Ultrasound-guided pathological sampling of cadaver lung tissue in cases of COVID-19

Sec62 Bridges the Gap from 3q Amplification to Molecular Cell Biology in Non–Small Cell Lung Cancer

Targeted Deletion of Jun/AP-1 in Alveolar Epithelial Cells Causes Progressive Emphysema and Worsens Cigarette Smoke–Induced Lung Inflammation

Ciprofloxacin (CIP) and metabolite concentrations in lung tissue, parietal pleura and bronchial tissue were assessed in 43 adult patients who underwent lung surgery. A single oral dose of CIP was given for prophylaxis of bacterial infections. Two to 6 h prior to tissues sampling, 23 patients received 250 mg and 20 subjects 500 mg of the substance. Blood plasma samples were obtained at the same time as the lung tissue samples. CIP and its metabolites were assayed chemically by high-pressure liquid chromatography (HPLC). After 250 mg CIP, the individual lung tissue CIP concentrations during the 2- to 6-hour post-dose period ranged from 0.5 to 4.8 mg/kg. In 20 of the 23 lung samples, the CIP concentrations were above 1 mg/kg. After 500 mg CIP, the corresponding lung CIP concentrations ranged from 1.6 to 6.0 mg/kg. The CIP lung concentrations were, irrespective of the dose size, between 2 and 7 times higher than the simultaneous blood plasma concentrations. This indicates an excellent penetration of CIP and its metabolites into lung tissue. Bronchial tissue was obtained in 9 cases. Penetration into bronchial mucosa tissue was good as well, as indicated by tissue/plasma ratio values between 1.5 and 4.4. Individual CIP concentrations in the patients given 250 mg CIP, ranged from 1.0 to 1.6 mg/kg. In the patients who received 500 mg, the range was from 1.7 to 3.4 mg/kg. Tissue/plasma ratio values between 0.8 and 2.1 indicated that penetration to pleural tissues was good as well. Metabolite concentrations in all of the tissues assayed (lung, bronchial mucosa, pleural tissue) were low when compared to the concentrations of CIP. The concentrations in lung, pleural and bronchial tissue will probably permit low doses in the treatment of most respiratory tract infections. The broad spectrum of antibacterial activity, the good tissue penetration, chemical stability and the good safety record of the substance means that the drug is potentially a useful agent for perioperative antibiotic prophylaxis.

To study the therapeutic effects and mechanism of saikosaponin-d (SSd) in mice with bleomycin (BLM)-induced pulmonary fibrosis. According to the random number table, 180 mice were randomly divided into 5 groups. Four groups were pulmonary fibrosis models. Fibrosis model mice were established by intratracheal injection of bleomycin (5 mgxkg(-1)). They were BLM, DXM, SSd and SSd + DXM groups (n = 40 each). At 1 hour post-modeling, DXM, SSd and SSd + DXM groups were injected ip with dexamethasone (DXM, 5 mgxkg(-1)xd(-1), 0.1 ml), SSd (1.8 mgxkg(-1)xd(-1), 0.18 ml), DXM + SSd (0.28 ml) respectively qd until Day 28. BLM group was similarly dosed with normal saline. In addition, a normal control group (NC group, n = 20) treated likewise. The mice were anesthetized and sacrificed at Days 3, 7, 14, 28 for harvests of serum and lung tissue samples. The conventional histopathological changes of lung tissue were observed. Except for NC group, modeling groups of mice were used to observe the natural survival rate. Such indices as superoxide dismutase (SOD) and malonaldehyde (MDA) were examined both in lung tissue and serum samples. And hydroxyproline (HYP) was tested only in lung tissue. SSd could markedly increase the survival rate (80.0% in SSd and SSd + DXM groups vs 50.0% in BLM group, P < 0.05) and reduce alveolitis and fibrosis in mice. In comparison with BLM group, the levels of HYP of three treatment groups (DXM, SSd and SSd + DXM) in lung tissue was significantly lower (P < 0.05) at Days 14 and 28. The levels of MDA both in serum and lung tissue were significantly lower at Days 3, 7 and 14 (P < 0.05). The serum level of SOD was significantly higher at Days 3, 7 and 14 while the level of SOD in lung tissue was significantly higher at Days 3 and 7 (P < 0.05, P < 0.01). SSd has marked therapeutic effects upon bleomycin-induced pulmonary fibrosis in mice. And the mechanism may be associated with its anti-lipid peroxidation effect.

Complement anaphylatoxins have been investigated extensively; however, the role of complement anaphylatoxin C4a in hyperoxic lung injury has yet to be investigated. To the best of our knowledge, the present study is the first to demonstrate the role of C4a in hyperoxic lung injury in vitro and in vivo. BALB/c mice were ventilated with 100% oxygen with or without C4a treatment for 36 h. The body weight and the relative lung weight of the mice were determined, along with any morphological changes in the lung. The expression levels of interleukin (IL)-1, IL-6 and tumor necrosis factor-α (TNF-α) were quantified in the lung tissue and bronchoalveolar lavage fluid (BALF) samples by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The total cell count and the number of macrophages, neutrophils and lymphocytes in the BALF were determined using cytocentrifuge slides and a hemocytometer. Histamine release from total cells in the BALF was also analyzed. The relative mRNA expression levels of CD68, F4/80, CD64, CD19 and CD3 in the murine lung tissue were assessed by reverse transcription-quantitative polymerase chain reaction. The results revealed that hyperoxia induced lung injury and morphological changes, and increased the expression levels of IL-1, IL-6 and TNF-α, histamine release, the number of inflammatory cells, and the expression levels of CD68, F4/80, CD64, CD19 and CD3. The hyperoxia-induced morphological changes and inflammatory reaction were significantly attenuated in mice treated with C4a. Treatment with C4a also attenuated the increase in the total cell count, decreased the number of macrophages in the BALF, and suppressed the elevated mRNA expression levels of CD68 and F4/80 in the lung tissue samples. Conversely, treatment with C4a did not affect the number of neutrophils or lymphocytes in the BALF or the mRNA expression of CD64, CD19 and CD3 in lung tissue. In conclusion, C4a attenuated hyperoxic lung injury via a macrophage-dependent but not a neutrophil/lymphocyte-dependent pathway.

To evaluate the possible use of mutant ras as a biomarker for lung cancer, we have analyzed "normal appearing" lung tissue, lung tumor, lung metastases and sputum samples from patients with non-small cell lung cancer (NSCLC). As a control, we used lung tissue and sputum samples from patients without oncological diseases or lung disorders. Our analyses were performed with the aid of enriched PCR (EPCR), a method which enables detection of ras mutation even if present at low incidence. EPCR identified K-ras codon 12 mutations in 10% of lung tissues obtained from patients with no lung diseases, whereas the same mutation was detected in 60% of samples of normal appearing lung tissues obtained from patients with NSCLC, 62% of NSCLC tumors and 80% of metastases. Analysis of sputum samples of patients with NSCLC identified 47% to harbor mutant ras allele, whereas 12.5% of controls diagnosed with non-oncological lung diseases carried this mutation. Most of these mutations were detected with the aid of EPCR only, indicating that a minority of cells in a given sample harbor this mutation. The ability to detect K-ras codon 12 mutation in 60% of lung tissue samples and in 47% of sputum samples taken from patients with lung cancer (as compared with 10% and 12.5% of respective controls) points to the potential use of ras mutation as a biomarker for exposure and possible identification of patients who may be at higher risk of developing lung cancer.

Delivery of Therapeutic siRNA to the Lung Endothelium via Novel Lipoplex Formulation DACC

Simple SummaryPulmonary hypertension is an unusual elevation of the blood pressure in pulmonary circulation. In dogs, pulmonary hypertension is commonly occurred as a complication of progressive degenerative mitral valve disease especially in senior small-breed dogs. Previous studies revealed that the serotonin signaling pathway is associated with the pathogenesis of pulmonary hypertension. However, research in dogs was scarce. Therefore, the present study aimed to illustrate the understanding of this point by assessing the expression of the targeted genes and proteins related to the serotonin pathway in lung tissues and pulmonary arteries of dogs. Our results showed that the pattern of gene and protein expression was different in canine lung and pulmonary arteries. The targeted proteins in pulmonary artery tissues of the degenerative mitral valve disease dogs with and without pulmonary hypertension tended to be upregulated. In addition, the expression of these protein was increased in the pneumocytes of the alveolar walls, pulmonary alveolar macrophages, and bronchial epithelial cells. A correlation between the targeted gene and protein expression and the echocardiographic data was also founded. Overall results pointed out that the serotonin pathway in lung and pulmonary artery tissues might have different roles in degenerative mitral disease with and without pulmonary hypertension.Pulmonary hypertension (PH) is defined as an increase in pulmonary vascular pressure. It is one of the most common complications that occur as a result of degenerative mitral valve disease (DMVD) in dogs. Serotonin (5-HT) can trigger the development of PH. Accordingly, this study investigated the changes in the expression of genes and proteins associated with local 5-HT signaling in the lungs and pulmonary arteries (PA) of dogs with PH secondary to DMVD. Lung and PA tissue samples were collected from the cadavers of fourteen small-breed dogs and divided into normal (n = 4), DMVD (n = 5) and DMVD with PH (n = 5) groups. Gene expression (tph1, slc6a4 and htr2a) was analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression of proteins (TPH-1, SERT, 5-HTR2A, ERK1/2 and pERK1/2) was examined by Western blot analysis and immunohistochemical staining. The results showed that the expression of genes and proteins evaluated by qRT-PCR and Western blot analysis in lung and PA tissues did not differ among groups. However, the expression of proteins related to 5-HT signaling tended to be upregulated in PA tissues from DMVD dogs with and without PH. Immunohistochemical examination revealed the overexpression of these proteins in the DMVD and DMVD with PH groups in lung tissue. These findings suggest a local effect of 5-HT signaling in DMVD dogs with and without PH.

IL-17-producing T lymphocytes in lung tissue and in the bronchoalveolar space after exposure to endotoxin from Escherichia coli in vivo – effects of anti-inflammatory pharmacotherapy

The objective of the present study was to examine the effects of zinc supplementation on the oxidant damage in lung and liver tissues in rats exposed to a 50-Hz frequency magnetic field for 5 minutes every other day over a period of 6 months. The study included 24 adult male Sprague-Dawley rats, which were divided into the three groups in equal numbers: Group 1, the control group (G1); Group 2, the group exposed to an electromagnetic field (G2); and Group 3, the group, which was exposed to an EMF and supplemented with zinc (G3). At the end of the 6-month procedures, the animals were decapitated to collect lung and liver tissue samples, in which MDA was analyzed using the "TBARS method (nmol/g/protein)", GSH by the "biuret method (mg/g/protein)" and zinc levels by atomic emission (µg/dl). MDA levels in lung and liver tissues in G2 were higher than those in G1 and G3, and the levels in G3 were higher than those in G1 (p<0.01). As for GSH levels, G3 had GSH levels higher than G1 and G2, and G2 had GSH levels higher than G1 (p<0.01). Zinc values in the liver and lung tissues were the highest in the Group 3 and the lowest in the Group 2 (p<0.01). Results of the study indicated that exposure to an electromagnetic field caused cellular damage in lung and liver tissues and zinc supplementation inhibited the inflicted cellular damage. Another important result of this study that needs emphasis was that exposure to an electromagnetic field led to a significant decrease in zinc levels in lung and liver tissues (Tab. 3, Ref. 23).

The aim of this study was to clarify the significance of DNA methylation alterations at precancerous stages of lung adenocarcinoma. Using single-CpG resolution Infinium array, genome-wide DNA methylation analysis was performed in 36 samples of normal lung tissue obtained from patients without any primary lung tumor, 145 samples of non-cancerous lung tissue (N) obtained from patients with lung adenocarcinomas, and 145 samples of tumorous tissue (T). Stepwise progression of DNA methylation alterations from normal lung tissue to non-cancerous lung tissue obtained from patients with lung adenocarcinomas, and then tumorous tissue samples, was observed at 3,270 CpG sites, suggesting that non-cancerous lung tissue obtained from patients with lung adenocarcinomas was at precancerous stages with DNA methylation alterations. At CpG sites of 2,083 genes, DNA methylation status in samples of non-cancerous lung tissue obtained from patients with lung adenocarcinomas was significantly correlated with recurrence after establishment of lung adenocarcinomas. Among such recurrence-related genes, 28 genes are normally unmethylated (average β-values based on Infinium assay in normal lung tissue samples was less than 0.2) and their DNA hypermethylation at precancerous stages was strengthened during progression to lung adenocarcinomas (ΔβT–N>0.1). Among these 28 genes, we focused on 6 for which implications in transcription regulation, apoptosis or cell adhesion had been reported. DNA hypermethylation of the ADCY5, EVX1, GFRA1, PDE9A, and TBX20 genes resulted in reduced mRNA expression in tumorous tissue samples. 5-Aza-2′-deoxycytidine treatment of lung cancer cell lines restored the mRNA expression levels of these 5 genes. Reduced mRNA expression in tumorous tissue samples was significantly correlated with tumor aggressiveness. These data suggest that DNA methylation alterations at precancerous stages determine tumor aggressiveness and outcome through silencing of specific genes.

Purpose We investigated plasma angiotensin-converting enzyme 2 (ACE2) concentration in a population sample and the ACE2 expression quantitated with the diaminobenzidine mean intensity in the lung tissue in patients who underwent lung surgery. Materials and methods The study participants were recruited from a residential area in the suburb of Shanghai for the plasma ACE2 concentration study (n = 503) and the lung tissue samples were randomly selected from the storage in Ruijin Hospital (80 men and 78 age-matched women). Results In analyses adjusted for covariables, men had a significantly higher plasma ACE2 concentration (1.21 vs. 0.98 ng/mL, p = 0.027) and the mean intensity of ACE2 in the lung tissue (55.1 vs. 53.9 a.u., p = 0.037) than women. With age increasing, plasma ACE2 concentration decreased (p = 0.001), while the mean intensity of ACE2 in the lung tissue tended to increase (p = 0.087). Plasma ACE2 concentration was higher in hypertension than normotension, especially treated hypertension (1.23 vs. 0.98 ng/mL, p = 0.029 vs. normotension), with no significant difference between users of RAS inhibitors and other classes of antihypertensive drugs (p = 0.64). There was no significance of the mean intensity of ACE2 in the lung tissue between patients taking and those not taking RAS inhibitors (p = 0.14). Neither plasma ACE2 concentration nor the mean intensity of ACE2 in the lung tissue differed between normoglycemia and diabetes (p ≥ 0.20). Conclusion ACE2 in the plasma and lung tissue showed divergent changes according to several major characteristics of patients.

Previous studies have shown that microRNAs (miRs), such as miR-146a play an important role in the pathogenesis of intestinal ischemia/reperfusion (I/R)-induced injury; however, the role of miR-146a in intestinal I/R-induced acute lung injury has not been elucidated. An intestinal I/R-induced injury mouse model was established in the present study by clamping the superior mesenteric artery and expression levels of miR-146a in intestinal and lung tissue samples were evaluated using reverse transcription-quantitative PCR (RT-qPCR). Intestinal and lung histopathological characteristics in mice with intestinal I/R-induced injury were assessed by hematoxylin and eosin staining, and mRNA and protein expression levels in intestinal and lung tissue samples were evaluated using RT-qPCR and western blotting, respectively. miR-146a expression was significantly downregulated in the intestinal and lung tissue samples of mice with intestinal I/R-induced injury. Intestinal I/R injury-induced histopathological changes in the lung and intestines, and pulmonary edema in mice transduced with an adenoviral miR-146a-overexpression vector (the miR-146a overexpression group) were alleviated. mRNA expression levels of TNF-α, IL-1β, IFN-γ and TGF-β1, and protein expression levels of TNF receptor-associated factor 6, phosphorylated-p65 NF-κB, cleaved caspase-3 and cleaved caspase-9 in lung and intestinal tissue samples were downregulated in I/R-miR-146a-overexpressing mice, compared with those from the I/R-negative control group. Thus, the present study identified that pre-treatment with the miR-146a overexpression vector alleviated intestinal I/R-induced acute lung injury in mice.

In recent years, Thelper (Th)9 cells have been demonstrated to be key mediators in immune responses in asthmatic lungs, and innate lymphoid cells2 (ILC2s) have been described as a novel type of innate immunocyte with the ability to enhance immunoglobulinE (IgE) production. However, the interaction between ILC2s and Th9 cells in the pulmonary system of a mouse model of asthma remains to be elucidated. In the present study, the response state of lung tissue with regards to Th9 and ILC2s in a mouse model of asthma was investigated by detecting Th9‑ and ILC2‑associated cytokine receptors. The present study also investigated the association between the expression levels of the cytokine receptors in lung tissue samples and the IgE levels in sera samples from mouse models of asthma. Results from the present study demonstrated that the frequency of ILC2s and Th9 cells was significantly increased in the lung tissue samples, indicating that a Th2-type immune response had occurred. In addition, high mRNA expression levels of RAR‑related orphan receptorα, interleukin1 receptor‑like1, transcription factor PU.1 and interleukin (IL)‑9 were observed. Furthermore, IL‑5Rα, IL‑13Rα2 and high‑affinity IgE receptor were increased in mouse models of asthma, and a positive association was observed between the expression levels of ILC2‑ or Th9‑associated receptors in tissue samples and IgE levels in the sera. This indicated that ILC2s and Th9 were in a state of polarization and may promote each other in the lung tissue of mouse models of asthma, and that the lung tissue was responding to the two types of cells via increased expression of receptors.

Top of pageAbstract Background: Neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3)] are growth factors that function as mediators for the development, survival and differentiation of neurons and other types of cells. During normal tissue morphogenesis, neurogenesis and myogenesis proceed as tightly linked processes. In the condition, congenital diaphragmatic hernia (CDH), there is failure of the diaphragm muscle to develop with consequent pulmonary hypoplasia and pulmonary hypertension. The probability that impaired neurogenesis, or coordination of neuro and myogenesis, underlies the disrupted development of the diaphragm muscle in CDH, needs to be explored. Objectives: To determine the expression of neurotrophic factors (NGF, NT-3 and BDNF) in neonatal lung disease; specifically congenital diaphragmatic hernia (CDH), persistent pulmonary hypertension (PPHN) and chronic lung disease (CLD) vs. normal lung tissue. Methods: Immunohistochemical studies for neurotrophin proteins (specific antibody staining using polyclonal rabbit anti-human NGF, BDNF and NT-3) were applied to human neonatal lung tissue samples. The samples included a control group of 18 samples ranging from 23wk EGA to term, a CDH group of 15 samples, a PPHN group of 6 samples and a CLD group of 12 samples. The tissue samples were studied and 4 representative slide fields of aveoli/saccules and 4 of bronchioles were recorded from each sample. These slide fields were then graded (from 0-3) by several blinded observers for intensity of staining. Results: BDNF, NGF and NT-3 immunostaining intensity scores were significantly less in the CDH lung tissue samples (n 15) compared to normal neonatal lung tissue (n 18) (p<0.001). Similarly, PPHN lung tissue (n 6) scored less than normal lung tissue, although not statistically significant; likely due to few samples studied. Chronic lung disease tissue (n 12) did not appear to differ in their neurotrophic staining intensity compared to normal neonatal lung tissue. Conclusion: Neurotrophin expression is decreased in CDH lungs. The decreased expression of neurotrophins in CDH lung tissue suggests they contribute to the abnormality in this condition. Further studies are needed to explore the role of deficient neurotrophin expression in the development of CDH.