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1.
Laryngoscope ; 2020 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32438526

RESUMO

OBJECTIVES: The objective was to explore the effect of the proneuronal transcription factor neurogenic differentiation 1 (Neurod1, ND1) on Schwann cells (SC) and schwannoma cell proliferation. METHODS: Using a variety of transgenic mouse lines, we investigated how expression of Neurod1 effects medulloblastoma (MB) growth, schwannoma tumor progression, vestibular function, and SC cell proliferation. Primary human vestibular schwannoma (VS) cell cultures were transduced with adenoviral vectors expressing Neurod1. Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine (EdU) uptake. STUDY DESIGN: Basic science investigation. RESULTS: Expression of Neurod1 reduced the growth of slow-growing but not fast-growing MB models. Gene transfer of Neurod1 in human schwannoma cultures significantly reduced cell proliferation in dose-dependent way. Deletion of the neurofibromatosis type 2 (Nf2) tumor-suppressor gene via Cre expression in SCs led to increased intraganglionic SC proliferation and mildly reduced vestibular sensory-evoked potentials (VsEP) responses compared to age-matched wild-type littermates. The effect of Neurod1-induced expression on intraganglionic SC proliferation in animals lacking Nf2 was mild and highly variable. Sciatic nerve axotomy significantly increased SC proliferation in wild-type and Nf2-null animals, and expression of Neurod1 reduced the proliferative capacity of both wild-type and Nf2-null SCs following nerve injury. CONCLUSION: Expression of Neurod1 reduces slow-growing MB progression and reduces human SC proliferation in primary VS cultures. In a genetic mouse model of schwannomas, we find some effects of Neurod1 expression; however, the high variability indicates that more tightly regulated Neurod1 expression levels that mimic our in vitro data are needed to fully validate Neurod1 effects on schwannoma progression. LEVEL OF EVIDENCE: NA Laryngoscope, 2020.

2.
Artigo em Inglês | MEDLINE | ID: mdl-32182423

RESUMO

Optical coatings with controllable ultralow refractive indices are of profound significance in optical areas. However, it remains a challenge to fabricate such coatings using a simple method. Here we develop an effective and simple approach to create ultra-low-index coatings. This approach was based on a modified sol-gel process, with a key process that involved the aggregation of silica nanoparticles via the addition of a polymer surfactant (e.g., polyvinylpyrrolydone) in sols before coating. The approach involves three steps: the synthesis of silica sols under ammonia catalysis in ethanol (Stöber method), the addition of polyvinylpyrrolydone in the silica sols to induce the aggregation of the silica nanoparticles, and the formation of ultra-low-index coatings by depositing the aggregated silica sols on substrates. Through varying the aggregation extent, this approach produced coatings with controllable refractive indices ranging from 1.17 to 1.07. To the best of our knowledge, the minimum index value of 1.07 from our coating is among the lowest refractive indices ever reported. The ultra-low-index coatings demonstrated excellent optical properties, with which perfect quarter-wavelength antireflection coatings (maximum transmittance ∼100%) and broadband antireflection coatings (transmittance >98% from 400 to 1100 nm) can be prepared. One advantage of the antireflection coatings is that their transmission is less dependent on the refractive index and the thickness of the stacking layer, which make it promising in large-scale production. Moreover, the coatings can be made hydrophobic (water contact angle 136°) by exposing the coatings to a hexamethyldisilazane atmosphere, exhibiting high environmental stability in a humid environment. The aggregation of silica nanoparticles in sol-gel processes provides a scalable alternative to the current approaches for creating ultra-low-index coatings.

3.
Int J Nanomedicine ; 15: 1021-1035, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32103954

RESUMO

Background and Aim: We have previously identified ubiquitinated proteins (UPs) from tumor cell lysates as a promising vaccine for cancer immunotherapy in different mouse tumor models. In this study, we aimed at developing a highly efficient therapeutic adjuvant built-in nanovaccine (α-Al2O3-UPs) by a simple method, in which UPs from tumor cells could be efficiently and conveniently enriched by α-Al2O3 nanoparticles covalently coupled with Vx3 proteins (α-Al2O3-CONH-Vx3). Methods: The α-Al2O3 nanoparticles were modified with 4-hydroxybenzoic acid followed by coupling with ubiquitin-binding protein Vx3. It was then used to enrich UPs from 4T1 cell lysate. The stability and the efficiency for the UPs enrichment of α-Al2O3-CONH-Vx3 were examined. The ability of α-Al2O3-UPs to activate DCs was examined in vitro subsequently. The splenocytes from the vaccinated mice were re-stimulated with inactivated tumor cells, and the IFN-γ secretion was detected by ELISA and flow cytometry. Moreover, the therapeutic efficacy of α-Al2O3-UPs, alone and in combination with chemotherapy, was examined in 4T1 tumor-bearing mice. Results: Our results showed that α-Al2O3-UPs were successfully synthesized and abundant UPs from tumor cell lysate were enriched by the new method. In vitro study showed that compared to the physical mixture of α-Al2O3 nanoparticles and UPs (α-Al2O3+UPs), α-Al2O3-UPs stimulation resulted in higher upregulations of CD80, CD86, MHC class I, and MHC class II on DCs, indicating the higher ability of DC activation. Moreover, α-Al2O3-UPs elicited a more effective immune response in mice, demonstrated by higher IFN-γ secretion than α-Al2O3+UPs. Furthermore, α-Al2O3-UPs also exhibited a more potent effect on tumor growth inhibition and survival prolongation in 4T1 tumor-bearing mice. Notably, when in combination with low dose chemotherapy, the anti-tumor effect was further enhanced, rather than using α-Al2O3-UPs alone. Conclusion: This study presents an adjuvant built-in nanovaccine generated by a new simple method that can be potentially applied to cancer immunotherapy and lays the experimental foundation for future clinical application.


Assuntos
Vacinas Anticâncer/farmacologia , Nanopartículas/química , Proteínas Ubiquitinadas/química , Adjuvantes Imunológicos/farmacologia , Óxido de Alumínio/química , Animais , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Interferon gama/metabolismo , Camundongos Endogâmicos BALB C , Nanopartículas/uso terapêutico , Neoplasias Experimentais/terapia , Parabenos/química , Proteínas Ubiquitinadas/imunologia
5.
Methods Mol Biol ; 2064: 61-71, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31565766

RESUMO

Mass spectrometry (MS) is an indispensable analytical technique for bioanalysis. Based on the measurement of mass/charge ratios (m/z) of ions, MS can be used for sensitive detection and accurate identification of species of interest. In traditional studies, MS is utilized to measure analytes in prepared solutions or gas-phase samples. Benefited from recent development of sampling and ionization approaches, MS has been extensively applied to the analysis of broad ranges of biological samples. We have developed a new device, the Single-probe, that can be used for in situ, real-time MS analysis of metabolites inside individual living cells. The Single-probe is a miniaturized multifunctional sampling and ionization device that is directly coupled to the mass spectrometer. With a sampling tip size smaller than 10 µm, we can insert the Single-probe tip into single cells to extract intracellular compounds, which are analyzed using MS in real-time. We have successfully used the Single-probe MS technique to detect a variety of endogenous and exogenous cellular metabolites in individual eukaryotic cells. Single cell mass spectrometry (SCMS) is a new scientific technology that has the potential to reshape approaches in biological and pharmaceutical bioanalytical research.

6.
Sci Rep ; 9(1): 19489, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862906

RESUMO

Transcription factor Neurod1 is required for enteroendocrine progenitor differentiation and maturation. Several earlier studies indicated that ectopic expression of Neurod1 converted non- neuronal cells into neurons. However, the functional consequence of ectopic Neurod1 expression has not been examined in the GI tract, and it is not known whether Neurod1 can similarly switch cell fates in the intestine. We generated a mouse line that would enable us to conditionally express Neurod1 in intestinal epithelial cells at different stages of differentiation. Forced expression of Neurod1 throughout intestinal epithelium increased the number of EECs as well as the expression of EE specific transcription factors and hormones. Furthermore, we observed a substantial reduction of Paneth cell marker expression, although the expressions of enterocyte-, tuft- and goblet-cell specific markers are largely not affected. Our earlier study indicated that Neurog3+ progenitor cells give rise to not only EECs but also Goblet and Paneth cells. Here we show that the conditional expression of Neurod1 restricts Neurog3+ progenitors to adopt Paneth cell fate, and promotes more pronounced EE cell differentiation, while such effects are not seen in more differentiated Neurod1+ cells. Together, our data suggest that forced expression of Neurod1 programs intestinal epithelial cells more towards an EE cell fate at the expense of the Paneth cell lineage and the effect ceases as cells mature to EE cells.

7.
Glob Chall ; 3(7): 1800082, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31565383

RESUMO

Cloth wearing seems so natural that everyone is self-deemed knowledgeable and has some expert opinions about it. However, to clearly explain the physics involved, and hence to make predictions for clothing design or selection, it turns out to be quite challenging even for experts. Cloth is a multiphased, porous, and anisotropic material system and usually in multilayers. The human body acts as an internal heat source in a clothing situation, thus forming a temperature gradient between body and ambient. But unlike ordinary engineering heat transfer problems, the sign of this gradient often changes as the ambient temperature varies. The human body also perspires and the sweat evaporates, an effective body cooling process via phase change. To bring all the variables into analysis quickly escalates into a formidable task. This work attempts to unravel the problem from a physics perspective, focusing on a few rarely noticed yet critically important mechanisms involved so as to offer a clearer and more accurate depiction of the principles in clothing thermal comfort.

8.
ACS Omega ; 4(13): 15348-15358, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31572833

RESUMO

Shape memory polymers (SMPs) are an exciting class of stimuli-responsive smart materials that demonstrate reactive and reversible changes in mechanical property, usually by switching between different states due to external stimuli. We report on the development of a polyurethane-based SMP foam for effective pressure redistribution that demonstrates controllable changes in dynamic pressure redistribution capability at a low transition temperature (∼24 °C)-ideally suited to matching modulations in body contact pressure for dynamic pressure relief (e.g., for alleviation or pressure ulcer effects). The resultant SMP material has been extensively characterized by a series of tests including stress-strain testing, compression testing, dynamic mechanical analysis, optical microscopy, UV-visible absorbance spectroscopy, variable-temperature areal pressure distribution, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray diffraction, differential scanning calorimetry, dynamic thermogravimetric analysis, and 1H nuclear magnetic resonance spectroscopy. The foam system exhibits high responsivity when tested for plantar pressure modulation with significant potential in pressure ulcers treatment. Efficient pressure redistribution (∼80% reduction in interface pressure), high stress response (∼30% applied stress is stored in fixity and released on recovery), and excellent deformation recovery (∼100%) are demonstrated in addition to significant cycling ability without performance loss. By providing highly effective pressure redistribution and modulation when in contact with the body's surface, this SMP foam offers novel mechanisms for alleviating the risk of pressure ulcers.

9.
Immunol Lett ; 216: 36-42, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31568811

RESUMO

As common features of human solid tumors, hypoxia and nutrient starvation play multifaceted roles in cancer progress. However, the mechanisms are far from clear. Our previous work has indicated that tumor cell-released autophagosomes (TRAPs) are sufficient to suppress anti-tumor immune response in mouse by inducing IL-10-producing B cells through high-mobility group B1 (HMGB1). Here, we hypothesized that hypoxia or starvation might exert immunosuppressive effect through upregulating HMGB1 on TRAPs. We found that HMGB1 on TRAPs from human hepatocellular carcinoma cell line HepG2 played a significant role in IL-10-producing B cell induction. HMGB1 in tumor cells was upregulated under hypoxia and starvation, but only hypoxia significantly enhanced the level of HMGB1 present on the surfaces of TRAPs. Moreover, hypoxic TRAPs induced more IL-10-producing B cells with suppressive activities on CD4+ and CD8+ T cells. The finding indicates the role of TRAPs as a messenger of hypoxic response to enhance immunosuppression in tumor microenvironment.

10.
Environ Pollut ; 253: 221-230, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31310872

RESUMO

There is a need to develop highly efficient materials for capturing uranium from nuclear wastewater. Here, 5-methylbenzotriazole modified graphene oxide (MBTA-GO) was used to adsorb U(VI) from aqueous solution. By the trials of different conditions, we found that the removal of U(VI) from acidic solution was strongly dependent on pH but independent of ionic strength. The U(VI) adsorption was perfectly conformed to the pseudo-second-order kinetics and the adsorption isotherms were simulated by the Langmuir model well. A high removal capacity (qmax = 264 mg/g) for U(VI) at pH 3.5 was obtained. XPS, EXAFS analyses and DFT calculations revealed that the mechanism of uranium capture was ascribed to (i) the surface complexation by benzotriazole and carboxyl groups (providing lone pair electrons) on MBTA-GO and (ii) enhanced synergistic coordination ability of delocalized π-bond of triazole group toward U due to the transfer of electrons from graphene sheet to benzotriazole. DFT calculations further demonstrated that benzotriazole displayed stronger binding with U(VI) compared to carboxyl group due to higher binding energy of [Side/Surface-U-MBTA-GO] (79.745, 54.986 kcal/mol) than [MBTA-GO-COOH-U] (27.131 kcal/mol). This work will provide valuable insight into designing novel nitrogen-containing adsorbents for practical application in wastewater treatment.


Assuntos
Grafite/química , Triazóis/química , Urânio/química , Poluentes Radioativos da Água/química , Adsorção , Cinética , Concentração Osmolar , Óxidos/química , Eliminação de Resíduos Líquidos/métodos , Águas Residuárias , Água
11.
J Immunother Cancer ; 7(1): 178, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31300052

RESUMO

BACKGROUND: CD4+ T cells are critical effectors of anti-tumor immunity, but how tumor cells influence CD4+ T cell effector function is not fully understood. Tumor cell-released autophagosomes (TRAPs) are being recognized as critical modulators of host anti-tumor immunity during tumor progression. Here, we explored the mechanistic aspects of TRAPs in the modulation of CD4+ T cells in the tumor microenvironment. METHODS: TRAPs isolated from tumor cell lines and pleural effusions or ascites of cancer patients were incubated with CD4+ T cells to examine the function and mechanism of TRAPs in CD4+ T cell differentiation and function. TRAPs-elicited CD4+ T cells were tested for their suppression of effector T cell function, induction of regulatory B cells, and promotion of tumorigenesis and metastasis in a mouse model. RESULTS: Heat shock protein 90α (HSP90α) on the surface of TRAPs from malignant effusions of cancer patients and tumor cell lines stimulated CD4+ T cell production of IL-6 via a TLR2-MyD88-NF-κB signal cascade. TRAPs-induced autocrine IL-6 further promoted CD4+ T cells secretion of IL-10 and IL-21 via STAT3. Notably, TRAPs-elicited CD4+ T cells inhibited CD4+ and CD8+ effector T cell function in an IL-6- and IL-10-dependent manner and induced IL-10-producing regulatory B cells (Bregs) via IL-6, IL-10 and IL-21, thereby promoting tumor growth and metastasis. Consistently, inhibition of tumor autophagosome formation or IL-6 secretion by CD4+ T cells markedly retarded tumor growth. Furthermore, B cell or CD4+ T cell depletion impeded tumor growth by increasing effector T cell function. CONCLUSIONS: HSP90α on the surface of TRAPs programs the immunosuppressive functions of CD4+ T cells to promote tumor growth and metastasis. TRAPs or their membrane-bound HSP90α represent important therapeutic targets to reverse cancer-associated immunosuppression and improve immunotherapy.

12.
Anal Chem ; 91(14): 9018-9024, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31246408

RESUMO

Analyzing cellular constituents on the single-cell level through mass spectrometry (MS) allows for a wide range of compounds to be studied simultaneously. However, there is a need for quantitative single-cell mass spectrometry (qSCMS) methods to fully characterize drug efficacy from individual cells within cell populations. In this study, qSCMS experiments were carried out using the Single-probe MS technique. The method was successfully used to perform rapid absolute quantifications of the anticancer drug irinotecan in individual mammalian cancer cells under ambient conditions in real time. Traditional liquid chromatography/mass spectrometry (LC/MS) quantifications of irinotecan in cell lysate samples were used to compare the results from Single-probe qSCMS. This technique showcases heterogeneity of drug efficacy on the single-cell level.

13.
Cardiovasc Eng Technol ; 10(3): 397-422, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31240664

RESUMO

PURPOSE: Hydrodynamic performance testing is one of the core in vitro assessments required by the ISO 5840 series of standards for all prosthetic heart valves. A round-robin study carried out in 2005 in accordance with ISO 5840:2005 revealed significant variabilities in prosthetic heart valve hydrodynamic performance measurements among the participating laboratories. In order to re-examine the inter-laboratory variability based on the "state-of-the-art" under ISO 5840-1 and 5840-2:2015, the ISO Cardiac Valve Working Groups decided in 2016 to repeat the round-robin study. METHODS: A total of 13 international laboratories participated in the study. The test valves were chosen to be the St. Jude Medical Masters Series mechanical valves (19 mm aortic, 25 mm aortic, 25 mm mitral, and 31 mm mitral), which were circulated among the laboratories. The testing was conducted according to a common test run sequence, with prespecified flow conditions. RESULTS: The study revealed improved, yet still significant variability among different laboratories as compared to the 2005 study. The coefficient of variation ranged from 7.7 to 21.6% for the effective orifice area, from 10.1 to 32.8% for the total regurgitant fraction, and from 14.7 to 45.5% for the mean transvalvular pressure gradient. CONCLUSIONS: The study revealed the ambiguities in the current versions of the ISO 5840 series of standards and the shortcomings of some participating laboratories. This information has allowed the ISO Working Group to incorporate additional clarifying language into the ISO 5840-1, -2, and -3 standards that are currently under revision to improve in vitro assessments. The results presented here can also be used by the testing laboratories to benchmark pulse duplicator systems and to train and certify testing personnel.


Assuntos
Próteses Valvulares Cardíacas/normas , Ensaio de Proficiência Laboratorial/normas , Desenho de Prótese/normas , Fluxo Pulsátil , Pressão Arterial , Débito Cardíaco , Frequência Cardíaca , Humanos , Hidrodinâmica , Teste de Materiais , Variações Dependentes do Observador , Sístole
14.
Macromol Rapid Commun ; 40(13): e1900078, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30969012

RESUMO

Polymer brushes exhibit functionalities useful for a large number of applications. Often these functionalities only emerge when the polymer brushes have a desired thickness. Here, a significant breakthrough is achieved in the synthesis of ultra-thick polymer brushes using polymer initiators in the approach of surface-initiated atom transfer radical polymerization, yielding polymer brushes with a controllable thickness up to 15.1 µm. This is reportedly the thickest polymer brush ever synthesized. This approach is applicable for several monomers such as acrylonitrile, methyl acrylate, and styrene, and for other types of polymer substrates such as fibers.


Assuntos
Acrilatos/química , Acrilonitrila/química , Polímeros/química , Estireno/química , Polimerização , Propriedades de Superfície
15.
Exp Ther Med ; 17(1): 943-947, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30651884

RESUMO

The aim of this randomized controlled trial was to evaluate the effect of tranexamic acid (TXA) on postoperative blood loss during transurethral resection of the prostate (TURP) for benign prostatic hyperplasia (BPH). A total of 60 patients with BPH and undergoing TURP were randomized into TXA and control groups. Patients were intravenously administered 1 g TXA or placebo (0.9% sodium chloride solution), respectively, after the induction of anesthesia for TURP. Intraoperative and postoperative bladder irrigation volumes and blood loss volumes were compared between the two groups. Coagulation function (measured by prothrombin, activated partial thromboplastin and thrombin time and fibrinogen levels) was measured before the operation and at 4 h post-operation. Complications from thromboembolic events, such as lower-limb and pulmonary embolisms, were also noted. The TXA group had significantly decreased blood loss intraoperatively and at 4 h postoperatively compared with the control group (P<0.05). The 24 h postoperative blood loss and coagulation function of the two groups were not significantly different. No thromboembolic events or other complications occurred in either group. In conclusion, a preoperative single dose of TXA was indicated to reduce perioperative blood loss in TURP without a notable increase in thrombosis risk.

16.
J Hazard Mater ; 365: 81-87, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30412810

RESUMO

The Bi-Bi2O3-TiO2-C composites were prepared by a sol-gel method and investigated for capturing iodine-129 (129I) in off-gas producing from spent fuel reprocessing. Firstly, the optimal process conditions were operated through the orthogonal experiments, showing that the capturing capacity of the optimal composite was calculated about 504.0 ± 19.5 mg/g, which is approximately 2.0-fold higher than that of the commercial silver-exchanged zeolites (AgX). Secondly, the structure and morphology of the Bi-Bi2O3-TiO2-C composite were characterized, suggesting that the Bi is regularly spherical in the shape, coating by the Bi2O3, TiO2 and amorphous carbon. Finally, the mechanism for the iodine adsorption in the Bi-Bi2O3-TiO2-C system was revealed, demonstrating that the iodine was captured by physisorption and chemisorption.

17.
J Immunother Cancer ; 6(1): 151, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30563569

RESUMO

BACKGROUND: Tumor-associated macrophages (TAMs) facilitate tumor progression via establishment of an immunosuppressive tumor microenvironment (TME). However, it is poorly understood how tumor cells could functionally modulate TAMs. Our previous work indicated that tumor cell-released autophagosomes (TRAPs), a type of LC3-II+ double-membrane extracellular vesicles (EVs) was sufficient to suppress anti-tumor immune responses by inducing IL-10-producing B cells and immune suppressive neutrophils. Here, we hypothesized that TRAPs may participate in regulating macrophage polarization. METHODS: TRAPs isolated from multiple murine tumor cell lines and pleural effusions or ascites of cancer patients were incubated with bone marrow-derived macrophages (BMDMs) and monocytes, respectively. Cellular phenotypes were examined by flow cytometry, ELISA and quantitative PCR. TRAPs treated BMDMs were tested for the ability to suppress T-cell proliferation in vitro, and for promotion of tumor growth in vivo. Transwell chamber and neutralization antibodies were added to ascertain the inhibitory molecules expressed on BMDMs exposed to TRAPs. Knockout mice were used to identify the receptors responsible for TRAPs-induced BMDMs polarization and the signaling mechanism was examined by western blot. Autophagy-deficient tumors were profiled for phenotypic changes of TAMs and IFN-γ secretion of T cells by flow cytometry. The phenotype of monocytes from pleural effusions or ascites of cancer patients was assessed by flow cytometry. RESULTS: TRAPs converted macrophages into an immunosuppressive M2-like phenotype characterized by the expression of PD-L1 and IL-10. These macrophages inhibited the proliferation of both CD4+ and CD8+ T cells in vitro, and promoted tumor growth mainly through PD-L1 in vivo. TRAPs-induced macrophage polarization was dependent on TLR4-mediated MyD88-p38-STAT3 signaling. In vivo studies indicated that disruption of autophagosome formation in B16F10 cells by silencing the autophagy gene Beclin1 resulted in a remarkable delay in tumor growth, which was associated with reduced autophagosome secretion, TAMs reprogramming and enhanced T cell activation. Moreover, the levels of LC3B+ EVs appeared to correlate significantly with up-regulation of PD-L1 and IL-10 in matched monocytes from effusions or ascites of cancer patients, and TRAPs isolated from these samples could also polarize monocytes to an M2-like phenotype with increased expression of PD-L1, CD163 and IL-10, decreased expression of HLA-DR, and T cell-suppressive function. CONCLUSIONS: These findings suggest the TRAPs-PD-L1 axis as a major driver of immunosuppression in the TME by eliciting macrophage polarization towards an M2-like phenotype, and highlight the potential novel therapeutic approach of simultaneously targeting autophagy and PD-L1.


Assuntos
Autofagossomos/imunologia , Autofagossomos/metabolismo , Antígeno B7-H1/metabolismo , Tolerância Imunológica , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Autofagossomos/ultraestrutura , Autofagia , Antígeno B7-H1/genética , Biomarcadores , Linhagem Celular Tumoral , Feminino , Humanos , Imunofenotipagem , Ativação Linfocitária/imunologia , Camundongos , Modelos Biológicos , Fator 88 de Diferenciação Mieloide/metabolismo , Neoplasias/patologia , Fenótipo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor 4 Toll-Like/metabolismo , Microambiente Tumoral/imunologia
18.
Anal Chem ; 90(18): 11078-11085, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30119596

RESUMO

The exploration of single cells reveals cell heterogeneity and biological principle of cellular metabolism. Although a number of mass spectrometry (MS) based single cell MS (SCMS) techniques have been dedicatedly developed with high efficiency and sensitivity, limitations still exist. In this work, we introduced a microscale multifunctional device, the T-probe, which integrates cellular contents extraction and immediate ionization, to implement online in situ SCMS analysis at ambient conditions with minimal sample preparation. With high sensitivity and reproducibility, the T-probe was employed for MS analysis of single HeLa cells under control and anticancer drug treatment conditions. Intracellular species and xenobiotic metabolites were detected, and changes of cellular metabolic profiles induced by drug treatment were measured. Combining SCMS experiments with statistical data analyses, including Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) and two-sample t-test, we provided biological insights into cellular metabolic response to drug treatment. Online MS/MS analysis was conducted at single cell level to identify species of interest, including endogenous metabolites and the drug compound. Using the T-probe SCMS technique combined with comprehensive data analyses, we provide an approach to understanding cellular metabolism and evaluate chemotherapies at the single cell level.


Assuntos
Metaboloma , Metabolômica/instrumentação , Análise de Célula Única/instrumentação , Espectrometria de Massas em Tandem/instrumentação , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Desenho de Equipamento , Células HeLa , Humanos
19.
Cell Tissue Res ; 374(2): 251-262, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29974252

RESUMO

RNAscope® technology provided by Advanced Cell Diagnostics (ACD) allows the detection and evaluation of coinciding mRNA expression profiles in the same or adjacent cells in unprecedented quantitative detail using multicolor fluorescent in situ hybridization (FISH). While already extensively used in thinly sectioned material of various pathological tissues and, to a lesser extent, in some whole mounts, we provide here a detailed approach to use the fluorescent RNAscope method in the mouse inner ear and thick brain sections by modifying and adapting existing techniques of whole mount fluorescent in situ hybridization (WH-FISH). We show that RNAscope WH-FISH can be used to quantify local variation in overlaying mRNA expression intensity, such as neurotrophin receptors along the length of the mouse cochlea. We also show how RNAscope WH-FISH can be combined with immunofluorescence (IF) of some epitopes that remain after proteinase digestion and, to some extent, with fluorescent protein markers such as tdTomato. Our WH-FISH technique provides an approach to detect cell-specific quantitative differences in developing and mature adjacent cells, an emerging issue revealed by improved cellular expression profiling. Further, the presented technique may be useful in validating single-cell RNAseq data on expression profiles in a range of tissue known or suspected to have locally variable mRNA expression levels.


Assuntos
Imunofluorescência/métodos , RNA Mensageiro/genética , Animais , Cóclea/metabolismo , Regulação da Expressão Gênica , Imageamento Tridimensional , Hibridização in Situ Fluorescente , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neurotrofina 3/metabolismo , RNA Mensageiro/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Receptor trkC/genética , Receptor trkC/metabolismo
20.
Oncoimmunology ; 7(6): e1438108, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29872581

RESUMO

Our previous studies have confirmed that tumor cell-released autophagosomes (TRAP) could induce the differentiation of B cells into IL-10+ regulatory B cells (Bregs) with suppressive activities on T lymphocytes. However, the mechanism of TRAP-mediated immune suppression is still largely unclear. Herein, we sought to assess the immunomodulatory effect of TRAPs on human neutrophils, a major immune cell type that infiltrates human tumor tissues. We found that TRAPs enriched from malignant effusions or ascites of cancer patients and tumor cell lines were rapidly and effectively phagocytized by neutrophils through macropinocytosis and promoted neutrophil apoptosis via reactive oxygen species (ROS) generation and caspase-3 activation. Moreover, the apoptotic neutrophils that have phagocytized TRAPs inhibited the proliferation and activation of CD4+ T and CD8+ T cells in a cell contact- and ROS-dependent manner. These findings define a novel TRAP-mediated mechanism in neutrophils that potentially suppresses the anti-tumor T cell immunity and highlight TRAPs as an important target for future tumor immunotherapy.

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