RESUMEN

INTRODUCTION: Device-related infections are difficult to treat due to biofilms. In this setting, optimizing antibiotic efficacy is difficult as most pharmacokinetic/pharmacdynamic (PK/PD) studies have been performed on planktonic cells, and therapies are limited when multi-drug-resistant bacteria are involved. This study aimed to analyse the PK/PD indices of meropenem predicting anti-biofilm efficacy against meropenem-susceptible and meropenem-resistant strains of Pseudomonas aeruginosa. MATERIALS AND METHODS: Pharmacodynamics of meropenem dosages mimicking those of clinical practice (intermittent bolus of 2 g every 8 h; extended infusion of 2 g over 4 h every 8 h), with and without colistin, were evaluated with the CDC Biofilm Reactor in-vitro model for susceptible (PAO1) and extensively-drug-resistant (XDR-HUB3) P. aeruginosa. Efficacy was correlated with the PK/PD indices for meropenem. RESULTS: For PAO1, both meropenem regimens were bactericidal, with higher killing for extended infusion [∆log10 colony-forming units (CFU)/mL 54-0h=-4.66±0.93 for extended infusion vs ∆log10 CFU/mL 54-0h=-3.4±0.41 for intermittent bolus; P<0.001]. For XDR-HUB3, the intermittent bolus regimen was non-active, but extended infusion showed bactericidal effect (∆log10 CFU/mL 54-0h=-3.65±0.29; P<0.001). Time above minimum inhibitory concentration (f%T>MIC) had the best correlation with efficacy for both strains. The addition of colistin always improved meropenem activity, and resistant strains did not emerge. CONCLUSION: f%T>MIC was the PK/PD index that best correlated with the anti-biofilm efficacy of meropenem; it was better optimized when using the extended infusion regimen, allowing recovery of bactericidal activity in monotherapy, including activity against meropenem-resistant P. aeruginosa. Combining meropenem by extended infusion with colistin offered the most effective therapy for both strains. Optimizing meropenem dosing by extended infusion should be encouraged when treating biofilm-related infections.

Asunto(s)

Colistina , Infecciones por Pseudomonas , Humanos , Meropenem/farmacología , Colistina/farmacología , Colistina/uso terapéutico , Pseudomonas aeruginosa , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Pruebas de Sensibilidad Microbiana

RESUMEN

Introduction: The use of antibiotics may induce the changes in gut microbiota. Previous studies have shown conflicting results on whether the changed gut microbiota by antibiotics can be recovered. Our study aims to investigate whether the gut microbiota could be recovered after a single dose of oral co-amoxiclav before transrectal ultrasound-guided transperineal prostate biopsy (TPPBx) in 5 weeks' time. Methods: Fifteen patients with elevated serum prostate-specific antigen (PSA) were recruited to provide pre-antibiotic and post-antibiotic fecal samples. The V4 region of 16S rRNA was sequenced. Analysis was performed by QIIME2. Alpha- and beta-diversities were analyzed, as well as the differential enrichment by Linear discriminant analysis Effect Size (LEfSe) analysis. Results: Both the alpha- and beta-diversities of the pre- and post-antibiotic fecal samples were significantly different. Genera that are associated with alleviation of inflammation were enriched in the pre-antibiotic fecal samples, while the inflammation-associated genera were more enriched in the post-antibiotic fecal samples. Conclusion: A single dose of oral co-amoxiclav before TPPBx could have led to a change of gut microbiota that cannot be recovered in 5 weeks' time. Microbiome studies on prostate cancer patients should be cautioned on the use of post-prostate biopsy fecal sampling. Further studies should be conducted for the impact on gut microbiome for TPPBx alone.

Asunto(s)

Microbioma Gastrointestinal , Combinación Amoxicilina-Clavulanato de Potasio/farmacología , Antibacterianos/farmacología , Biopsia , Heces , Humanos , Inflamación/patología , Masculino , Próstata , ARN Ribosómico 16S/genética

RESUMEN

OBJECTIVE: Angiotensin II (Ang II)-induced endothelial dysfunction plays an important role in the pathogenesis of cardiovascular diseases such as systemic hypertension, cardiac hypertrophy and atherosclerosis. Recently, long noncoding RNAs (lncRNAs) have been shown to play an essential role in the pathobiology of cardiovascular diseases; however, the effect of Ang II on lncRNAs and coding RNAs expression in endothelial cells has not been evaluated. Accordingly, we sought to evaluate the expression profiles of lncRNAs and coding RNAs in endothelial cells following treatment with Ang II. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured and treated with Ang II (10-6 mol/l) for 24 h. The cells were then profiled for the expression of lncRNAs and mRNAs using the Arraystar Human lncRNA Expression Microarray V3.0. RESULTS: In HUVECs following Ang II treatment, from a total of 30 584 lncRNA targets screened, 25 targets were significantly upregulated, while 69 were downregulated. In the same HUVECs samples, from 26 106 mRNA targets screened, 28 targets were significantly upregulated and 67 were downregulated. Of the differentially expressed lncRNAs, RP11-354P11.2 and RP11-360F5.1 were the most upregulated (11-fold) and downregulated (three-fold) lncRNAs, respectively. Assigning the differentially regulated genes into functional groups using bioinformatics reveals numerous genes involved in the nucleotide excision repair and ECM-receptor interaction. CONCLUSION: This is the first study to profile the Ang II-induced differentially expressed lncRNAs and mRNAs in human endothelial cells. Our results reveal novel targets and substantially extend the list of potential candidate genes involved in Ang II-induced endothelial dysfunction and cardiovascular diseases.

Asunto(s)

Enfermedades Cardiovasculares , ARN Largo no Codificante , Angiotensina II/metabolismo , Angiotensina II/farmacología , Enfermedades Cardiovasculares/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcriptoma

RESUMEN

The emergence of multidrug-resistant (MDR) Gram-negative pathogens is an urgent global medical challenge. The old polymyxin lipopeptide antibiotics (polymyxin B and colistin) are often the only therapeutic option due to resistance to all other classes of antibiotics and the lean antibiotic drug development pipeline. However, polymyxin B and colistin suffer from major issues in safety (dose-limiting nephrotoxicity, acute toxicity), pharmacokinetics (poor exposure in the lungs) and efficacy (negligible activity against pulmonary infections) that have severely limited their clinical utility. Here we employ chemical biology to systematically optimize multiple non-conserved positions in the polymyxin scaffold, and successfully disconnect the therapeutic efficacy from the toxicity to develop a new synthetic lipopeptide, structurally and pharmacologically distinct from polymyxin B and colistin. This resulted in the clinical candidate F365 (QPX9003) with superior safety and efficacy against lung infections caused by top-priority MDR pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.

Asunto(s)

Colistina , Polimixina B , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Colistina/farmacología , Farmacorresistencia Bacteriana Múltiple , Lipopéptidos/farmacología , Lipopéptidos/uso terapéutico , Pruebas de Sensibilidad Microbiana , Polimixinas/farmacología , Polimixinas/uso terapéutico , Pseudomonas aeruginosa

RESUMEN

BACKGROUND: Androgen deprivation therapy (ADT), either by medical or surgical castration, is the backbone for standard treatment of locally advanced or metastatic prostate cancer, yet it is also associated with various metabolic and cardiovascular complications. Recent evidence have shown that obesity, insulin resistance, or metabolic disturbances can be associated with changes in the gut microbiome, while animal studies also show that castration is associated with changes in the gut microbiome. This study aims to investigate whether the fecal microbiota in prostate cancer patients who had undergone prostatectomy or ADT are different, and explore changes in phylogeny and pathways that may lead to side effects from ADT. METHODS: A total of 86 prostate cancer patients (56 patients on ADT and 30 patients with prostatectomy) were recruited. The fecal microbiota was analyzed by the 16S rRNA gene for alpha- and beta-diversities by QIIME2, as well as the predicted metabolic pathways by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2. RESULTS: The alpha-diversity was significantly lower in the ADT group. The beta-diversity was significantly different between the groups, in which Ruminococcus gnavus and Bacteroides spp were having higher relative abundance in the ADT group, whereas Lachnospira and Roseburia were reduced. The Firmicutes-to-Bacteroidetes ratio is noted to be lower in the ADT group as well. The functional pathway prediction showed that the biosynthesis of lipopolysaccharide (endotoxin) and propanoate was enriched in the ADT as well as the energy cycle pathways. This study is limited by the cross-sectional design and the clinical heterogeneity. CONCLUSIONS: There is a significant difference in gut microbiome between prostate cancer patients on ADT and prostatectomy. We theorize that this difference may contribute to the development of metabolic complications from ADT. Further longitudinal studies are awaited.

Asunto(s)

Antagonistas de Andrógenos/uso terapéutico , Heces/microbiología , Microbioma Gastrointestinal , Prostatectomía , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/cirugía , Anciano , Técnicas de Tipificación Bacteriana , Estudios Transversales , Humanos , Masculino

RESUMEN

Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and nonhuman primates naturally controlling HIV/SIV infection express more of the transcription factor TCF-1 than noncontrollers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and expansion capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 contributes to the regulation of the stem-like memory property of secondary expansion capacity of HIV-specific CD8+ T cells, and they provide a rationale for exploring the enhancement of this pathway in T cell-based therapeutic strategies for HIV.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Factor 1 de Transcripción de Linfocitos T/inmunología , Adulto , Anciano , Animales , Femenino , Técnicas de Inactivación de Genes , Antígenos VIH/genética , Antígenos VIH/inmunología , VIH-1/genética , Humanos , Memoria Inmunológica , Macaca mulatta , Masculino , Persona de Mediana Edad , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/inmunología , Factor 1 de Transcripción de Linfocitos T/antagonistas & inhibidores , Factor 1 de Transcripción de Linfocitos T/genética , Carga Viral/inmunología

RESUMEN

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

Asunto(s)

Neoplasias del Colon/patología , Células Mieloides/metabolismo , Análisis de la Célula Individual/métodos , Adulto , Anciano , Anciano de 80 o más Años , Animales , Secuencia de Bases/genética , Linfocitos T CD8-positivos/inmunología , China , Neoplasias del Colon/terapia , Neoplasias Colorrectales/patología , Células Dendríticas/inmunología , Femenino , Humanos , Inmunoterapia , Macrófagos/inmunología , Masculino , Ratones , Persona de Mediana Edad , Análisis de Secuencia de ARN/métodos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología

RESUMEN

The lateral septum (LS) plays an important role in regulating aggression. It is well recognized that LS lesions lead to a dramatic increase in aggressive behaviors. A better understanding of LS neurophysiology and its functional output is therefore important to assess LS involvement in regulating aggression. The LS is a heterogeneous structure that maintains inputs and outputs with multiple brain regions, and is also divided into subregions that innervate one another. Thus, it is challenging to identify the exact cell type and projections for characterization. In this study, we determined the expression pattern of the calcium-activated chloride channel, TMEM16B, in the LS of both male and female mice. We then investigated the physiological contribution of the calcium-activated chloride channel to LS neuronal signaling. By performing whole-cell patch-clamp recording, we showed that TMEM16B alters neurotransmitter release at the hippocampal-LS synapse, and regulates spike frequency and spike frequency adaptation in subpopulations of LS neurons. We further demonstrated that loss of TMEM16B function promotes lengthened displays of aggressive behaviors by male mice during the resident intruder paradigm. In conclusion, our findings suggest that TMEM16B function contributes to neuronal excitability in subpopulations of LS neurons and the regulation of aggression in male mice.SIGNIFICANCE STATEMENT Aggression is a behavior that arose evolutionarily from the necessity to compete for limited resources and survival. One particular brain region involved in aggression is the lateral septum (LS). In this study, we characterized the expression of the TMEM16B calcium-activated chloride channel in the LS and showed that TMEM16B regulates the action potential firing frequency of LS neurons. We discovered that loss of TMEM16B function lengthens the displays of aggressive behaviors in male mice. These findings suggest that TMEM16B plays an important role in regulating LS neuronal excitability and behaviors associated with LS function, thereby contributing to our understanding of how the LS may regulate aggression.

Asunto(s)

Potenciales de Acción , Agresión , Anoctaminas/metabolismo , Núcleos Septales/fisiología , Animales , Anoctaminas/genética , Femenino , Hipocampo/citología , Hipocampo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/fisiología , Núcleos Septales/citología , Núcleos Septales/metabolismo , Factores Sexuales , Sinapsis/metabolismo , Sinapsis/fisiología , Potenciales Sinápticos

RESUMEN

Perturbations of ganglioside homeostasis have been observed following stroke whereby toxic simple gangliosides GM2 and GM3 accumulate, while protective complex species GM1 and GD1 are reduced. Thus, there is a need for therapeutic interventions which can prevent ganglioside dysregulation after stroke. A pharmacological intervention using chloroquine was selected for its transient lysosomotropic properties which disrupt the activity of catabolic ganglioside enzymes. Chloroquine was administered both in vitro (0.1 µM), to primary cortical neurons exposed to GM3 toxicity, and in vivo (45 mg/kg i.p.), to 3-month-old male Wistar rats that underwent a severe stroke injury. Chloroquine was administered for seven consecutive days beginning 3 days prior to the stroke injury. Gangliosides were examined using MALDI imaging mass spectrometry at 3 and 21 days after the injury, and motor deficits were examined using the ladder task. Chloroquine treatment prevented ganglioside dysregulation 3 days post-stroke and partially prevented complex ganglioside depletion 21 days post-stroke. Exogenous GM3 was found to be toxic to primary cortical neurons which was protected by chloroquine treatment. Motor deficits were prevented in the forelimbs of stroke-injured rats with chloroquine treatment and was associated with decreased inflammation, neurodegeneration, and an increase in cell survival at the site of injury. Chloroquine administration prevents ganglioside dysregulation acutely, protects against GM3 toxicity in neurons, and is associated with long-term functional and pathological improvements after stroke in the rat. Therefore, targeting lipid dysregulation using lysosomotropic agents such as chloroquine may represent a novel therapeutic avenue for stroke injuries.

Asunto(s)

Conducta Animal , Cloroquina/farmacología , Gangliósidos/metabolismo , Homeostasis/efectos de los fármacos , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Animales , Conducta Animal/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Miembro Anterior/patología , Miembro Anterior/fisiopatología , Masculino , Actividad Motora/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Ratas Wistar , Accidente Cerebrovascular/fisiopatología

RESUMEN

Orexin (also known as hypocretin) neurons in the hypothalamus play an essential role in sleep-wake control, feeding, reward, and energy homeostasis. The likelihood of anesthesia and sleep sharing common pathways notwithstanding, it is important to understand the processes underlying emergence from anesthesia. In this study, we investigated the role of the orexin system in anesthesia emergence, by specifically activating orexin neurons utilizing the designer receptors exclusively activated by designer drugs (DREADD) chemogenetic approach. With injection of adeno-associated virus into the orexin-Cre transgenic mouse brain, we expressed the DREADD receptor hM3Dq specifically in orexin neurons and applied the hM3Dq ligand clozapine to activate orexin neurons. We monitored orexin neuronal activities by c-Fos staining and whole-cell patch-clamp recording and examined the consequence of orexin neuronal activation via EEG recording. Our results revealed that the orexin-DREADD mice with activated orexin neurons emerged from anesthesia with significantly shorter latency than the control mice. As an indication of reduced pain sensitivity, these orexin-DREADD mice took longer to respond to the 55 °C thermal stimuli in the hot plate test and exhibited significantly less frequent licking of the formalin-injected paw in the formalin test. Our study suggests that approaches to activate the orexin system can be beneficial in postoperative recovery.

Asunto(s)

Periodo de Recuperación de la Anestesia , Hipotálamo/metabolismo , Neuronas/metabolismo , Receptores de Orexina/genética , Orexinas/genética , Dolor/genética , Anestésicos por Inhalación , Animales , Clozapina/farmacología , Dependovirus/genética , Dependovirus/metabolismo , Electroencefalografía , Regulación de la Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Calor , Hipotálamo/efectos de los fármacos , Hipotálamo/fisiopatología , Isoflurano , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/patología , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Dolor/fisiopatología , Dolor/prevención & control , Dimensión del Dolor , Técnicas de Placa-Clamp , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Antagonistas de la Serotonina/farmacología , Técnicas Estereotáxicas

RESUMEN

Three decades ago, the Garlands postulated that vitamin D3 produced in the skin by ultraviolet radiation (UVR)-induced conversion of 7-dehydrocholesterol to pre-D3 has anticancer effects, thus triggering more than 9,500 publications on D3 and cancer. Here, we report that UVR treatment of transgenic mice of the well-established C3(1)/SV40 Tag mammary cancer model significantly inhibits both autochthonous carcinogenesis and allograft tumor growth, but in contrast neither dietary nor topical D3 influences mammary carcinogenesis in this specific mouse model. Furthermore, UVR's inhibitory effects occur irrespective of whether or not the treatment increases circulating D3 in the mice. The inhibitory effect of UVR on autochthonous tumors occurs at or before the stage of ductal carcinoma in situ. Our studies indicate clearly that UVR can exert D3-independent anticancer effects in C3(1)/SV40 Tag mice. Therefore, supplemental D3 may not mimic all possible beneficial effects of UVR, and uncovering non-D3-mediated mechanisms of UVR tumor inhibition may lead to novel strategies for cancer prevention. Cancer Prev Res; 11(7); 383-92. ©2018 AACR.

Asunto(s)

Carcinogénesis/efectos de la radiación , Carcinoma Intraductal no Infiltrante/prevención & control , Neoplasias Mamarias Experimentales/prevención & control , Receptores de Estrógenos/metabolismo , Rayos Ultravioleta , Animales , Carcinoma Intraductal no Infiltrante/patología , Línea Celular Tumoral/trasplante , Colecalciferol/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Piel/metabolismo , Piel/efectos de la radiación

RESUMEN

Calcium-activated chloride channels (CaCCs) formed by TMEM16A or TMEM16B are broadly expressed in the nervous system, smooth muscles, exocrine glands, and other tissues. With two calcium-binding sites and a pore within each monomer, the dimeric CaCC exhibits voltage-dependent calcium sensitivity. Channel activity also depends on the identity of permeant anions. To understand how CaCC regulates neuronal signaling and how CaCC is, in turn, modulated by neuronal activity, we examined the molecular basis of CaCC gating. Here, we report that voltage modulation of TMEM16A-CaCC involves voltage-dependent occupancy of calcium- and anion-binding site(s) within the membrane electric field as well as a voltage-dependent conformational change intrinsic to the channel protein. These gating modalities all critically depend on the sixth transmembrane segment.

Asunto(s)

Anoctamina-1/química , Anoctamina-1/metabolismo , Canales de Cloruro/química , Canales de Cloruro/metabolismo , Activación del Canal Iónico/fisiología , Secuencia de Aminoácidos , Animales , Anoctamina-1/genética , Canales de Cloruro/genética , Células HEK293 , Humanos , Ratones , Unión Proteica/fisiología , Estructura Secundaria de Proteína

RESUMEN

Neurons within different brain regions have varying levels of vulnerability to external stress and respond differently to injury. A potential reason to explain this may lie within a key lipid class of the cell's plasma membrane called gangliosides. These glycosphingolipid species have been shown to play various roles in the maintenance of neuronal viability. The purpose of this study is to use electrospray ionization mass spectrometry (ESI-MS) and immunohistochemistry to evaluate the temporal expression profiles of gangliosides during the course of neurodegeneration in rat primary cortical neurons exposed to glutamate toxicity. Primary embryonic (E18) rat cortical neurons were cultured to DIV (days in vitro) 14. Glutamate toxicity was induced for 1, 3, 6, and 24 h to injure and kill neurons. Immunofluorescence was used to stain for GM1 and GM3 species, and ESI-MS was used to quantify the ganglioside species expressed within these injured neurons. ESI-MS data revealed that GM1, GM2, and GM3 were up-regulated in neurons exposed to glutamate. Interestingly, using immunofluorescence, we demonstrated that the GM1 increase following glutamate exposure occurred in viable neurons, possibly indicating a potential intrinsic neuroprotective response. To test this potential neuroprotective property, neurons were pretreated with GM1 for 24 h prior to glutamate exposure. Pretreatment with GM1 conferred significant neuroprotection against glutamate-induced cell death. Overall, work from this study validates the use of ESI-MS for cell-derived gangliosides and supports the further development of lipid based strategies to protect against neuron cell death.

Asunto(s)

Gangliósidos/análisis , Ácido Glutámico/toxicidad , Neuronas/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Embrión de Mamíferos/metabolismo , Gangliósidos/aislamiento & purificación , Gangliósidos/farmacología , Microscopía Fluorescente , Neuronas/citología , Neuronas/metabolismo , Ratas , Extracción en Fase Sólida , Esfingosina/química

RESUMEN

Chronic inflammation is associated with immunosuppression and downregulated expression of the TCR CD247. In searching for new biomarkers that could validate the impaired host immune status under chronic inflammatory conditions, we discovered that sorting nexin 9 (SNX9), a protein that participates in early stages of clathrin-mediated endocytosis, is downregulated as well under such conditions. SNX9 expression was affected earlier than CD247 by the generated harmful environment, suggesting that it is a potential marker sensing the generated immunosuppressive condition. We found that myeloid-derived suppressor cells, which are elevated in the course of chronic inflammation, are responsible for the observed SNX9 reduced expression. Moreover, SNX9 downregulation is reversible, as its expression levels return to normal and immune functions are restored when the inflammatory response and/or myeloid-derived suppressor cells are neutralized. SNX9 downregulation was detected in numerous mouse models for pathologies characterized by chronic inflammation such as chronic infection (Leishmania donovani), cancer (melanoma and colorectal carcinoma), and an autoimmune disease (rheumatoid arthritis). Interestingly, reduced levels of SNX9 were also observed in blood samples from colorectal cancer patients, emphasizing the feasibility of its use as a diagnostic and prognostic biomarker sensing the host's immune status and inflammatory stage. Our new discovery of SNX9 as being regulated by chronic inflammation and its association with immunosuppression, in addition to the CD247 regulation under such conditions, show the global impact of chronic inflammation and the generated immune environment on different cellular pathways in a diverse spectrum of diseases.

Asunto(s)

Complejo CD3/biosíntesis , Huésped Inmunocomprometido/inmunología , Inflamación/inmunología , Células Mieloides/inmunología , Nexinas de Clasificación/biosíntesis , Animales , Artritis Reumatoide/inmunología , Biomarcadores de Tumor/sangre , Proliferación Celular , Células Cultivadas , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Inflamación/patología , Leishmania donovani/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/parasitología , Masculino , Melanoma/diagnóstico , Melanoma/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Nexinas de Clasificación/sangre , Células TH1/inmunología , Células Th2/inmunología

RESUMEN

PURPOSE: We aimed to determine differentially expressed genes relevant to orbital inflammation and orbital fat expansion in thyroid orbitopathy (TO) using microarray gene profiling in a case-control study. METHODS: Human orbital adipose samples were obtained from individuals with active TO (n = 12), inactive TO (n = 21), and normal controls (n = 21). Gene expression profiles were examined using microarray analysis and were compared between active and inactive TO, and between active TO and normal controls. Top ranked differentially expressed genes were validated by real-time RT-PCR in an additional eight active TO, 13 inactive TO, and 11 normal controls and correlated with gene set enrichment analysis (GSEA) and molecular pathways analysis. RESULTS: Seven hundred twenty-one probes (683 genes) and 806 probes (735 genes) were significantly differentially expressed in comparing active to inactive TO and in comparing active TO to healthy controls, respectively. All selected genes were confirmed to be differentially expressed by real-time RT-PCR. Multiple top ranked genes in active versus inactive TO comparison are overrepresented by immune and inflammatory response genes. They include defensins (DEFA1, DEFA1B, DEFA3), which were overexpressed by 3.05- to 4.14-fold and TIMD4 by 4.20-fold. Markers for adipogenesis were overexpressed including SCD, FADS1, and SCDP1. Gene set enrichment analysis revealed dysregulation of epigenetic signatures, T-cell activation, Th1 differentiation, defensin pathway, cell adhesion, cytoskeleton organization, apoptosis, cell cycling, and lipid metabolism in active TO. CONCLUSIONS: Active TO is characterized by upregulation of genes involved in cell-mediated immune, innate immune, and inflammatory response and enhanced orbital adipogenesis. TIMD4, DEFA1, DEFA1B, and DEFA3 genes may be involved in the innate immune-mediated orbital inflammation in TO. Epigenetic mechanisms may play a role in the pathogenesis of TO.

Asunto(s)

Adipogénesis/genética , Tejido Adiposo/metabolismo , Defensinas/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Oftalmopatía de Graves/genética , ARN/genética , Tejido Adiposo/patología , Defensinas/biosíntesis , delta-5 Desaturasa de Ácido Graso , Oftalmopatía de Graves/metabolismo , Oftalmopatía de Graves/patología , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

INTRODUCTION: Sexual health care remains an unmet need for women with cancer. Many barriers are described, such as provider discomfort and lack of training; however, there is little evidence-based guidance regarding how to effectively address these obstacles. AIM: This pilot study was performed to determine whether brief, targeted sexual health training for oncology providers results in improved provider comfort level and frequency of addressing female cancer-related sexual issues. METHODS: A brief (30-45 minute), targeted sexual health training program focused on improving comfort level, knowledge and communication skills when addressing breast cancer-related sexual issues was developed by the primary author. Using a pretest-posttest format, this educational program was provided to oncology providers (physicians and nurses/other allied health) from a suburban health-care system. Surveys based on 5-point Likert scales were provided before and 3-6 month post training. MAIN OUTCOME MEASURES: Primary endpoints were changes in mean Likert scores for provider comfort level and self-reported frequency of addressing sexual issues. A secondary endpoint was change in mean Likert scores for perception of access to sexual health resources/referrals. RESULTS: Eligible respondents included 8 oncologists, 4 surgeons, and 62 nurses/other allied health. For total respondents, comparison of mean Likert scores for survey 1 (n = 71) and survey 2 (n = 36) demonstrated statistically significant increases for all parameters queried, including provider comfort level with bringing up (Pre mean Likert score = 3.4, Post = 4.3, P < 0.0001) and coordinating care (Pre = 3.5, Post = 4.6, P < 0.0001), and frequency of addressing sexual issues for both diagnosis/treatment and surveillance phase (Pre = 2.4, Post = 3.3, P ≤ 0.0052). CONCLUSION: Brief, targeted sexual health training for oncology providers positively correlated with improved provider comfort level and frequency of addressing female cancer-related sexual issues.

RESUMEN

BACKGROUND: Physicians in China face heavy demands from patients and the government for services but deal with the threat of unpredictable legal and physical conflicts with patients, some ending with the death of doctors. More than 40 doctors and nurses have been killed by patients since 2001. QUESTIONS/PURPOSES: We sought to evaluate (1) the demographics of orthopaedic practice, (2) duty periods, (3) practice support, and (4) job satisfaction among orthopaedic surgeons in China. METHODS: Questionnaires were posted online at www.OrthoChina.org for download by orthopaedic surgeons in 2006 to 2007, and sent to those attending meetings in 2013. In 2013, a total of 1350 surgeons were invited and 456 participated in the survey at meetings. In 2007, during the period of the survey, 9759 individuals were qualified orthopaedic surgeons, and 334 participated in the survey at www.OrthoChina.org . RESULTS: Ninety-one percent of orthopaedic surgeons work in public and 9% in private hospitals. Ninety-four percent work more than 8 hours per day 6 to 7 days a week. Twenty-five percent work more than 12 hours per day 6 to 7 days a week without extra compensation. The majority of orthopaedic surgeons must work on national statutory holidays. Almost none received contractually mandated income for weekends and national holidays. Approximately 80% of participants reported an attack of some kind, including physical or psychologic harm. With respect to job satisfaction, 73% stated they would not choose to be a physician again and 86% reported that they do not want their children to become a physician. CONCLUSIONS: China's rapid economic growth and resulting demands for modern health care have resulted in heavy pressure on orthopaedic surgeons, financially and personally. Chinese orthopaedic surgeons are overworked, suffer lack of respect, and face the possibility of serious personal harm. As a consequence, they are demoralized and unsatisfied. Significant reforms are needed.

Asunto(s)

Monitoreo del Ambiente/estadística & datos numéricos , Satisfacción en el Trabajo , Ortopedia/organización & administración , Ortopedia/estadística & datos numéricos , Pautas de la Práctica en Medicina/organización & administración , Pautas de la Práctica en Medicina/estadística & datos numéricos , Medio Social , Adulto , Agresión , China , Disentimientos y Disputas , Femenino , Humanos , Masculino , Relaciones Médico-Paciente , Vigilancia de la Población , Encuestas y Cuestionarios , Carga de Trabajo/estadística & datos numéricos , Lugar de Trabajo/estadística & datos numéricos , Adulto Joven

RESUMEN

TCR-mediated activation induces receptor microclusters that evolve to a defined immune synapse (IS). Many studies showed that actin polymerization and remodeling, which create a scaffold critical to IS formation and stabilization, are TCR mediated. However, the mechanisms controlling simultaneous TCR and actin dynamic rearrangement in the IS are yet not fully understood. Herein, we identify two novel TCR ζ-chain motifs, mediating the TCR's direct interaction with actin and inducing actin bundling. While T cells expressing the ζ-chain mutated in these motifs lack cytoskeleton (actin) associated (cska)-TCRs, they express normal levels of non-cska and surface TCRs as cells expressing wild-type ζ-chain. However, such mutant cells are unable to display activation-dependent TCR clustering, IS formation, expression of CD25/CD69 activation markers, or produce/secrete cytokine, effects also seen in the corresponding APCs. We are the first to show a direct TCR-actin linkage, providing the missing gap linking between TCR-mediated Ag recognition, specific cytoskeleton orientation toward the T-cell-APC interacting pole and long-lived IS maintenance.

Asunto(s)

Citoesqueleto/metabolismo , Sinapsis Inmunológicas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Actinas/metabolismo , Secuencias de Aminoácidos/genética , Animales , Células Cultivadas , Citocinas/metabolismo , Femenino , Sinapsis Inmunológicas/inmunología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Mutantes , Mutación/genética , Agregación de Receptores/genética , Receptores de Antígenos de Linfocitos T/genética

RESUMEN

The mammalian neocortex undergoes dramatic transformation during development, from a seemingly homogenous sheet of neuroepithelial cells into a complex structure that is tangentially divided into discrete areas. This process is thought to be controlled by a combination of intrinsic patterning mechanisms within the cortex and afferent axonal projections from the thalamus. However, roles of thalamic afferents in the formation of areas are still poorly understood. In this study, we show that genetically increasing or decreasing the size of the lateral geniculate nucleus of the mouse thalamus resulted in a corresponding change in the size of the primary visual area. Furthermore, elimination of most thalamocortical projections from the outset of their development resulted in altered areal gene expression patterns, particularly in the primary visual and somatosensory areas, where they lost sharp boundaries with adjacent areas. Together, these results demonstrate the critical roles of thalamic afferents in the establishment of neocortical areas.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica/fisiología , Neocórtex/embriología , Neocórtex/crecimiento & desarrollo , Tálamo/fisiología , Vías Aferentes/fisiología , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Recuento de Células , Tamaño de la Célula , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Hedgehog/genética , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Mutación/genética , Neocórtex/metabolismo , Proteínas/genética , ARN Mensajero/metabolismo , ARN no Traducido

RESUMEN

BACKGROUND: The size and cell number of each brain region are influenced by the organization and behavior of neural progenitor cells during embryonic development. Recent studies on developing neocortex have revealed the presence of neural progenitor cells that divide away from the ventricular surface and undergo symmetric divisions to generate either two neurons or two progenitor cells. These 'basal' progenitor cells form the subventricular zone and are responsible for generating the majority of neocortical neurons. However, not much has been studied on similar types of progenitor cells in other brain regions. RESULTS: We have identified and characterized basal progenitor cells in the embryonic mouse thalamus. The progenitor domain that generates all of the cortex-projecting thalamic nuclei contained a remarkably high proportion of basally dividing cells. Fewer basal progenitor cells were found in other progenitor domains that generate non-cortex projecting nuclei. By using intracellular domain of Notch1 (NICD) as a marker for radial glial cells, we found that basally dividing cells extended outside the lateral limit of radial glial cells, indicating that, similar to the neocortex and ventral telencephalon, the thalamus has a distinct subventricular zone. Neocortical and thalamic basal progenitor cells shared expression of some molecular markers, including Insm1, Neurog1, Neurog2 and NeuroD1. Additionally, basal progenitor cells in each region also expressed exclusive markers, such as Tbr2 in the neocortex and Olig2 and Olig3 in the thalamus. In Neurog1/Neurog2 double mutant mice, the number of basally dividing progenitor cells in the thalamus was significantly reduced, which demonstrates the roles of neurogenins in the generation and/or maintenance of basal progenitor cells. In Pax6 mutant mice, the part of the thalamus that showed reduced Neurog1/2 expression also had reduced basal mitosis. CONCLUSIONS: Our current study establishes the existence of a unique and significant population of basal progenitor cells in the thalamus and their dependence on neurogenins and Pax6. These progenitor cells may have important roles in enhancing the generation of neurons within the thalamus and may also be critical for generating neuronal diversity in this complex brain region.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas del Ojo/fisiología , Proteínas de Homeodominio/fisiología , Células-Madre Neurales/fisiología , Factores de Transcripción Paired Box/fisiología , Proteínas Represoras/fisiología , Tálamo/citología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteínas del Ojo/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Masculino , Ratones , Ratones Transgénicos , Neocórtex/citología , Neocórtex/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Embarazo , Proteínas Represoras/genética , Tálamo/embriología , Tálamo/metabolismo , Factores de Transcripción/metabolismo