Establishing a Microfiber Recovery Rate in Human Lung Tissue

A new modification of the 32P postlabelling method was described for the analysis of lipophilic DNA in human tissues. To isolate these DNA adducts the method applied nuclease P1 enrichment before labelling and butanol extraction after labelling, followed by high performance liquid chromatography HPLC separation and flow through radioactivity detection. These enrichment methods are known to work for many adducts of polycyclic aromatic hydrocarbons PAHs. In human peripheral lung tissue fro m smokers the apparent level of the DNA adducts observed was 25-244 adducts per 108 nucelotides; in two alleged non smokers the level of adducts was 17 and 109 adducts per 108 nucleotides. When the same samples were analysed by thin layer chromatography TLC, as in the conventional postlabelling assay, the recovery was 5 of that of the HPLC method. Nevertheless, the results from the two methods correlated. In TLC the adducts were lost because they did not remain in the origin in D1 of the TLC development. There was no large difference in recovery between the two techniques for the PAH-DNA adduct standards used. The present results are underestimates of the true adduct levels because there is no way to correct for labelling efficiency and recovery of unknown adducts. Yet they give a lower estimate of the level of lipophilic DNA adducts in human lung tissue.

Dear Editor, Inhalational exposure to toxic chemicals present in humidifier disinfectant (HD), such as polyhexamethylene guanidine (PHMG), was identified as a cause of the serious lung injury,1-3 and this fatal humidifier disinfectant-associated lung injury (HDLI), as characterized by rapid progression of respiratory failure with lung fibrosis and frequent air leak syndrome with high mortality, was classified as a subcategory of interstitial lung diseases (ILDs).4, 5 However, differences in clinical progression, high mortality (44–58%), and pathology between HDLI and previously identified ILDs have aroused interest over the mechanisms of these serious lung diseases.1, 2, 6 The aim of the present study was to explore regulatory molecules and gain insight into the comprehensive biological processes associated with HDLI using integrated multispecies multi-omics of human and rat lung tissues exposed to PHMG. Study subjects and their clinical characteristics are summarized in Tables S1 and S2. The most common symptoms at admission were coughing and tachypnea in children with HDLI, and chest wall retraction and cyanosis in adult patients with HDLI. A global expression showed a distinct clustering of patients with HDLI from controls (Figure 1A,B). Among the differentially expressed genes (DEGs) as shown in Table S3, given the overproduction of collagen during pulmonary fibrosis, significant up-regulation of collagen-related gene of top COL1A2 (log2 fold change = 2.58, PFDR = 0.04) in the present study was notable. Further comparison of gene expressions between human and rat lung tissues (Tables S4 and S5, Figure S1) relative to controls identified eight overlapping genes (Table S6, Figure 1C). Among them, several genes (including MMP2, SERPINF1, and A2M) have been previously reported to be associated with lung diseases such as idiopathic pulmonary fibrosis (IPF). In the ingenuity pathway analysis (IPA), significant networks, canonical pathway, and disease/bio-function were predicted in human lung tissues with HDLI and rat lung tissues exposed to PHMG (Tables S7–S9). Notably, hepatic "fibrosis" in canonical pathway and respiratory disease in network and disease/bio-function were predicted to be related to HDLI. Interactome network analysis revealed higher DEG enrichment in interacting together rather than control genes (Figure S2), indicating that genes engaging in significant interactions with DEGs might play a disease-associated role. Moreover, this interaction landscape reflected the proteome results (Figure 1D, Figure S3, Table S10): the interaction enrichment of DEGs showed a strong positive correlation (r = 0.637, p < 0.001) with interaction enrichment of differentially expressed proteins (DEPs) based on protein expression analysis (Figure S4, Table S11). Intriguingly, MMP2, which was a core gene and consistently identified in both human and rat transcriptome datasets, was found to interact strong with DEGs in both children and adults (Figure 2A), and increased MMP2 protein levels were observed in formalin-fixed paraffin-embedded (FFPE) specimens, along with hypo-methylation in transcription start site and promoter regions of MMP2 (Figure 2B, Figure S5). In further re-analysis based on gene expression profiles from children (Table S12) and adults (Table S13), similarly and differentially enriched pathways between children and adults were predicted (Figure 2C,D, Table S14). The well-established fibrosis-related TGF-β/SMAD signaling was the major pathway identified in adults (Figures 2D and 3A,B, Tables S14 and S15), while other potential pathways were identified in children, of which integrin signaling (Figures 2D and 3C,D; p = 0.002, adjusted p = 0.02 in Tables S14 and S16) appeared to be the most important among child-specific signaling pathways, as also shown in gene set enrichment analysis (GSEA) analysis (Figure 3E). Based on our transcriptomic/proteomic profiling and network analysis results, different MMP2-mediated mechanisms appear to operate in children and adults during HDLI development, suggesting that pre-exposure to toxic materials in the environment may affect apoptosis signaling in adults. Furthermore, exposure to toxic PHMG-containing HD may promote activation of TGF-β/SMAD signaling in adults. Meanwhile, different signaling pathways were observed in children with HDLI and integrin (also known as a potential activator of MMPs and mediator of fibrosis and TGF-β activation)7 was identified to be crucial in children with HDLI (Figure 4A). The present study further compared pathways identified from interactome network analysis of HDLI and IPF (the most common subtype of ILDs in adults) using previously reported IPF transcriptome data (Table S17) compared to public datasets.8 Extracellular matrix (ECM)–receptor interaction and integrin signaling pathways were enriched in the patients with HDLI (Figure 4B). The integrin signaling pathway plays a role in fibrosis associated with human diseases and has a possible relationship to the CCKR and EFG receptor pathways (highly significant in both adults and children, Table S14) associated with fibrosis.9, 10 These differences in pathways relevant to HDLI and IPF, along with highly replicated enrichment of transcriptomes in the lung tissues of humans and rats exposed to PHMG (Figure 4C), imply that the potential mechanisms related to HDLI may differ between IPF and other types of pulmonary fibrosis. The predictions from the lung transcriptomes of PHMG-exposed rats revealed similar signaling pathways with those previously reported in pulmonary fibrosis (Table S18); however, there were differences between the PHMG-exposed rats and clinical cases of fibroblast activation and pulmonary fibrosis regarding STAT3, endothelin-1, and Wnt/β-catenin signaling pathways, suggesting that PHMG exposure has a distinct pathogenesis from other fibrotic lung diseases. Limitations of the present study are the relatively small number of samples, due to the scarcity of HDLI patients and the low availability of human lung tissues, and insufficient evaluation of the functions of the identified candidates. Although disease severity, stage, and progression during tissue acquisition might affect gene expression, we were unable to account for this factor in the present work. However, to minimize this, we analyzed lung samples during the acute phase, and replicated human expression changes in the rat model following exposure to PHMG. In addition, we integrated multispecies multi-omics (human transcriptomics, methylation, proteomics, and rat transcriptomics) approaches, and performed interactome network analysis, to avoid issues with its low coverage in analyzed data. In summary, this study identified several DEGs and further validated DEPs in HDLI, along with differences in mechanisms between children and adults and human–rat commonality, hoping that these results may provide new insight into the HDLI pathogenesis and chemical-induced lung injury and fibrosis. We offer our sincerest sympathy to patients with HDLI and their families, and thank them for participating in the study. This study was funded by the Korea Ministry of Environment (MOE) as the Environmental Health Action Program (2016001360006), and was supported by the Environmental Health Center for Hazardous Chemical Exposure (2017) funded by the Ministry of Environment (MOE) of the Republic of Korea. The authors declare that there is no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Microplastics are a pressing global concern. Inhalation of microplastic fibers has been associated with interstitial lung disease related to alveolar epithelial damage in nylon flock workers. However, the means by which fibers affect lung tissue and epithelial growth remains unknown. Our aim was to assess the effects of nylon and polyester textile microplastic fibers on epithelial growth and differentiation using airway and alveolar lung organoids cultured from epithelial cell progenitors, isolated both from murine lungs and human lung tissue obtained from COPD patients. Exposure to nylon (11x30 µm) or polyester (15x53 µm) microfibers resulted in significantly fewer and smaller human and murine airway organoids after 14 days of culture, the effect being most profound with nylon. Alveolar organoids were not affected by these fibers. Incubation with nylon- or polyester-conditioned medium also resulted in fewer airway organoids. Effects were mainly observed in developing airway organoids; exposure of developed organoids from day 14 to day 21 to fibers or fiber-conditioned medium had no significant effect on organoid number or size. In conclusion, airway organoid formation is negatively impacted by the presence of textile microplastic fibers and this effect appears to be mediated by leaching additives. Our results suggest that microplastic fibers may especially harm the developing airways or airways undergoing repair. Further studies will focus on identifying these additives and the mechanism behind their effect. Importantly, wider investigations into the presence of microplastic fibers in human lung tissue are urgently needed to determine the actual risk of these fibers to human health.

Microplastics are a pressing global concern. Inhalation of microplastic fibers has been associated with interstitial lung disease related to alveolar epithelial damage in nylon flock workers. However, the means by which fibers affect lung tissue and epithelial growth remains unknown. Our aim was to assess the effects of nylon and polyester textile microplastic fibers on epithelial growth and differentiation using airway and alveolar lung organoids cultured from epithelial cell progenitors, isolated both from murine lungs and human lung tissue obtained from COPD patients. Exposure to nylon (11x30 µm) or polyester (15x53 µm) microfibers resulted in significantly fewer and smaller human and murine airway organoids after 14 days of culture, the effect being most profound with nylon. Alveolar organoids were not affected by these fibers. Incubation with nylon- or polyester-conditioned medium also resulted in fewer airway organoids. Effects were mainly observed in developing airway organoids; exposure of developed organoids from day 14 to day 21 to fibers or fiber-conditioned medium had no significant effect on organoid number or size. In conclusion, airway organoid formation is negatively impacted by the presence of textile microplastic fibers and this effect appears to be mediated by leaching additives. Our results suggest that microplastic fibers may especially harm the developing airways or airways undergoing repair. Further studies will focus on identifying these additives and the mechanism behind their effect. Importantly, wider investigations into the presence of microplastic fibers in human lung tissue are urgently needed to determine the actual risk of these fibers to human health.

Formation of leukotrienes and prostaglandins in human lung tissue, measured by HPLC and RIA

Late-breaking abstract: Preclinical testing of therapeutic WNT/β-catenin activation in human 3D lung tissue cultures

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging coronavirus that has resulted in nearly 1,000,000 laboratory-confirmed cases including over 50,000 deaths. Although SARS-CoV-2 and SARS-CoV share a number of common clinical manifestations, SARS-CoV-2 appears to be highly efficient in person-to-person transmission and frequently cause asymptomatic infections. However, the underlying mechanism that confers these viral characteristics on high transmissibility and asymptomatic infection remain incompletely understood.MethodsWe comprehensively investigated the replication, cell tropism, and immune activation profile of SARS-CoV-2 infection in human lung tissues with SARS-CoV included as a comparison.ResultsSARS-CoV-2 infected and replicated in human lung tissues more efficiently than that of SARS-CoV. Within the 48-hour interval, SARS-CoV-2 generated 3.20 folds more infectious virus particles than that of SARS-CoV from the infected lung tissues (P<0.024). SARS-CoV-2 and SARS-CoV were similar in cell tropism, with both targeting types I and II pneumocytes, and alveolar macrophages. Importantly, despite the more efficient virus replication, SARS-CoV-2 did not significantly induce types I, II, or III interferons in the infected human lung tissues. In addition, while SARS-CoV infection upregulated the expression of 11 out of 13 (84.62%) representative pro-inflammatory cytokines/chemokines, SARS-CoV-2 infection only upregulated 5 of these 13 (38.46%) key inflammatory mediators despite replicating more efficiently.ConclusionsOur study provided the first quantitative data on the comparative replication capacity and immune activation profile of SARS-CoV-2 and SARS-CoV infection in human lung tissues. Our results provided important insights on the pathogenesis, high transmissibility, and asymptomatic infection of SARS-CoV-2.

Adipocytes secrete a number of molecules such as tumor necrosis factor-alpha, leptin and free fatty acids that can influence the ability of the body to metabolize glucose. Recently, a novel 12.5 kDa cysteine-rich protein, termed resistin, was shown to be secreted by adipocytes. Resistin expression was markedly induced during the conversion of 3T3-L1 cells to mature adipocytes. Expression of resistin has been studied in human, mouse and rat; however, sequence information about an alternative splicing variant (ASV) of resistin mRNA has not been reported. In the present study, we investigated the occurrence of a novel ASV of the resistin gene in human normal tissues. We identified a novel ASV of resistin mRNA in human lung tissue by RT-PCR analysis in human lung tissue. We then investigated a novel ASV of resistin mRNA by real-time PCR analysis in 26 different types of normal human tissues. We identified a novel deletion variant of the resistin transcript in the normal human tissues. The deleted transcript of resistin was characterized by an in-frame deletion of 78 bp, corresponding to the complete loss of exon 2 (resistin delta2 ASV). Thus, resistin delta2 ASV causes protein truncation. Our results provide the basis for more detailed studies on the regulation of resistin activity, and should assist in the development of clinical trials with resistin for the central regulation of adipogenesis and adipocyte metabolism.

Heavy metals, including chromium (Cr), cadmium (Cd), lead (Pb), nickel (Ni), and arsenic (As), are pervasive environmental pollutants that accumulate in human lung tissue through inhalation from sources such as smoking, industrial emissions, and ambient air pollution. This systematic review synthesizes evidence on metal concentrations in lung tissues across diverse human populations and their associations with respiratory diseases. To systematically review and synthesize data from studies conducted between 1975 and 2025 on the concentrations of heavy metals in human lung tissues, their sources of exposure, and associated health implications. Adhering to PRISMA guidelines, a comprehensive search was conducted across databases including PubMed, Scopus, Web of Science, and Embase, utilizing keywords related to heavy metals and lung tissue. Inclusion criteria encompassed human studies reporting quantifiable metal concentrations and health outcomes. Two independent reviewers screened titles, abstracts, and full texts, with data extracted on population characteristics, metal concentrations, key findings, and study quality. From an initial pool of 1,256 records, 39 studies were included after excluding duplicates, animal studies, and those not meeting quality thresholds (32 human epidemiological/autopsy studies and 7 in vitro studies using human cell lines). Elevated levels of Cd and Pb were strongly associated with smoking and impaired lung function, including chronic obstructive pulmonary disease (COPD) with a pooled odds ratio from meta-analysis of 2.85 (95% CI: 1.34–6.10) for Cd (highest vs. lowest exposure quartile) and 1.73 (95% CI: 1.37–2.18) for Pb. Chromium and Ni exhibited significant correlations with occupational exposures and inflammatory responses. Combined metal exposures demonstrated amplified health risks. The accumulation of heavy metals in lung tissue contributes significantly to respiratory morbidity. Mitigation strategies, including regulatory measures and smoking cessation initiatives, are imperative. Further longitudinal research on the effects of mixed-metal exposures is warranted.

β2-Adrenoceptors in Human Lung and Peripheral Mononuclear Leukocytes of Untreated and Terbutaline-treated Patients

While human autopsy samples have provided insights into pulmonary immune mechanisms associated with severe viral respiratory diseases, the mechanisms that contribute to a clinically favorable resolution of viral respiratory infections remain unclear due to the lack of proper experimental systems. Using mice co-engrafted with a genetically matched human immune system and fetal lung xenograft (fLX), we mapped the immunological events defining successful resolution of SARS-CoV-2 infection in human lung tissues. Viral infection is rapidly cleared from fLX following a peak of viral replication, histopathological manifestations of lung disease and loss of AT2 program, as reported in human COVID-19 patients. Infection resolution is associated with the activation of a limited number of hematopoietic subsets, including inflammatory monocytes and non-canonical double-negative T-cells with cytotoxic functions, which are highly enriched in viral RNA and dissipate upon infection resolution. Activation of specific human fibroblast and endothelial subsets also elicit robust antiviral and monocyte chemotaxis signatures, respectively. Notably, systemic depletion of human CD4+ cells, but not CD3+ cells, abrogates infection resolution in fLX and induces persistent infection, supporting evidence that peripheral CD4+ monocytes are important contributors to SARS-CoV-2 infection resolution in lung tissues. Collectively, our findings unravel a comprehensive picture of the immunological events defining effective resolution of SARS-CoV-2 infection in human lung tissues, revealing markedly divergent immunological trajectories between resolving and fatal COVID-19 cases.

Microplastics (MPs) are ubiquitous in the environment, in the human food chain, and have been recently detected in blood and lung tissues. To undertake a pilot analysis of MP contamination in human vein tissue samples with respect to their presence (if any), levels, and characteristics of any particles identified. This study analysed digested human saphenous vein tissue samples (n = 5) using μFTIR spectroscopy (size limitation of 5 μm) to detect and characterise any MPs present. In total, 20 MP particles consisting of five MP polymer types were identified within 4 of the 5 vein tissue samples with an unadjusted average of 29.28 ± 34.88 MP/g of tissue (expressed as 14.99 ± 17.18 MP/g after background subtraction adjustments). Of the MPs detected in vein samples, five polymer types were identified, of irregular shape (90%), with alkyd resin (45%), poly (vinyl propionate/acetate, PVAc (20%) and nylon-ethylene-vinyl acetate, nylon-EVA, tie layer (20%) the most abundant. While the MP levels within tissue samples were not significantly different than those identified within procedural blanks (which represent airborne contamination at time of sampling), they were comprised of different plastic polymer types. The blanks comprised n = 13 MP particles of four MP polymer types with the most abundant being polytetrafluoroethylene (PTFE), then polypropylene (PP), polyethylene terephthalate (PET) and polyfumaronitrile:styrene (FNS), with a mean ± SD of 10.4 ± 9.21, p = 0.293. This study reports the highest level of contamination control and reports unadjusted values alongside different contamination adjustment techniques. This is the first evidence of MP contamination of human vascular tissues. These results support the phenomenon of transport of MPs within human tissues, specifically blood vessels, and this characterisation of types and levels can now inform realistic conditions for laboratory exposure experiments, with the aim of determining vascular health impacts.

The accumulation of mitochondrial DNA (mtDNA) mutations has been suggested to be an important contributor to human aging and degenerative diseases. The lung is exposed to ambient air and makes direct contact with the external environment. Numerous potentially noxious agents may damage lung tissues directly or indirectly through free-radical-mediated reactions. In previous studies, we demonstrated an age-dependent increase of mtDNA mutations in various human tissues. We hypothesize that the accumulation of the 4,977 bp (base pairs) deleted mtDNA in human lung tissues is also age-dependent. Using the polymerase chain reaction technique, we determined the incidence of the 4,977 bp-deleted mtDNA in 127 human lung specimens from 34-wk gestation to 79 yr of age. The results showed that 77 lung biopsies (60.6%) contained the 4,977 bp-deleted mtDNA, which started to appear in lung tissues after the fourth decade of life. The incidence apparently increased from 14.3% (one of seven) of the subjects in the 30- to 39-yr age group to 77.8% (two of 27) of the subjects in the 70- to 79-yr age group (p < 0.0001). The mean (+/- SEM) proportion of the 4,977 bp-deleted mtDNA in lung tissues significantly increased from 0.007 +/- 0.007% of the subjects in the 30- to 39-yr age group to 0.833 +/- 0.330% of those in the 70- to 79-yr age group (p < 0.005). Other factors such as sex, pulmonary function indices, and smoking status did not have statistically significant impact on the amount of the deleted mtDNA. These findings suggest that the accumulation of the 4,977 bp-deleted mtDNA is associated with aging human lung.

Part of the soluble cyclic nucleotide phosphodiesterase activity of crude human lung tissue can be attributed to a thermosensitive (37 degrees) enzyme with a high apparent affinity for both adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP). The enzyme can be partially purified by DEAE-Sephadex chromatography. In the presence of 0.1 mM EDTA or ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), it is eluted from the column immediately before a cyclic GMP-specific phosphodiesterase, but in the presence of 0.2 mM Ca2+, the elution follows that of the cyclic GMP-specific enzyme. The two forms of the nonspecific phosphodiesterase activity are referred to as DEAD-Sephadex Fractions Ia and Ic, respectively. Their apparent molecular weights, recorded at gel filtration, vary with different preparations from 230,000 to 150,000. Occasionally, corresponding recordings for main peaks of activity also cluster round the values 120,000, 105,000, and 78,000. The enzymatic properties of Fractions Ia and Ic closely resemble each other. The enzyme activity is blocked by EDTA, partially inhibited in the presence of 1,10-phenanthroline, but only slightly affected by EGTA. The inhibitory effect of EDTA can be overcome by Mg2+ and Mn2+ and that of 1,10-phenanthroline, in part, by Zn2+; this cation in itself is inhibitory at millimolar concentrations. With submicromolar substrate concentrations, the activity of either fraction obeys linear kinetics displaying an apparent Km of approximately 0.4 micron for both substrates. Reciprocal inhibition experiments suggest that hydrolysis of both cyclic AMP and cyclic GMP is performed by the same active site. Examination of the activity using extended substrate concentration ranges indicates nonlinear kinetics; Hill plots of such data also show nonlinear curvature. The activity is inhibited by micromolar concentrations of inosine 3':5'-monophosphate (cyclic IMP), 3-isobutyl-1-methylxanthine, papervine, and some antiallergic agents. Theophylline and disodium cromoglycate are less potent inhibitors. Inhibition of activity by Lubrol PX follows a biphasic dose response curve. The activity of Fraction Ia can be enhanced 2- to 3-fold by a Ca2+-dependent activator prepared from lung tissue, whose action is counteracted by chlorpromazine, and by lysophosphatidylcholine. It is initially enhanced but subsequently decreased at exposure to trypsin. Fraction Ic is less prone to activation by these agents. The results indicate that the present activity represents an enzyme form that differs from three previously described phosphodiesterases of human lung tissue. It is apparently related to, but also shows distinct differences from the Ca2+-dependent enzyme(s) of brain and heart tissue.

Presence, enzymatic activity, and subcellular localization of paraoxonases 1, 2, and 3 in human lung tissues