hoxa1a-Null Zebrafish as a Model for Studying HOXA1-Associated Heart Malformation in Bosley-Salih-Alorainy Syndrome.

Mutations in HOXA1 can lead to diseases such as Bosley-Salih-Alorainy syndrome, involving severe cardiovascular malformations. However, the role of HOXA1 in cardiac morphogenesis remains unclear. hoxa1a is a homologous gene to human HOXA1 in zebrafish. We used CRISPR to make hoxa1a-null zebrafish that exhibited multiple heart malformations. In situ hybridization and sections revealed the morphological changes in mutants: enlarged ventricle with thickened myocardium and increased trabeculae, intensified OFT and inadequate heart looping, with electrocardiography supporting these pathological changes. High-speed photography captured cardiac pumping and revealed a significant decrease in cardiac output. Furthermore, lacking hoxa1a led to posterior body abnormality that affected movement ability, corresponding with the motor development delay in patients. Upregulation of hox paralogues in hoxa1a-null fish implied a compensatory mechanism between hox genes. Accordingly, we successfully constructed a hoxa1a-null model with a cardiac disease pattern which occurred in human HOXA1-associated heart malformation. The study of hoxa1a in zebrafish can further promote the understanding of hox genes and related diseases.

Baseline Insulin Resistance Is a Determinant of the Small, Dense Low-Density Lipoprotein Response to Diets Differing in Saturated Fat, Protein, and Carbohydrate Contents.

Individual responses to diet vary but causes other than genetics are poorly understood. This study sought to determine whether baseline values of homeostasis model assessment (HOMA-IR) was related to changes in small, dense low-density lipoprotein (sdLDL, i.e., LDL4, d = 1.044-1.063 g/mL) amounts quantified by isopycnic density profiling, in mildly hypercholesterolemic subjects (n = 27) consuming one of three low saturated fatty acid (SFA) diets: Dietary Approaches to Stop Hypertension (DASH), Beef in an Optimal Lean Diet (BOLD) and BOLD plus extra protein (BOLD+) when compared to a higher-SFA healthy American diet (HAD). The diets were consumed in random order for 5 wk, with 1 wk between diets. BOLD+ reduced fractional abundance (%) LDL4 (p < 0.05) relative to HAD, DASH and BOLD, and reductions in % LDL4 correlated with reductions in triglycerides (p = 0.044), total cholesterol (p = 0.014), LDL cholesterol (p = 0.004) and apolipoprotein B (p < 0.001). Responses to the four diets were similar (~12% decrease in % LDL4, p = 0.890) in the lower (<2.73 median) HOMA-IR subgroup but differed across diet conditions in the higher HOMA-IR subgroup (p = 0.013), in which % LDL4 was reduced with BOLD+ (-11%), was unchanged in BOLD and increased with the HAD (8%) and DASH (6%) diets (p < 0.05 for BOLD+ vs. HAD). Individual responses to diet interventions are influenced by presence and degree of insulin resistance as measured by HOMA-IR.

Early Use of Corticosteroid May Prolong SARS-CoV-2 Shedding in Non-Intensive Care Unit Patients with COVID-19 Pneumonia: A Multicenter, Single-Blind, Randomized Control Trial.

BACKGROUND: There is still no clinical evidence available to support or to oppose corticosteroid treatment for coronavirus disease 2019 (COVID-19) pneumonia. OBJECTIVE: To investigate the efficacy and safety of corticosteroid given to the hospitalized patients with COVID-19 pneumonia. METHODS: This was a prospective, multicenter, single-blind, randomized control trial. Adult patients with COVID-19 pneumonia who were admitted to the general ward were randomly assigned to either receive methylprednisolone or not for 7 days. The primary end point was the incidence of clinical deterioration 14 days after randomization. RESULTS: We terminated this trial early because the number of patients with COVID-19 pneumonia in all the centers decreased in late March. Finally, a total of 86 COVID-19 patients underwent randomization. There was no difference of the incidence of clinical deterioration between the methylprednisolone group and control group (4.8 vs. 4.8%, p = 1.000). The duration of throat viral RNA detectability in the methylprednisolone group was 11 days (interquartile range, 6-16 days), which was significantly longer than that in the control group (8 days [2-12 days], p = 0.030). There were no significant differences between the 2 groups in other secondary outcomes. Mass cytometry discovered CD3+ T cells, CD8+ T cells, and NK cells in the methylprednisolone group which were significantly lower than those in the control group after randomization (p < 0.05). CONCLUSIONS: From this prematurely closed trial, we found that the short-term early use of corticosteroid could suppress the immune cells, which may prolong severe acute respiratory syndrome coronavirus 2 shedding in patients with COVID-19 pneumonia. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04273321.

Rare Earth Elements Lanthanum and Praseodymium Adversely Affect Neural and Cardiovascular Development in Zebrafish (Danio rerio).

Increasing rare earth element (REE) mining and refining activities have led to a considerable release of these substances into aquatic environment, yet the knowledge of their impacts on aquatic organisms is still limited. Here, we explored the developmental effects of 16 REEs (concentration ranged from 0.46 to 1000 mg/L) to zebrafish embryos and highlighted the adverse effects of lanthanum (La) and praseodymium (Pr). Among the multiple developmental parameters measured, the significant effects on swimming behavior and cardiac physiology were the most prominent. Transcriptomic analysis of La and Pr at concentrations of 1.1 to 10 mg/L revealed their rather uniform effects at molecular levels. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis revealed that among others, notch, glutamate, and serotonin signaling, as well as cardiac hypertrophy and cardiac muscle contraction, were significantly affected. These changes of neural signaling were consistent with behavior effects observed and supported by neurotransmitter changes and thus provide a reasonable molecular mechanistic explanation. Furthermore, increased DNA damage and apoptotic activity at high concentrations were observed, especially in the heart. They may contribute to explain the observed adverse morphological and physiological outcomes, such as pericardial edema. The effect concentrations observed in the present study were comparable to the concentrations of REE residues at highly contaminated sites (several mg/L), indicating ecotoxicological effects at environmentally relevant concentrations. Overall, the present data help to clarify the potential developmental toxicity of REEs that was not yet fully recognized and thus contribute to their environmental risk assessment.

TSAd Plays a Major Role in Myo9b-Mediated Suppression of Malignant Pleural Effusion by Regulating TH1/TH17 Cell Response.

Emerging evidence indicates that Myo9b is a cancer metastasis-related protein and functions in a variety of immune-related diseases. However, it is not clear whether and how Myo9b functions in malignant pleural effusion (MPE). In this study, our data showed that Myo9b expression levels correlated with lung cancer pleural metastasis, and nucleated cells in MPE from either patients or mice expressed a lower level of Myo9b than those in the corresponding blood. Myo9b deficiency in cancer cells suppressed MPE development via inhibition of migration. Myo9b deficiency in mice suppressed MPE development by decreasing TH1 cells and increasing TH17 cells. CD4+ naive T cells isolated from Myo9b-/- mouse spleens exhibited less TH1 cell differentiation and more TH17 cell differentiation in vitro. mRNA sequencing of nucleated cells showed that T cell-specific adaptor protein (TSAd) was downregulated in Myo9b-/- mouse MPE, and enrichment of the H3K27me3 mark in the TSAd promoter region was found in the Myo9b-/- group. Naive T cells purified from wild type mouse spleens transfected with TSAd-specific small interfering RNAs (siRNAs) also showed less TH1 cell differentiation and more TH17 cell differentiation than those from the siRNA control group. Furthermore, downregulation of TSAd in mice using cholesterol-conjugated TSAd-specific siRNA suppressed MPE development, decreased TH1 cells, and increased TH17 cells in MPE in vivo. Taken together, Myo9b deficiency suppresses MPE development not only by suppressing pleural cancer metastasis but also by regulating TH1/TH17 cell response via a TSAd-dependent pathway. This work suggests Myo9b and TSAd as novel candidates for future basic and clinical investigations of cancer.

IL-10 promotes malignant pleural effusion by regulating TH 1 response via an miR-7116-5p/GPR55/ERK pathway in mice.

IL-10, produced by a wide variety of cells, is a highly pleiotropic cytokine that plays a critical role in the control of immune responses. However, its regulatory activity in tumor immunity remains poorly understood. In this study, we report that IL-10 deficiency robustly suppressed the formation of malignant pleural effusion (MPE) and significantly enhanced miR-7116-5p expression in pleural CD4+ T cells. We demonstrated that miR-7116-5p suppressed IL-10-mediated MPE formation by inhibiting pleural vascular permeability as well as tumor angiogenesis and tumor growth. IL-10 promoted MPE formation by suppressing miR-7116-5p that enhances TH 1 response. We identified G protein-coupled receptor 55 (GPR55) as a potential target of miR-7116-5p, and miR-7116-5p promoted TH 1 cell function by downregulating GPR55. Moreover, GPR55 promoted MPE formation by inhibiting TH 1 cell expansion through the ERK phosphorylation pathway. These results uncover an IL-10-mediated pathway controlling TH 1 cells and demonstrate a central role for miR-7116-5p/GPR55/ERK signaling in the physiological regulation of IL-10-driven pro-malignant responses.

Influence of age on the diagnostic accuracy of soluble biomarkers for tuberculous pleural effusion: a post hoc analysis.

BACKGROUND: Accurately diagnosing pleural effusion is a frequent and significant problem in clinical practice. Combining pleural biomarkers with patients' age may be a valuable method for diagnosing TPE. We sought to evaluate the influence of age on diagnostic values of pleural adenosine deaminase (ADA), interferon-gamma (IFN-γ), and interleukin 27 (IL-27) for tuberculous pleural effusion (TPE). METHODS: Two hundred seventy-four consecutive adult patients with pleural effusion were selected from Beijing and Wuhan between January 1, 2014 and June 30, 2015, and their pleural fluid concentrations of ADA, IFN-γ, and IL-27 were tested. Biomarker performance was analyzed by standard receiver operating characteristic (ROC) curves according to different ages. RESULTS: Data from the Beijing cohort showed that ADA, IFN-γ, and IL-27 could all accurately diagnose TPE in young patients (≤ 40 years of age). With a cutoff of 21.4 U/L, the area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ADA for diagnosing TPE were 1.000 (95% confidence interval: 0.884-1.000), 100.0, 100.0%, 100.0, and 100.0, respectively. In older patients (> 40 years of age), IL-27 and IFN-γ were excellent biomarkers for discriminating TPE versus non-TPE cases. With a cutoff of 591.4 ng/L, the AUC, sensitivity, specificity, PPV, and NPV of IL-27 for diagnosing TPE were 0.976 (95% confidence interval: 0.932-0.995), 96.3, 99.0%, 96.3, and 99.0, respectively. Similar diagnostic accuracy among the three pleural biomarkers was validated in the Wuhan cohort. CONCLUSIONS: Among young patients, ADA is reliable for diagnosing TPE. Conversely, in older patients, IL-27 and IFN-γ are excellent biomarkers to differentiate TPE versus non-TPE cases.

Diagnostic value of CD206+CD14+ macrophages in diagnosis of lung cancer originated malignant pleural effusion.

BACKGROUND: Pleural effusions are common complications of various diseases. Patients with malignant pleural effusion (MPE) usually face poor prognosis and short life expectancy. Discriminating between MPE and benign pleural effusion remains to be difficult. The aim of our current study was to evaluate whether CD206+CD14+ macrophages could be a diagnostic biomarker for MPE. METHODS: The percentages of CD14+, CD86+CD14+ and CD206+CD14+ macrophages in pleural effusions were determined by flow cytometry in 34 patients with MPE and 26 with benign pleural effusion, and their diagnostic performances were evaluated by receiver operating characteristic (ROC) curves. RESULTS: The percentages of CD14+, CD86+CD14+ and CD206+CD14+ macrophages were remarkably higher in MPE than in benign pleural effusion (all P<0.05). With a cutoff value of 39.8%, a high sensitivity of 88.2% and high specificity of 100.0% were found in CD206+CD14+ macrophages to diagnose MPE. The area under the curve, positive predictive value and negative predictive value of CD206+CD14+ macrophages were 0.980 (95% CI, 0.905 to 0.999), 100.0 (88.4 to 100.0) and 86.7 (69.3 to 96.2), respectively. The diagnostic performance of CD206+CD14+ macrophages was more accurate than those of CD14+ and CD86+CD14+ macrophages. CONCLUSIONS: CD206+CD14+ macrophages could be used to discriminate MPE from benign pleural effusion.

Development and validation of the PET-CT score for diagnosis of malignant pleural effusion.

PURPOSE: Although some parameters of positron emission tomography with 18F-fluorodeoxyglucose (18F-FDG) and computed tomography (PET-CT) are somehow helpful in differentiating malignant pleural effusion (MPE) from benign effusions, no individual parameter offers sufficient evidence for its implementation in the clinical practice. The aim of this study was to establish the diagnostic accuracy of a scoring system based on PET-CT (the PET-CT score) in diagnosing MPE. METHODS: One prospective derivation cohort of patients with pleural effusions (84 malignant and 115 benign) was used to develop the PET-CT score for the differential diagnosis of malignant pleural effusion. The PET-CT score was then validated in another independent prospective cohort (n = 74). RESULTS: The PET-CT parameters developed for discriminating MPE included unilateral lung nodules and/or masses with increased 18F-FDG uptake (3 points); extrapulmonary malignancies (3 points); pleural thickening with increased 18F-FDG uptake (2 points); multiple nodules or masses (uni- or bilateral lungs) with increased 18F-FDG uptake (1 point); and increased pleural effusion 18F-FDG uptake (1 point). With a cut-off value of 4 points in the derivation cohort, the area under the curve, sensitivity, specificity, positive likelihood ratio, and negative likelihood ratio of the PET-CT score to diagnose MPE were 0.949 (95% CI: 0.908-0.975), 83.3% (73.6%-90.6%), 92.2% (85.7%-96.4%), 10.7 (5.6-20.1), and 0.2 (0.1-0.3), respectively. CONCLUSIONS: A simple-to-use PET-CT score that uses PET-CT parameters was developed and validated. The PET-CT score can help physicians to differentiate MPE from benign pleural effusions.

IL-10 promotes malignant pleural effusion in mice by regulating TH 1- and TH 17-cell differentiation and migration.

The role of IL-10 in malignant pleural effusion (MPE) remains unknown. By using murine MPE models, we observed that an increase in pleural IL-10 was a significant predictor of increased risk of death. We noted that TH 1- and TH 17-cell content in MPE was higher in IL-10-/- mice than in WT mice, and IL-10 deficiency promoted differentiation into TH 1 but not into TH 17 cells. A higher fraction of TH 1 and TH 17 cells in the MPE of IL-10-/- mice expressed CXCR3 compared with WT mice. We also demonstrated that Lewis lung cancer and colon adenocarcinoma cells secreted large amounts of CXCL10, a ligand of CXCR3, which induced the migration of TH 1 and TH 17 cells into the MPE, and IFN-γ could promote this signaling cascade. Furthermore, intrapleural injection of mice with CXCL10-deficient tumor cells led to decreased TH 1- and TH 17-cell content in MPE, increased MPE volume, and reduced survival of MPE-bearing mice. Taken together, we demonstrated that IL-10 deficiency promoted T-cell differentiation into TH 1 cells and upregulated the CXCR3-CXCL10 signaling pathway that recruits TH 1 and TH 17 cells into MPE, ultimately resulting in decreased MPE formation and longer survival time of mice-bearing MPE.

IL-17A-Producing γδT Cells Inhibit the Formation of Malignant Pleural Effusions.

γδT cells are an important source of IL-17A and play an anti- or protumor role depending on the surrounding microenvironment. The precise role of γδT cells in the development of malignant pleural effusions (MPE) remains unknown. Using flow cytometry, we analyzed the distribution and differentiation of γδT cells in wild-type (WT) and IL-10-∕- mice. We carefully elucidated the influence of γδT cells on the formation of MPE by depleting γδT cells from WT, IL-10-∕-, and IL-17a-∕- mice. The distribution of γδT17 cells in human MPE and peripheral blood was also determined. Our data showed that both γδT cells and IL-17A-producing γδT (γδT17) cells accumulated in murine MPE, and IL-10 deficiency enhanced their accumulation. γδT cells were the main source of IL-17A in MPE for both WT and IL-10-∕- mice. IL-10 inhibited the chemotactic response of γδT cells to MPE and the proliferation of these cells. IL-10 suppressed γδT cell secretion of IL-17A via RORγt. The ablation of γδT cells accelerated MPE accumulation in both WT and IL-10-∕- mice, but it did not influence MPE accumulation in IL-17a-∕- mice. Patients with higher frequencies of γδT17 cells had significantly longer survival times than patients with lower frequencies of γδT17 cells. Taken together, our data demonstrate that γδT17 cells play an inhibitory role in the progression of MPE, and the accumulation of γδT17 cells in MPE is suppressed by IL-10.

Tumor necrosis factor-alpha-308 gene promoter polymorphism associates with survival of cancer patients: A meta-analysis.

BACKGROUND: Tumor necrosis factor-alpha (TNF-α) is involved in cancer pathogenesis, and TNF-α-308G>A, a single-nucleotide polymorphism, is associated with cancer prognosis; however, different studies have reported inconsistent results. This meta-analysis aimed to determine the correlation between TNF-α-308G>A polymorphism and the survival of cancer patients. METHODS: PubMed, Web of Science, Embase, Wanfang database, VIP database, and China National Knowledge Infrastructure database were used to obtain articles on association between TNF-α-308G>A polymorphism and cancer survival, published until April 2018. A meta-analysis was carried out using Stata 12.0 software to determine the pooled hazard ratio (HR) and 95% confidence intervals (95% CI). Furthermore, publication bias was assessed, and sensitivity analysis was performed to validate the analysis. RESULTS: In total, 13 retrospective cohort studies including 2559 cancer patients were reviewed to estimate the association between TNF-α-308G>A polymorphism and overall survival (OS) of cancer patients. The pooled results suggested that within TNF-α-308G>A polymorphism, genotypes GA+AA/GG (HR = 1.39, 95% CI: 0.90-2.14, P < .001, I = 78.1%), GA/GG (HR = 1.06, 95% CI: 0.83-1.36, P = .072, I = 53.5%), and AA/AG+GG (HR = 3.28, 95% CI: 0.92-11.72, P = .001, I = 85.9%) were not associated with the OS of cancer patients. However, interestingly, the HR was greater for patients with the AA genotype than for those with the GG genotype, suggesting an association between TNF-α-308G>A polymorphism and OS among cancer patients (AA/GG, HR = 2.16, 95% CI: 1.36-3.43, P = .281, I = 21.5%). CONCLUSION: TNF-α-308G>A polymorphism affects the OS of cancer patients and is a potential therapeutic target for cancer.

Activated naïve B cells promote development of malignant pleural effusion by differential regulation of TH1 and TH17 response.

Inflammatory signaling networks between tumor cells and immune cells contribute to the development of malignant pleural effusion (MPE). B cells have been found in MPE; however, little is known about their roles there. In the present study, by using mouse MPE models, we noted that although the total B cells in MPE were decreased as compared with the corresponding blood and spleen, the percentage of activated naïve B cells expressing higher levels of CD80, CD86, myosin heavy chain-II, CD44, CD69, and programmed cell death-ligand 1 (PD-L1) molecules were increased in wild-type mouse MPE. Compared with wild-type mice, decreased T helper (TH)1 cells and increased TH17 cells were present in B cell-deficient mouse MPE, which paralleled to the reduced MPE volume and longer survival time. Adoptive transfer of activated naïve B cells into B cell-deficient mice was able to increase TH1 cells and decrease TH17 cells in MPE and shorten the survival of mice bearing MPE. Furthermore, we demonstrated that activated naïve B cells inhibited TH17-cell expansion via the PD-1/PD-L1 pathway and promoted naïve CD4+ T-cell differentiation into TH1/TH17 cells through secreting IL-27/IL-6 independent of the PD-1/PD-L1 pathway. Collectively, our data uncovered a mechanism by which naïve B cells promote MPE formation by regulating TH1/TH17 cell responses, making these B cells an attractive target for therapeutic intervention in the fight against cancer.

Immune Regulation of Toll-Like Receptor 2 Engagement on CD4+ T Cells in Murine Models of Malignant Pleural Effusion.

Toll-like receptor (TLR) 2 has a well-known role in sensing multiple ligands that include microbial products, endotoxin, and some extracellular matrix molecules; however, its role in the development of malignant pleural effusion (MPE) remains unknown. We performed the present study to explore the impact of TLR2 signaling on the development of MPE and to define the underlying mechanisms by which TLR2 works. Development of MPE was compared between TLR2-/- and wild-type (WT) mice. The effect of TLR2 on differentiation of T helper type 17 (Th17), Th9, and Th2 cells in MPE was explored. The mechanisms of TLR2 on survival of mice bearing MPE were also investigated. MPE volume in TLR2-/- mice was lower than that in WT mice, and the survival of TLR2-/- mice bearing MPE was longer than that of WT mice. TLR2 deficiency increased, and TLR2 activation decreased, Th17 cells in MPE, whereas TLR2 signaling showed the contrary effects on Th2 cells. Th9 cells were increased in MPE of TLR2-/- mice but were not influenced by TLR2 signaling. Intraperitoneal injection of anti-IL-17 monoclonal antibody (mAb), anti-IL-9 mAb, or recombinant mouse IL-4 accelerated the death of TLR2-/- mice bearing MPE, and intraperitoneal injection anti-IL-17 mAb in TLR2-/- mice was associated with a significantly shorter survival time than in WT mice. We have demonstrated, for the first time, that TLR2 signaling promotes the development of MPE and accelerates the death of mice bearing MPE by directly suppressing Th17 cell differentiation and directly promoting Th2 cell differentiation, and also by indirectly suppressing Th9 cell differentiation via an IL-17-dependent mechanism.

Interplay of Th1 and Th17 cells in murine models of malignant pleural effusion.

RATIONALE: IFN-γ-producing CD4(+) T (Th1) cells and IL-17-producing CD4(+) T (Th17) cells have been found to be involved in multiple malignancies; however, the reciprocal relationship between Th1 and Th17 cells in malignant pleural effusion (MPE) remains to be elucidated. OBJECTIVES: To explore the differentiation and immune regulation of Th1 and Th17 cells in the development of MPE in murine models. METHODS: The distribution and differentiation of Th1 and Th17 cells in MPE were investigated in IFN-γ(-/-), IL-17(-/-), and wild-type mice. The effects of Th1 and Th17 cells on the development of MPE and the survival of mice bearing MPE were also investigated. MEASUREMENTS AND MAIN RESULTS: We have demonstrated that increased Th1 and Th17 cells could be found in MPE as compared with blood and spleen. Compared with wild-type mice, Th17 cells were markedly augmented in MPE from IFN-γ(-/-) mice, and improved survival could be seen in IFN-γ(-/-) mice. Th1 cell numbers were elevated in MPE from IL-17(-/-) mice, and decreased survival could be seen in IL-17(-/-) mice. The in vitro experiments showed that IFN-γ deficiency promoted Th17-cell differentiation by suppressing the STAT3 pathway and that IL-17 deficiency promoted Th1-cell differentiation by suppressing the STAT1 pathway. CONCLUSIONS: In mouse models of MPE, IFN-γ inhibited Th17-cell differentiation, whereas IL-17 inhibited Th1-cell differentiation. IL-17 inhibited the formation of MPE and improved the survival of mice bearing MPE; in contrast, IFN-γ promoted MPE formation and mouse death.

[Effects of corticosteroid on eosinophils and expression of transforming growth factor beta 1 in nasal polyps].

OBJECTIVE: To observe the effects of systemic steroids on the infiltration of eosinophils and the expression of TGF beta 1 in nasal polyps. METHOD: The SP immunohistochemical methods were applied to explore the expression of TGF beta 1 in nasal polyps before and after corticosteroids use; the optical density of positive cells in mucosal epithelia, eosinophils, glandular epithelium, vascular endothelial, matrix and mucous membrane was measured by using HPIAS-1,000 image-conduct system. RESULT: The expression of TGF beta 1 was positive in nasal polyps. The number of eosinophils and the expression of TGF beta 1 was significantly reduced in nasal polyps after treated with corticosteroids compared with pre-treated. CONCLUSION: The effects of systemical steroid use on the nasal polyps may depend on decreasing the infiltration of eosinophils and the expression of TGF beta 1.