The clinical relevance of neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio in chronic obstructive pulmonary disease with lung cancer.

Background: Chronic obstructive pulmonary disease (COPD) coexisting with lung cancer is associated with severe mortality and a worse prognosis. Inflammation plays an important role in common pathogenic pathways and disease progression. However, a few studies have identified the clinical value of the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) in COPD with lung cancer, which are systemic inflammatory response markers in the blood. This study aimed to determine the association of the NLR or PLR with clinical characteristics and whether NLR or PLR can be diagnostic markers for COPD with lung cancer. Methods: Between 2015 and 2021, we conducted a retrospective analysis of 236 COPD patients with lung cancer and 500 patients without lung cancer (control group). Clinical information, blood routine examination, and spirometry results were collected and analyzed. The receiver operating characteristic (ROC) curve was used to identify the best cutoff point of NLR or PLR. Multivariate logistic regression analysis was performed to evaluate the association of NLR or PLR with the diagnosis and prognosis of COPD with lung cancer. Results: Compared to patients in the COPD-only group, patients in the lung cancer group had a higher percentage of current smoking and emphysema, and it was found that NLR or PLR was significantly higher in the lung cancer group. Multivariate analysis showed that age, smoking status, FEV1%pred, emphysema, NLR, and PLR were independent risk factors for lung cancer development in COPD. Furthermore, the high level of NLR or PLR was associated with age over 70 years old, current smoking status, and ineligible surgery treatment. The level of PLR or NLR markedly increased with hypercoagulation status, the severity of airflow limitation, and advanced progression of lung cancer. Additionally, the ROC analysis also revealed that elevated NLR or PLR was an independent predictor of COPD in lung cancer patients, TNM stages IIIB-IV at first diagnosis in lung cancer, and ineligible surgery in lung cancer patients. Conclusion: Increased NLR or PLR values might be an important and easily measurable inflammation biomarker to predict the diagnosis and severity of lung cancer with COPD.

Acute exacerbations of chronic obstructive pulmonary disease in a cohort of Chinese never smokers goes along with decreased risks of recurrent acute exacerbation, emphysema and comorbidity of lung cancer as well as decreased levels of circulating eosinophils and basophils.

Acute exacerbations show a significant impact on disease morbidity and mortality in chronic obstructive pulmonary disease (COPD). In contrast to stable COPD, the association of smoking status with clinical and laboratory characteristics in patients with acute exacerbations of COPD (AECOPD) has not been well studied. In this retrospective study, we compared never smokers and ever smokers on their demographic, clinical, and laboratory characteristics in a Chinese clinical cohort of AECOPD. In this cohort comprising 1,034 consecutive patients with AECOPD, never smokers were older (75 vs 70.5 years, padjusted < 0.001) and had a higher body mass index than smokers (21.1 ± 4.0 vs 20.3 ± 3.4, padjusted = 0.028). Furthermore, never smokers showed a decreased risk of recurrent acute exacerbation (13.0 vs 21.8%, padjusted = 0.029), a lower risk of development of emphysema (77.8 vs 89.1%, padjusted < 0.001), a lower prevalence of the co-morbidity of lung cancer (0.5 vs 6.6%, padjusted < 0.001), lower levels of circulating eosinophils (EO; 0.04 × 109/L vs 0.10 × 109/L, padjusted = 0.007) and basophils (BA; 0.02 × 109/L vs 0.03 × 109/L, padjusted = 0.019), and a higher plasma levels of D-dimer (0.62 µg/ml vs 0.51 µg/ml, padjusted = 0.02). Furthermore, multivariate logistic regression analysis identified several risk factor for the recurrent acute exacerbation, such as smoking [odds ratio (OR) = 1.84, 95% CI: 1.03-3.40, p = 0.044], urban residential area (OR = 1.43, 95% CI: 1.01-2.05, p = 0.045), and the presence of emphysema (OR = 2.31, 95% CI: 1.25-4.69, p = 0.012). In conclusion, this study demonstrates that the smoking status of patients is associated with recurrent acute exacerbations, emphysema, lung cancer, and levels of circulating EO and BA in AECOPD. Identification of cigarette smoking as a risk factor for recurrent acute exacerbation supports behavioral intervention of smoking cessation in the management of patients with AECOPD.

Platycodon D-induced A549 Cell Apoptosis through RRM1-Regulated p53/VEGF/ MMP2 Pathway.

BACKGROUND: Lung cancer is one of the leading causes of cancer-related deaths worldwide. Platycodin D (PD), a major pharmacological constituent from the Chinese medicinal herb named Platycodonis Radix, has shown potent anti-tumor activity. Also, it is reported that PD could inhibit cellular growth in the non-small-cell lung carcinoma (NSCLC) A549 cell line. However, the underlying mechanism is not fully clarified. METHODS: Cell proliferation was measured by MTT assay. Annexin V and propidium iodide (PI) assay were employed to study the apoptosis effects of PD on A549 cells. Western blot analysis was used to evaluate protein expression. Also, we used a siRNA against p53, as well as a plasmid-based RRM1 over-expression to investigate their functions. RESULTS: It is demonstrated that PD inhibited A549 cell proliferation in a dose- and time-dependent manner. Further investigations showed that PD induced cell apoptosis, which was supported by dose-dependent and time-dependent caspase-3 activation and p53/VEGF/MMP2 pathway regulation. Also, PD demonstrated the inhibition effect of ribonucleotide reductase M1 (RRM1), whose role in various tumors is contradictory. Remarkably, in this work, RRM1 overexpression in A549 cells could have a negative impact on the regulation of the p53/VEGF/MMP2 pathway induced by PD treatment. Note that RRM1 overexpression also attenuated cell apoptosis and inhibition of cell proliferation of A549 treated with PD. CONCLUSION: The results suggested that PD could inhibit A549 cell proliferation and induce cell apoptosis by regulating p53/VEGF/MMP2 pathway, in which RRM1 plays an important role directly.

The role of HOTAIR/miR-152-3p/LIN28B in regulating the progression of endometrial squamous carcinoma.

INTRODUCTION: There is growing evidence that long non-coding RNAs (lncRNAs) are correlated with malignancy in the modulation of tumor progression. This study aims to investigate the effect of homeobox protein (HOX) transcript antisense RNA (HOTAIR) on the migration and invasion of ESC. MATERIAL AND METHODS: Starbase was used to identify miRNAs with complementary base pairing with HOTAIR. RNA pull-down and qRT-PCR were employed to investigate the effect of HOTAIR on miR-152-3p. In vitro cell migration and invasion assays were performed to assess the effects of HOTAIR and miR-152-3p on ESC. Computational software, TargetScan, was then used to identify the potential target of miR-152-3p, and their relationship was verified by immunoblotting analysis, qRT-PCR and luciferase reporter assay. RESULTS: Starbase predicted a potential miR-152-3p binding site in HOTAIR, which was validated by RNA pull-down assay. HOTAIR was negatively correlated with miR-152-3p in ESC. Moreover, HOTAIR promoted migration and invasion of ESC. The oncogenic activity of HOTAIR was partly through its negative regulation of miR-152-3p. LIN28B was identified to be a direct target of miR-152-3p. A negative correlation between LIN28B and miR-152-3p was observed in ESC. In addition, overexpression of miR-152-3p suppressed the progression of ESC by directly targeting and regulating LIN28B. CONCLUSIONS: Our results reveal that HOTAIR may be a driver of ESC through inhibiting miR-152-3p, a tumor suppressor, suggesting that miR-152-3p may be a potential target for advanced ESC therapeutic treatment.

High Levels of Circulating IL-8 and Soluble IL-2R Are Associated With Prolonged Illness in Patients With Severe COVID-19.

Objectives: The coordinated immune response of the host is the key of the successful combat of the body against SARS-CoV-2 infection and is decisive for the development and progression of COVID-19. In this study, we aimed to investigate whether the immunological phenotype of patients are associated with duration of illness in patients with severe COVID-19. Method: In this single-center study, 69 patients with severe or critical COVID-19 were recruited retrospectively. Immunological parameters including counts of white blood cells, neutrophils, lymphocytes, the neutrophil-to-lymphocyte ratio, and levels of circulating cytokines and cytokine receptors were screened for their association with disease severity, survival and duration of illness of COVID-19. Results: Our data confirmed previous results that neutrophil-to-lymphocyte ratio and circulating levels of IL-6 represent prominent biomarker for the prediction of disease severity and survival of COVID-19. However, this study shows for the first time that duration of illness in patients with severe COVID-19 is positively associated with serum levels of IL-8 (P=0.004) and soluble IL-2Rα (P=0.025). Conclusion: The significant association of duration of illness with circulating levels of IL-8 and soluble IL-2Rα in patients with severe COVID-19 implicates that neutrophils and T cells are involved in the evolution of COVID-19.

Identification of Potential Biomarkers of Polycystic Ovary Syndrome via Integrated Bioinformatics Analysis.

Polycystic ovary syndrome (PCOS) is a life-long reproductive, endocrine, and metabolic disorder that affects up to 17% of women of reproductive age. However, the effect of granulosa cells (GCs) transcriptome changes on oocyte capacity and follicular development in patients with PCOS has not been elucidated. This study aims to analyze transcriptome changes in GCs of PCOS from different perspectives and explore potential biomarkers for the diagnosis and treatment of PCOS. The gene expression profiles of GSE34526 and GSE107746 were obtained from the GEO database. Differentially expressed genes (DEGs) and key signaling pathways were identified. Gene Set Enrichment Analysis (GSEA) revealed that Toll-like receptors, NOD-like receptors, and NOTCH signaling pathways were obviously enriched in GCs of PCOS. We further analyzed DEGs from three aspects: transcription factors (TFs), secreted proteins, and follicular development. Compared with normal GCs, the DEGs encoding TFs and secretory proteins in GCs of PCOS remarkably changed. Besides, HAS2 and CBLN1, which are highly expressed in preovulatory follicular GCs and may trigger ovulation, were significantly decreased in GCs of PCOS. This study found candidate genes and signaling pathways in PCOS, providing new insights and foundations for the etiology of PCOS. Besides, HSA2 and CBLN1 may be potential therapeutic biomarkers for ovulation disorders in PCOS.

Torin2 inhibits the EGFR-TKI resistant Non-Small Lung Cancer cell proliferation through negative feedback regulation of Akt/mTOR signaling.

It is known that mammalian target of rapamycin (mTOR) signaling plays an important role in NSCLC cells proliferation. Torin2 is a second-generation ATP-competitive inhibitor which is selective for mTOR activity. In this study, we investigated whether torin2 was effective against lung cancer cells, especially EGFR-TKIs resistant NSCLC cells. We found that torin2 dramatically inhibited EGFR-TKI resistant cells viability in vitro. In xenograft model, torin2 treatment significantly reduced the volume and weight of xenograft tumor in the erlotinib resistant PC9/E cells. Additionally, autophagy protein of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3II/I (LC3II/I) increased in PC9/E after torin2 treatment. Torin2 blocked the level of phosphorylated S6 and the phosphorylation of Akt at both T308 and S473 sites compared with erlotinib treatment. Furthermore, TUNEL assay showed that apoptosis of tumor tissue increased significantly in the torin2 treatment group. Immunohistochemical analysis demonstrated that tumor angiogenesis was obviously inhibited by torin2 treatment in EGFR-TKI resistant group. Collectively, our results suggested that torin2 could inhibit the NSCLC cells proliferation by negative feedback regulation of Akt/mTOR signaling and inducing autophagy. This suggests that torin2 could be a novel therapeutic approach for EGFR-TKI resistant NSCLC.

Ivermectin Augments the In Vitro and In Vivo Efficacy of Cisplatin in Epithelial Ovarian Cancer by Suppressing Akt/mTOR Signaling.

BACKGROUND: The poor outcomes in epithelial ovarian cancer necessitate new treatments. In this work, we systematically analyzed the inhibitory effects of ivermectin and the molecular mechanism of its action in ovarian cancer. METHODS: The effects of ivermectin alone and its combination with cisplatin on growth and survival were examined using cultured ovarian cancer cells and a xenograft mouse model. The molecular mechanism of action of ivermectin, focusing on Akt/mTOR signaling, was elucidated. RESULTS: Ivermectin arrested growth in the G2/M phase and induced caspase-dependent apoptosis in ovarian cancer, regardless of specific cellular and molecular differences. Ivermectin significantly augmented the inhibitory effect of cisplatin on ovarian cancer cells in a dose-dependent manner. Mechanistically, ivermectin suppressed the phosphorylation of key molecules in the Akt/mTOR signaling pathway in ovarian cancer cells. In addition, overexpression of constitutively active Akt restored ivermectin-induced inhibition of Akt/mTOR, growth arrest and apoptosis. In an ovarian cancer xenograft mouse model, ivermectin alone significantly inhibited tumor growth. In combination with cisplatin, tumor growth was completely reversed over the entire duration of drug treatment without any toxicity. Furthermore, the concentrations of ivermectin used in our study are pharmacologically achievable. CONCLUSIONS: Our work suggests that ivermectin may be a useful addition to the treatment armamentarium for ovarian cancer and that targeting Akt/mTOR signaling is a therapeutic strategy to increase chemosensitivity in ovarian cancer.

Serum Levels of Autoantibodies Against Extracellular Antigens and Neutrophil Granule Proteins Increase in Patients with COPD Compared to Non-COPD Smokers.

Background: Chronic obstructive pulmonary disease (COPD) is a highly prevalent disease leading to irreversible airflow limitation and is characterized by chronic pulmonary inflammation, obstructive bronchiolitis and emphysema. Etiologically, COPD is mediated by toxic gases and particles, eg, cigarette smoke, while the pathogenesis of the disease is largely unknown. Several lines of evidence indicate a link between COPD and autoimmunity but comprehensive studies are lacking. Methods: By using a protein microarray assaying more than 19,000 human proteins we determined in this study the autoantibody profiles of COPD and non-COPD smokers. The discovery cohort included 5 COPD patients under acute exacerbation (AECOPD) and 5 age- and gender-matched non-COPD smokers. One putative candidate autoantibody, anti-lactoferrin IgG, was further investigated by using immunoblotting with a large validation cohort containing 124 healthy controls, 92 patients with AECOPD and 52 patients with stable COPD. Results: We show that i) autoantigens targeted by autoantibodies with higher titers in COPD patients were enriched in extracellular regions, while those with lower titers in COPD patients were enriched in intracellular compartments. ii) levels of IgG autoantibodies against many neutrophil granule proteins were significantly higher in COPD patients than in non-COPD smokers. Furthermore, increased levels of anti-lactoferrin antibodies in COPD patients were confirmed in a cohort with a large number of samples. Conclusion: The comprehensive autoantibody profiles from COPD patients established in this study demonstrated for the first time a shift in the cellular localization of antigens targeted by autoantibodies in COPD.

Association between H19 polymorphisms and NSCLC risk in a Chinese Population.

PURPOSE: Long non-coding RNAs (lncRNA) polymorphisms have been reported to associate with the carcinogenicity mechanisms. The association between lncRNA H19 polymorphisms and the risk of non-small cell lung cancer (NSCLC) in Chinese population has not been reported yet. We designed this case-control study to evaluate the effects of H19 polymorphisms on NSCLC susceptibility. METHODS: In this case-control study, four single nucleotide polymorphisms (SNPs) (rs2067051, rs217727, rs2839698 and rs4929984) in H19 gene were genotyped in a Chinese population which consisted of 564 NSCLC cases and 1536 controls. RESULTS: rs2067051 was associated with a significantly decreased risk of NSCLC in our population [AA vs. GG: adjusted odds ratio (OR) = 0.75, 95% confidence interval (CI)=0.60-0.93; Additive model: OR=0.79, 95%CI=0.67-0.93)]. rs217727 was related to significantly increased NSCLC susceptibility (TT vs. CC: OR=1.16, 95%CI=1.01-1.33; Additive model: OR=1.16, 95%CI=1.01-1.33). However, no significant association was observed between rs2839698, rs4929984 and NSCLC risk. CONCLUSIONS: H19 polymorphism rs2067051 and rs217727 might influence NSCLC susceptibility and the mechanism warrants further exploration.

[Association between Severity of Scrub Typhus and Serum Procalcitonin Level].

Objective To explore the association between the severity of scrub typhus (ST) and the serum procalcitonin (PCT) level. Methods The clinical data of 58 ST patients who were treated in the First Affiliated Hospital of Xiamen University and confirmed by Xiamen Municipal Center for Disease Prevention and Control (CDC) from January 2016 to October 2017,were retrospectively analyzed. According to clinical manifestations and related laboratory tests,these patients were divided into four grades:Ⅰ,Ⅱ,Ⅲ,and Ⅳ. These four grade groups were compared in terms of age,interval from symptom onset to hospital presentation,hospitalization days,and serum PCT level. Results These 58 patients were divided into four grade groups:grade Ⅰ group (n=17),grade Ⅱ group (n=17),grade Ⅲ group (n=11),and grade Ⅳ group (n=13). No significant difference was found among these four groups in terms of age (F=0.618,P=0.606),interval from symptom onset to hospital presentation (F=1.744,P=0.169),and hospitalization days (F=0.398,P=0.755).However,the median serum PCT level in the grade Ⅳ patients[2.60(1.33,61.08)ng/ml] was significantly higher than those in grade I[0.24(0.10,0.33)ng/ml;Z=-4.63,P=0.000], grade Ⅱ[0.29(0.21,0.51)ng/ml;Z=-4.63,P=0.000], and grade Ⅲ[1.33(0.89,2.41)ng/ml;Z=-2.09,P=0.040].The median serum PCT level in the grade Ⅲ patients was also significantly higher than grade Ⅰ (Z=-4.16,P=0.000)and grade Ⅱ(Z=-3.83,P=0.000).There was no significant difference between the grade Ⅰ patients and grade Ⅱ patients(Z=-1.37,P=0.170).There was significantly positive correlation between PCT level and the severity of ST (r=0.804,P=0.000).Conclusion There is positive correlation between serum PCT level and the severity of ST,and serum PCT level may be a biomarker in assessing the severity of ST.

Novel peptide screened from a phage display library antagonizes the activity of CC chemokine receptor 9.

CC chemokine receptor 9 (CCR9) serves a role in the drug resistance and metastasis of tumors. In the present study, a peptide specifically bound to CCR9 was obtained and the effect on tumor cells was observed. A Ph.D.-12 phage display peptide library was used to screen for peptides binding specifically to the second extracellular loop of CCR9. The ratios of the input and output of phage clones increased gradually following three rounds of biopanning. A total of 8 positive phage clones were identified from DNA analysis. A phage clone, C-4, was identified which exhibited higher affinity and specificity for the second extracellular loop of CCR9 in vitro compared with other clones. A peptide (P1; VHWDFRQWWQPS) was identified which may inhibit the corresponding phage, C-4, binding to the second extracellular loop of CCR9. Furthermore, P1 was able to bind specifically with MOLT4 cells which exhibit marked expression of CCR9. In addition, P1 promoted the apoptosis of MOLT4 cells induced by doxorubicin, and inhibited the migration of MOLT4 cells in the presence of chemokine (C-C motif) ligand 25. It was suggested that decreased activity in the phosphorylation of protein kinase B in MOLT4 cells may be responsible for the inhibition. In conclusion, the peptide P1 derived from a screened phage is able to specifically bind to CCR9 and inhibit the activity of CCR9. It has potential use as an antagonist in the treatment of CCR9-overexpressed carcinoma.

mTOR Overactivation and Compromised Autophagy in the Pathogenesis of Pulmonary Fibrosis.

The mammalian target of rapamycin (mTOR) signaling pathway in pulmonary fibrosis was investigated in cell and animal models. mTOR overactivation in alveolar epithelial cells (AECs) was achieved in the conditional and inducible Tsc1 knock-down mice SPC-rtTA/TetO-Cre/Tsc1(fx/+) (STT). Doxycycline caused Tsc1 knock-down and consequently mTOR activation in AECs for the STT mice. Mice treated with bleomycin exhibited increased mortality and pulmonary fibrosis compared with control mice. In wild-type C57BL/6J mice, pretreatment with rapamycin attenuated the bleomycin-mediated mortality and fibrosis. Rapamycin-mediated mouse survival benefit was inhibited by chloroquine, an autophagy inhibitor. Autophagosomes were decreased in the lungs after bleomycin exposure. Rapamycin induced the production of autophagosomes and diminished p62. We concluded that mTOR overactivation in AECs and compromised autophagy in the lungs are involved in the pathogenesis of pulmonary fibrosis. The suppression of mTOR and enhancement of autophagy may be used for treatment of pulmonary fibrosis.

[Serum markers in patients with idiopathic pulmonary alveolar proteinosis].

OBJECTIVE: Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by PAS positive lipoproteinaceous material accumulation in the pulmonary alveoli, of which autoimmune (APAP) or idiopathic type (IPAP) accounts for about 90%. Several serum markers were reported to be valuable in IPAP diagnosis. However, data in Chinese patients was not available. Here we analyzed the diagnostic value of serum markers in Chinese IPAP patients. METHODS: Thirty-one IPAP patients, 25 healthy volunteers and 15 patients with other lung diseases were enrolled in the study. Anti-GM-CSF antibody, SP-A, SP-D and YKL-40 levels of the serum samples were tested by ELISA method. LDH, CEA, arterial blood gas analysis and pulmonary function test results were collected and analyzed. Then the difference between the groups and the relationship between the 2 of the markers were tested. RESULTS: The level of serum anti-GM-CSF antibody, SP-A and SP-D [29.80(13.02-53.86) mg/L, (47 ± 30) and (77 ± 57) µg/L respectively] were higher in IPAP patients than in healthy volunteers and disease control patients (P < 0.05). IPAP and disease control groups had higher YKL-40 and LDH levels than the healthy control group (P < 0.05), but no statistical difference between the former 2 groups (P > 0.05). The CEA level of IPAP patients was higher than that of the disease control group (P < 0.05). The sensitivity and specificity of anti-GM-CSF antibody in IPAP diagnosis were both 100% at the cut-off value of 2.46 µg/mL. The LDH, CEA and SP-A levels correlated positively with P(A-a) O2 and negatively with TL(CO)%, PaO2 and SaO2 (P < 0.05). The SP-D level correlated negatively with SaO2 (P < 0.05), and positively with LDH and CEA level (P < 0.05), so did SP-A (P < 0.05). YKL-40 correlated positively with SP-A but not with other markers. Anti-GM-CSF antibody did not correlate with any of the markers. CONCLUSIONS: Serum anti-GM-CSF antibody testing showed high sensitivity and specificity for the diagnosis of APAP, though it did not correlate with disease severity. Serum LDH, CEA, SP-A and SP-D levels were elevated in IPAP patients and correlated well with disease severity. YKL-40 level was also higher in IPAP patients, but it could not be used as a disease severity marker.

mTORC1 is involved in hypoxia-induced pulmonary hypertension through the activation of Notch3.

Hypoxia-induced pulmonary hypertension (HPH) is a clinical syndrome associated with high morbidity and mortality. However, the underlying mechanisms remain unclear. Both the mammalian target of rapamycin (mTOR) and the Notch3 signaling pathways have been reported to be involved in HPH; however, it is unknown whether there is a connection between these two signaling pathways in HPH. This study was designed to investigate the relationship between mTOR and Notch3 in HPH. After treatment with 10% O2 for 4 weeks, male C57BL/6 mice developed HPH with gradually increased right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), and pulmonary arteriolar remodeling accompanied by the activation of mTOR complex 1 (mTORC1) and Notch3 in the lung tissue and pulmonary arterioles. Pretreatment with the mTORC1 inhibitor rapamycin not only alleviated pulmonary arterial pressure and pulmonary arteriolar remodeling but also suppressed hypoxia-induced mTORC1 and Notch3 activation. Prophylactic N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) administration, a Notch signaling inhibitor, protected against the effects of hypoxia. These in vivo data were confirmed by in vitro experiments on human pulmonary arterial smooth muscle cell (PASMC) exposed to 3% O2 . Furthermore, overexpression of Notch3 intracellular domain partially abrogated the inhibitory effects of rapamycin on human PASMC proliferation. These data indicate that both mTORC1 and Notch3 signaling are involved in HPH and the downstream effects of mTORC1 activation in HPH are partially dependent on the activation of Notch3 signaling.

Tsc1 deficiency-mediated mTOR hyperactivation in vascular endothelial cells causes angiogenesis defects and embryonic lethality.

This is a study on the role of tuberous sclerosis complex1 (TSC1) mutation and mTOR activation in endothelial cells during angiogenic and embryonic development. Past studies had shown that Tsc1/Tsc2 mutant genes lead to overactivation of mTOR in the regulating pathways in developing fetus. We used conditional Cre-loxp gene knockout approach to delete Tsc1 in mice's endothelial cells in our experimental models. Similarly, activation of mTOR signaling in endothelial cells of these embryos (Tie2-Cre/Tsc1(-/-)) was found. Majority of Tie2-Cre/Tsc1(-/-) embryos died at embryonic day 14.5 in utero. Cardiovascular defects, subcutaneous edema and hemorrhage were present among them. Whole-mount immunostaining in these embryos revealed a disorganized vascular network, defective sprouting of vessels in yolk sac and thickening of the labyrinth layer in the placenta. A thinner ventricular wall with disorganized trabeculae was present in the hearts of Tie2-Cre/Tsc1(-/-) embryos. Endothelial cells in Tsc1-deficient mice showed defective mitochondrial and endoplasmic reticular morphology, but no significant change was observed in cell junctions. The mutant embryos displayed significantly reduced cell proliferation, increased apoptosis and disturbed expression of angiogenic factors. A cohort of mice was treated prenatally with mTOR inhibitor rapamycin. The offspring of these mutant mice survived up to 22 days after birth. It was concluded that physiological TSC1-mTOR signaling in endothelial cells is crucial for vascular development and embryogenesis. We postulated that disruption of normal angiogenic pathways through hyperactive mTOR signaling maybe the mechanism that lead to deranged vascular pathogenesis in the tuberous sclerosis complex.

Characteristics of serum lipid metabolism in patients with autoimmune pulmonary alveolar proteinosis.

OBJECTIVE: To study the serum lipid panels in consecutive autoimmune pulmonary alveolar proteinosis(APAP)patients and analyze their relationship with anti-granulocyte macrophage-colony stimulating factor(GM-CSF)antibody and other markers. METHODS: Thirty-two non-diabetic APAP patients were enrolled in the study. Serum lipids of these patients and 100 healthy volunteers were tested after an overnight fasting. Anti-GM-CSF antibody levels were measured with enzyme-linked immunosorbent assay. The correlation of serum lipids with lactate dehydrogenase,carcinoembryonic antigen,pulmonary function,and artery blood gas parameters were analyzed. RESULTS: Total cholesterol and low-density lipoprotein cholesterol levels [(5.54±0.99)and(3.73±0.83)mmol/L respectively] were significantly higher in APAP patients than in healthy volunteers [(5.05±0.97)and(3.17±0.89)mmol/L respectively](all P<0.05). High-density lipoprotein cholesterol(HDL-C)level of the APAP group [(1.10±0.18)mmol/L ]was significantly lower than that of the healthy group(P<0.05). Low-density lipoprotein/HDL and total cholesterol/HDL ratios in the APAP group(3.47±0.90 and 5.14±1.12 respectively)were significantly higher than those in the healthy group[(2.63±0.87)and(4.18±1.12)](all P<0.05). There was no significant difference in triglyceride level between the two groups(P>0.05). HDL-C level was negatively correlated with alveolar-arterial oxygen pressure difference(r=-0.436,P<0.05)and positively correlated with arterial oxygen saturation(r=0.459,P<0.05). None of the lipid markers correlated with serum anti-GM-CSF antibody levels(all P>0.05). CONCLUSIONS: APAP patients were likely to suffer from disturbed lipid metabolism,which was correlated with disease severity to some degree. Lipid markers deserved more attention in the management of APAP patients.

The role of vascular endothelial growth factor-D in diagnosis of lymphangioleiomyomatosis (LAM).

BACKGROUND: Definite diagnosis of lymphangioleiomyomatosis (LAM) depends on either transbronchial lung biopsy or video-assisted thoracic surgery, unless there is a history of chylothorax, kidney angiomyolipoma (AML), or tuberous sclerosis complex (TSC). Vascular endothelial growth factor-D (VEGF-D) was recently considered as a novel diagnostic marker for LAM. Herein, we evaluated diagnostic value of serum VEGF-D in LAM patients. METHODS: Serum samples were obtained from 78 cases of LAM (50 definite and 28 probable LAM based on European Respiratory Society guidelines), and 40 healthy female volunteers. VEGF-D was measured using enzyme-linked immunosorbant assay according to product instruction (R&D). RESULTS: Serum VEGF-D was significantly increased in definite LAM group, compared with that of health control (median: 3841.9 pg/mL vs 405.5 pg/mL respectively, p < 0.001). The optimal cut-off point for definite LAM diagnosis was 850.7 pg/mL. In probable LAM group, the majority of patients (92.9%) had serum VEGF-D level over 850.7 pg/mL. The serum levels of VEGF-D in LAM patients with pulmonary cystic lesions only were lower than that in patients with any of evidences of AML, chylous effusions, adenopathy, lymphangioleiomyomas, or TSC, but higher than that in the health control. In addition, VEGF-D levels were correlated with disease severity measured as LAM CT grade, and presentations of chylous effusions and/or lymphatic involvement (p < 0.05). CONCLUSION: Serum VEGF-D should be added to the current diagnosis algorithm to enhance definitive diagnosis for LAM.

SPC-Cre-ERT2 transgenic mouse for temporal gene deletion in alveolar epithelial cells.

Although several Cre-loxP-based gene knockout mouse models have been generated for the study of gene function in alveolar epithelia in the lung, their applications are still limited. In this study, we developed a SPC-Cre-ER(T2) mouse model, in which a tamoxifen-inducible Cre recombinase (Cre-ER(T2)) is under the control of the human surfactant protein C (SPC) promoter. The specificity and efficiency of Cre-ER(T2) activity was first evaluated by crossing SPC-Cre-ER(T2) mouse with ROSA26R mouse, a ß-galactosidase reporter strain. We found that Cre-ER(T2) was expressed in 30.7% type II alveolar epithelial cells of SPC-Cre-ER(T2)/ROSA26R mouse lung tissues in the presence of tamoxifen. We then tested the tamoxifen-inducible recombinase activity of Cre-ER(T2) in a mouse strain bearing TSC1 conditional knockout alleles (TSC1(fx/fx)). TSC1 deletion was detected in the lungs of tamoxifen treated SPC-Cre-ER(T2)/TSC1(fx/fx) mice. Therefore this SPC-Cre-ER(T2) mouse model may be a valuable tool to investigate functions of genes in lung development, physiology and disease.