Improved HPLC method for the determination of ribavirin concentration in red blood cells and its application in patients with COVID-19.

Ribavirin is a synthetic, broad-spectrum antiviral drug. Ribavirin is recommended as an antiviral drug in the Interim Guidance for Diagnosis and Treatment (the seventh edition) of COVID-19. The ribavirin levels in red blood cells may be closely related to both its efficacy and adverse drug reactions. In this study, a simple and fast HPLC-UV method was established to determine the concentrations of total ribavirin in the red blood cells of 13 patients with COVID-19. Phosphorylated ribavirin was dephosphorylated by phosphatase incubation to obtain the total amount of ribavirin in red blood cells. The chromatographic column was an Atlantis C18 . The recoveries were 85.45-89.05% at three levels. A good linear response was from 1 to 200 µg/ml, with a correlation coefficient of r2 = 0.9991. The concentration of total ribavirin in the red blood cells of the patients ranged from 30.83 to 133.34 µg/ml. The same samples without phosphatase incubation ranged from 4.07 to 20.84 µg/ml. About 85% of ribavirin was phosphorylated in red blood cells. In addition, we observed changes in these patients' hematological parameters and found that the erythrocyte, hemoglobin and hematocrit declined to the lowest levels on the fifth day after discontinuation of ribavirin (p < 0.05).

Effects of Atmospheric Fine Particulate Matter and Its Carrier Microbes on Pulmonary Microecology in Patients with COPD.

Objective: The aim of this paper was to analyse the influence of atmospheric fine particulate matter (AFPM) and atmospheric microorganisms on the pulmonary microecology of chronic obstructive pulmonary disease (COPD) patients in northeast China. Methods: Collected bronchoalveolar lavage fluid (BALF) of COPD patients in the high-risk period (group A) and low-risk period (group B) of AFPM inhalation and samples of AFPM in the same time range (group C) were collected. DNA sample sequencing, the bacterial abundance, and diversity bioinformatics of BALFs were performed by methods of Illumina MiSeq™ platform and Mothur and Uclust. Results: A total of 58 samples were sequenced, including 22 samples from group A, 26 samples from group B and 10 samples from group C. A total of 2,005,790 bacterial sequences and 34,256 bacterial numbers were detected. Group B had the highest bacterial diversity of the three groups. Group B also had the highest bacterial abundance index value. There were differences in the classification of bacterial colonies for the three groups at the genus level. The types of bacteria in group C were more numerous than other groups, and group B was higher than group A, which indicates that there were more bacteria in BALF during the high-risk period of AFPM inhalation. The detection rates of Streptococcus, Mycoplasma, Roche, Pushia, Chlamydia trachomatis and Brucella for group C were significantly higher than group A. The COG and KEGG databases' difference analysis results for the bacterial gene function abundance of group A and group B were 40.7% in group A and 38.9% in group B (R=0.098, P=0.006). The human disease abundance in group A and group B was 1.16% and 1.12%, respectively (P>0.05). Conclusion: The increase in the concentration of AFPM can increase the diversity and abundance of bacteria in the BALF of stable COPD patients. Clinical Trial Registration Number: 2020XS04-02.

Environmental and Genetic Factors in the Pathogenesis of COPD in the Road-Working Population.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a typical heterogeneous condition caused by environmental and genetic risk factors. OBJECTIVES: We investigated extrinsic (environmental) and intrinsic (genetic) factors contributing to the development of COPD in a nonsmoker road-working population in Northeast China. METHOD: The target population was divided into a COPD group and an exposed control group. Another healthy nonroad working nonsmoker control group was also included for environmental factor comparison. Peripheral blood was collected and analyzed using inductively coupled plasma mass spectrometry for inorganic elements of PM2.5, and microarray, rt-PCR, and Multiplex ELISA for genetic factors. RESULTS: Forty-three COPD road workers, thirty-nine non-COPD road workers, and 52 age and gender-matched healthy nonroad workers were enrolled. There were significantly higher levels in all 24 inorganic elements in the COPD group compared with the healthy control group except potassium and manganese, while the majority of inorganic elements were similar between the COPD group and the exposed control group except in aluminum and cobalt. There were 39 genes showing significant differences between the COPD group and the exposed control group. Collagen, type XV, alpha 1 (COL15A1), Meis homeobox 1 (MEIS1), carbonyl reductase 3 (CBR3), and amine oxidase, copper containing 3 (AOC3) were confirmed by rt-PCR to be differentially expressed. Their correlations with blood cytokines were also evaluated. CONCLUSIONS: Aluminum might contribute to the development of COPD in the road-working population. CBR3 and AOC3 seem expressed in different patterns than previously reported, evidenced by their correlation with proinflammatory and anti-inflammatory cytokines.

Seasonal characteristic composition of inorganic elements and polycyclic aromatic hydrocarbons in atmospheric fine particulate matter and bronchoalveolar lavage fluid of COPD patients in Northeast China.

OBJECTIVE: To explore the composition characteristics of atmospheric fine particulate matter (PM2.5) and bronchoalveolar lavage fluid (BALF), and their impact on the development of chronic obstructive pulmonary disease (COPD). METHODS: The atmospheric PM2.5 samples and BALF samples from COPD patients were collected from June 2, 2017 to October 30, 2018, and allocated into a high-risk of PM2.5 inhalation group and a low-risk PM2.5 inhalation group according to the heating season in Harbin. Inorganic elements were detected by ICP-MS, and polycyclic aromatic hydrocarbons (PAHs) were detected by GC/MS. RESULTS: Twenty-six inorganic elements were found in 54 BALF specimens. There was a high correspondence in inorganic elements between BALF and atmospheric PM2.5. Trace elements Cr, Mn, V, and Co, and toxic trace elements Al, Pb, Cd, As, and Ag were above the upper limit of normal blood. There were significant higher K, Ti, Fe, Co, Cu, Se, Rb, Ag, and Sb in BALF of the high-risk PM2.5 inhalation group (p < 0.05). Sixteen PAHs were detected in 32 BALF samples. The main components of BALF and atmospheric PM2.5 were the high molecular weight PAHs, and the species and concentration of PAHs in BALF and atmospheric PM2.5 are highly consistent. CONCLUSION: The types and concentrations of inorganic elements and PAHs in BALF of COPD patients are highly consistent with those of atmospheric PM2.5. The sustained high concentrations of Benzo(a)anthracene, Chrysene, Benzo(b)Fluoranthene, Benzo(k)Fluoranthene, Indeno(123-c,d)Pyrene, and Benzo(a)Pyrene in BALF of COPD patients may have long-term adverse effects on COPD patients.

Argon gas knife combined with cryotherapy for amyloidosis leading to severe airway stenosis.

OBJECTIVE: This case report shows that bronchoscopy is an important method to treat severe airway stenosis caused by bronchial amyloidosis. Bronchoscopic forceps were used to incise the intra-tracheal lump repeatedly. The incision was frozen with a cryosurgery probe, argon knife was used to stop the bleeding until the airway lumen stenosis was reduced to approximately 40%, after which, it continued to enter the lumen. We used bronchoscopic biopsy forceps to repeatedly clamp the lumps in the tracheal carina and left and right main bronchial tumors until the lumen was completely unobstructed. RESULTS: The symptoms of severe dyspnea and wheezing were significantly improved after two interventions with the bronchoscope.

Combined pulmonary fibrosis and emphysema: a retrospective analysis of clinical characteristics, treatment and prognosis.

BACKGROUND: Combined pulmonary fibrosis and emphysema (CPFE) is increasingly acknowledged as a separate syndrome with distinct clinical, physiological and radiological characteristics. We sought to identify physiologic and radiographic indices that predict mortality in CPFE. METHODS: Data on clinical characteristics, pulmonary function, high-resolution computed tomography (HRCT) and treatment were compared between patients with usual interstitial pneumonia (UIP) plus emphysema (CPFE group) and those with IPF alone (IPF group). Composite physiologic index (CPI) and HRCT scores at diagnosis and during follow-up were assessed. RESULTS: CPFE group (N = 87) was characterized by the predominance of males and smokers, who were less likely to have viral infection prior to the diagnosis, and display basal crackles, finger clubbing and wheeze, as compared to that in the IPF group (N = 105). HRCT and CPI scores increased over time in both groups. Moreover, CPFE group had a poorer prognosis, lower 5-year survival rate (43.42 % vs. 65.56 %; P < 0.05), and higher mortality (39.47 % vs. 23.33 %; P < 0.05) as compared to that in the IPF group. All CPFE patients received oxygen therapy, antibiotics and oral N-acetylcysteine; > 50 % received bronchodilators, 40 % received corticosteroids and 14 % needed noninvasive mechanical ventilation. On survival analyses, pulmonary arterial hypertension (PAH) and ≥ 5-point increase in CPI score per year were predictors of mortality in the CPFE group (hazard ratio [HR]: 10.29, 95 % Confidence Interval [CI]: 2.69-39.42 and HR: 21.60, 95 % CI: 7.28-64.16, respectively). CONCLUSION: Patients with CPFE were predominantly male and smokers and exhibited distinct clinical, physiological and radiographic characteristics. They had a poorer prognosis than IPF. PAH and ≥ 5-point increase in CPI score per year were predictors of mortality in these patients. Future studies are needed to identify the optimal treatment approach to CPFE.

Clinical efficacy and safety of ICS/LABA in patients with combined idiopathic pulmonary fibrosis and emphysema.

The study aim was to explore the clinical efficacy and safety of inhaled corticosteroids (ICS)/long-acting beta2-agonists (LABA) in combined with idiopathic pulmonary fibrosis and emphysema. 45 patients with combined idiopathic pulmonary fibrosis and emphysema (CPFE) who were treated with ICS/LABA (Group A), 24 patients with CPFE who were treated without ICS/LABA (Group B) and 35 patients with idiopathic pulmonary fibrosis (IPF) (Group C) were enrolled into this study. Then, clinical efficacy and safety of ICS/LABA was analyzed through lung function scores and lung high-resolution computed tomography (HRCT) scans. Compared with baseline levels, the FEV1%, FVC% and DLCO% levels were increased 11.2%, 13.53% and 12.8% respectively in group A, but declined 14.21%, 16.8% and 21.25% respectively in group B, meanwhile, lung HRCT score was declined 9.31 in group A but increased 14.87 in group B, and there was significant difference between group A and group B (P<0.01). Furthermore, the acute outbreak frequency was 44.4% and 75% in group A and B respectively within 12 months (P<0.05); moreover, CPI index and HRCT score were both lower in group A than those in group B in acute episode period (P<0.05), but there was no significant difference of PO2 value between group A and B (P>0.05). The incidence of adverse reaction was higher in group A than that in group B during this study, but there was no significant difference (P>0.05). ICS/LABA therapy could improve lung function condition in patients with CPFE and declined acute out-break frequency and severity of diseases during acute episode period.

Inhibition of bleomycin-induced pulmonary fibrosis by bone marrow-derived mesenchymal stem cells might be mediated by decreasing MMP9, TIMP-1, INF-γ and TGF-ß.

The study was aimed to investigate the mechanism and administration timing of bone marrow-derived mesenchymal stem cells (BMSCs) in bleomycin (BLM)-induced pulmonary fibrosis mice. Thirty-six mice were divided into six groups: control group (saline), model group (intratracheal administration of BLM), day 1, day 3 and day 6 BMSCs treatment groups and hormone group (hydrocortisone after BLM treatment). BMSCs treatment groups received BMSCs at day 1, 3 or 6 following BLM treatment, respectively. Haematoxylin and eosin and Masson staining were conducted to measure lung injury and fibrosis, respectively. Matrix metalloproteinase (MMP9), tissue inhibitor of metalloproteinase-1 (TIMP-1), γ-interferon (INF-γ) and transforming growth factor ß1 (TGF-ß) were detected in both lung tissue and serum. Histologically, the model group had pronounced lung injury, increased inflammatory cells and collagenous fibres and up-regulated MMP9, TIMP-1, INF-γ and TGF-ß compared with control group. The histological appearance of lung inflammation and fibrosis and elevation of these parameters were inhibited in BMSCs treatment groups, among which, day 3 and day 6 treatment groups had less inflammatory cells and collagenous fibres than day 1 treatment group. BMSCs might suppress lung fibrosis and inflammation through down-regulating MMP9, TIMP-1, INF-γ and TGF-ß. Delayed BMSCs treatment might exhibit a better therapeutic effect. Highlights are as follows: 1. BMSCs repair lung injury induced by BLM. 2. BMSCs attenuate pulmonary fibrosis induced by BLM. 3. BMSCs transplantation down-regulates MMP9 and TIMP-1. 4. BMSCs transplantation down-regulates INF-γ and TGF-ß. 5. Delayed transplantation timing of BMSCs might exhibit a better effect against BLM.

RNA interference targeting enhancer of polycomb1 exerts anti-tumor effects in lung cancer.

BACKGROUND AND AIM: Lung cancer is one of leading malignant tumor worldwide with a high mortality rate. A new therapy target, enhancer of polycomb1 (EPC1) knocked down by short hairpin RNA (shRNA) interference technology, for lung cancer was established to investigate its effects on lung cancer in present study. METHODS: RNA interference technology was applied to down-regulate the expression of EPC1 by specific-shRNA with lentivirus vector in neoplastic human alveolar basal epithelial cells (A549 cells). The survival rate and apoptosis were respectively measured by MTT and Flow Cytometry to evaluate the effects of shRNA EPC1 on cells. Mice xenografts of HCT116 cells with shRNA EPC1 were also established to assess the effect on tumor growth. The levels of AKT and p65 were detected by western blotting. RESULTS: The down-regulation of EPC1 by specific-shRNA with lentivirus vector was significantly decreased the survival rate and apoptosis of A549 cells, and the tumors in EPC1 shRNA transfection group had a significant lower size and weight compared with the ones with control shRNA. The protein expression of p-AKT and p65 was reduced by EPC1 shRNA in both in vitro and in vivo experiments. CONCLUSION: Silencing EPC1 by shRNA technology had the inhibition effects on cell proliferation and tumor growth in lung cancer, which provided a new potential target for treatment of cancers.

Array analysis for potential biomarker of gemcitabine identification in non-small cell lung cancer cell lines.

Gemcitabine is one of the most widely used drugs for the treatment of advanced Non-small cell lung cancer (NSCLC), but modest objective response rate of patients to gemcitabine makes it necessary to identify novel biomarkers for patients who can benefit from gemcitabine-based therapy and to improve the effect of clinical therapy. In this work, 3 NSCLC cell lines displaying different sensitivities to gemcitabine were applied for mRNA and microRNA (miR) expression chips to figure out the biomarkers for gemcitabine sensitivity. Genes whose expression increased dramatically in sensitive cell lines were mainly enriched in cell adhesion (NRP2, CXCR3, CDK5R1, IL32 and CDH2) and secretory granule (SLC11A1, GP5, CD36 and IGF1), while genes with significantly upregulated expression in resistant cell line were mainly clustered in methylation modification (HIST1H2BF, RAB23 and TP53) and oxidoreductase (TP53I3, CYP27B1 and SOD3). The most intriguing is the activation of Wnt/ß-catenin signaling in gemcitabine resistant NSCLC cell lines. The miR-155, miR-10a, miR-30a, miR-24-2* and miR-30c-2* were upregulated in sensitive cell lines, while expression of miR-200c, miR-203, miR-885-5p, miR-195 and miR-25* was increased in resistant cell line. Genes with significantly altered expression and putatively mediated by the expression-changed miRs were mainly enriched in chromatin assembly (MAF, HLF, BCL2, and IGSF3), anti-apoptosis (BCL2, IGF1 and IKBKB), protein kinase (NRP2, PAK7 and CDK5R1) (all the above genes were upregulated in sensitive cells) and small GTPase mediated signal transduction (GNA13, RAP2A, ARHGAP5 and RAB23, down-regulated in sensitive cells). Our results might provide potential biomarkers for gemcitabine sensitivity prediction and putative targets to overcome gemcitabine resistance in NSCLC patients.

DNA microarray reveals different pathways responding to paclitaxel and docetaxel in non-small cell lung cancer cell line.

The wide use of paclitaxel and docetaxel in NSCLC clinical treatment makes it necessary to find biomarkers for identifying patients who can benefit from paclitaxel or docetaxel. In present study, NCI-H460, a NSCLC cell line with different sensitivity to paclitaxel and docetaxel, was applied to DNA microarray expression profiling analysis at different time points of lower dose treatment with paclitaxel or docetaxel. And the complex signaling pathways regulating the drug response were identified, and several novel sensitivity-realted markers were biocomputated.The dynamic changes of responding genes showed that paclitaxel effect is acute but that of docetaxel is durable at least for 48 hours in NCI-H460 cells. Functional annotation of the genes with altered expression showed that genes/pathways responding to these two drugs were dramatically different. Gene expression changes induced by paclitaxel treatment were mainly enriched in actin cytoskeleton (ACTC1, MYL2 and MYH2), tyrosine-protein kinases (ERRB4, KIT and TIE1) and focal adhesion pathway (MYL2, IGF1 and FLT1), while the expression alterations responding to docetaxel were highly co-related to cell surface receptor linked signal transduction (SHH, DRD5 and ADM2), cytokine-cytokine receptor interaction (IL1A and IL6) and cell cycle regulation (CCNB1, CCNE2 and PCNA). Moreover, we also confirmed some different expression patterns with real time PCR. Our study will provide the potential biomarkers for paclitaxel and docetaxel-selection therapy in clinical application.

[Coexpression and clinical significance of multi-drug resistance factors in lung cancer].

BACKGROUND: To study the levels of expression, coexpression, and clinical significancer of four multidrug resistance factors in lung cancer. METHODS: The P glycoprotein (P-gp), multidrug-resistance-associated protein (MRP), lung resistance protein (LRP), glutathione S-transferase (GST-π) of 60 lung cancer patients were detected by using immunohistochemical method. RESULTS: The positive rate of the drug resistance factor was 53.3% (32/60), 63.3% (38/60), 70.0% (42/60), and 80.0% (48/60) for P-gp, MRP, LRP, and GST-π respectively. Patients with NSCLC had significantly higher expression of the drug resistance factors than those with SCLC. On the other hand, no relationship was observed between the expression of drug resistance factors and TNM stage and cell differentiation. The coexpression rate was as follows: P-gp+MRP, 41.6% ; P-gp+LRP, 35.0%; MRP+LRP, 53.3%; MRP+GST-π, 50.0%; LRP+GST-π, 58.3%; P-gp+GST-π, 45.0%; P-gp+MRP+LRP+GST-π, 20.0%. Among them, a relationship was detected between P-gp and MRP ( rs =0.756, P < 0.01), between P-gp and LRP ( rs =0.686, P < 0.01), between MRP and LRP ( rs =0.669, P < 0.01), between MRP and GST-π( rs =0.546, P < 0.01), between LRP and GST-π ( rs =0.848, P < 0.01), between P-gp and LRP ( rs =0.689, P < 0.01), and between P-gp and GST-π ( rs = 0.535 , P < 0.01). CONCLUSIONS: The MDR in lung cancer patients is affected by various multidrug resistance factors. The drug resistance factors' expression is related to histology, but not to TNM stage and cell differentiation.