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Introduction: Pulmonary fibrosis is a severe disease which can be familial. A genetic cause can only be found in ∼40% of families. Searching for shared novel genetic variants may aid the discovery of new genetic causes of disease., Methods: Whole-exome sequencing was performed in 152 unrelated patients with a suspected genetic cause of pulmonary fibrosis from the St Antonius interstitial lung disease biobank. Variants of interest were selected by filtering for novel, potentially deleterious variants that were present in at least three unrelated pulmonary fibrosis patients., Results: The novel c.586G>A p.(E196K) variant in the ZCCHC8 gene was observed in three unrelated patients: two familial patients and one sporadic patient, who was later genealogically linked to one of the families. The variant was identified in nine additional relatives with pulmonary fibrosis and other telomere-related phenotypes, such as pulmonary arterial venous malformations, emphysema, myelodysplastic syndrome, acute myeloid leukaemia and dyskeratosis congenita. One family showed incomplete segregation, with absence of the variant in one pulmonary fibrosis patient who carried a PARN variant. The majority of ZCCHC8 variant carriers showed short telomeres in blood. ZCCHC8 protein was located in different lung cell types, including alveolar type 2 (AT2) pneumocytes, the culprit cells in pulmonary fibrosis. AT2 cells showed telomere shortening and increased DNA damage, which was comparable to patients with sporadic pulmonary fibrosis and those with pulmonary fibrosis carrying a telomere-related gene variant, respectively., Discussion: The ZCCHC8 c.586G>A variant confirms the involvement of ZCCHC8 in pulmonary fibrosis and short-telomere syndromes and underlines the importance of including the ZCCHC8 gene in diagnostic gene panels for these diseases., Competing Interests: Conflict of interest: C.I. de Bie received a one-time fee for a presentation from Boehringer Ingelheim. Conflict of interest: Leonie J.M. Vergouw received, via her insititution, a grant from the Longfibrose patiëntenvereniging/Pendersfonds. Conflict of interest: C.H.M. van Moorsel received, via her institution, funding from Boehringer Ingelheim for digital auscultation in pre-ILD and a lecture fee. Additionally, she is co-chair of the ClinGen ILD-GCEP. Conflict of interest: The remaining authors have nothing to disclose., (Copyright ©The authors 2024.)

In sporadic idiopathic pulmonary fibrosis (sIPF) and pulmonary fibrosis caused by a mutation in telomere (TRG-PF) or surfactant related genes (SRG-PF), there are a number of aberrant cellular processes known that can lead to fibrogenesis. We investigated whether RNA expression of genes involved in these processes differed between sIPF, TRG-PF, and SRG-PF and whether expression levels were associated with survival. RNA expression of 28 genes was measured in lung biopsies of 26 sIPF, 17 TRG-PF, and 6 SRG-PF patients. Significant differences in RNA expression of TGFBR2 ( p = 0.02) and SFTPA2 ( p = 0.02) were found between sIPF, TRG-PF, and SRG-PF. Patients with low (

The unknown etiology of sarcoidosis, along with the variability in organ involvement and disease course, complicates the effective treatment of this disease. Based on recent studies, the cellular inflammatory pathways involved in granuloma formation are of interest regarding possible new treatment options, such as the mechanistic (formerly mammalian) target of rapamycin complex 1 (mTORC1) pathway, the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, and the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome pathway. The aim of this study was to explore the potential coexpression of these three inflammatory pathways in patients with sarcoidosis and see whether possible differences were related to disease outcome. The tissue of 60 patients with sarcoidosis was used to determine the activity of these three signaling pathways using immunohistochemistry. The activation of NLRP3 was present in 85% of all patients, and the activation of mTORC1 and JAK/STAT was present in 49% and 50% of patients, respectively. Furthermore, the presence of NLRP3 activation at diagnosis was associated with a chronic disease course of sarcoidosis. Our finding of different new conceptual inflammatory tissue phenotypes in sarcoidosis could possibly guide future treatment studies using the available inhibitors of either NLRP3, JAK-STAT, and mTORC1 inhibitors in a more personalized medicine approach.

In only around 40% of families with pulmonary fibrosis (PF) a suspected genetic cause can be found. Genetic overlap analysis of Whole Exome Sequencing (WES) data may be a powerful tool to discover new shared variants in novel genes for PF. As a proof of principle, we first selected unrelated PF patients for whom a genetic variant was detected (n = 125) in established PF genes and searched for overlapping variants. Second, we performed WES (n = 149) and identified novel potentially deleterious variants shared by at least two unrelated PF patients. These variants were genotyped in validation cohorts (n = 2748). In 125 unrelated patients, a potentially deleterious variant was detected in known PF genes of which 15 variants in six genes overlapped, involving 51 patients. Overlap analysis of WES data identified two novel variants of interest: TOM1L2 c.421T > C p.(Y141H) and TDP1 c.1373dupG p.(S459fs*5), neither gene had been related to pulmonary fibrosis before. Both proteins were present in the alveolar epithelium. No apparent characteristics of telomere disease were observed. This study underlines the potential of searching for overlapping rare potentially deleterious variants to identify disease-associated variants and genes. A previously unreported variant was found in two putative new PF genes, but further research is needed to determine causality.

Background and Objective: Patients with progressive fibrosing interstitial lung disease (PF-ILD) are prone to early mortality compared with other phenotypes of ILD. The possible effect of smoking on survival has not been investigated yet. Furthermore, it is unknown what the effect of quantity of smoking is in PF-ILD. In this study, it was determined if quantity of smoking is associated with worse survival in patients with PF-ILD., Methods: Patients meeting the INBUILD trial-criteria for PF-ILD were included in this retrospective cohort study. Pack year (py) was tested as a prognostic variable with a multivariable Cox proportional hazard model. Also, median transplant-free survival was compared between heavy (≥20 pys) and mild-moderate smokers (0.1-19.9 pys)., Results: In PF-ILD (N = 377), the unadjusted and adjusted hazard ratio for py were significant, (1.014, 95% confidence interval (CI): 1.006-1.022, P < 0.001; 1.011, CI:1.002-1.021, P = 0.022 respectively). This translates to an 11%, 22%, or 44% higher risk for mortality for patients accumulating 10, 20 or 40 pys, respectively. Heavy smokers demonstrated a median transplant-free survival of 3.0 years, which was significantly reduced compared with mild-moderate smokers (3.8 years, P = 0.035). Additionally, more patients with emphysema were heavy smokers (N = 68) than never (N = 5, P < 0.001) or mild-moderate smokers (n = 21, p < 0.001)., Conclusion: In PF-ILD, a pack year is associated with an increased risk of mortality. Furthermore, quantity of smoking is associated with worse survival and higher prevalence of emphysema. Our data indicates that limiting amount of pys will provide a survival benefit in patients developing PF-ILD., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease, in which inflammation is thought to only play a secondary role. Several factors associated with acute exacerbations of IPF (AE-IPF) have been identified, including infections. This study investigated whether humoral immunodeficiency or increased inflammatory markers at diagnosis were associated with AE-IPF and survival., Methods: Four-hundred-and-nine patients diagnosed with IPF between 2011 and 2017 were retrospectively included. Immune status investigations at diagnosis included measurement of serum immunoglobulins (available in 38%), leukocyte and lymphocyte subsets in blood and bronchoalveolar lavage (BAL) fluid (available in 58%), as well as response to pneumococcal vaccination (available in 64%)., Results: Serum immunoglobulins or IgG subclass levels were below the lower limit of normal in 6%. The response to pneumococcal vaccination was severely impaired in 1%. Thirteen percent of patients developed an AE-IPF (4.7% per year). AE-IPF were associated with elevated lymphocytes in BAL fluid at diagnosis (p = 0.03). Higher serum IgA and IgG at diagnosis were associated with worse survival (p = 0.01; and p = 0.04), as were an increased BAL lymphocyte percentage (p = 0.005), and higher blood leukocytes and neutrophils (p = 0.01; and p = 0.0005). In a multivariate model, only BAL lymphocyte count retained statistical significance (p = 0.007)., Conclusion: The prevalence of humoral immunodeficiencies was low in patients with IPF and not associated with AE-IPF or survival. Elevated lymphocytes in BAL were associated with the development of AE-IPF and worse survival. Higher serum immunoglobulins and immune cells in blood were also associated with worse survival. The local immune response in the lungs may be a target for future therapies., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Familial pulmonary fibrosis (FPF) is a monogenic disease most commonly involving telomere- ( TERT ) or surfactant- ( SFTP ) related mutations. These mutations have been shown to alter lymphocytic inflammatory responses, and FPF biopsies with histological lymphocytic infiltrates have been reported. Recently, a model of a surfactant mutation in mice showed that the disease initially started with an inflammatory response followed by fibrogenesis. Since inflammation and fibrogenesis are targeted by different drugs, we investigated whether the degree of these two features co-localize or occur independently in different entities of FPF, and whether they influence survival. We quantified the number of lymphocyte aggregates per surface area, the extent of diffuse lymphocyte cell infiltrate, the number of fibroblast foci per surface area, and the percentage of fibrotic lung surface area in digitally scanned hematoxylin and eosin (H&E) sections of diagnostic surgical biopsies of patients with TERT- related FPF (TERT-PF; n = 17), SFTP -related FPF (SFTP-PF; n = 7), and sporadic idiopathic pulmonary fibrosis (sIPF; n = 10). For comparison, we included biopsies of patients with cellular non-specific interstitial pneumonia (cNSIP; n = 10), an inflammatory interstitial lung disease with high lymphocyte influx and usually responsive to immunosuppressive therapy. The degree of inflammatory cell infiltrate and fibrosis in TERT-PF and SFTP-PF was not significantly different from that in sIPF. In comparison with cNSIP, the extent of lymphocyte infiltrates was significantly lower in sIPF and TERT-PF, but not in SFTP-PF. However, in contrast with cNSIP, in sIPF, TERT-PF, and SFTP-PF, diffuse lymphocyte cell infiltrates were predominantly present and lymphocyte aggregates were only present in fibrotic areas (p < 0.0001). Furthermore, fibroblast foci and percentage of fibrotic lung surface were associated with survival ( p = 0.022 and p = 0.018, respectively), while this association was not observed for lymphocyte aggregates or diffuse lymphocytic infiltration. Inflammatory cells in diagnostic lung biopsies of TERT-PF, SFTP-PF, and sIPF were largely confined to fibrotic areas. However, based on inflammation and fibrosis, no differences were found between FPF and sIPF, substantiating the histological similarities between monogenic familial and sporadic disease. Furthermore, the degree of fibrosis, rather than inflammation, correlates with survival, supporting that fibrogenesis is the key feature for therapeutic targeting of FPF., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 van Batenburg, van Oosterhout, Knoppert, Kazemier, van der Vis, Grutters, Goldschmeding and van Moorsel.)

C-C chemokine receptor 5 (CCR5) and polymorphisms in CCR5 gene are associated with sarcoidosis and Löfgren's syndrome. Löfgren's syndrome is an acute and usually self-remitting phenotype of sarcoidosis. We investigated whether the single nucleotide polymorphism (SNP) rs1799987 is associated with susceptibility for Löfgren's syndrome and has an effect on CCR5 expression on monocytes and function of CCR5. A total of 106 patients with Löfgren's syndrome and 257 controls were genotyped for rs1799987. Expression of CCR5 on monocytes was measured by flowcytometry. We evaluated calcium influx kinetics following stimulation upon N-formylmethionyl-leucyl-phenylalanine (fMLP) and macrophage inflammatory protein-1α (MIP-1α) on monocytes by measuring the median fluorescence intensity (MFI). The frequency of the G allele of rs1799987 was significantly higher in Löfgren's syndrome than in healthy controls ( p = 0.0015, confidence interval (CI) 1.22-2.32, odds ratio (OR) 1.680). Patients with a GG genotype showed higher CCR5 expression on monocytes than patients with the AA genotype ( p = 0.026). A significantly ( p = 0.027) lower count of patients with the GG genotype showed a calcium influx reaction to simulation upon MIP-1 α, compared with patients with the AA genotype. The rs1799987 G allele in CCR5 gene is associated with susceptibility to Löfgren's syndrome and with quantitative and qualitative changes in CCR5, potentially effecting the inflammatory response.

Pulmonary fibrosis is strongly associated with telomere shortening and increased DNA damage. Key cells in the pathogenesis involve alveolar type 2 (AT2) cells, club cells and myofibroblasts; however, to what extent these cells are affected by telomere shortening and DNA damage is not yet known. We sought to determine the degree of, and correlation between, telomere shortening and DNA damage in different cell types involved in the pathogenesis of progressive fibrosing interstitial lung disease. Telomere length and DNA damage were quantified, using combined fluorescence in situ hybridisation and immunofluorescence staining techniques, in AT2 cells, club cells and myofibroblasts of controls and patients with pulmonary fibrosis and a telomerase reverse transcriptase mutation (TERT-PF), idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (fHP). In IPF and TERT-PF lungs, AT2 cells contained shorter telomeres and expressed higher DNA damage signals than club cells and myofibroblasts. In fHP lungs, club cells contained highly elevated levels of DNA damage, while telomeres were not obviously short. In vitro , we found significantly shorter telomeres and higher DNA damage levels only in AT2 surrogate cell lines treated with telomerase inhibitor BIBR1532. Our study demonstrated that in IPF and TERT-PF lungs, telomere shortening and accumulation of DNA damage primarily affects AT2 cells, further supporting the importance of AT2 cells in these diseases, while in fHP the particularly high telomere-independent DNA damage signals in club cells underscores its bronchiolocentric pathogenesis. These findings suggest that cell type-specific telomere shortening and DNA damage may help to discriminate between different drivers of fibrogenesis., Competing Interests: Conflict of interest: A.A. van Batenburg has nothing to disclose. Conflict of interest: K.M. Kazemier has nothing to disclose. Conflict of interest: M.F.M. van Oosterhout reports personal fees from Roche outside the submitted work. Conflict of interest: J.J. van der Vis has nothing to disclose. Conflict of interest: J.C. Grutters has nothing to disclose. Conflict of interest: R. Goldschmeding has nothing to disclose. Conflict of interest: C.H.M. van Moorsel has nothing to disclose., (Copyright ©ERS 2021.)

Rationale: A subset of patients with idiopathic pulmonary fibrosis (IPF) contains short leukocyte telomeres or telomere related mutations. We previously showed that alveolar type 2 cells have short telomeres in fibrotic lesions. Our objectives were to better understand how telomere shortening associates with fibrosis in IPF lung and identify a subset of patients with telomere-related disease., Methods: Average telomere length was determined in multiple organs, basal and apical lung, and diagnostic and end-stage fibrotic lung biopsies. Alveolar type 2 cells telomere length was determined in different areas of IPF lungs., Results: In IPF but not in controls, telomere length in lung was shorter than in other organs, providing rationale to focus on telomere length in lung. Telomere length did not correlate with age and no difference in telomere length was found between diagnostic and explant lung or between basal and apical lung, irrespective of the presence of a radiological apicobasal gradient or fibrosis. Fifteen out of 28 IPF patients had average lung telomere length in the range of patients with a telomerase (TERT) mutation, and formed the IPFshort group. Only in this IPFshort and TERT group telomeres of alveolar type 2 cells were extremely short in fibrotic areas. Additionally, whole exome sequencing of IPF patients revealed two genetic variations in RTEL1 and one in PARN in the IPFshort group., Conclusions: Average lung tissue telomere shortening does not associated with fibrotic patterns in IPF, however, approximately half of IPF patients show excessive lung telomere shortening that is associated with pulmonary fibrosis driven by telomere attrition., Competing Interests: The authors have declared that no competing interests exist.

Telomeres are small repetitive DNA sequences at the ends of chromosomes which act as a buffer in age-dependent DNA shortening. Insufficient telomere repeats will be recognized as double-strand breaks. Presently, it is becoming more evident that telomere attrition, whether or not caused by mutations in telomere maintenance genes, plays an important role in many inflammatory and age-associated diseases. In this report, a method to (semi)quantitatively assess telomere length and DNA double-strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue is described. Therefore, a novel combination of quantitative fluorescence in situ hybridization, tissue elution, and immunofluorescence staining techniques was developed. Caveolin-1 (type 1 pneumocytes), pro-surfactant protein C (type 2 pneumocytes), club cell-10 (club cells), and alpha smooth muscle actin (smooth muscle cells) markers were used to identify cell types. To visualize all the different probes, restaining the tissue by heat-mediated slide elution is essential. Fluorescent signals of telomeres and DNA double-strand breaks were quantified using the Telometer plugin of ImageJ. As example, we analyzed lung tissue from a familial pulmonary fibrosis patient with a mutation in the telomere-associated gene poly(A)-specific ribonuclease ( PARN). The protocol displays a novel opportunity to directly quantitatively link DNA double-strand breaks to telomere length in specific FFPE cells.

Telomere maintenance dysfunction has been implicated in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). However, the mechanism of how telomere length is related to fibrosis in the lungs is unknown. Surgical lung biopsies of IPF patients typically show a heterogeneous pattern of non-fibrotic and fibrotic areas. Therefore, telomere length (TL) in both lung areas of patients with IPF and familial interstitial pneumonia was compared, specifically in alveolar type 2 (AT2) cells. Fluorescent in situ hybridization was used to determine TL in non-fibrotic and fibrotic areas of 35 subjects. Monochrome multiplex quantitative polymerase chain reaction (MMqPCR) was used for 51 whole lung biopsies and blood TL measurements. For sporadic IPF subjects, AT2 cell TL in non-fibrotic areas was 56% longer than in fibrotic areas. No such difference was observed in the surrounding lung cells. In subjects carrying a telomerase reverse transcriptase (TERT) mutation, AT2 cell TL was significantly shorter than in sporadic subjects. However, no difference in surrounding cell TL was observed between these subject groups. Finally, using biopsy MMqPCR TL measurements, it was determined that IPF subjects with shortest lung TL had a significantly worse survival than patients with long TL. This study shows that shortening of telomeres critically affects AT2 cells in fibrotic areas, implying TL as a cause of fibrogenesis. Furthermore, short lung telomere length is associated with decreased survival.

Background and Objective: A common polymorphism in the MUC5B gene (rs35705950) is associated with susceptibility to idiopathic pulmonary fibrosis (IPF) and familial interstitial pneumonia (FIP). We investigated predisposition of the MUC5B polymorphism to fibrotic interstitial pneumonias in Dutch Caucasian patient cohorts. Furthermore, we investigated the correlation between MUC5B genotype and survival in these cohorts., Methods: Sporadic IPF (spIPF, n = 115), FIP (n = 55), idiopathic non-specific interstitial pneumonia (iNSIP, n = 43), connective tissue disease associated interstitial pneumonia (CTD&#95;IP, n = 35) and a control cohort (n = 249) were genotyped for rs35705950., Results: Rs35705950 minor allele frequency (MAF) in controls was 0.09. Case-control analysis showed significant allelic association with spIPF (MAF = 0.27; P = 5.0 × 10(-10)), FIP (MAF = 0.30; P = 2.7 × 10(-9)) and iNSIP (MAF = 0.22; P = 3.4 × 10(-4)). No association was observed in CTD&#95;IP (MAF = 0.07). FIP subgroup analysis revealed an association between MUC5B and telomerase mutated FIP (P = 0.003), and between MUC5B and FIP with unknown genetic cause (P = 1.2 × 10(-8)). In spIPF carriership of MUC5B minor allele did not influence survival. In FIP MUC5B minor allele carriers had better survival (non-carriers 37 vs carriers 53 months, P = 0.01). In iNSIP survival analysis showed an opposite effect. Worse survival was found in iNSIP patients that carried the MUC5B minor allele (non-carriers 118 vs carriers 46 months, P = 0.027) CONCLUSION: This study showed that MUC5B minor allele predisposes to spIPF, FIP and iNSIP. In spIPF, survival is not influenced by MUC5B alleles. In FIP, MUC5B minor allele predicts better survival, pointing towards a subgroup of FIP patients with a milder, MUC5B-driven form of pulmonary fibrosis., (© 2015 Asian Pacific Society of Respirology.)

Background: Interstitial lung disease (ILD) is a heterogeneous group of rare diseases that primarily affect the pulmonary interstitium. Studies have implicated a role for telomere length (TL) maintenance in ILD, particularly in idiopathic interstitial pneumonia (IIP). Here, we measure TL in a wide spectrum of sporadic and familial cohorts of ILD and compare TL between patient cohorts and control subjects., Methods: A multiplex quantitative polymerase chain reaction method was used to measure TL in 173 healthy subjects and 359 patients with various ILDs, including familial interstitial pneumonia (FIP). The FIP cohort was divided into patients carrying TERT mutations, patients carrying SFTPA2 or SFTPC mutations, and patients without a proven mutation (FIP-no mutation)., Results: TL in all cases of ILD was significantly shorter compared with those of control subjects (P range: .038 to < .0001). Furthermore, TL in patients with idiopathic pulmonary fibrosis (IPF) was significantly shorter than in patients with other IIPs (P = .002) and in patients with sarcoidosis (P < .0001). Within the FIP cohort, patients in the FIP-telomerase reverse transcriptase (TERT) group had the shortest telomeres (P < .0001), and those in the FIP-no mutation group had TL comparable to that of patients with IPF (P = .049). Remarkably, TL of patients with FIP-surfactant protein (SFTP) was significantly longer than in patients with IPF, but similar to that observed in patients with other sporadic IIPs., Conclusions: The results show telomere shortening across all ILD diagnoses. The difference in TL between the FIP-TERT and FIP-SFTP groups indicates the distinction between acquired and innate telomere shortening. Short TL in the IPF and FIP-no mutation groups is indicative of an innate telomere-biology defect, while a stress-induced, acquired telomere shortening might be the underlying process for the other ILD diagnoses.

Idiopathic pulmonary fibrosis (IPF) is a rare and devastating lung disease of unknown aetiology. Genetic variations in the IL1RN gene, encoding the interleukin-1 receptor antagonist (IL-1Ra), have been associated with IPF susceptibility. Several studies investigated the variable number tandem repeat (VNTR) or single nucleotide polymorphisms rs408392, rs419598 and rs2637988, with variable results. The aim of this study was to elucidate the influence of polymorphisms in IL1RN on IPF susceptibility and mRNA expression. We performed a meta-analysis of the five case-control studies that investigated an IL1RN polymorphism in IPF in a Caucasian population. In addition, we investigated whether IL1RN mRNA expression was influenced by IL1RN polymorphisms. The VNTR, rs408392 and rs419598 were in tight linkage disequilibrium, with D' > 0.99. Furthermore, rs2637988 was in linkage disequilibrium with the VNTR (D' = 0.90). A haploblock of VNTR*2 and the minor alleles of rs408392and rs419598 was constructed. Meta-analysis revealed that this VNTR*2 haploblock is associated with IPF susceptibility both with an allelic model (odds ratio = 1.42, p = 0.002) and a carriership model (odds ratio = 1.60, p = 0.002). IL1RN mRNA expression was significantly influenced by rs2637988, with lower levels found in carriers of the (minor) GG genotype (p < 0.001). From this meta-analysis, we conclude that the VNTR*2 haploblock is associated with susceptibility to IPF. In addition, polymorphisms in IL1RN influence IL-1Ra mRNA expression, suggesting that lower levels of IL-1Ra predispose to developing IPF. Together these findings demonstrate that the cytokine IL-1Ra plays a role in IPF pathogenesis.

Idiopathic pulmonary fibrosis (IPF) is a devastating and progressive lung disease. Its aetiology is thought to involve damage to the epithelium and abnormal repair. Alveolar epithelial cells near areas of remodelling show an increased expression of proapoptotic molecules. Therefore, we investigated the role of genes involved in cell cycle control in IPF. Genotypes for five single nucleotide polymorphisms (SNPs) in the tumour protein 53 (TP53) gene and four SNPs in cyclin-dependent kinase inhibitor 1A (CDKN1A), the gene encoding p21, were determined in 77 IPF patients and 353 controls. In peripheral blood mononuclear cells (PBMC) from 16 healthy controls mRNA expression of TP53 and CDKN1A was determined. Rs12951053 and rs12602273, in TP53, were significantly associated with survival in IPF patients. Carriers of a minor allele had a 4-year survival of 22% versus 57% in the non-carrier group (p = 0.006). Rs2395655 and rs733590, in CDKN1A, were associated with an increased risk of developing IPF. In addition, the rs2395655 G allele correlated with progression of the disease as it increased the risk of a rapid decline in lung function. Functional experiments showed that rs733590 correlated significantly with CDKN1A mRNA expression levels in healthy controls. This is the first study to show that genetic variations in the cell cycle genes encoding p53 and p21 are associated with IPF disease development and progression. These findings support the idea that cell cycle control plays a role in the pathology of IPF. Variations in TP53 and CDKN1A can impair the response to cell damage and increase the loss of alveolar epithelial cells.

Background: Caveolin 1 (Cav-1) is the primary structural component of cell membrane invaginations called 'caveolae'. Expression of Cav-1 is implicated in the pathogenesis of pulmonary fibrosis. Genetic polymorphisms in the CAV1 gene influence the function of Cav-1 in malignancies and associate with renal allograft fibrosis. Chronic allograft rejection after lung transplantation, called 'bronchiolitis obliterans syndrome' (BOS), is also characterised by the development of fibrosis.In this study, we investigated whether CAV1 genotypes associate with BOS and whether Cav-1 serum levels are influenced by the CAV1 genotype and can be used as a biomarker to predict the development of BOS., Methods: Twenty lung transplant recipients with BOS (BOSpos), ninety without BOS (BOSneg) and four hundred twenty-two healthy individuals donated DNA samples. Four SNPs in CAV1 were genotyped. Serial Cav-1 serum levels were measured in a matched cohort of 10 BOSpos patients and 10 BOSneg patients. Furthermore, single-time point Cav-1 serum levels were measured in 33 unmatched BOSneg patients and 60 healthy controls., Results: Homozygosity of the minor allele of rs3807989 was associated with an increased risk for BOS (odds ratio: 6.13; P = 0.0013). The median Cav-1 serum level was significantly higher in the BOSpos patients than in the matched BOSneg patients (P = 0.026). Longitudinal analysis did not show changes in Cav-1 serum levels over time in both groups. The median Cav-1 serum level in the group of 43 BOSneg patients was lower than that in the healthy control group (P = 0.046).In lung transplant recipients, homozygosity of the minor allele of rs3807989 and rs3807994 was associated with increased Cav-1 serum levels., Conclusion: In lung transplant recipients, the CAV1 SNP rs3807989 was associated with the development of BOS and Cav-1 serum levels were influenced by the CAV1 genotype.

Pulmonary fibrosis is defined by an overgrowth of fibroblasts and extracellular matrix deposition, and results in respiratory dysfunction that is often fatal. It is the end stage in many chronic inflammatory interstitial lung diseases (ILD) such as sarcoidosis and idiopathic pulmonary fibrosis (IPF). The myeloid-related proteins (MRPs) belong to the S100 family of calcium-binding proteins and are highly expressed by neutrophils, macrophages and epithelial cells during chronic inflammation. MRP14 stimulates fibroblast proliferation in vitro and is expressed in granulomas from sarcoidosis patients. We hypothesized that MRP14 may be a biomarker for fibrotic interstitial lung diseases. The objective of this study was to investigate whether levels of MRP14 in the bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis and IPF correlate with clinical parameters. We used an enzyme-linked immunosorbent assay (ELISA) to measure MRP14 in BALF of 74 sarcoidosis patients, 54 IPF patients and 19 controls. Mean BALF levels of MRP14 were elevated significantly in IPF (P < 0.001) and sarcoidosis (P < 0.05) patients compared to controls. MRP14 levels were associated linearly with sarcoidosis disease severity based on chest radiographic stage. Moreover, BALF MRP14 levels were correlated inversely with diffusion capacity and forced vital capacity in sarcoidosis patients. In IPF patients, a correlation with BALF neutrophil percentage was found. In conclusion, BALF MRP14 levels are elevated in IPF and sarcoidosis and are associated with disease severity in sarcoidosis. The results support the need for further studies into the role of MRP14 in the pathogenesis of lung fibrosis.

The integrin alpha(E)beta(7) is believed to play a key role in retention of lymphocytes in mucosal tissues of gut, urogenital tract and lung. Five common single nucleotide polymorphisms spanning ITGAE, the gene encoding the alpha(E) (CD103) unit, were genotyped in 556 sarcoidosis patients and 465 controls. The -1088 A/G polymorphism was associated with sarcoidosis (P=0.004). An increased risk of disease was found for homozygous carriers of the A allele vs. carriers of the G allele (P=0.001, odds ratio=1.63 [1.22-2.17]). Analysis of lymphocytes from bronchoalveolar lavage and in vitro functional tests showed higher percentages of CD103+CD4+ T cells for the sarcoidosis risk genotype. Radiographic staging at disease outcome revealed prevalence of -1088 AA genotype in patients with fibrosis (P=0.01). A higher proportion of CD103+CD4+ T cells and ITGAE -1088 AA genotype might be associated with fibrosis formation in pulmonary sarcoidosis.

Systemic inflammation can be investigated by changes in expression profiles of neutrophil receptors. Application of this technology for analysis of neutrophil phenotypes in diseased tissues is hampered by the absence of information regarding the modulation of neutrophil phenotypes after extravasation to tissues under non-inflammatory conditions. To fill this gap we measured the expression of neutrophil receptors in bronchoalveolar lavage fluid (BALF) and in the peripheral blood of healthy volunteers, which included both smokers and non-smokers. Blood and BALF neutrophils were identified by CD16(bright)/CD45(dim) cells, and triple-stained with antibodies directed against integrins, chemokine- and Fc gamma-receptors. BALF neutrophils of healthy volunteers showed an activated phenotype characterized by Mac-1 (CD11b)(bright), L-selectin (CD62L)(dim), intercellular adhesion molecule 1 (ICAM-1) (CD54)(bright), Fc gamma RII (CD32)(bright), C5a receptor (CD88)(bright) and CD66b(bright). A similar phenotype was observed for BALF neutrophils of patients affected by sarcoidosis. Furthermore, our results demonstrate a modulated expression of C5a receptor (CD88) and ICAM-1 (CD54) in neutrophils of sarcoidosis patients. In conclusion, our data indicate that neutrophils found in the lung exhibit an activated phenotype under both homeostatic and inflammatory conditions.

Treatment failure in pediatric acute lymphoblastic leukemia (ALL) is related to cellular resistance to glucocorticoids (eg, prednisolone). Recently, we demonstrated that genes associated with glucose metabolism are differentially expressed between prednisolone-sensitive and prednisolone-resistant precursor B-lineage leukemic patients. Here, we show that prednisolone resistance is associated with increased glucose consumption and that inhibition of glycolysis sensitizes prednisolone-resistant ALL cell lines to glucocorticoids. Treatment of prednisolone-resistant Jurkat and Molt4 cells with 2-deoxy-D-glucose (2-DG), lonidamine (LND), or 3-bromopyruvate (3-BrPA) increased the in vitro sensitivity to glucocorticoids, while treatment of the prednisolone-sensitive cell lines Tom-1 and RS4; 11 did not influence drug cytotoxicity. This sensitizing effect of the glycolysis inhibitors in glucocorticoid-resistant ALL cells was not found for other classes of antileukemic drugs (ie, vincristine and daunorubicin). Moreover, down-regulation of the expression of GAPDH by RNA interference also sensitized to prednisolone, comparable with treatment with glycolytic inhibitors. Importantly, the ability of 2-DG to reverse glucocorticoid resistance was not limited to cell lines, but was also observed in isolated primary ALL cells from patients. Together, these findings indicate the importance of the glycolytic pathway in glucocorticoid resistance in ALL and suggest that targeting glycolysis is a viable strategy for modulating prednisolone resistance in ALL.

L-asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia (ALL). The effectiveness is thought to result from depletion of asparagine in serum and cells. We investigated the clinical response in vivo of 1000 IU/m(2) pegylated (PEG)-asparaginase and its pharmacokinetic, pharmacodynamic and intracellular effects in children with newly diagnosed ALL before start of combination chemotherapy. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common/pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (P=0.01) and BCRABL-positive ALL (P=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-asparaginase (P=0.02) and both were prognostic factors for long-term event-free survival (hazard ratio 6.4, P=0.004; hazard ratio 3.7, P=0.01). After administration of one in vivo dose of PEG-asparaginase no changes in apoptotic parameters or in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. One additional dose of PEG-asparaginase upfront ALL treatment did not lead to other severe toxicities.

New prognostic factors may result in better risk classification and improved treatment of children with acute lymphoblastic leukemia (ALL). Recently, high expression of a gene named OPAL1 (outcome predictor in acute leukemia) was reported to be associated with favorable prognosis in ALL. Therefore, we investigated whether OPAL1 expression was of prognostic importance in 2 independent cohorts of children with ALL treated on Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL)-92/97 (n = 180) and St Jude Total 13 protocols (n = 257). We observed a consistently higher (2.8-fold) expression of OPAL1 in TEL-AML1-positive ALL compared with TEL-AML1-negative ALL in both cohorts, but higher OPAL1 expression was not consistently associated with other favorable prognostic indicators such as age and white blood cell count, or ALL genetic subtype. Lower OPAL1 expression was also not associated with increased in vitro drug resistance. Multivariate analyses including known risk factors showed that OPAL1 expression was not independently related to prognosis in either the COALL or St Jude cohorts. In conclusion, OPAL1 expression may not be an independent prognostic feature in childhood ALL, and its previously reported prognostic impact appears to be treatment dependent.

Childhood acute lymphoblastic leukemia (ALL) consists of various subtypes that respond differently to cytotoxic drugs and therefore have a markedly different clinical outcome. We used microarrays to investigate, in 190 children with ALL at initial diagnosis, whether 70 key apoptosis genes were differentially expressed between leukemic subgroups defined by lineage, genetic subtype, in vitro drug resistance, and clinical outcome. The expression of 44 of 70 genes was significantly different in T-versus B-lineage ALL, 22 genes differed in hyperdiploid versus nonhyperdiploid, 16 in TEL-AML1-positive versus-negative, and 13 in E2A-rearranged versus germ-line B-lineage ALL. Expression of MCL1 and DAPK1 was significantly associated with prednisolone sensitivity, whereas BCL2L13, HRK, and TNF were related to L-asparaginase resistance. BCL2L13 overexpression was also associated with unfavorable clinical outcome (P < .001). Multivariate analysis including known risk factors revealed that BCL2L13 expression was an independent prognostic factor (P = .011). The same trend was observed in a validation group of 92 children with ALL treated on a different protocol at St Jude (P = .051). In conclusion, ALL subtypes have a unique expression pattern of apoptosis genes and our data suggest that selective genes are linked to cellular drug resistance and prognosis in childhood B-lineage ALL.

Drug resistance in childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) is associated with impaired ability to induce apoptosis. To elucidate causes of apoptotic defects, we studied the protein expression of Apaf-1, procaspases-2, -3, -6, -7, -8, -10, and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) in cells from children with acute lymphoblastic leukemia (ALL; n = 43) and acute myeloid leukemia (AML; n = 10). PARP expression was present in all B-lineage samples, but absent in 4 of 15 T-lineage ALL samples and 3 of 10 AML cases, which was not caused by genomic deletions. PARP expression was a median 7-fold lower in T-lineage ALL (P < .001) and 10-fold lower in AML (P < .001) compared with B-lineage ALL. PARP expression was 4-fold lower in prednisolone, vincristine and L-asparaginase (PVA)-resistant compared with PVA-sensitive ALL patients (P < .001). Procaspase-2 expression was 3-fold lower in T-lineage ALL (P = .022) and AML (P = .014) compared with B-lineage ALL. In addition, procaspase-2 expression was 2-fold lower in PVA-resistant compared to PVA-sensitive ALL patients (P = .042). No relation between apoptotic protease-activating factor 1 (Apaf-1), procaspases-3, -6, -7, -8, -10, and drug resistance was found. In conclusion, low baseline expression of PARP and procaspase-2 is related to cellular drug resistance in childhood acute lymphoblastic leukemia.

Resistance of leukemic cells to chemotherapeutic agents is associated with an unfavorable outcome in pediatric acute lymphoblastic leukemia (ALL). To investigate the underlying mechanisms of cellular drug resistance, the activation of various apoptotic parameters in leukemic cells from 50 children with ALL was studied after in vitro exposure with 4 important drugs in ALL therapy (prednisolone, vincristine, l-asparaginase, and daunorubicin). Exposure to each drug resulted in early induction of phosphatidylserine (PS) externalization and mitochondrial transmembrane (Deltapsim) depolarization followed by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) inactivation in the majority of patients. For all 4 drugs, a significant inverse correlation was found between cellular drug resistance and (1) the percentage of cells with PS externalization (<.001 < P <.008) and (2) the percentage of cells with Deltapsim depolarization (.002 < P <.02). However, the percentage of cells with caspase-3 activation and the percentage of cells with PARP inactivation showed a significant inverse correlation with cellular resistance for prednisolone (P =.001; P =.001) and l-asparaginase (P =.01; P =.001) only. This suggests that caspase-3 activation and PARP inactivation are not essential for vincristine- and daunorubicin-induced apoptosis. In conclusion, resistance to 4 unrelated drugs is associated with defect(s) upstream or at the level of PS externalization and Deltapsim depolarization. This leads to decreased activation of apoptotic parameters in resistant cases of pediatric ALL.

Purpose: To confirm the prognostic value of a drug resistance profile combining prednisolone, vincristine, and l-asparaginase (PVA) cytotoxicity in an independent group of children with acute lymphoblastic leukemia (ALL) treated with a different protocol and analyzed at longer follow-up compared with our previous study of patients treated according to the Dutch Childhood Leukemia Study Group (DCLSG) ALL VII/VIII protocol., Patients and Methods: Drug resistance profiles were determined in 202 children (aged 1 to 18 years) with newly diagnosed ALL who were treated according to the German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL)-92 protocol., Results: At a median follow-up of 6.2 years (range, 4.1 to 9.3 years), the 5-year disease-free survival probability (pDFS) rate +/- SE was 69% +/- 7.0%, 83% +/- 4.4%, and 84% +/- 6.8% for patients with resistant (PVA score 7 to 9), intermediate-sensitive (PVA score 5 to 6), and sensitive (SPVA score 3 to 4) profiles, respectively (sensitive and intermediate-sensitive v resistant, P

Contradictory data have been reported about the prognostic value of myeloid antigen co-expression (My+) in childhood acute lymphoblastic leukaemia (ALL). In the present study the methyl thiazol tetrazoliumbromide (MTT) assay was used to compare the in vitro cytotoxicity of 14 drugs between 60 My+ (CD13+ and/or CD33+) and 107 My- ALL children at initial diagnosis. P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), major vault protein/lung resistance protein (LRP) and the intracellular daunorubicin concentration were studied by flow cytometry. My+ ALL samples were significantly more resistant, i.e. between 1.1- and 2.9-fold, to daunorubicin, doxorubicin, idarubicin, mitoxantrone, vincristine, 6-thioguanine, 6-mercaptopurine, teniposide, etoposide and ifosfamide compared with My- ALL samples. My- and My+ ALL did not significantly differ in sensitivity to prednisolone, dexamethasone, L-asparaginase and cytarabine. Comparable results were found when only common and preB ALL cases were analysed. Drug resistance in My+ ALL was not related to increased expression of P-gp, MRP or LRP compared with My- ALL (ratio My+/My-:P-gp 0.8, MRP 1.0, LRP 1.1). Accumulation and retention of daunorubicin did not significantly differ between My- and My+ ALL cells (ratio My+/My-: accumulation 1.2, retention 1.3). Therefore the nature of drug resistance in My+ ALL remains unknown. The lack of prognostic value for My+ in childhood ALL may be explained by the responsiveness of My+ ALL to glucocorticoids, L-asparaginase and cytarabine. In addition, the currently intensive treatment regimens may apply drug doses which are simply high enough to overcome the mild resistance to anthracyclines, mitoxantrone, vincristine, thiopurines, epipodophyllotoxins and ifosfamide in childhood My+ ALL.

Expression of three major classes of glutathione S-transferases (GSTs), i.e. alpha, mu and pi class, P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) were studied in childhood acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and normal peripheral blood lymphocytes by flow cytometry. In vitro cytotoxicity of 4-hydroxy-ifosfamide (IFOS), daunorubicin (DNR) and prednisolone (PRED) was assessed by the MTT assay. Expression of alpha, mu and pi class GST did not significantly differ between leukaemic cells from 100 initial and 14 unrelated relapse ALL patients (GSTalpha P=026; GSTmu P=O009; GSTpi P=0.13). The expression of GSTalpha (1.4-fold, P=0.0004), GSTpi (13-fold, P = 0001) and to a lesser extent also GSTmu (1.1-fold, P=0.03) was higher in ALL compared with normal peripheral blood lymphocytes. Expression of GSTmu and GST7pi was significantly higher in 18 AML compared with 100 ALL patients at initial diagnosis (respectively 1.3-fold, P=0.0005 and 2-fold, P<0.0001). In contrast, GSTalpha was median 2-fold lower expressed in the AML samples (P< 0.0001). Expression levels of alpha, mu and pi class GSTs were not related to the degree of resistance to IFOS, DNR and PRED nor to immunophenotype, white blood cell count or age at presentation of childhood ALL. One exception was a remarkably low expression of GSTalpha in IFOS-sensitive samples compared with a heterogenous expression in IFOS-resistant samples (P= 0.02). Expression of GSTpi, but not of GSTalpha or GSTmu, weakly correlated with the expression of MRP (Rs 0.36, P = 0.002, n = 74) but not with P-gp. However, a high expression of both GSTpi and MRP was not associated with in vitro resistance to IFOS, DNR or PRED. The present data suggest that expression of GSTs is not linked to the degree of resistance to IFOS, DNR and PRED or clinical risk factors in childhood ALL. Whether the high expression of GSTmu and GSTpi in AML cells contributes to the relative resistance to IFOS, DNR and PRED compared with ALL samples (P < or = 0.0001) warrants further study.

Cellular drug resistance is related to a poor prognosis in childhood leukemia, but little is known about the underlying mechanisms. We studied the expression of P-glycoprotein (P-gp), multidrug resistance (MDR)-associated protein (MRP), and major vault protein/lung resistance protein (LRP) in 141 children with acute lymphoblastic leukemia (ALL) and 27 with acute myeloid leukemia (AML) by flow cytometry. The expression was compared between different types of leukemia and was studied in relation with clinical risk indicators and in vitro cytotoxicity of the MDR-related drugs daunorubicin (DNR), vincristine (VCR), and etoposide (VP16) and the non-MDR-related drugs prednisolone (PRD) and L-asparaginase (ASP). In ALL, P-gp, MRP, and LRP expression did not differ between 112 initial and 29 unrelated relapse samples nor between paired initial and relapse samples from 9 patients. In multiple relapse samples, LRP expression was 1.6-fold higher compared with both initial (P = .026) and first relapse samples (P = .050), which was not observed for P-gp and MRP. LRP expression was weakly but significantly related to in vitro resistance to DNR (Spearman's rank correlation coefficient 0.25, P = .016) but not to VCR, VP16, PRD, and ASP. No significant correlations were found between P-gp or MRP expression and in vitro drug resistance. Samples with a marked expression of two or three resistance proteins did not show increased resistance to the tested drugs compared with the remaining samples. The expression of P-gp, MRP, and LRP was not higher in initial ALL patients with prognostically unfavorable immunophenotype, white blood cell count, or age. The expression of P-gp and MRP in 20 initial AML samples did not differ or was even lower compared with 112 initial ALL samples. However, LRP expression was twofold higher in the AML samples (P < .001), which are more resistant to a variety of drugs compared with ALL samples. In conclusion, P-gp and MRP are unlikely to be involved in drug resistance in childhood leukemia. LRP might contribute to drug resistance but only in specific subsets of children with leukemia.

Cytosine arabinoside (Ara-C) activation to cytosine arabinoside triphosphate (Ara-CTP) and subsequent incorporation into DNA is regulated by the pyrimidine nucleotides UTP, CTP and dCTP. Inhibition of the de novo synthesis of these pyrimidine nucleotides by N-(phosphon)-acetyl-L-aspartate (PALA) may enhance the cytotoxicity of Ara-C. We therefore studied the effect of PALA on Ara-C cytotoxicity and on Ara-CTP accumulation and incorporation into DNA on cell lines and patient samples. Fifty micromolar PALA increased the growth inhibitory effect of Ara-C in U937 cells several fold both with pre- and coincubation. Ara-C cytotoxicity was not potentiated by PALA in Hl60 cells. However, coincubation with PALA did not enhance Ara-CTP accumulation both in HL60 and U937 cells, nor affect Ara-C incorporation into DNA. Ara-C cytotoxicity to leukemic blast cells from 11 untreated patients with different types of leukemia was only modulated to a small extent by high PALA concentrations in only two cases. Ara-CTP accumulation in leukemic blast cells varied from non-detectable levels to 200 pmol/10(6) cells. Fifty micromolar PALA enhanced the accumulation of Ara-CTP significantly in only one patient with no apparent effect on UTP and CTP levels. Raising PALA to 500 microM decreased UTP and CTP levels to 50% but had no effect on Ara-CTP levels. In conclusion, modulation by PALA of Ara-C cytotoxicity and metabolism is limited in leukemic cells, both in culture and from patients. This suggests the possibility for selective modulation of other agents by PALA on non-hematological cells.

The effect of low (25 per cent of energy) and high (35 per cent of energy) fat diets with either low (less than 0.4) or high (greater than 1.0) polyunsaturated/saturated fatty acid (P/S) ratios on fatty acid compositions of plasma cholesterol esters and neutrophil phospholipids, and leukotriene production was studied in four groups of healthy volunteers supplemented with 6 g fish oil daily for four weeks. Except for three subjects, eicosapentaenoic acid (20:5n-3) content markedly increased from baseline in the plasma cholesterol ester fraction and to a lesser extent in the neutrophil phospholipid fraction. The increase in the plasma cholesterol ester fraction was inversely, though weakly related to the dietary intake of linoleic acid (18:2n-6). At supplementation endpoints, the 20:5n-3/20:4n-6 ratio in both plasma cholesterol esters and neutrophil phospholipids was highest in the groups consuming diets with low P/S ratio. In vitro leukotriene B5 production by neutrophils, did not differ between groups and there was no consistent suppression of LTB4 production in this four-week study. It is suggested that factors other than the actual dietary 18:2n-6 intake additionally influence the accumulation of 20:5n-3 in tissue during dietary fish oil supplementation.