Soluble TREM-like Transcript-1 Acts as a Damage-Associated Molecular Pattern through the TLR4/MD2 Pathway Contributing to Immune Dysregulation during Sepsis.

Studies have shown that elevated plasma levels of platelet-derived soluble TREM-like transcript-1 (sTLT-1) are associated with an unfavorable outcome in patients with septic shock. However, the underlying molecular mechanisms are not well defined. This research aimed to study the role of sTLT-1 in mediating immune dysfunction during the development of sepsis. Our study demonstrated that patients with septic shock have significantly higher plasma concentrations of sTLT-1, whereas sTLT-1 is not detectable in healthy subjects. Plasma concentrations of sTLT-1 were correlated with the degree of immunosuppressive parameters in monocytes from patients with septic shock. sTLT-1 can first activate monocytes by binding to the TLR4/MD2 complex but subsequently induce immunosuppressive phenotypes in monocytes. Blocking Abs against TLR4 and MD2 led to a significant decrease in sTLT-1-induced activation. Treatment with an anti-TLT-1 Ab also significantly reduces sTLT-1 binding to monocytes and proinflammatory cytokine secretion in a mouse model of endotoxemia. sTLT-1 acts as an endogenous damage-associated molecular pattern molecule, triggering the activation of monocytes through the TLR4/MD2 complex followed by sustained immune suppression. This process plays a crucial role in the development of sepsis-associated pathophysiology. Our findings outline, to our knowledge, a novel pathway whereby platelets counteract immune dynamics against infection through sTLT-1.

A Microfluidic Flip-Chip Combining Hydrodynamic Trapping and Gravitational Sedimentation for Cell Pairing and Fusion.

Cancer cell-immune cell hybrids and cancer immunotherapy have attracted much attention in recent years. The design of efficient cell pairing and fusion chips for hybridoma generation has been, subsequently, a subject of great interest. Here, we report a three-layered integrated Microfluidic Flip-Chip (MFC) consisting of a thin through-hole membrane sandwiched between a mirrored array of microfluidic channels and saw-tooth shaped titanium electrodes on the glass. We discuss the design and operation of MFC and show its applicability for cell fusion. The proposed device combines passive hydrodynamic phenomenon and gravitational sedimentation, which allows the transportation and trapping of homotypic and heterotypic cells in large numbers with pairing efficiencies of 75~78% and fusion efficiencies of 73%. Additionally, we also report properties of fused cells from cell biology perspectives, including combined fluorescence-labeled intracellular materials from THP1 and A549, mixed cell morphology, and cell viability. The MFC can be tuned for pairing and fusion of cells with a similar protocol for different cell types. The MFC can be easily disconnected from the test setup for further analysis.

Author response to "lack of benefit from low dose computed tomography in screening for lung cancer".

We explain to Dr. Benjamin (corresponding author) about why low-dose computed tomography reduce lung cancer mortality without significantly reducing all-cause mortality. We also conduct an up-to-date meta-analysis to evaluate low-dose computed tomography clinical effectiveness compared with usual care of lung cancer screening.

Fenofibrate Facilitates Post-Active Tuberculosis Infection in Macrophages and is Associated with Higher Mortality in Patients under Long-Term Treatment.

BACKGROUND: Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects and persists in macrophages. This study aimed to investigate the effects of long-term fenofibrate treatment in patients with tuberculosis (TB), and the intracellular viability of Mtb in human macrophages. METHODS: Epidemiological data from the National Health Insurance Research Database of Taiwan were used to present outcomes of TB patients treated with fenofibrate. In the laboratory, we assessed Mtb infection in macrophages treated with or without fenofibrate. Mtb growth, lipid accumulation in macrophages, and expression of transcriptional genes were examined. RESULTS: During 11 years of follow-up, TB patients treated with fenofibrate presented a higher risk of mortality. Longer duration of fenofibrate use was associated with a significantly higher risk of mortality. Treatment with fenofibrate significantly increased the number of bacilli in human macrophages in vitro. Fenofibrate did not reduce, but induced an increasing trend in the intracellular lipid content of macrophages. In addition, dormant genes of Mtb, icl1, tgs1, and devR, were markedly upregulated in response to fenofibrate treatment. Our results suggest that fenofibrate may facilitate intracellular Mtb persistence. CONCLUSIONS: Our data shows that long-term treatment with fenofibrate in TB patients is associated with a higher mortality. The underlying mechanisms may partly be explained by the upregulation of Mtb genes involved in lipid metabolism, enhanced intracellular growth of Mtb, and the ability of Mtb to sustain a nutrient-rich reservoir in human macrophages, observed during treatment with fenofibrate.

Effects of low-dose computed tomography on lung cancer screening: a systematic review, meta-analysis, and trial sequential analysis.

BACKGROUND: The Nelson mortality results were presented in September 2018. Four other randomized control trials (RCTs) were also reported the latest mortality outcomes in 2018 and 2019. We therefore conducted a meta-analysis to update the evidence and investigate the benefits and harms of low-dose computed tomography (LDCT) in lung cancer screening. METHODS: Detailed electronic database searches were performed to identify reports of RCTs that comparing LDCT to any other type of lung cancer screening. Pooled risk ratios (RRs) were calculated using random effects models. RESULTS: We identified nine RCTs (n = 97,244 participants). In pooled analyses LDCT reduced lung cancer mortality (RR 0.83, 95% CI 0.76-0.90, I2 = 1%) but had no effect on all-cause mortality (RR 0.95, 95% CI 0.90-1.00). Trial sequential analysis (TSA) confirmed the results of our meta-analysis. Subgroup defined by high quality trials benefitted from LDCT screening in reducing lung cancer mortality (RR 0.82, 95% CI 0.73-0.91, I2 = 7%), whereas no benefit observed in other low quality RCTs. LDCT was associated with detection of a significantly higher number of early stage lung cancers than the control. No significant difference (RR 0.64, 95% CI 0.30-1.33) was found in mortality after invasive procedures between two groups. CONCLUSIONS: In meta-analysis based on sufficient evidence demonstrated by TSA suggests that LDCT screening is superiority over usual care in lung cancer survival. The benefit of LDCT is expected to be heavily influenced by the risk of lung cancer in the different target group (smoking status, Asian) being screened.

Synthesis of novel C4-benzazole naphthalimide derivatives with potent anti-tumor properties against murine melanoma.

Novel C4-benzazole naphthalimide derivatives were synthesized and tested in vitro and in vivo as anti-cancer drugs. Among these synthetic molecules, compounds 9 and 10 exhibited cytotoxicity against murine B16F10 melanoma cells. In addition, the above-mentioned compounds significantly suppressed lung tumor metastasis with no visible sign of toxicity.

Potential effect of ezetimibe against Mycobacterium tuberculosis infection in type II diabetes.

BACKGROUND AND OBJECTIVE: Tuberculosis (TB) risk might be increased in patients with diabetes by factors other than hyperglycaemia, such as dyslipidaemia. Host lipids are essential energy sources used by mycobacteria to persist in a latent TB state. A potential therapy targeting cholesterol catabolism of mycobacteria has been proposed, but the potential of cholesterol-lowering drugs as anti-TB therapy is unclear. The purpose of this study was to determine the effects of ezetimibe, a 2-azetidinone cholesterol absorption inhibitor, on intracellular mycobacteria survival and dormancy. METHODS: Intracellular mycobacteria survival was determined by measurements of ATP activity and colony-formation units (CFUs). Gene expression profiles of hypoxia-induced dormant Mycobacterium tuberculosis (Mtb) were analysed by real-time PCR. Flow cytometry and microscopy analysis were used to measure the lipid loads of human macrophages with or without ezetimibe treatment. QuantiFERON-TB Gold In-Tube (QFT-G-IT) assays were performed to diagnose latent TB infection. The levels of intracellular cholesterol/ triglyceride were measured by an enzymatic fluorometric method. RESULTS: Ezetimibe was capable of effectively lowering intracellular growth of Mtb and hypoxia-induced dormant Mtb. There was a significant decrease in Mtb growth in leucocytes from ezetimibe-treated patients with diabetes in terms of ATP levels of intracellular mycobacteria and CFU formation. Also, patients receiving ezetimibe therapy had a lower prevalence of latent TB and had lower intracellular lipid contents. CONCLUSION: Ezetimibe, which is a currently marketed drug, could hold promise as an adjunctive, host-directed therapy for TB.

Analysis of the immune response of human dendritic cells to Mycobacterium tuberculosis by quantitative proteomics.

BACKGROUND: The cellular immune response for Mycobacterium tuberculosis (M. tuberculosis) infection remained incompletely understood. To uncover membrane proteins involved in this infection mechanism, an integrated approach consisting of an organic solvent-assisted membrane protein digestion, stable-isotope dimethyl labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to comparatively profile the membrane protein expression of human dendritic cells upon heat-killed M. tuberculosis (HKTB) treatment. RESULTS: Organic solvent-assisted trypsin digestion coupled with stable-isotope labeling and LC-MS/MS analysis was applied to quantitatively analyze the membrane protein expression of THP-1 derived dendritic cells. We evaluated proteins that were upregulated in response to HKTB treatment, and applied STRING website database to analyze the correlations between these proteins. Of the investigated proteins, aminopeptidase N (CD13) was found to be largely expressed after HKTB treatment. By using confocal microscopy and flow cytometry, we found that membranous CD13 expression was upregulated and was capable of binding to live mycobacteria. Treatment dendritic cell with anti-CD13 antibody during M. tuberculosis infection enhanced the ability of T cell activation. CONCLUSIONS: Via proteomics data and STRING analysis, we demonstrated that the highly-expressed CD13 is also associated with proteins involved in the antigen presenting process, especially with CD1 proteins. Increasing expression of CD13 on dendritic cells while M. tuberculosis infection and enhancement of T cell activation after CD13 treated with anti-CD13 antibody indicates CD13 positively involved in the pathogenesis of M. tuberculosis.

A microfluidic approach towards hybridoma generation for cancer immunotherapy.

Dendritic cells/tumor fusions have shown to elicit anti-cancer immunity in different cancer types. However, the application of these vaccines for human cancer immunotherapy are limited by the instable quality and insufficient quanity of fusion cells. We present a cell electrofusion chip fabricated using soft lithography technique, which combines the rapid and precise cell pairing microstructures and the high yield electrofusion micro-electrodes to improve the cell fusion. The design uses hydrodynamic trapping in combination with positive dielectrophoretic force (pDEP) to achieve cell fusion. The chip consists of total 960 pairs of trapping channels, which are capable of pairing and fusing both homogeneous and heterogeneous types of cells. The fused cells can be easily taken out of the chip that makes this device a distinguishable from other designs. We observe pairing efficiency of 68% with fusion efficiency of 64%.

Agenesis of right upper lobe of lung.

Agenesis of the right upper lobe of the lung is a very uncommon congenital anomaly and may be referred to chest clinics in adulthood for an incidental finding of abnormal chest radiograph. The presentations of chest radiograph may imitate many common situations such as right upper lobe collapse presenting as an ipsilateral shifting of the mediastinum or elevation of the right hemidiaphragm due to eventration or subdiaphragmatic lesions. A chest computed tomography is considered the most conclusive examination used to diagnose lung agenesis. Three-dimensional reconstructed images can be particularly helpful in delineating abnormalities of the bronchi and associated arterial and venous structures. We describe here a young woman with allergic rhinitis and bronchial asthma since her early childhood. She was referred to our clinic for an incidental finding of abnormal chest radiograph after a school health checkup. Right upper lobe atelectasis or intra-abdominal lesions were initially suspected. After a thorough image study, she was diagnosed as a case of agenesis of the right upper lobe. Our report emphasizes the importance that a high index of suspicion and adequate image investigation are necessary to diagnose congenital lung anomalies.

Aminopeptidase N facilitates entry and intracellular survival of Mycobacterium tuberculosis in monocytes.

BACKGROUND AND OBJECTIVE: Aminopeptidase N (CD13) is an ectoenzyme located in the outer membrane of a variety of cells. Proteomic profiling indicates an increased expression of CD13 in phagocytes during Mycobacterium tuberculosis infection. The purpose of this study was to investigate the role of CD13 on the internalization and intracellular survival of M. tuberculosis in monocytes. METHODS: Magnetic nanoparticles and confocal microscopy were used to observe interactions between CD13 and M. tuberculosis. Mycobacterial entry and intracellular survival in monocytes were assessed with and without anti-CD13 antibody (WM15 and WM47) using flow cytometry and colony formation assay. RESULTS: By using magnetic nanoparticles and confocal microscopy, M. tuberculosis was found to be capable of binding to either soluble CD13 or membranous CD13 on monocytes. Flow cytometry showed that pretreatment of monocytes with WM15 or WM47 reduced the number of intracellular M. tuberculosis. Collectively, the data suggest that CD13 is a binding and entry receptor for M. tuberculosis on monocytes. Treatment of infected monocytes showed a greater effect of WM47 than WM15 in reducing the intracellular colonization of M. tuberculosis, suggesting that specific epitopes of CD13 may play an important role modulating intracellular M. tuberculosis survival. CONCLUSIONS: CD13 acts as a receptor for M. tuberculosis on human monocytes. The molecule facilitates internalization, and interaction of CD13 with an anti-CD13 antibody reduces intracellular M. tuberculosis survival.

Mycobacterium tuberculosis DNA detection using surface plasmon resonance modulated by telecommunication wavelength.

A surface plasmon resonance sensor for Mycobacterium tuberculosis (MTB) deoxyribonucleic acid (DNA) is developed using repeatable telecommunication wavelength modulation based on optical fiber communications laser wavelength and stability. MTB DNA concentrations of 1 µg/mL and 10 µg/mL were successfully demonstrated to have the same spectral half-width in the dip for optimum coupling. The sensitivity was shown to be -0.087 dB/(µg/mL) at all applied telecommunication wavelengths and the highest sensitivity achieved was 115 ng/mL without thiolated DNA immobilization onto a gold plate, which is better than the sensor limit of 400 ng/mL possible with commercial biosensor equipment.

Detecting small lung tumors in mouse models by refractive-index microradiology.

Refractive-index (phase-contrast) radiology was able to detect lung tumors less than 1 mm in live mice. Significant micromorphology differences were observed in the microradiographs between normal, inflamed, and lung cancer tissues. This was made possible by the high phase contrast and by the fast image taking that reduces the motion blur. The detection of cancer and inflammation areas by phase contrast microradiology and microtomography was validated by bioluminescence and histopathological analysis. The smallest tumor detected is less than 1 mm(3) with accuracy better than 1 × 10(-3) mm(3). This level of performance is currently suitable for animal studies, while further developments are required for clinical application.

Prognostic value of dynamic soluble triggering receptor expressed on myeloid cells in bronchoalveolar lavage fluid of patients with ventilator-associated pneumonia.

BACKGROUND AND OBJECTIVE: The aim of this study was to investigate the time course, and correlation with prognosis, of BAL fluid concentrations of soluble triggering receptor expressed on myeloid cells (sTREM-1) in patients with ventilator-associated pneumonia (VAP). METHODS: The study included 35 patients with clinically diagnosed VAP, eight of whom were BAL fluid culture-negative and 27 BAL fluid culture-positive (16 survivors, 11 non-survivors). sTREM-1 levels were measured in BAL fluid of these mechanically ventilated patients, at the time of diagnosis, on days 4-5 and on days 7-9. The time course of this biomarker and its prognostic value for outcome in patients with culture-positive VAP were assessed. RESULTS: sTREM-1 concentrations were significantly greater in culture-positive VAP patients than in culture-negative VAP patients. sTREM-1 levels decreased significantly with time in surviving patients with culture-positive VAP, but increased significantly with time in non-survivors. In contrast, PaO(2)/fraction of inspired oxygen (FiO(2)) increased significantly with time in survivors and decreased significantly with time in non-survivors. At a cut-off value of -10 pg/mL 7-9 days after initial diagnosis, sTREM levels had a sensitivity of 90% and a specificity of 87.5% for predicting mortality. CONCLUSIONS: sTREM-1 concentrations in BAL fluid are of potential prognostic value in patients with VAP.

Immune impairment in patients with terminal cancers: influence of cancer treatments and cytomegalovirus infection.

Although immunodeficiency is usually considered a prerequisite of oncogenesis, a detailed immune pro- file in cancer has not yet been described. Without such profiling, it is not surprising that there is a vast discrepancy in the responses of cancer patients to immunotherapy. Our results show that the integrity of the immune system deteriorates with cancer progression by displaying a trend toward decreasing levels of functional T cells, including CD4, naïve, and central memory T cells, and an expansion of hyporesponsive populations such as CD28⁻ and CMV-specific T cells. One hundred and one patients constitute the study group for the observational study reported in this paper. Forty-eight patients with newly diagnosed stages III and IV and 53 patients with extensively treated stage IV disease. The costimulatory molecules CD27 and CD28 were downregulated in all patients. Among the proinflammatory cytokines (IL-6, TNF-α, IFN-γ), only IL-6 differed significantly among the groups, increasing as the cancer stage progressed. Plasma IL-7 did not diVer among the participants. The relative deficits of naïve T cells in cancer patients may be associated with the downregulation of IL-7Rα expression rather than changes in the circulating levels of IL-7. The downregulation of IL-7Rα expression was shown to be associated with increased levels of intracellular CMV. The present study suggests that the immune impairment in patients with cancer is associated with multiple factors, such as the stage of cancer, consequence of CMV infection and impact of treatment.

Bronchodilatation effects of a small volume spacer used with a metered-dose inhaler.

BACKGROUND AND OBJECTIVE: In an effort to improve the delivery of drugs to the lungs, various spacer devices have been developed to attach to metered-dose inhalers (MDIs). The aim of the study was to determine whether use of a small volume tube spacer with MDI is associated with better bronchodilatation. METHODS: We assessed bronchodilatation by measuring forced expiratory volume in 1 second (FEV(1)) before and after inhalation of fenoterol 0.4 mg (2 puffs) delivered by using a MDI in four different ways: with or without a spacer alone or with a mouth rinse of 100 mL of water immediately after inhalation with or without a spacer. Results. A total of 303 patients who had a positive bronchodilator test were studied. There was no significant difference in the Delta FEV(1) (mL or %) with or without a spacer (MDI + spacer vs. MDI, mean +/- SD, 365.1 +/- 146.5 mL vs. 356.3 +/- 131.1 mL, p = 0.696; and 21.4 +/- 9.4% vs. 21.4 +/- 9.5%, p = 0.968, respectively). When patients rinsed their mouth after inhalation, bronchodilatation was significantly less in those using an MDI alone compared with MDI + spacer (302.6 +/- 116.5 mL vs. 367.6 +/- 128.3 mL, p = 0.002; and 18.0% +/- 7.9% vs. 21.7% +/- 9.5%, p = 0.013, respectively). CONCLUSIONS: When patients correctly use an MDI, addition of a spacer does not significantly improve bronchodilatation. However, if the mouth is rinsed after inhalation, a spacer does yield better bronchodilatation. Our results suggest that systemic effects from bronchodilator inhalation may not be negligible.

Involvement of p29 in DNA damage responses and Fanconi anemia pathway.

Human p29 is a chromatin-associated protein and the silencing of p29 expression increases cell population in G(1) phase and decreases phosphorylation levels of Chk1 and Chk2 in response to UV treatment. To further characterize the function of p29, U2OS and Fanconi anemia complementation group G (FA-G) cells with constitutive p29 expression have been established. Analyses of these cells identified increased phosphorylation levels of Chk1 and Chk2, which were accompanied by elevated amounts of chromatin-associated Mre11-Rad50-Nbs1 complex and ATR-IP. Monoubiquitination of the FA ID complex was restored in p29 stably expressing FA-G cells. Moreover, lower tumor incidence was observed in mp29 transgenic mice after UV irradiation. These results suggest the involvement of p29 in the DNA damage responses and Fanconi anemia pathway.

Amplification of immune responses against a DNA-delivered idiotypic lymphoma antigen by fusion to the B subunit of E. coli heat labile toxin.

The pentameric B-subunit of Escherichia coli heat-labile enterotoxin (EtxB) is not only highly immunogenic itself, but can also act as a potent adjuvant or carrier to increase immune responses to other antigens. In this study, we investigated the ability of EtxB to promote anti-tumor immune responses using a fusion DNA vaccine design. EtxB was genetically linked to a single chain Fv sequence derived from the idiotypic immunoglobulin antigen (Id) of the mouse BCL1 B-cell lymphoma. We found that the EtxB-BCL1scFv fusion protein with a specifically selected linker retained the ability to pentamerize and to bind the GM1 ganglioside. Immunization of mice with the pEtxB-BCL1scFv DNA construct generated high levels of Id-specific antibody and protected against lethal tumor challenge. The immuno-enhancing activities of EtxB were highly dependent on GM1 binding, since fusion constructs unable to pentamerize or to bind to GM1 were less effective. Thus, the EtxB fusion vaccine approach may be an attractive strategy to increase the potency of tumor vaccines.

Rac1 regulates peptidoglycan-induced nuclear factor-kappaB activation and cyclooxygenase-2 expression in RAW 264.7 macrophages by activating the phosphatidylinositol 3-kinase/Akt pathway.

Previously, we found that peptidoglycan (PGN), a cell wall component of the gram-positive bacterium Staphylococcus aureus, may activate the Ras/Raf-1/extracellular signal-regulated kinase (ERK) pathway, which in turn initiates IkappaB kinases alpha/beta (IKKalpha/beta) and nuclear factor-kappaB (NF-kappaB) activation, and ultimately induces cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. In this study, we further investigated the roles of Rac1, phosphatidylinositol 3-kinase (PI3K), and Akt in PGN-induced NF-kappaB activation and COX-2 expression in RAW 264.7 macrophages. PGN-induced COX-2 expression was attenuated by a Rac1 dominant negative mutant (RacN17), PI3K inhibitors (wortmannin and LY 294002), and an Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate]). PGN-induced PGE(2) release was also inhibited by RacN17. Treatment of RAW 264.7 macrophages with PGN caused the activation of Rac and Akt. PGN-induced Akt activation was inhibited by RacN17, LY 294002, and the Akt inhibitor. Stimulation of RAW 264.7 macrophages with PGN resulted in an increase in IKKalpha/beta phosphorylation and p65 Ser536 phosphorylation; these effects were inhibited by RacN17, LY 294002, an Akt inhibitor, and an Akt dominant negative mutant (AktDN). The PGN-induced increases in kappaB-luciferase activity were also inhibited by RacN17, wortmannin, LY 294002, an Akt inhibitor, and AktDN. Treatment of macrophages with PGN induced the recruitment of p85alpha and Rac1 to Toll-like receptor 2 (TLR2) in a time-dependent manner. These results indicate that PGN may activate the Rac1/PI3K/Akt pathway through the recruitment of p85alpha and Rac1 to TLR2 to mediate IKKalpha/beta activation and p65 phosphorylation, which in turn induces NF-kappaB transactivation, and ultimately causes COX-2 expression in RAW 264.7 macrophages.

Proteomic profiles of bronchoalveolar lavage fluid from patients with ventilator-associated pneumonia by gel-assisted digestion and 2-D-LC/MS/MS.

Development of ventilator-associated pneumonia (VAP) imposes a significant financial burden to the health care system, yet the therapeutic decisions rely on the conventional, less sensitive microbiological examination. Characterization of proteins secreted into bronchoalveolar lavage fluid (BALF) provides an opportunity for discovery of diagnostic marker candidates for accurate therapeutic decision-making. We report the first global description of the BALF proteome from patients with VAP. Our approach combining gel-assisted digestion followed by SCX fractionation and nano-LC-MS/MS effectively overcame the interference of high salt concentrations in BALF. Semi-quantitative analysis by a simple, label-free approach based on chromatographic peak area intensity revealed that the protein constituents were dramatically different between the non-VAP controls and the VAP group. To our knowledge, the 206 identified proteins present the most comprehensive global proteome map of BALF. The expression of four selected proteins with unique roles, including gelsolin, serum amyloid P-component, vitamin D-binding protein and pyruvate kinase, were significantly higher in BALF from patients with VAP (p <0.05). We demonstrate that this proteomic approach provides in-depth profiling of the BALF proteome, which comprises proteins functionally associated with the pathogenesis of VAP, including those expressed due to stress-induced injury and host immune response to local inflammation.