-causing ABCA3 mutations collected between 2001 and 2015.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.Overall long-term (5 years) survival of subjects with two disease-causing ABCA3 mutations was20%. Response to therapies needs to be ascertained in randomised controlled trials.

Idiopathic pulmonary fibrosis (IPF) is a severe interstitial lung disorder characterized by a pattern of Usual Interstitial Pneumonia where the presence of fibroblastic foci is the hallmark of the disease.In the present study, we analyzed the migration inhibitory factor (MIF) expression in lung tissue of IPF patients compared with healthy controls and organizing pneumonia (OP) patients focusing into MIF potential role in fibroblastic foci development.The immunohistochemical analysis was performed in 10 IPF patients (7 male), 3 OP patients (2 male), and 3 healthy controls (all male) using the streptavidin-biotin method (Dako).In IPF samples, MIF resulted overexpressed in the areas of active fibrosis and, in particular, in the alveolar epithelium, bronchiolar epithelium, and in the peripheral zones of fibroblastic foci. Bronchiolar epithelium from organizing pneumonia patients resulted only weakly positive for MIF while no evidence of MIF expression was reported for alveolar epithelium. In the control subject group, MIF was unexpressed except for a weak presence in the bronchiolar epithelium.In conclusion, MIF is a pleiotropic cytokine involved in the pathogenesis of IPF being mainly expressed in the areas of remodeling and active fibrosis, in bronchiolar and alveolar epithelium, and in the peripheral zone of fibroblastic foci.

Rationale: We identified a 6-year-old girl with pulmonary alveolar proteinosis (PAP), impaired granulocyte-macrophage colony–stimulating factor (GM-CSF) receptor function, and increased GM-CSF. Objectives: Increased serum GM-CSF may be useful to identify individuals with PAP caused by GM-CSF receptor dysfunction. Methods: We screened 187 patients referred to us for measurement of GM-CSF autoantibodies to diagnose autoimmune PAP. Five were children with PAP and increased serum GM-CSF but without GM-CSF autoantibodies or any disease causing secondary PAP; all were studied with family members, subsequently identified patients, and controls. Measurement and Main Results: Eight children (seven female, one male) were identified with PAP caused by recessive CSF2RA mutations. Six presented with progressive dyspnea of insidious onset at 4.8 ± 1.6 years and two were asymptomatic at ages 5 and 8 years. Radiologic and histopathologic manifestations were similar to those of autoimmune PAP. Molecular analysis demonstrated that GM-CSF signaling was absent in six and severely reduced in two patients. The GM-CSF receptor β chain was detected in all patients, whereas the α chain was absent in six and abnormal in two, paralleling the GM-CSF signaling defects. Genetic analysis revealed multiple distinct CSF2RA abnormalities, including missense, duplication, frameshift, and nonsense mutations; exon and gene deletion; and cryptic alternative splicing. All symptomatic patients responded well to whole-lung lavage therapy. Conclusions: CSF2RA mutations cause a genetic form of PAP presenting as insidious, progressive dyspnea in children that can be diagnosed by a combination of characteristic radiologic findings and blood tests and treated successfully by whole-lung lavage.

Objectives: Recent evidences suggest that the activation of peroxisome proliferator-activated receptor (PPAR)-γ2, which plays an important role in vascular homeostasis, also regulates the expression of the Receptor for Advanced Glycation End products (RAGE). In turn, low levels of soluble RAGE (sRAGE) have recently emerged as a valuable biomarker of vascular inflammation. The potential alterations in sRAGE concentrations in peripheral arterial disease (PAD), however, have not been yet investigated. The aim of the present study was to clarify whether the Pro12Ala polymorphism of the PPAR-γ2 gene is related to plasma sRAGE levels and the presence of PAD in nondiabetic Italian individuals. Design and methods: A total of 201 patients with PAD and 201 PAD-free control subjects were investigated. Genotyping of the Pro12Ala polymorphism of the PPAR-γ2 gene was performed by means of PCR-RFLPs. Plasma sRAGE levels were determined by ELISA. Results: Subjects carrying at least one Ala12 allele of the PPAR-γ2 gene had lower sRAGE levels (all p valuesb0.001). The prevalence rate of the Ala12 allele was significantly higher in PAD patients (14.0%) than in controls (8.0%, p=0.009). In multivariate logistic regression analysis after adjustment for potential confounders, the Ala12 allele was significantly and independently associated with the risk of PAD (OR=1.57, 95% CI=1.11–2.65, p=0.021). Conclusions: Our data indicate that the Ala12 allele of the PPAR-γ2 gene is associated with lower levels of the soluble decoy receptor sRAGE and the presence of PAD.

SummaryTargeted detection programmes are recommended to identify subjects affected by severe alpha1-antitrypsin deficiency (AATD). Guidelines are available to address physicians towards subjects at high risk for AATD. We wanted to investigate the clinical characteristics of subjects enrolled in the programme, who result as not being affected by severe AATD; this information is not available in the present literature. We elaborated data contained in the questionnaires accompanying the samples of 2127 Italian subjects submitted for AATD detection in a period spanning 11 years (1996–2006).A total of 588 subjects were eligible to enter this study: PI*MM subjects and subjects with intermediate AATD, referred for lung disease, were characterised by a relatively young mean age, and a high proportion (31.2% and 28.6%, respectively) were never smokers. Fifty percent or more had symptoms of chronic bronchitis, but without obstruction. Only a minority belonged to most severe GOLD stages. The mean levels of AAT varied as a function of the presence or absence of airflow obstruction in intermediate AATD subjects, but not in PI*MM. Individuals enrolled in AATD detection programmes represent an interesting cohort both for public health and research purposes.

Rationale: Whole lung lavage (WLL) is the current standard of care for PAP; WLL is not a standardized procedure, moreover it is highly invasive and is performed under general anesthesia. During the lavage, chest wall percussion is added when the outflow, initially milky, becomes clear. Lavage and percussion are continued until the outflow fluid became completely clear, which may take 3 hrs and an average of 15L saline for a single lung. The complications are rare, consisting mostly of hypoxemia, hydropneumothorax, ARDS, and post-procedure infections. Aims: Our aim is to demonstrate that the efficacy of WLL remains unaltered by using a reduced volume of saline. Methods: Functional data (FEV1, FVC, TLC, FEV1/VC, DLCO) and PaO2 were collected from 15 PAP patients who underwent WLL, at the baseline and 1,3,6 months after the lavage. The amount of 15L saline for single lung was used in 8 patients. In order to optimize WLL procedure, in 7 patients the infusion was reduced to 8L and chest percussion was started in concomitance with the first aliquot infusion. Results: We compared the parameters at the baseline and 1,3,6 months after the lavage and we did not find any statistically significant difference, although we can identify a positive trend of all the functional parameters considered, in the patients group lavaged with 8L saline. Conclusions: Our preliminary data suggest the possibility to reduce the infusion volume (with an earlier clapping) and consequently the time of the procedure. Considering the complexity of the WLL and the risk of a prolonged anesthesia, it could be an advantage in terms of safety of the procedure.

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.

Rationale: PAP is a rare disorder characterized by the accumulation of lipoproteinaceous material within alveolar spaces. The clinical course is variable, ranging from an asymptomatic presentation to respiratory failure and death. WLL is the current standard treatment for PAP patients and although it is effective in the majority of cases, disease persistence is not an unusual outcome. Although the introduction of the WLL has changed the natural history of PAP, little is known about the intimate mechanisms of action of WLL. Aims: Our aim is to identify blood biomarkers able to predict the outcome of the WLL: total resolution/persistent improvement (positive outcome) or transient resolution/progressive deterioration (negative outcome). Methods: We collected total RNA from peripheral mononuclear cells of 8 PAP patients who underwent WLL, at the baseline and 24 months from the lavage. After the 24-month follow up, they were dichotomized in positive vs negative outcome and a microarray analysis, with around 20000 genes screened (SurePrint G3 Human Gene, Agilent) was performed. Results: Our preliminary data show 86 genes significantly differentially expressed, between PAP patients with a positive outcome (n=4) vs negative outcome (n=4), which could be predictive of the response to the lavage; whereas WLL seems not to induce difference in gene expression, in the same subject, at least after two years. Conclusion: The identification of an expression signature, able to discriminate between PAP patients with a positive outcome vs negative outcome, will lead us to select biomarkers useful to follow up the clinical response to WLL. Funding: AIFA(FARM7MCPK4), eRARE(EuPAPNet).

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.

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.

SummaryClinical trials to evaluate patients affected by rare diseases are often hampered by the difficulty of recruiting a critical sample size. Registries for rare conditions are thus extremely powerful tools for overcoming recruitment problems. Here we present and discuss the international experience with alpha1-antitrypsin deficiency achieved by the Alpha One International Registry, and national experience obtained with a large series of patients with pulmonary alveolar proteinosis.