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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.

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., A concise educational review of pulmonary alveolar proteinosis (PAP), a rare respiratory syndrome with various and heterogeneous aetiologies, caused by the impairment of pulmonary surfactant clearance or by abnormal surfactant production https://bit.ly/3aFpQm9

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 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 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.

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.

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).

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.

Whole lung lavage (WLL) is currently the standard therapy for pulmonary alveolar proteinosis (PAP). Nevertheless, some PAP patients respond poorly to WLL or require it frequently. The present paper reports a patient with autoimmune PAP with persistent disease despite three WLL treatments over 10 months. Plasmapheresis with ten 1.5-L plasma exchanges was performed, which lowered the serum granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody level from 250 microg mL(-1) to 156 microg mL(-1) but did not improve respiratory impairment. Further WLL therapy was required and transiently effective. Serum GM-CSF autoantibody levels declined progressively, reaching a value of 56 microg mL(-1) 80 weeks after completion of plasmapheresis. However, this decrease was not accompanied by clinical improvement and the patient required additional WLL therapy. The results confirm that minor reductions in serum granulocyte-macrophage colony-stimulating factor autoantibody levels from plasmapheresis are not reflected in clinical improvement in the severity of lung disease in pulmonary alveolar proteinosis.

Background Interstitial lung disease occurring in children is a condition characterized by high frequency of cases due to genetic aberrations of pulmonary surfactant homeostasis, that are also believed to be responsible of a fraction of familial pulmonary fibrosis. To our knowledge, ABCA3 gene was not previously reported as causative agent of fibrosis affecting both children and adults in the same kindred. Methods We investigated a large kindred in which two members, a girl whose interstitial lung disease was first recognized at age of 13, and an adult, showed a diffuse pulmonary fibrosis with marked differences in terms of morphology and imaging. An additional, asymptomatic family member was detected by genetic analysis. Surfactant abnormalities were investigated at biochemical, and genetic level, as well as by cell transfection experiments. Results Bronchoalveolar lavage fluid analysis of the patients revealed absence of surfactant protein C, whereas the gene sequence was normal. By contrast, sequence of the ABCA3 gene showed a novel homozygous G > A transition at nucleotide 2891, localized within exon 21, resulting in a glycine to aspartic acid change at codon 964. Interestingly, the lung specimens from the girl displayed a morphologic usual interstitial pneumonitis-like pattern, whereas the specimens from one of the two adult patients showed rather a non specific interstitial pneumonitis-like pattern. Conclusions We have detected a large kindred with a novel ABCA3 mutation likely causing interstitial lung fibrosis affecting either young and adult family members. We suggest that ABCA3 gene should be considered in genetic testing in the occurrence of familial pulmonary fibrosis.

Background. Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease of unknown etiology characterized by interstitial fibrosis determining irreversible distortion of pulmonary architecture. Reactive oxygen species (ROS) and markers of oxidative stress play a pivotal role in human IPF pathology, possibly through induction of epithelial-mesenchymal transition (EMT).Methods. We investigated by immunohistochemistry, in UIP and COP tissue samples, the expression of most relevant markers of the molecular interplay involving RAGE, oxidant/antioxidant balance regulation, tissue nitrosylation, and mediators of EMT.Results. In both UIP and COP, the degree of RAGE expression was similarly high, while SODs and i-NOS, diffusely present in COP endoalveolar plugs, were almost absent in UIP fibroblast foci. A lower degree of tissue nitrosilation was observed in UIP than in COP.Conclusions. Fibroblast lesions of UIP and of COP share a similar degree of activation of RAGE, while antioxidant enzyme expression markedly reduced in UIP.

Pulmonary alveolar proteinosis is a rare condition characterized by the accumulation of lipoproteinaceous material within the airspaces, resulting in impaired gas transfer, and clinical manifestations ranging from asymptomatic to severe respiratory failure. To the best of the authors’ knowledge, there are only a few conditions whose natural history has been so dramatically changed by the influence of advances in basic science, clinical medicine, and translational research in therapeutic approaches. Whole-lung lavage is the current standard of care and it plays a critical role as a modifier factor of the natural history of proteinosis. That notwithstanding, the identification of autoantibodies neutralizing granulocyte-macrophage colony-stimulating factor in serum and lung of patients affected by the form of proteinosis previously referred to as idiopathic, has opened the way to novel therapeutic options, such as supplementation of exogenous granulocyte-macrophage colony-stimulating factor, or strategies aimed at reducing the levels of the autoantibodies. The aim of this paper is to provide an updated review of the current therapeutic approach to proteinosis.

Background AATD is one of the most common inherited disorders in the World. However, it is generally accepted that AATD in North African populations is not a risk factor for lung and/or liver disease, based on a number of small studies. We therefore planned a screening study for detection of AATD in patients with OLD in a cohort of patients from Kairouan in central Tunisia. Methods: One hundred twenty patients with OLD (asthma, emphysema, COPD) were enrolled in the screening programme. Laboratory diagnosis for AATD was performed according to current diagnostic standards. Results We found that 6/120 OLD patients carried an AAT deficient allele, 1 PI*MZ, 1 PI*MPlowel, 3 PI*MMmalton, 1 PI*MMwurzburg. Conclusion this pilot study demonstrated that alleles related to deficiency of AAT are not absent in the Tunisian population, and that rare AATD variants prevailed over commonest PI*Z variant. These results would support a larger scale screening for AATD in Tunisia.

The laboratory diagnosis of alpha 1 -antitrypsin (AAT) deficiency (AATD) has evolved over the last 40 years since the first cases of the disorder were reported. It is currently performed in specialized centers, and it requires a combination of different biochemical methods: nephelometric AAT concentration, isoelectric focusing, genotyping, and sequencing. The availability of matrices such as the dried blood spot have facilitated the implementation of laboratory analyses for AATD, but they have also challenged laboratories to develop more reliable and reproducible techniques starting from dried blood. In this article, we describe the protocols we have optimized for AATD diagnosis from dried blood spot, in an attempt to hopefully provide useful information for physicians and scientists involved in this diagnostic line. We also describe the diagnostic flowchart for AATD detection that we have developed accordingly.

The receptor for advanced glycation end products (RAGE) engages a number of ligands implicated in inflammatory processes. The RAGE coding gene maps to the 6p21.32 region, close to the genes DRB1 and BTNL2, which are associated with sarcoidosis.We investigated a possible implication of RAGE in sarcoid granulomas.RAGE and major ligands (N-epsilon-carboxy-methyl-lysine [CML], S100A12, and S100B) expression was investigated by immunostaining of 99 paraffin-embedded biopsies of sarcoid tissues, and expression patterns were determined. Among the three RAGE gene single-nucleotide polymorphisms investigated, -374 T/A was selected, characterized in terms of transcriptional effect (immunocytochemistry and real-time polymerase chain reaction), and its frequency was determined in DNA extracted from biopsies.RAGE, CML, S100A12, and S100B immunoreactivity was observed in all sarcoid granulomas, although at different intensities. The degree of RAGE expression significantly correlated with the degree of S100A12 expression. The -374 TT/AT genotypes, associated with higher RAGE transcriptional activity, were more frequent in the sarcoidosis biopsy group than in control subjects, and the association was confirmed in a second, independent series of 101 patients with sarcoidosis.We showed the association of RAGE and its ligands with sarcoidosis and suggest that an intrinsic genetic factor could be in part involved in its expression. In Italian patients, the -374 T/A polymorphism seems to be significantly associated with this disease.

Alpha1-antitrypsin deficiency (AATD) is a genetic condition associated with an increased risk of developing chronic obstructive pulmonary disease (COPD) early in life and, to a lesser extent, liver disease.1 Significant advances have been made during the last decades in understanding its epidemiology and it has been recently suggested that AATD is one of the commonest inherited disorders not only in Caucasians but also among other ethnic groups worldwide.2,3 Although AAT is a highly pleomorphic glycoprotein, with approximately 100 variants having being identified,4 two major deficient variants, namely Z and S, account for most cases of AATD, since the vast majority of such individuals carry the PI*ZZ or PI*SZ genotype, coding for approximately 15% and 25%, respectively, of normal AAT plasma levels. The establishment of international registries, including large series of AATD individuals,5,6 has allowed not only better definition of the epidemiology of AATD, but also more precise definition of the associated clinical phenotypes.5,7,8 Nevertheless, there are at least 30 AAT alleles other than the PI*Z and the PI*S alleles which are associated with significantly reduced or absent plasma AAT levels.9 Given the extreme rarity of such variants, often described in the literature as single case reports, little is known about their epidemiology and even less is known about the associated clinical phenotypes. The Italian Registry for Severe AATD was established in 1996 as a result of a nationwide screening programme sponsored by the two major Italian scientific respiratory societies.10 Although Italy is considered a country with a medium-low prevalence of AATD (mean PI*Z gene frequency: 0.0013),11 the programme succeeded in identifying a relatively large cohort of AATD individuals. During the development of the screening program, we noticed that, in addition to the groups of AATD individuals …

Background We have recently reported that homozygosity for the minor A-allele of RAGE (receptor for advanced glycation end products) −374T/A polymorphism may exert a protective effect toward the development of angiographic coronary artery disease (CAD). Here we focused on the putative involvement of this functional RAGE polymorphism on the severity of coronary atherosclerosis as assessed by angiography. Methods In a total of 234 consecutive Caucasian patients with angiographically proven CAD, the severity of coronary atherosclerosis was assessed by the number of diseased vessels (greater than 50% stenosis). Genotyping for the −374T/A variant was performed by means of PCR-RFLPs. Results The mean number of diseased vessels was significantly lower in patients with the AA genotype (1.47±0.68) than in those with the AT or TT genotype (1.88±0.82, p =0.029). After confounding variables were controlled for, the number of diseased vessels remained significantly different in the AA genotype carriers from that in the AT or TT carriers ( p =0.041, ANCOVA). Conclusions Our data suggest that the RAGE −374T/A polymorphism is one of the likely candidate determinants for the genetic variance of disease phenotype in coronary atherosclerosis.

The receptor for advanced glycation end products (RAGE) is thought to play a critical role in diabetic atherosclerosis. Accordingly, a functional -374T/A polymorphism in RAGE gene promoter has been associated with macrovascular complications in type 1 diabetic patients. However, the extent to which this common variant influences the risk of coronary artery disease (CAD) in the general population remains to be determined. We genotyped the -374T/A RAGE polymorphism in 259 non-diabetic individuals, of whom 175 had angiographically documented coronary artery disease (CAD patients) and 84 had normal coronary angiography (CAD-free control subjects). Homozygosity for the -374A allele was found in 9.7% of the CAD patients versus 22.6% of the CAD-free subjects (p=0.005). By means of a multiple logistic regression analysis, the AA genotype of the -374T/A polymorphism was shown to be independently associated with a reduced risk of CAD (adjusted odds ratio 0.33, 95% CI 0.15 to 0.73; p=0.006). Our observations suggest that the -374T/A polymorphism of the RAGE gene may reduce susceptibility to CAD, thus exerting a protective effect on coronary risk. Future pathophysiological studies may be worthwhile to clarify the mechanisms behind this association.

Inflammation plays an important role in the pathogenesis of atherosclerosis and coronary syndromes; moreover, various lines of evidence suggest that genetic factors contribute significantly to the risk of coronary artery disease (CAD). Through its effects on endothelial function, coagulation, insulin resistance and lipid metabolism, the proinflammatory cytokine TNF could be involved in cardiovascular pathophysiology. The aim of our study is to analyze whether TNF gene promoter (-308 G/A; −857 G/A) and TNF receptor polymorphisms (TNFR1 MspA1 I exon 1 and TNFR2 Nla III exon 6) show involvement in CAD predisposition in a group of Italian patients compared with healthy controls. Genotyping was performed by PCR-RFLP. Consecutive Italian patients with angiographically proven CAD (n= 248) were compared with controls (n=241), matched for age, sex and geographical origins. CAD patients showed a higher frequency of the TNF −308 A allele than healthy controls (p=0.046). After stratification according to risk factors for CAD, our analysis revealed that CAD patients with diabetes (p=0.042) and CAD patients without hypertension (p=0.0495) displayed a higher frequency of the TNF −308 AA genotype compared with healthy controls. Our data stress the inflammatory nature of CAD and show a possible involvement of TNF −308G/A promoter polymorphisms in the predisposition to the development of this disease. The less frequent A allele seems to be a predisposing factor for development of CAD in particular pathological settings associated with the disease itself, such as diabetes.