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ConspectusLaboratory-based experimental astrochemistry regularly entails simulation of astrophysical environments whereby low-temperature condensed ices are exposed to radiation from ultraviolet (UV) photons or energetic charged particles. Here, excited atoms/radicals are generated that are not in thermal equilibrium with their surroundings (i.e., they are nonthermal, or suprathermal). These species can surpass typical reaction barriers and partake in unusual chemical processes leading to novel molecular species. Often, these are uniquely observable under low-temperature conditions where the surrounding ice matrix can stabilize excited intermediates that would otherwise fall apart. Fourier-transform infrared (FTIR) spectroscopy is traditionally utilized to monitor the evolution of chemical species within ices in situ during radiolysis. Yet, the characterization and quantification of novel species and radicals formed within astrophysical ices is often hindered since many of these cannot be synthesized by traditional synthetic chemistry. Computational approaches can provide fundamental vibrational frequencies and isotopic shifts to help aid in assignments alongside infrared intensities and Raman activities to quantify levels of production. In this Account, we begin with a brief history and background regarding the composition and radiation of interstellar ices. We review details of some of the early work on carbon oxides produced during the radiolysis of pure carbon dioxide ices and contention around the carrier of an absorption feature that could potentially be a product of radiation. We then provide an overview of current and emerging experimental methodologies and some of the chemistries that occur via nonthermal processes during radiolysis of low-temperature ices. Next, we detail computational approaches to reliably predict vibrational frequencies, infrared intensities, and Raman activities based on our recent work. Our focus then turns to studies on the formation of complex organics and carbon oxides, highlighting those aided by computational approaches and their role in astrochemistry. Some recent controversies regarding assignments alongside our recent results on the characterization of novel carbon oxide species are discussed. We present an argument for the potential role of carbon oxides within cometary ices as parent molecular species for small volatiles. We provide an overview of some of the complex organic species that can be formed within interstellar and cometary ices that contain either carbon monoxide or carbon dioxide. We examine how Raman spectroscopy could potentially be leveraged to help determine and characterize carbon oxides in future experiments as well as how computational approaches can aid in these assignments. We conclude with brief remarks on future directions our research group is taking to unravel astrochemically relevant carbon oxides using combined computational and experimental approaches.
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Recent studies have implicated a role for adenosine-dependent immunosuppression in head and neck tumor microenvironments. We describe expression of CD73, an enzyme critical to the generation of adenosine from extracellular AMP, in T cells and other cell types within human head and neck tumors. Flow cytometric analyses of tumor-infiltrating cells indicate that CD3+ cells are the predominant source of CD73 among immune infiltrating cells and that CD73 expression, especially among CD8+ T cells, is inversely related to indices of T cell infiltration and T cell activation in the microenvironment of head and neck tumors. We provide evidence that CD73 expression on peripheral T cells and levels of soluble CD73 in circulation are correlated with CD73 expression on CD8+ T cells in tumors. Moreover, fluorescent microscopy studies reveal that CD8+ CD73+ cells are observed in close proximity to tumor cells as well as in surrounding tissue. In vitro studies with peripheral blood T cells indicate that anti-CD3-stimulation causes loss of CD73 expression, especially among cells that undergo proliferation and that exogenous AMP can impair T cell proliferation, while sustaining CD73 expression. These data suggest that CD8+ CD73+ T cells may be especially important mediators of immunosuppression in human head and neck cancer.
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5'-Nucleotidase/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Linfócitos do Interstício Tumoral/imunologia , Microambiente Tumoral/imunologia , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células/fisiologia , Feminino , Proteínas Ligadas por GPI/imunologia , Humanos , Tolerância Imunológica/imunologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
AIMS: To investigate the immunoglobulin (Ig) G response after being fed upon by Cimex lectularius L. METHODS AND RESULTS: Participants were fed upon by three male C lectularius insects weekly for a month. Blood was obtained before the feeding and at the last feeding, which was used for immunoblots against bed bug salivary gland extract, with antihuman Immunoglobulin G (IgG) secondary antibodies. No consistent IgG changes developed in 11 humans serially fed upon by C lectularius. Two participants had new IgG responses to proteins at molecular weights of approximately 12-13 kDa, and one had an IgG response to a protein at approximately 40 kDa. At the last study visit, more intense IgG bands to proteins at molecular weights of 12-13 kDa had developed in 55% of participants (6/11) and at molecular weights of ≈30, ≈40 and ≈70 kDa in 45% (5/11) compared with the first study visit. Nitrophorin and apyrase were the most common C lectularius proteins identified with liquid chromatography-tandem mass spectrometry in both crushed bed bug salivary gland extract and post-bed bug feeding extract. CONCLUSIONS: Human participants did not have consistent IgG responses to crushed C lectularius salivary gland extract.
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Percevejos-de-Cama/imunologia , Imunoglobulina G/imunologia , Mordeduras e Picadas de Insetos/imunologia , Saliva/imunologia , Adolescente , Adulto , Animais , Feminino , Humanos , Imunoglobulina G/sangue , Mordeduras e Picadas de Insetos/sangue , Masculino , Pessoa de Meia-Idade , Saliva/química , Glândulas Salivares/química , Proteínas e Peptídeos Salivares/análise , Proteínas e Peptídeos Salivares/imunologia , Adulto JovemRESUMO
Human ß defensin-3 (hBD-3), an epithelial cell-derived antimicrobial peptide, mediates chemotaxis and activation of myeloid cells. In this study, we provide evidence that hBD-3 induces the costimulatory molecule CD86 on primary human monocytes by a mechanism involving autocrine activation of ionotropic P2X7 receptors (P2X7R) by ATP. Incubation of monocytes with hBD-3 resulted in increased expression of both the CD80 and CD86 costimulatory molecules. Treatment of monocytes with a selective P2X7R antagonist inhibited the ability of hBD-3 to induce expression of CD86 but not CD80. The hBD-3-dependent upregulation of CD86 was also attenuated in monocytes incubated with apyrase, a potent scavenger of extracellular ATP. Finally, direct activation of monocyte P2X7R by exogenous ATP mimicked the ability of hBD-3 to induce CD86 expression. These data suggest that hBD-3 induces monocyte activation by both P2X7-dependent (CD86 upregulation) and P2X7-independent (CD80 upregulation) signaling mechanisms and raise the possibility that activation of P2X7R could play an important role in shaping the inflammatory microenvironment in conditions where hBD-3 is highly expressed, such as psoriasis or oral carcinoma.
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Trifosfato de Adenosina/farmacologia , Antígeno B7-2/metabolismo , Monócitos/efeitos dos fármacos , Receptores Purinérgicos P2X7/metabolismo , beta-Defensinas/farmacologia , Apirase/metabolismo , Apirase/farmacologia , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/genética , Cálcio/metabolismo , Células Cultivadas , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Monócitos/metabolismo , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
In the interstellar medium, six molecules have been conclusively detected in the solid state in interstellar ices, and a few dozen have been hypothesized and modeled to be present in the solid state as well. The icy mantles covering micrometer-sized dust grains are, in fact, thought to be at the core of complex molecule formation as a consequence of the local high density of molecules that are simultaneously adsorbed. From a structural perspective, the icy mantle is considered to be layered, with an amorphous water-rich inner layer surrounding the dust grain, covered by an amorphous CO-rich outer layer. Moreover, recent studies have suggested that the CO-rich layer might be crystalline and possibly even be segregated as a single crystal atop the ice mantle. If so, there are far-reaching consequences for the formation of more complex organic molecules, such as methanol and sugars, that use CO as a backbone. Validation of these claims requires further investigation, in particular on acquiring atomistic insight into surface processes, such as adsorption, diffusion, and reactivity on CO ices. Here, we present the first detailed computational study toward treating the weak interaction of (pure) CO ices. We provide a benchmark of the performance of various density functional theory methods in treating the binding of pure CO ices. Furthermore, we perform an atomistic and in-depth study of the binding energy of CO on amorphous and crystalline CO ices using a pair-potential-based force field. We find that CO adsorption is represented by a large distribution of binding energies (200-1600 K) on amorphous CO, including a significant amount of weak binding sites (<350 K). Increasing both the cluster size and the number of neighbors increases the mean of the observed binding energy distribution. Finally, we find that CO binding energies are dominated by dispersion and, as such, exchange-correlation functionals need to include a treatment of dispersion to accurately simulate surface processes on CO ices. In particular, we find the ωB97M-V functional to be a strong candidate for such simulations.
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While platelets are the essential mediators of hemostasis, they are being increasingly recognized for their potential of contributing to host defenses. Here, using immunofluorescent microscopy, western blot, and ELISA, we found that human ß-defensin 3 (hBD-3), an important antimicrobial peptide produced by epithelial cells, can be detected in human platelets and megakaryocytes. Flow cytometry and immuno-electron microscopy revealed hBD-3 on the surface of thrombin activated platelets. Moreover, hBD-3 was also found in platelet derived extracellular vesicles (p-EVs), isolated from platelet poor plasma and from platelet supernatants following thrombin stimulation. Incubation of platelets with hBD-3 peptide resulted in modest platelet activation and pre-incubation of platelets with synthetic hBD-3 prior to exposure to thrombin appeared to increase hBD-3 content in platelet lysates as well as in p-EVs, suggesting that hBD-3 can be initially taken up by platelets, perhaps via their open canalicular system. Interestingly, in vitro exposure of primary human endothelial cells to either hBD-3 peptide or purified p-EVs, caused significant endothelial dysfunction as documented by diminished levels of phosphorylated endothelial nitric oxide synthase (eNOS), Krüppel like factor-2 (KLF-2), and elevated relative expression of von Willebrand Factor (vWF). Pre-incubation of platelets with hBD-3 appeared to augment endothelial dysfunction caused by p-EVs. Overall, the current study provides evidence that hBD-3 enriched EVs can be released by activated platelets and may play a role in positive feedback of platelet activation as well as in endothelial dysfunction. Theoretically, these effects could contribute to both cellular recruitment to the endothelium creating a pro-thrombotic vascular microenvironment which serve as a bridge between innate immunity and hemostasis.
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People living with HIV (PLWH) who are immune nonresponders (INRs) are at greater risk of comorbidity and mortality than are immune responders (IRs) who restore their CD4+ T cell count after antiretroviral therapy (ART). INRs have low CD4+ T cell counts (<350 c/µL), heightened systemic inflammation, and increased CD4+ T cell cycling (Ki67+). Here, we report the findings that memory CD4+ T cells and plasma samples of INRs from several cohorts are enriched in gut-derived bacterial solutes p-cresol sulfate (PCS) and indoxyl sulfate (IS) that both negatively correlated with CD4+ T cell counts. In vitro PCS or IS blocked CD4+ T cell proliferation, induced apoptosis, and diminished the expression of mitochondrial proteins. Electron microscopy imaging revealed perturbations of mitochondrial networks similar to those found in INRs following incubation of healthy memory CD4+ T cells with PCS. Using bacterial 16S rDNA, INR stool samples were found enriched in proteolytic bacterial genera that metabolize tyrosine and phenylalanine to produce PCS. We propose that toxic solutes from the gut bacterial flora may impair CD4+ T cell recovery during ART and may contribute to CD4+ T cell lymphopenia characteristic of INRs.
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Toxinas Bacterianas , Infecções por HIV , HIV-1 , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos , Humanos , Linfopenia , MitocôndriasRESUMO
SARS-CoV-2 infection can cause compromised respiratory function and thrombotic events. SARS-CoV-2 binds to and mediates downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. Theoretically, diminished enzymatic activity of ACE2 may result in increased concentrations of pro-inflammatory molecules, angiotensin II, and Bradykinin, contributing to SARS-CoV-2 pathology. Using immunofluorescence microscopy of lung tissues from uninfected, and SARS-CoV-2 infected individuals, we find evidence that ACE2 is highly expressed in human pulmonary alveolar epithelial cells and significantly reduced along the alveolar lining of SARS-CoV-2 infected lungs. Ex vivo analyses of primary human cells, indicated that ACE2 is readily detected in pulmonary alveolar epithelial and aortic endothelial cells. Exposure of these cells to spike protein of SARS-CoV-2 was sufficient to reduce ACE2 expression. Moreover, exposure of endothelial cells to spike protein-induced dysfunction, caspase activation, and apoptosis. Exposure of endothelial cells to bradykinin caused calcium signaling and endothelial dysfunction (increased expression of von Willibrand Factor and decreased expression of Krüppel-like Factor 2) but did not adversely affect viability in primary human aortic endothelial cells. Computer-assisted analyses of molecules with potential to bind bradykinin receptor B2 (BKRB2), suggested a potential role for aspirin as a BK antagonist. When tested in our in vitro model, we found evidence that aspirin can blunt cell signaling and endothelial dysfunction caused by bradykinin in these cells. Interference with interactions of spike protein or bradykinin with endothelial cells may serve as an important strategy to stabilize microvascular homeostasis in COVID-19 disease. IMPORTANCE SARS-CoV-2 causes complex effects on microvascular homeostasis that potentially contribute to organ dysfunction and coagulopathies. SARS-CoV-2 binds to, and causes downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. It is thought that reduced ACE2 enzymatic activity can contribute to inflammation and pathology in the lung. Our studies add to this understanding by providing evidence that spike protein alone can mediate adverse effects on vascular cells. Understanding these mechanisms of pathogenesis may provide rationale for interventions that could limit microvascular events associated with SARS-CoV-2 infection.
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COVID-19/fisiopatologia , Células Endoteliais/virologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/virologia , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Aorta/citologia , Aorta/metabolismo , Aorta/virologia , Apoptose , Bradicinina/química , Bradicinina/metabolismo , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Homeostase , Humanos , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/virologia , Microcirculação , Receptores da Bradicinina/química , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
BACKGROUND: HIV-infected patients who fail to normalize CD4 T cells despite suppressive antiretroviral therapy have impaired immune homeostasis: diminished naive T-cell numbers, elevated T-cell turnover, senescence, and inflammation. METHODS: Blood samples from immune failures (n = 60), immune successes (n = 20), and healthy controls (n = 20) were examined for plasma interleukin (IL)-7 levels, for cellular expression of the IL-7Rα chain (CD127), for the exhaustion and senescence markers programed death 1 (PD-1) and CD57, and for the survival factor Bcl2. Because both inflammatory and homeostatic cytokines can induce T-cell cycling, we also examined the effects of these mediators on exhaustion and senescence markers. RESULTS: Plasma levels of IL-7 were elevated and both CD4 and CD8 T-cell CD127 expression was decreased in immune failure. Plasma levels of IL-7 correlated directly with naive CD4 T-cell counts in immune success and inversely with T-cell cycling (Ki67) in healthy controls and immune success, but not in immune failure. CD4 T-cell density of PD-1 was increased and Bcl2+ CD4 T cells were decreased in immune failure but not in immune success, whereas the proportion of T cells expressing CD57 was increased in immune failure. PD-1 and CD57 were induced on CD4 but not CD8 T cells by stimulation in vitro with inflammatory IL-1ß or homeostatic (IL-7) cytokines. CONCLUSIONS: Perturbation of the IL-7/IL-7 receptor axis, increased T-cell turnover, and increased senescence may reflect dysregulated responses to both homeostatic and inflammatory cytokines in immune failure patients.
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Infecções por HIV/imunologia , Interleucina-7/sangue , Adulto , Fármacos Anti-HIV , Biomarcadores/metabolismo , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Antígenos CD57/metabolismo , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Morte Celular/fisiologia , Senescência Celular/imunologia , Feminino , Humanos , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-7/sangue , Ativação Linfocitária/imunologia , Masculino , Falha de Tratamento , Proteína de Morte Celular Associada a bcl/sangueRESUMO
BACKGROUND: Despite suppressive antiretroviral therapy (ART), increased levels of immune activation persist in HIV-infected subjects. Statins have anti-inflammatory effects and may reduce immune activation in HIV disease. METHODS: Stopping Atherosclerosis and Treating Unhealthy bone with RosuvastatiN in HIV (SATURN-HIV) is a randomized, double-blind placebo-controlled trial assessing the effect of rosuvastatin (10 mg daily) on markers of cardiovascular risk and immune activation in ART-treated patients. T-cell activation was measured by expression of CD38, HLA-DR, and PD1. Monocyte activation was measured with soluble markers (sCD14 and sCD163) and by enumeration of monocyte subpopulations and tissue factor expression. Markers of systemic and vascular inflammation and coagulation were also measured. SATURN-HIV is registered on clinicaltrials.gov (identifier: NCT01218802). RESULTS: Rosuvastatin, compared with placebo, reduced sCD14 (-10.4% vs 0.5%, P = 0.006), lipoprotein-associated phospholipase A2 (-12.2% vs -1.7%, P = 0.0007), and IP-10 (-27.5 vs -8.2%, P = 0.03) levels after 48 weeks. The proportion of tissue factor-positive patrolling (CD14CD16) monocytes was also reduced by rosuvastatin (-41.6%) compared with placebo (-18.8%, P = 0.005). There was also a greater decrease in the proportions of activated (CD38HLA-DR) T cells between the arms (-38.1% vs -17.8%, P = 0.009 for CD4 cells, and -44.8% vs -27.4%, P = 0.003 for CD8 cells). CONCLUSIONS: Forty-eight weeks of rosuvastatin treatment reduced significantly several markers of inflammation and lymphocyte and monocyte activation in ART-treated subjects.
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Antirretrovirais/uso terapêutico , Anticolesterolemiantes/uso terapêutico , Fluorbenzenos/uso terapêutico , Infecções por HIV/patologia , Monócitos/imunologia , Pirimidinas/uso terapêutico , Sulfonamidas/uso terapêutico , Linfócitos T/imunologia , Vasculite/prevenção & controle , Adulto , Antígenos CD/análise , Feminino , Infecções por HIV/tratamento farmacológico , Antígenos HLA-DR/análise , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/química , Receptor de Morte Celular Programada 1/análise , Rosuvastatina Cálcica , Linfócitos T/química , Resultado do Tratamento , Vasculite/patologiaRESUMO
BACKGROUND: HIV infection is associated with increased cardiovascular risk, and this risk correlates with markers of monocyte activation. We have shown that HIV is associated with a prothrombotic monocyte phenotype, which can be partially mitigated by statin therapy. We therefore explored the relationship between oxidized low-density lipoprotein (oxLDL) particles and monocyte activation. METHODS: We performed phenotypic analysis of monocytes using flow cytometry on fresh whole blood in 54 patients with HIV and 24 controls without HIV. Plasma levels of oxLDL, soluble CD14, IL-6, and soluble CD163 were measured by enzyme-linked immunosorbent assay. In vitro experiments were performed using flow cytometry. RESULTS: Plasma levels of oxLDL were significantly increased in HIV infection compared with controls (60.1 units vs. 32.1 units, P < 0.001). Monocyte expression of the oxLDL receptors, CD36 and Toll-like receptor 4, was also increased in HIV. OxLDL levels correlated with markers of monocyte activation, including soluble CD14, tissue factor expression on inflammatory monocytes, and CD36. In vitro stimulation with oxLDL, but not to low-density lipoprotein, resulted in expansion of inflammatory monocytes and increased monocyte expression of tissue factor, recapitulating the monocyte profile we find in HIV disease. CONCLUSIONS: OxLDL may contribute to monocyte activation, and further study in the context of HIV disease is warranted.