Functional assessment and phenotypic heterogeneity of SFTPA1 and SFTPA2 mutations in interstitial lung diseases and lung cancer

2020 ◽  
Vol 56 (6) ◽  
pp. 2002806 ◽  
Author(s):  
Marie Legendre ◽  
Afifaa Butt ◽  
Raphaël Borie ◽  
Marie-Pierre Debray ◽  
Diane Bouvry ◽  
...  
Keyword(s):  
Lung Transplantation ◽  
Lung Tissue ◽  
Lung Diseases ◽  
Ex Vivo ◽  
Phenotypic Expression ◽  
Surfactant Protein ◽  
Interstitial Lung Diseases ◽  
Incomplete Penetrance ◽  
Tissue Samples

IntroductionInterstitial lung diseases (ILDs) can be caused by mutations in the SFTPA1 and SFTPA2 genes, which encode the surfactant protein (SP) complex SP-A. Only 11 SFTPA1 or SFTPA2 mutations have so far been reported worldwide, of which five have been functionally assessed. In the framework of ILD molecular diagnosis, we identified 14 independent patients with pathogenic SFTPA1 or SFTPA2 mutations. The present study aimed to functionally assess the 11 different mutations identified and to accurately describe the disease phenotype of the patients and their affected relatives.MethodsThe consequences of the 11 SFTPA1 or SFTPA2 mutations were analysed both in vitro, by studying the production and secretion of the corresponding mutated proteins and ex vivo, by analysing SP-A expression in lung tissue samples. The associated disease phenotypes were documented.ResultsFor the 11 identified mutations, protein production was preserved but secretion was abolished. The expression pattern of lung SP-A available in six patients was altered and the family history reported ILD and/or lung adenocarcinoma in 13 out of 14 families (93%). Among the 28 SFTPA1 or SFTPA2 mutation carriers, the mean age at ILD onset was 45 years (range 0.6–65 years) and 48% underwent lung transplantation (mean age 51 years). Seven carriers were asymptomatic.DiscussionThis study, which expands the molecular and clinical spectrum of SP-A disorders, shows that pathogenic SFTPA1 or SFTPA2 mutations share similar consequences for SP-A secretion in cell models and in lung tissue immunostaining, whereas they are associated with a highly variable phenotypic expression of disease, ranging from severe forms requiring lung transplantation to incomplete penetrance.

scholarly journals Modern problems of interstitial lung diseases in children

2021 ◽  
Vol 66 (3) ◽  
pp. 5-11
Author(s):  
Yu. L. Mizernitskiy ◽  
N. S. Lev
Keyword(s):  
Respiratory Failure ◽  
Lung Transplantation ◽  
Lung Tissue ◽  
Lung Diseases ◽  
Clinical Medicine ◽  
Interstitial Lung Diseases ◽  
Severe Respiratory Failure ◽  
Large Group ◽  
Current State

Interstitial lung diseases refer to a large group of diseases with a severe course and unfavorable prognosis, since the most forms of these diseases cause irreversible fibrosis of the lung tissue and severe respiratory failure. Lung transplantation remains the only way to save the patient’s.The article highlights the current state of the problem, achievements and prospects in the study, diagnosis and approaches to the treatment of interstitial lung diseases in children. The authors emphasize that this direction is one of the most relevant in pediatric pulmonology and clinical medicine in general.

scholarly journals In vitro models of fetal lung development to enhance research into congenital lung diseases

2021 ◽  
Author(s):  
Soichi Shibuya ◽  
Jessica Allen-Hyttinen ◽  
Paolo De Coppi ◽  
Federica Michielin
Keyword(s):  
Lung Development ◽  
Lung Diseases ◽  
Ex Vivo ◽  
Fetal Lung ◽  
Branching Morphogenesis ◽  
In Vitro Models ◽  
Normal Lung ◽  
Novel Therapeutic Strategies ◽  
Pseudoglandular Stage

Abstract Purpose This paper aims to build upon previous work to definitively establish in vitro models of murine pseudoglandular stage lung development. These can be easily translated to human fetal lung samples to allow the investigation of lung development in physiologic and pathologic conditions. Methods Lungs were harvested from mouse embryos at E12.5 and cultured in three different settings, i.e., whole lung culture, mesenchyme-free epithelium culture, and organoid culture. For the whole lung culture, extracted lungs were embedded in Matrigel and incubated on permeable filters. Separately, distal epithelial tips were isolated by firstly removing mesothelial and mesenchymal cells, and then severing the tips from the airway tubes. These were then cultured either in branch-promoting or self-renewing conditions. Results Cultured whole lungs underwent branching morphogenesis similarly to native lungs. Real-time qPCR analysis demonstrated expression of key genes essential for lung bud formation. The culture condition for epithelial tips was optimized by testing different concentrations of FGF10 and CHIR99021 and evaluating branching formation. The epithelial rudiments in self-renewing conditions formed spherical 3D structures with homogeneous Sox9 expression. Conclusion We report efficient protocols for ex vivo culture systems of pseudoglandular stage mouse embryonic lungs. These models can be applied to human samples and could be useful to paediatric surgeons to investigate normal lung development, understand the pathogenesis of congenital lung diseases, and explore novel therapeutic strategies.

Surfactant metabolism in surfactant protein A-deficient mice

1997 ◽  
Vol 272 (3) ◽  
pp. L479-L485 ◽  
Author(s):  
M. Ikegami ◽  
T. R. Korfhagen ◽  
M. D. Bruno ◽  
J. A. Whitsett ◽  
A. H. Jobe
Keyword(s):  
Lung Tissue ◽  
Protein A ◽  
Surfactant Protein ◽  
Normal Lung ◽  
Tissue Slices ◽  
Normal Lung Function ◽  
Deficient Mice ◽  
Surfactant Metabolism

In the present study we asked if surfactant metabolism was altered in surfactant protein (SP) A-deficient mice in vivo. Although previous studies in vitro demonstrated that SP-A modulates surfactant secretion and reuptake by type II cells, mice made SP-A deficient by homologous recombination grow and reproduce normally and have normal lung function. Alveolar and lung tissue saturated phophatidylcholine (Sat PC) pools were 50 and 26% larger, respectively, in SP-A(-/-) mice than in SP-A(+/+) mice. Radiolabeled choline and palmitate incorporation into lung Sat PC was similar both in vivo and for lung tissue slices in vitro from SP-A(+/+) and SP-A(-/-) mice. Percent secretion of radiolabeled Sat PC was unchanged from 3 to 15 h, although SP-A(-/-) mice retained more labeled Sat PC in the alveolar lavages at 48 h (consistent with the increased surfactant pool sizes). Clearance of radiolabeled dipalmitoylphosphatidylcholine and SP-B from the air spaces after intratracheal injection was similar in SP-A(-/-) and SP-A(+/+) mice. Lack of SP-A had minimal effects on the overall metabolism of Sat PC or SP-B in mice.

scholarly journals Evaluation of the efficacy of a novel perfusion solution for normothermic ex vivo lung perfusion compared with Steen solution™ (animal experimental study)

2021 ◽  
Vol 23 (3) ◽  
pp. 82-89
Author(s):  
S. V. Gautier ◽  
O. M. Tsirulnikova ◽  
I. V. Pashkov ◽  
N. V. Grudinin ◽  
D. O. Oleshkevich ◽  
...  
Keyword(s):  
Lung Transplantation ◽  
Lung Diseases ◽  
Ex Vivo ◽  
Infectious Complications ◽  
Lung Perfusion ◽  
Ex Vivo Lung Perfusion ◽  
Perfusion Solution ◽  
Number Of Patients ◽  
Donor Lung ◽  
End Stage

Respiratory diseases, together with infectious complications and hereditary lung diseases, rank third in international mortality statistics. Today, lung transplantation is a recognized method of treating end-stage lung diseases. However, the number of transplant surgeries performed is not much. This is down to the high requirements on the condition of a potential lung donor and directly on the quality of the donor lung. This has significantly limited the number of optimal donors. Rehabilitation of donor lungs to optimal gas exchange indicators can be achieved and objectively assessed in the course of ex vivo lung perfusion (EVLP). The EVLP procedure is widespread in leading transplantation centers in Europe and North America. It allows to significantly expand the pool of donor lungs, thereby serving a greater number of patients in need of lung transplantation. The possibility of EVLP procedure using publicly available perfusion equipment was demonstrated. The optimized protocol fully demonstrated its reliability and efficiency. The developed perfusion solution had no statistically significant differences in comparison with the Steen SolutionTM, which in the future will serve as an alternative for EVLP procedure.

scholarly journals SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Gabrielle Sherella Dijksteel ◽  
Peter H. Nibbering ◽  
Magda M. W. Ulrich ◽  
Esther Middelkoop ◽  
Bouke K. H. L. Boekema
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptides ◽  
Antimicrobial Agents ◽  
Ex Vivo ◽  
Residual Activity ◽  
Gram Negative Bacteria ◽  
Tissue Samples ◽  
Gram Negative ◽  
Viable Bacteria

Abstract Background Accurate determination of the efficacy of antimicrobial agents requires neutralization of residual antimicrobial activity in the samples before microbiological assessment of the number of surviving bacteria. Sodium polyanethol sulfonate (SPS) is a known neutralizer for the antimicrobial activity of aminoglycosides and polymyxins. In this study, we evaluated the ability of SPS to neutralize residual antimicrobial activity of antimicrobial peptides [SAAP-148 and pexiganan; 1% (wt/v) in PBS], antibiotics [mupirocin (Bactroban) and fusidic acid (Fucidin) in ointments; 2% (wt/wt))] and disinfectants [2% (wt/wt) silver sulfadiazine cream (SSD) and 0.5% (v/v) chlorhexidine in 70% alcohol]. Methods Homogenates of human skin models that had been exposed to various antimicrobial agents for 1 h were pipetted on top of Methicillin-resistant Staphylococcus aureus (MRSA) on agar plates to determine whether the antimicrobial agents display residual activity. To determine the optimal concentration of SPS for neutralization, antimicrobial agents were mixed with PBS or increasing doses of SPS in PBS (0.05–1% wt/v) and then 105 colony forming units (CFU)/mL MRSA were added. After 30 min incubation, the number of viable bacteria was assessed. Next, the in vitro efficacy of SAAP-148 against various gram-positive and gram-negative bacteria was determined using PBS or 0.05% (wt/v) SPS immediately after 30 min incubation of the mixture. Additionally, ex vivo excision wound models were inoculated with 105 CFU MRSA for 1 h and exposed to SAAP-148, pexiganan, chlorhexidine or PBS for 1 h. Subsequently, samples were homogenized in PBS or 0.05% (wt/v) SPS and the number of viable bacteria was assessed. Results All tested antimicrobials displayed residual activity in tissue samples, resulting in a lower recovery of surviving bacteria on agar. SPS concentrations at ≥0.05% (wt/v) were able to neutralize the antimicrobial activity of SAAP-148, pexiganan and chlorhexidine, but not of SSD, Bactroban and Fucidin. Finally, SPS-neutralization in in vitro and ex vivo efficacy tests of SAAP-148, pexiganan and chlorhexidine against gram-positive and gram-negative bacteria resulted in significantly higher numbers of CFU compared to control samples without SPS-neutralization. Conclusions SPS was successfully used to neutralize residual activity of SAAP-148, pexiganan and chlorhexidine and this prevented an overestimation of their efficacy.

scholarly journals Vitamin D alleviates oxidative stress in adipose tissue and mesenteric vessels from obese patients with subclinical inflammation

2020 ◽  
Vol 98 (2) ◽  
pp. 85-92 ◽  
Author(s):  
Mihaela Ionica ◽  
Oana M. Aburel ◽  
Adrian Vaduva ◽  
Alexandra Petrus ◽  
Sonia Rațiu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Vitamin D ◽  
Adipose Tissue ◽  
Vascular Function ◽  
Ex Vivo ◽  
Active Form ◽  
Reactive Oxygen Species Generation ◽  
Obese Patients ◽  
Tissue Samples

Obesity is an age-independent, lifestyle-triggered, pandemic disease associated with both endothelial and visceral adipose tissue (VAT) dysfunction leading to cardiometabolic complications mediated via increased oxidative stress and persistent chronic inflammation. The purpose of the present study was to assess the oxidative stress in VAT and vascular samples and the effect of in vitro administration of vitamin D. VAT and mesenteric artery branches were harvested during abdominal surgery performed on patients referred for general surgery (n = 30) that were randomized into two subgroups: nonobese and obese. Serum levels of C-reactive protein (CRP) and vitamin D were measured. Tissue samples were treated or not with the active form of vitamin D: 1,25(OH)2D3 (100 nmol/L, 12 h). The main findings are that in obese patients, (i) a low vitamin D status was associated with increased inflammatory markers and reactive oxygen species generation in VAT and vascular samples and (ii) in vitro incubation with vitamin D alleviated oxidative stress in VAT and vascular preparations and also improved the vascular function. We report here that the serum level of vitamin D is inversely correlated with the magnitude of oxidative stress in the adipose tissue. Ex vivo treatment with active vitamin D mitigated obesity-related oxidative stress.

Leveraging RB status to define therapy for castrate-resistant prostate cancer.

2014 ◽  
Vol 32 (4_suppl) ◽  
pp. 96-96
Author(s):  
Renee de Leeuw ◽  
Clay de Comstock ◽  
Daniela de Pollutri ◽  
Matthew Joseph Schiewer ◽  
Stephen J Ciment ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Tissue Samples ◽  
Ki67 Staining ◽  
Cell Architecture ◽  
Castrate Resistant Prostate Cancer ◽  
Castrate Resistant

96 Background: Loss of retinoblastoma (RB) tumor suppressor is overrepresented in castrate-resistant prostate cancer (CRPC) compared to primary PCa. We previously showed using analyses of human tissue and in vitro and in vivo modeling that RB constrains androgen receptor (AR) function, and that loss of RB is sufficient promote resistance to castration and AR antagonists. Thus, novel strategies are needed to treat RB-deficient tumor. By contrast, in tumors retaining RB, suppressing enhancing RB activity would be of therapeutic advantage, and may be accomplished through next-generation Cdk4/6 inhibitors. Methods: Stable isogenic pairs of prostate cancer cell lines either retaining RB or RB depleted (by shRNA) were assessed in vitro and in xenografts for response to Cdk4/6 kinase inhibitors or the cabazitaxel. In addition, using an ex vivo explant assay, fresh tumor tissue samples from radical prostatectomy were exposed to the Cdk4/6 inhibitor or cabazitaxel for up to 7 days, and evaluated by IHC for Ki67, Caspase-3, and AR. Results: Cdk4/6 inhibition blocks tumor cell proliferation dependent on RB status. This was further confirmed ex vivo, as evidenced by a marked reduction in Ki67 staining in Cdk4/6 inhibitor treated explant tissue from two prostate cancer patients. Conversely, in vitro studies revealed a modest sensitization of RB-depleted tumors to cabazitaxel that was dramatically enhanced in vivo and after castration. Cabazitaxel, like docetaxel, targets the cell architecture and induces cell death, but also induces a distinct gene expression profile that may partially explain efficacy in docetaxel-resistant tumors. Neither taxane showed affects on AR nuclear localization using in vivoor explant studies. Conclusions: These results strongly support our hypothesis that RB status can be used as a metric to define therapeutic response to cabazitaxel, as such that loss of RB function induces sensitization taxanes, whereas RB proficient tumors give an enhanced response to Cdk4/6 kinase inhibitors.

scholarly journals Lung Tissue Concentrations of Pyrazinamide among Patients with Drug-Resistant Pulmonary Tuberculosis

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Russell R. Kempker ◽  
M. Tobias Heinrichs ◽  
Ketino Nikolaishvili ◽  
Irina Sabulua ◽  
Nino Bablishvili ◽  
...  
Keyword(s):  
Pulmonary Tuberculosis ◽  
Lung Tissue ◽  
Ex Vivo ◽  
Inverse Correlation ◽  
Drug Resistant ◽  
Tissue Penetration ◽  
Serum Samples ◽  
Tissue Samples ◽  
Content Type ◽  
Antituberculosis Treatment

ABSTRACT Improved knowledge regarding the tissue penetration of antituberculosis drugs may help optimize drug management. Patients with drug-resistant pulmonary tuberculosis undergoing adjunctive surgery were enrolled. Serial serum samples were collected, and microdialysis was performed using ex vivo lung tissue to measure pyrazinamide concentrations. Among 10 patients, the median pyrazinamide dose was 24.7 mg/kg of body weight. Imaging revealed predominant lung lesions as cavitary (n = 6 patients), mass-like (n = 3 patients), or consolidative (n = 1 patient). On histopathology examination, all tissue samples had necrosis; eight had a pH of ≤5.5. Tissue samples from two patients were positive for Mycobacterium tuberculosis by culture (pH 5.5 and 7.2). All 10 patients had maximal serum pyrazinamide concentrations within the recommended range of 20 to 60 μg/ml. The median lung tissue free pyrazinamide concentration was 20.96 μg/ml. The median tissue-to-serum pyrazinamide concentration ratio was 0.77 (range, 0.54 to 0.93). There was a significant inverse correlation between tissue pyrazinamide concentrations and the amounts of necrosis (R = −0.66, P = 0.04) and acid-fast bacilli (R = −0.75, P = 0.01) identified by histopathology. We found good penetration of pyrazinamide into lung tissue among patients with pulmonary tuberculosis with a variety of radiological lesion types. Our tissue pH results revealed that most lesions had a pH conducive to pyrazinamide activity. The tissue penetration of pyrazinamide highlights its importance in both drug-susceptible and drug-resistant antituberculosis treatment regimens.

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