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1.
Nat Methods ; 17(4): 422-429, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-32203389

RESUMEN

Brain circuits comprise vast numbers of interconnected neurons with diverse molecular, anatomical and physiological properties. To allow targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.


Asunto(s)
Genómica/métodos , Optogenética , Recombinasas/metabolismo , Animales , Encéfalo/citología , Regulación de la Expresión Génica , Ingeniería Genética , Ratones , Neuronas/metabolismo , Recombinasas/genética , Pez Cebra
2.
Biochemistry ; 60(15): 1178-1190, 2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33749246

RESUMEN

Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.


Asunto(s)
Fosfolipasas/metabolismo , Dominio Catalítico , Simulación de Dinámica Molecular , Fosfolipasas/química , Especificidad por Sustrato , Temperatura
3.
Carcinogenesis ; 42(1): 38-47, 2021 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-32832992

RESUMEN

Tumor cells promote immune evasion through upregulation of programmed death-ligand 1 (PD-L1) that binds with programmed cell death protein 1 (PD1) on cytotoxic T cells and promote dysfunction. Though therapeutic efficacy of anti-PD1 antibody has remarkable effects on different type of cancers it is less effective in breast cancer (BC). Hence, more details understanding of PD-L1-mediated immune evasion is necessary. Here, we report BC cells secrete extracellular vesicles in form of exosomes carry PD-L1 and are highly immunosuppressive. Transforming growth factor beta (TGF-ß) present in tumor microenvironment orchestrates BC cell secreted exosomal PD-L1 load. Circulating exosomal PD-L1 content is highly correlated with tumor TGF-ß level. The later also found to be significantly associated with CD8+CD39+, CD8+PD1+ T-cell phenotype. Recombinant TGF-ß1 dose dependently induces PD-L1 expression in Texos in vitro and blocking of TGF-ß dimmed exosomal PD-L1 level. PD-L1 knocked down exosomes failed to suppress effector activity of activated CD8 T cells like tumor exosomes. While understanding its effect on T-cell receptor signaling, we found siPD-L1 exosomes failed to block phosphorylation of src family proteins, linker for activation of T cells and phosphoinositide phospholipase Cγ of CD8 T cells more than PD-L1 exosomes. In vivo inhibition of exosome release and TGF-ß synergistically attenuates tumor burden by promoting Granzyme and interferon gamma release in tumor tissue depicting rejuvenation of exhausted T cells. Thus, we establish TGF-ß as a promoter of exosomal PD-L1 and unveil a mechanism that tumor cells follow to promote CD8 T-cell dysfunction.


Asunto(s)
Antígeno B7-H1/metabolismo , Neoplasias de la Mama/inmunología , Linfocitos T CD8-positivos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Compuestos de Anilina/administración & dosificación , Animales , Antígeno B7-H1/sangre , Antígeno B7-H1/genética , Benzamidas/administración & dosificación , Compuestos de Bencilideno/administración & dosificación , Mama/patología , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Ehrlich/inmunología , Carcinoma de Ehrlich/patología , Línea Celular Tumoral , Dioxoles/administración & dosificación , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Femenino , Técnicas de Inactivación de Genes , Granzimas/metabolismo , Voluntarios Sanos , Humanos , Interferón gamma/metabolismo , Ratones , Persona de Mediana Edad , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Cultivo Primario de Células , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal/inmunología , Escape del Tumor/efectos de los fármacos , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología
5.
Microcirculation ; : e12734, 2021 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-34741773

RESUMEN

BACKGROUND: It remains unknown whether the cutaneous microvascular responses are different between patients with scleroderma-associated pulmonary arterial hypertension (SSc-PAH) and SSc without pulmonary hypertension (PH). METHODS: We included 59 patients with SSc between March 2013 and September 2019. We divided patients into 4 groups: (a) no PH by right heart catheterization (RHC) (n = 8), (b) no PH by noninvasive screening tests (n = 16), (c) treatment naïve PAH (n = 16), and (d) PAH under treatment (n = 19). Microvascular studies using laser Doppler flowmetry (LDF) were done immediately after RHC or at the time of an outpatient clinic visit (group b). RESULTS: The median (IQR) age was 59 (54-68) years, and 90% were females. The responses to local thermal stimulation and postocclusive reactive hyperemia, acetylcholine, and sodium nitroprusside iontophoresis were similar among groups. The microvascular response to treprostinil was more pronounced in SSc patients without PH by screening tests (% change: 340 (214-781)) compared with SSc-PAH (naïve + treatment) (Perfusion Units (PU) % change: 153 (94-255) % [p = .01]). The response to A-350619 (a soluble guanylate cyclase (sGC) activator) was significantly higher in patients with SSc without PH by screening tests (PU % change: 168 (46-1,296)) than those with SSc-PAH (PU % change: 22 (15-57) % [p = .006]). The % change in PU with A350619 was directly associated with cardiac index and stroke volume index (R: 0.36, p = .03 and 0.39, p = .02, respectively). CONCLUSIONS: Patients with SSc-PAH have a lower cutaneous microvascular response to a prostacyclin analog treprostinil and the sGC activator A-350619 when compared with patients with SSc and no evidence of PH on screening tests, presumably due to a peripheral reduction in prostacyclin receptor expression and nitric oxide bioavailability.

6.
J Immunol ; 203(4): 972-980, 2019 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-31253726

RESUMEN

Failure of the most recent tuberculosis (TB) vaccine trial to boost bacillus Calmette-Guérin-mediated anti-TB immunity despite the induction of Th1-specific central memory cell and effector memory cell responses highlights the importance of identifying optimal T cell targets for protective vaccines. In this study, we describe a novel, Mycobacterium tuberculosis-specific IFN-γ+CD4+ T cell population expressing surface markers characteristic of naive-like memory T cells (TNLM), which were induced in both human (CD45RA+CCR7+CD27+CD95-) and murine (CD62L+CD44-Sca-1+CD122-) systems in response to mycobacteria. In bacillus Calmette-Guérin-vaccinated subjects and those with latent TB infection, TNLM were marked by the production of IFN-γ but not TNF-α and identified by the absence of CD95 expression and increased surface expression CCR7, CD27, the activation markers T-bet, CD69, and the survival marker CD74. Increased tetramer-positive TNLM frequencies were noted in the lung and spleen of ESAT-61-20-specific TCR transgenic mice at 2 wk postinfection with M. tuberculosis and progressively decreased at later time points, a pattern not seen with TNF-α+CD4+ T cells expressing naive cell surface markers. Importantly, adoptive transfer of highly purified TNLM alone, from vaccinated ESAT-61-20-specific TCR transgenic mice, conferred equivalent protection against M. tuberculosis infection in the lungs of Rag-/- mice when compared with total memory populations (central and effector memory cells). Thus, TNLM may represent a memory T cell population that, if optimally targeted, may significantly improve future TB vaccine responses.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica/inmunología , Interferón gamma/inmunología , Subgrupos de Linfocitos T/inmunología , Tuberculosis Pulmonar/inmunología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/inmunología , Fenotipo , Vacunas contra la Tuberculosis/inmunología
7.
J Environ Manage ; 283: 111986, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33486195

RESUMEN

Constructed wetland (CW) represents an efficient eco-technological conglomerate interweaving water security, energy possibility and environmental protection. In the context of wastewater treatment technologies requiring substantial efficiency at reduced cost, chemical input and low environmental impact, applications of CW is being demonstrated at laboratory and field level with reasonably high contaminant removal efficiency and ecological benefits. However, along with the scope of applications, role of individual wetland component has to be re-emphasized through related research interventions. Hence, this review distinctively explores the concerns for extracting maximum benefit of macrophyte (focusing on interface of pollutant removal, root radial oxygen loss, root iron plaque, endophyte-macrophyte assisted treatment in CW, and prospects of energy harvesting from macrophyte) and role of biofilm (effect on treatment efficiency, composition and factors affecting) in a CW. Another focus of the review is on recent advances and developments in alternative low-cost substrate materials (including conventional type, industrial by-products, organic waste, mineral based and hybrid type) and their effect on target pollutants. The remainder of this review is organized to discuss the concerns of CW with respect to wastewater type (municipal, industrial, agricultural and farm wastewater). Attempt is made to analyze the practical relevance and significance of these aspects incorporating all recent developments in the areas to help making informed decisions about future directions for research and development related to CW.


Asunto(s)
Aguas Residuales , Purificación del Agua , Oxígeno , Tecnología , Eliminación de Residuos Líquidos , Aguas Residuales/análisis , Humedales
8.
Resour Conserv Recycl ; 164: 105156, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32921917

RESUMEN

Along with outbreak of the pandemic COVID-19 caused by SARS-CoV-2, the problem of biomedical wastewater disposal has caused widespread public concern, as reportedly the presence is confirmed in wastewater. Keeping in mind (i) available evidence indicating need to better understand potential of wastewater mediated transmission and (ii) knowledge gaps in its occurrence, viability, persistence, and inactivation in wastewater, in this present work, we wanted to re-emphasize some strategies for management of SARS-CoV-2 contaminated wastewater to minimise any possible secondary transmission to human and environment. The immediate challenges to consider while considering wastewater management are uncertainty about this new biothreat, relying on prediction based treatments options, significant population being the latent asymptomatic carrier increased risk of passing out of the virus to sewage network, inadequacy of wastewater treatment facility particularly in populated developing countries and increased generation of wastewater due to increased cleanliness concern. In absence of regulated central treatment facility, installation of decentralized wastewater treatment units with single or multiple disinfection barriers in medical units, quarantine centre, isolation wards, testing facilities seems to be urgent for minimizing any potential risk of wastewater transmission. Employing some emerging disinfectants (peracetic acid, performic acid, sodium dichloro isocyanurate, chloramines, chlorine dioxide, benzalconium chloride) shows prospects in terms of virucidal properties. However, there is need of additional research on coronaviruses specific disinfection data generation, regular monitoring of performance considering all factors influencing virus survival, performance evaluation in actual water treatment, need of augmenting disinfection dosages, environmental considerations to select the most appropriate disinfection technology.

9.
Environ Monit Assess ; 193(2): 80, 2021 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-33486600

RESUMEN

2,4,6-trinitrotoluene or TNT, a commonly used explosive, can pollute soil and groundwater. Conventional remediation practices for the TNT-contaminated sites are neither eco-friendly nor cost-effective. However, exploring bacteria to biodegrade TNT into environment-friendly compound(s) is an interesting area to explore. In this study, an indigenous bacterium, Pseudarthrobacter chlorophenolicus, strain S5-TSA-26, isolated from explosive contaminated soil, was investigated for potential aerobic degradation of TNT for the first time. The isolated strain of P. chlorophenolicus was incubated in a minimal salt medium (MSM) containing 120 mg/L TNT for 25 days at specified conditions. TNT degradation pattern by the bacterium was monitored at regular interval using UV-Vis spectrophotometry, high-performance liquid chromatography, and liquid chromatography mass spectrophotometric, by estimating nitrate, nitrite, and ammonium ion concentration and other metabolites such as 2,4-dinitrotoluene (DNT), 2-amino-4,6-dinitrotoluene (2-ADNT), and 2,4-diamino-6-nitrotoluene (2-DANT). It was observed that, in the presence of TNT, there was no reduction in growth of the bacterium although it multiplied well in the presence of TNT along with no considerable morphological changes. Furthermore, it was found that TNT degraded completely within 15 days of incubation. Thus, from this study, it may be concluded that the bacterium has the potential for degrading TNT completely with the production of non-toxic by-products and might be an important bacterium for treating TNT (i.e., a nitro-aromatic compound)-contaminated sites.


Asunto(s)
Sustancias Explosivas , Micrococcaceae , Trinitrotolueno , Biodegradación Ambiental , Monitoreo del Ambiente
10.
Hepatology ; 69(5): 1873-1884, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-29443378

RESUMEN

A comprehensive strategy to control hepatitis C virus (HCV) infection needs a vaccine. Our phase I study with recombinant HCV E1/E2 envelope glycoprotein (EnvGPs) as a candidate vaccine did not induce a strong immune response in volunteers. We analyzed the interactions of HCV EnvGPs with human monocyte-derived macrophages as antigen-presenting cells. HCV E2 induced immune regulatory cytokine interleukin (IL)-10 and soluble CD163 (sCD163) protein expression in macrophages from 7 of 9 blood donors tested. Furthermore, HCV E2 enhanced Stat3 and suppressed Stat1 activation, reflecting macrophage polarization toward M2 phenotype. E2-associated macrophage polarization appeared to be dependent of its interaction with CD81 leading endothelial growth factor receptor (EGFR) activation. Additionally, E2 suppressed the expression of C3 complement, similar to HCV-exposed dendritic cells (DCs), implying potential impairment of immune cell priming. Conclusion: Our results suggest that E2 EnvGP may not be an ideal candidate for HCV vaccine development, and discrete domains within E2 may prove to be more capable of elliciting a protective immune response. (Hepatology 2018).


Asunto(s)
Interacciones Huésped-Patógeno/inmunología , Macrófagos/metabolismo , Proteínas del Envoltorio Viral/fisiología , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Complemento C3/metabolismo , Células Dendríticas/metabolismo , Receptores ErbB/metabolismo , Humanos , Interleucina-10/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Superficie Celular/metabolismo , Tetraspanina 28/metabolismo
11.
Pestic Biochem Physiol ; 170: 104680, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32980059

RESUMEN

The progression of neurodegenerative disease is very complex biological process and the molecular crosstalk of inflammatory cytokines during neurodegeneration is associated with multiple cascade signalling. Few evidences suggest that environmental toxin, Paraquat (PQ) administration activates the microglia and intensify the release of proinflamatory cytokines during progression of Parkinson''s disease (PD) but the proper aetiology remained unknown. However, the fundamental role of anti-inflammatory molecule Decapentaplegic (Dpp), homologue of the secreted mammalian Transforming growth factor-ß (TGF-ß) signalling molecule during neurodegeneration of invertebrate fly model is yet to establish. To elucidate the molecular processes during early stage of Parkinson's disease, we observed neuro-toxin plays a determining role in the increased vulnerability to a particular PQ exposure that is attended by decreased lifespan, severe locomotor deficits, and more loss of dopaminergic (DA) neuron in PQ-treated Dpp deficient fly than wild type (WT). Simultaneously, activated microglia induced the inflammatory response with the release of pro-inflammatory and anti-inflammatory cytokine in Drosophila during neurodegeneration. Moreover, neuro-toxin exposure altered the expression of innate immune genes in both WT and mutant fly compared to the respective PQ-treated flies. Interestingly, PQ exposure reduced the expression of innate immune genes in mutant fly compared to WT. It may indicate that PQ exposure had broken down the immune defence response in mutant fly than WT whereas, without PQ exposure the innate immune tolerance level was higher in fly with reduced Dpp expression than WT. Thus, we observed the conserve anti-inflammatory factor TGF-ß may exhibit a crucial defensive role during inflammation mediated neurodegeneration in invertebrate Drosophila melanogaster.


Asunto(s)
Proteínas de Drosophila/genética , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/genética , Animales , Modelos Animales de Enfermedad , Drosophila , Drosophila melanogaster/genética , Inmunidad Innata/genética , Inflamación/inducido químicamente , Inflamación/genética , Neuroglía , Paraquat/toxicidad
12.
Br J Cancer ; 121(8): 679-689, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31481734

RESUMEN

BACKGROUND: Cancer-associated fibroblasts (CAFs) are known to impact on tumour behaviour, but the mechanisms controlling this are poorly understood. METHODS: Breast normal fibroblasts (NFs) or CAFs were isolated from cancers by laser microdissection or were cultured. Fibroblasts were transfected to manipulate miR-222 or Lamin B receptor (LBR). The fibroblast-conditioned medium was collected and used to treat epithelial BC lines MDA-MB-231 and MDA-MB-157. Migration, invasion, proliferation or senescence was assessed using transwell, MTT or X-gal assays, respectively. RESULTS: MiR-222 was upregulated in CAFs as compared with NFs. Ectopic miR-222 expression in NFs induced CAF-like expression profiles, while miR-222 knockdown in CAFs inhibited CAF phenotypes. LBR was identified as a direct miR-222 target, and was functionally relevant since LBR knockdown phenocopied miR-222 overexpression and LBR overexpression phenocopied miR-222 knockdown. MiR-222 overexpression, or LBR knockdown, was sufficient to induce NFs to show the CAF characteristics of enhanced migration, invasion and senescence, and furthermore, the conditioned medium from these fibroblasts induced increased BC cell migration and invasion. The reverse manipulations in CAFs inhibited these behaviours in fibroblasts, and inhibited paracrine influences on BC cells. CONCLUSION: MiR-222/LBR have key roles in controlling pro-progression influences of CAFs in BC. This pathway may present therapeutic opportunities to inhibit CAF-induced cancer progression.


Asunto(s)
Neoplasias de la Mama/genética , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma/genética , MicroARNs/genética , Receptores Citoplasmáticos y Nucleares/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma/metabolismo , Carcinoma/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Técnicas de Reprogramación Celular , Senescencia Celular , Medios de Cultivo Condicionados , Femenino , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Captura por Microdisección con Láser , Invasividad Neoplásica , Metástasis de la Neoplasia , Receptor de Lamina B
14.
J Clin Gastroenterol ; 53(3): 184-190, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-29356781

RESUMEN

GOALS: This study was carried out to assess the clinical characteristics and associated systemic diseases seen in patients diagnosed with absent contractility as per the Chicago Classification version 3.0, allowing us to propose a diagnostic algorithm for their etiologic testing. BACKGROUND: The Chicago Classification version 3.0 has redefined major and minor esophageal motility disorders using high-resolution esophageal manometry. There is a dearth of publications based on research on absent contractility, which historically has been associated with myopathic processes such as systemic sclerosis (SSc). STUDY: We conducted a retrospective, multicenter study. Data of patients diagnosed with absent contractility were pooled from Cleveland Clinic, Cleveland, OH (January 2006 to July 2016) and Metrohealth Medical Center, Cleveland, OH (July 2014 to July 2016) and included: age, gender, associated medical conditions, surgical history, medications, and specific antibody testing. RESULTS: A total of 207 patients, including 57 male individuals and 150 female individuals, with mean age of 56.1 and 60.0 years, respectively, were included. Disease distribution was as follows: SSc (diffuse or limited cutaneous) 132, overlap syndromes 7, systemic lupus erythematosus17, Sjögren syndrome 4, polymyositis 3, and dermatomyositis 3. Various other etiologies including gastroesophageal reflux disease, postradiation esophagitis, neuromuscular disorders, and surgical complications were seen in the remaining cohort. CONCLUSIONS: Most practitioners use the term "absent contractility" interchangeably with "scleroderma esophagus"; however, only 63% of patients with absent contractility had SSc. Overall, 20% had another systemic autoimmune rheumatologic disease and 16% had a nonrheumatologic etiology for absent contractility. Therefore, alternate diagnosis must be sought in these patients. We propose an algorithm for their etiologic evaluation.


Asunto(s)
Algoritmos , Trastornos de la Motilidad Esofágica/diagnóstico , Enfermedades Reumáticas/complicaciones , Técnicas de Diagnóstico del Sistema Digestivo , Trastornos de la Motilidad Esofágica/complicaciones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ohio , Estudios Retrospectivos
15.
Biol Blood Marrow Transplant ; 24(10): 1961-1964, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29953945

RESUMEN

Systemic sclerosis is a progressive inflammatory disease that is frequently fatal and has limited treatment options. High-dose chemotherapy with autologous hematopoietic cell transplantation (AHCT) has been evaluated as treatment for this disease in observational studies, multicenter randomized controlled clinical trials, and meta-analyses. On behalf of the American Society for Blood and Marrow Transplantation (ASBMT), a panel of experts in transplantation and rheumatology was convened to review available evidence and make a recommendation on AHCT as an indication for systemic sclerosis. Three randomized trials have compared the efficacy of AHCT with cyclophosphamide only, and all demonstrated benefit for the AHCT arm for their primary endpoint (improvement in the American Scleroderma Stem Cell versus Immune Suppression Trial, event-free survival in Autologous Stem Cell Transplantation International Scleroderma trial, and change in global rank composite score in Scleroderma: Cyclophosphamide or Transplantation trial). AHCT recipients also had better overall survival and a lower rate of disease progression. These findings have been confirmed in subsequent meta-analyses. Based on this high-quality evidence, the ASBMT recommends systemic sclerosis should be considered as a "standard of care" indication for AHCT. Close collaboration between rheumatologists and transplant clinicians is critical for optimizing patient selection and patient outcomes. Transplant centers in the United States are strongly encouraged to report patient and outcomes data to the Center for International Blood and Marrow Transplant Research on their patients receiving AHCT for this indication.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Esclerodermia Sistémica/terapia , Autoinjertos , Trasplante de Médula Ósea , Ciclofosfamida/uso terapéutico , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Esclerodermia Sistémica/mortalidad , Esclerodermia Sistémica/patología , Sociedades Médicas , Estados Unidos
16.
J Biomol NMR ; 71(2): 69-77, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29860650

RESUMEN

Understanding of the molecular mechanisms of protein function requires detailed insight into the conformational landscape accessible to the protein. Conformational changes can be crucial for biological processes, such as ligand binding, protein folding, and catalysis. NMR spectroscopy is exquisitely sensitive to such dynamic changes in protein conformations. In particular, Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments are a powerful tool to investigate protein dynamics on a millisecond time scale. CPMG experiments that probe the chemical shift modulation of 15N in-phase magnetization are particularly attractive, due to their high sensitivity. These experiments require high power 1H decoupling during the CPMG period to keep the 15N magnetization in-phase. Recently, an improved version of the in-phase 15N-CPMG experiment was introduced, offering greater ease of use by employing a single 1H decoupling power for all CPMG pulsing rates. In these experiments however, incomplete decoupling of off-resonance amide 1H spins introduces an artefactual dispersion of relaxation rates, the so-called slow-pulsing artifact. Here, we analyze the slow-pulsing artifact in detail and demonstrate that it can be suppressed through the use of composite pulse decoupling (CPD). We report the performances of various CPD schemes and show that CPD decoupling based on the 90x-240y-90x element results in high-quality dispersion curves free of artifacts, even for amides with high 1H offset.


Asunto(s)
Artefactos , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Amidas , Magnetismo , Protones
17.
18.
Cell Biol Int ; 41(9): 991-1000, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28557137

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is a deadly, progressive lung disease with very few treatment options till now. Bleomycin-induced pulmonary fibrosis (BIPF) is a commonly used mice model in IPF research. TGF-ß1 has been shown to play a key role in pulmonary fibrosis (PF). Dendritic cell (DC) acts as a bridge between innate and adaptive immune systems. The coexistence of chronic inflammation sustained by mature DCs with fibrosis suggests that inflammatory phenomenon has key importance in the pathogenesis of pulmonary fibrosis. Here, we investigated the modulation of DCs phenotypic maturation, accumulation in lung tissue, and expression of other lung DC subsets in respect to TGF-ß in PF. First, we established BIPF model in mice and blocked TGF-ß expression by the use of inhibitor SB431542. Accumulation of lung CD11c+ DCs is significantly higher in both inflammatory and fibrotic phases of the disease but that percentages got reduced in the absence of TGF-ß. TGF-ß initiates up-regulation of costimulatory molecules CD86 and CD80 in the inflammatory phases of the disease but not so at fibrotic stage. Expression of lung DC subset CD11c+CD103+ is significantly increased in inflammatory phase and also in fibrotic phase of BIPF. Blocking of TGF-ß causes decreased expression of CD11c+CD103+ DCs. Another important lung DC subset CD11c+CD11b+ expression is suppressed by the absence of TGF-ß after bleomycin administration. CD11c+CD103+ DCs might have anti-inflammatory as well as anti-fibrotic nature in PF. All these data demonstrate differential modulation of CD11c+ lung DCs by TGF-ß in experimental PF.


Asunto(s)
Antígeno CD11c/inmunología , Células Dendríticas/inmunología , Fibrosis Pulmonar Idiopática/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Benzamidas/farmacología , Bleomicina , Antígeno CD11c/metabolismo , Células Dendríticas/metabolismo , Dioxoles/farmacología , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/terapia , Cadenas alfa de Integrinas/inmunología , Cadenas alfa de Integrinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismo , Regulación hacia Arriba/efectos de los fármacos
20.
J Immunol ; 194(6): 2706-14, 2015 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-25667413

RESUMEN

Exaggerated CD4(+) T helper 2-specific cytokine producing memory T cell responses developing concomitantly with a T helper 1 response might have a detrimental role in immunity to infection caused by Mycobacterium tuberculosis. To assess the dynamics of Ag-specific memory T cell compartments in the context of filarial infection, we used multiparameter flow cytometry on PBMCs from 25 microfilaremic filarial-infected (Inf) and 14 filarial-uninfected (Uninf) subjects following stimulation with filarial Ag (BmA) or with the M. tuberculosis-specific Ag culture filtrate protein-10 (CFP-10). Our data demonstrated that the Inf group had a marked increase in BmA-specific CD4(+)IL-4(+) cells (median net frequency compared with baseline [Fo] = 0.09% versus 0.01%; p = 0.038) but also to CFP-10 (Fo = 0.16% versus 0.007%; p = 0.04) and staphylococcal enterotoxin B (Fo = 0.49% versus 0.26%; p = 0.04). The Inf subjects showed a BmA-specific expansion of CD4(+)CD45RO(+)IL-4(+) producing central memory (TCM, CD45RO(+)CCR7(+)CD27(+); Fo = 1.1% versus 0.5%; p = 0.04) as well as effector memory (TEM, CD45RO(+)CCR7(-)CD27(-); Fo = 1.5% versus 0.2%; p = 0.03) with a similar but nonsignificant response to CFP-10. In addition, there was expansion of CD4(+)IL-4(+)CD45RA(+)CCR7(+)CD27(+) (naive-like) in Inf individuals compared with Uninf subjects. Among Inf subjects with definitive latent tuberculosis, there were no differences in frequencies of IL-4-producing cells within any of the memory compartments compared with the Uninf group. Our data suggest that filarial infection induces Ag-specific, exaggerated IL-4 responses in distinct T cell memory compartments to M. tuberculosis-specific Ags, which are attenuated in subjects who are able to mount a delayed type hypersensitivity reaction to M. tuberculosis.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Memoria Inmunológica/inmunología , Interleucina-4/inmunología , Tuberculosis Latente/inmunología , Loa/inmunología , Loiasis/inmunología , Mycobacterium tuberculosis/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos Bacterianos/inmunología , Antígenos Helmínticos/inmunología , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Femenino , Citometría de Flujo , Humanos , Interleucina-4/metabolismo , Tuberculosis Latente/microbiología , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Loa/fisiología , Loiasis/parasitología , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/fisiología , Receptores CCR7/inmunología , Receptores CCR7/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Adulto Joven
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