RESUMEN
This study comprehensively evaluated the association between known circulating tumor necrosis factor (TNF) superfamily ligands and receptors and the development of early progressive kidney decline (PKD) leading to end-stage kidney disease (ESKD) in Type 1 diabetes. Participants for the study were from the Macro-Albuminuria Study (198 individuals), and the Micro-Albuminuria Study (148 individuals) of the Joslin Kidney Study. All individuals initially had normal kidney function and were followed for seven-fifteen years to determine the slope of the estimate glomerular filtration rate and to ascertain onset of ESKD. Plasma concentrations of 25 TNF superfamily proteins were measured using proximity extension assay applied in the OLINK proteomics platform. In the both studies risk of early PKD, determined as estimated glomerular filtration rate loss greater than or equal to three ml/min/1.73m2/year, was associated with elevated circulating levels of 13 of 19 TNF receptors examined. In the Macro-Albuminuria Study, we obtained similar findings for risk of progression to ESKD. These receptors comprised: TNF-R1A, -R1B, -R3, -R4, -R6, -R6B, -R7, -R10A, -R10B, -R11A, -R14, -R21, and -R27. Serial measurements showed that circulating levels of these TNF receptors had increased before the onset of PKD. In contrast, none of the six measured TNF ligands showed association with risk of early PKD. Of significance, the disease process that underlies PKD leading to ESKD in Type 1 diabetes has a profile also seen in autoimmune disorders. The mechanisms of this enrichment may be causally related to the development of PKD in Type 1 diabetes and must be investigated further. Thus, some of these receptors may be used as new risk predictors of ESKD.
Asunto(s)
Diabetes Mellitus Tipo 1 , Nefropatías Diabéticas , Albuminuria , Diabetes Mellitus Tipo 1/complicaciones , Nefropatías Diabéticas/diagnóstico , Nefropatías Diabéticas/etiología , Progresión de la Enfermedad , Tasa de Filtración Glomerular , Humanos , Riñón , Receptores del Factor de Necrosis Tumoral , Factores de RiesgoRESUMEN
BACKGROUND: People with human immunodeficiency virus (PWH) demonstrate increased atherosclerotic cardiovascular disease (ASCVD). Statins are being studied to prevent ASCVD in human immunodeficiency virus (HIV), but little is known regarding the effects of statins on a broad range of inflammatory and cardiovascular proteins in this population. METHODS: We used a highly specific discovery proteomic approach (Protein Extension Assay), to determine statin effects on over 350 plasma proteins in relevant ASCVD pathways among HIV and non-HIV groups. Responses to pitavastatin calcium were assessed in 89 PWH in the INTREPID trial and 46 non-HIV participants with features of central adiposity and insulin resistance. History of cardiovascular disease was exclusionary for both studies. RESULTS: Among participants with HIV, PCOLCE (enzymatic cleavage of type I procollagen) significantly increased after pitavastatin therapy and PLA2G7 (systemic marker of arterial inflammation) decreased. Among participants without HIV, integrin subunit alpha M (integrin adhesive function) and defensin alpha-1 (neutrophil function) increased after pitavastatin therapy and PLA2G7 decreased. At baseline, comparing participants with and without HIV, differentially expressed proteins included proteins involved in platelet and endothelial function and immune activation. CONCLUSIONS: Pitavastatin affected proteins important to platelet and endothelial function and immune activation, and effects differed to a degree within PWH and participants without HIV.
Asunto(s)
Biomarcadores/sangre , Proteínas Sanguíneas , Infecciones por VIH/sangre , Infecciones por VIH/virología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Proteoma , Proteómica , Anciano , Anciano de 80 o más Años , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/prevención & control , Estudios de Casos y Controles , Femenino , Infecciones por VIH/complicaciones , Infecciones por VIH/inmunología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Masculino , Persona de Mediana Edad , Proteómica/métodos , Proyectos de Investigación , Carga ViralRESUMEN
The hematopoietic stem cell (HSC) niche provides essential microenvironmental cues for the production and maintenance of HSCs within the bone marrow. During inflammation, hematopoietic dynamics are perturbed, but it is not known whether changes to the HSC-niche interaction occur as a result. We visualize HSCs directly in vivo, enabling detailed analysis of the 3D niche dynamics and migration patterns in murine bone marrow following Trichinella spiralis infection. Spatial statistical analysis of these HSC trajectories reveals two distinct modes of HSC behavior: (a) a pattern of revisiting previously explored space and (b) a pattern of exploring new space. Whereas HSCs from control donors predominantly follow pattern (a), those from infected mice adopt both strategies. Using detailed computational analyses of cell migration tracks and life-history theory, we show that the increased motility of HSCs following infection can, perhaps counterintuitively, enable mice to cope better in deteriorating HSC-niche microenvironments following infection. Stem Cells 2017;35:2292-2304.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Infecciones/genética , Animales , Movimiento Celular , Células Madre Hematopoyéticas/citología , Ratones , Modelos Teóricos , FenotipoRESUMEN
Mass cytometry (CyTOF), a mass spectrometry-based single cell phenotyping technology, allows utilization of over 35 antibodies in a single sample and is a promising tool for translational human immunology studies. Although several analysis tools are available to interpret the complex data sets generated, a robust method for standardization and quality control within and across studies is needed. Here we report an efficient and easily adaptable method to monitor quality of individual samples in human immunology studies and to facilitate reproducible data analysis. Samples to be assessed are spiked with a defined amount of reference peripheral blood mononuclear cells from a healthy donor, derived from a single large blood draw. The presence of known standardized numbers and phenotypic profiles of these reference cells greatly facilitates sample analysis by allowing for: 1) quality control for consistent staining of each antibody in the panel, 2) identification of potential batch effects, and 3) implementation of a robust gating strategy. We demonstrate the utility of this method using peripheral blood and bronchoalveolar lavage samples from HIV+ patients by characterizing their CD8+ T-cell phenotypes and cytokine expression, respectively. Our results indicate that this method allows quality control of experimental conditions and results in highly reproducible population frequencies through a robust gating strategy. © 2016 International Society for Advancement of Cytometry.
Asunto(s)
Citometría de Flujo/métodos , Espectrometría de Masas/métodos , Anticuerpos/metabolismo , Lavado Broncoalveolar/métodos , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Infecciones por VIH/metabolismo , Humanos , Inmunofenotipificación/métodos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Control de Calidad , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Hematopoietic stem cells (HSCs) maintain the turnover of mature blood cells during steady state and in response to systemic perturbations such as infections. Their function critically depends on complex signal exchanges with the bone marrow (BM) microenvironment in which they reside, but the cellular mechanisms involved in HSC-niche interactions and regulating HSC function in vivo remain elusive. We used a natural mouse parasite, Trichinella spiralis, and multipoint intravital time-lapse confocal microscopy of mouse calvarium BM to test whether HSC-niche interactions may change when hematopoiesis is perturbed. We find that steady-state HSCs stably engage confined niches in the BM whereas HSCs harvested during acute infection are motile and therefore interact with larger niches. These changes are accompanied by increased long-term repopulation ability and expression of CD44 and CXCR4. Administration of a CXCR4 antagonist affects the duration of HSC-niche interactions. These findings suggest that HSC-niche interactions may be modulated during infection.
Asunto(s)
Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Nicho de Células Madre/fisiología , Triquinelosis/metabolismo , Animales , Médula Ósea/inmunología , Médula Ósea/metabolismo , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Receptores de Hialuranos/inmunología , Receptores de Hialuranos/metabolismo , Ratones , Microscopía Confocal , Receptores CXCR4/inmunología , Receptores CXCR4/metabolismo , Imagen de Lapso de Tiempo , Trichinella spiralis , Triquinelosis/inmunologíaRESUMEN
Adult hematopoietic stem cells (HSCs) reside in the bone marrow in stable microenvironments known as the stem cell niche. One key component of the stem cell niche is cells of the osteoblastic lineage. Factors that are known to affect osteoblast activity, such as parathyroid hormone (PTH), have also been shown to affect the HSCs. Treatment of mice with PTH has led to beneficial effects on the HSC pool, which have led to clinical trials of PTH treatment to enhance HSC-based therapies.
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Células Madre Hematopoyéticas/citología , Hormona Paratiroidea/fisiología , Nicho de Células Madre/citología , Animales , Diferenciación Celular/fisiología , Células Madre Hematopoyéticas/fisiología , Humanos , Modelos Animales , Osteoblastos/citología , Osteoblastos/fisiología , Transducción de Señal/fisiología , Nicho de Células Madre/fisiología , Trasplante de Células MadreRESUMEN
For more than five years, high-dimensional mass cytometry has been employed to study immunology. However, these studies have typically been performed in one laboratory on one or few instruments. We present the results of a six-center study using healthy control human peripheral blood mononuclear cells (PBMCs) and commercially available reagents to test the intra-site and inter-site variation of mass cytometers and operators. We used prestained controls generated by the primary center as a reference to compare against samples stained at each individual center. Data were analyzed at the primary center, including investigating the effects of two normalization methods. All six sites performed similarly, with CVs for both Frequency of Parent and median signal intensity (MSI) values<30%. Increased background was seen when using the premixed antibody cocktail aliquots at each site, suggesting that cocktails are best made fresh. Both normalization methods tested performed adequately for normalizing MSI values between centers. Clustering algorithms revealed slight differences between the prestained and the sites-stained samples, due mostly to the increased background of a few antibodies. Therefore, we believe that multicenter mass cytometry assays are feasible.
Asunto(s)
Citometría de Flujo/métodos , Leucocitos Mononucleares/fisiología , Espectrometría de Masas/métodos , Anticuerpos/metabolismo , Voluntarios Sanos , Humanos , Inmunofenotipificación , Proyectos Piloto , Estándares de ReferenciaRESUMEN
Recent demonstrations of the anticonvulsant properties of agmatine suggest it may be considered as a potential adjunct for protection against seizure. We investigated the possibility of an additive anticonvulsant effect between low doses of agmatine and morphine. The thresholds for the clonic seizures induced by the intravenous administration of gamma-aminobutyric acid (GABA)-antagonist, pentylenetetrazole (PTZ) were assessed in mice. Morphine at lower doses (1-3mg/kg) increased and at higher doses (30, 60 mg/kg) decreased the seizure threshold. Pretreatment with a per se non-effective dose of agmatine (1mg/kg) potentiated the anticonvulsant effect of morphine. The combination of subeffective doses of agmatine and morphine led to potent anticonvulsant effects. The pro-convulsant effect of morphine was attenuated by agmatine. Yohimbine with a dose (1mg/kg) incapable of affecting seizure threshold reversed the effect of agmatine on both anticonvulsant and pro-convulsant effects of morphine. These results suggest that agmatine potentiates the anticonvulsant effect of morphine and alpha 2-adrenoceptors may be involved in this effect.
Asunto(s)
Agmatina/uso terapéutico , Anticonvulsivantes/uso terapéutico , Morfina/uso terapéutico , Receptores Adrenérgicos alfa 2/fisiología , Convulsiones/tratamiento farmacológico , Antagonistas Adrenérgicos alfa/farmacología , Análisis de Varianza , Animales , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Masculino , Ratones , Pentilenotetrazol , Convulsiones/inducido químicamente , Yohimbina/farmacologíaRESUMEN
Measuring three-dimensional (3D) localization of hematopoietic stem cells (HSCs) within the bone marrow microenvironment using intravital microscopy is a rapidly expanding research theme. This approach holds the key to understanding the detail of HSC-niche interactions, which are critical for appropriate stem cell function. Due to the complex tissue architecture of the bone marrow and to the progressive introduction of scattering and signal loss at increasing imaging depths, there is no ready-made software to handle efficient segmentation and unbiased analysis of the data. To address this, we developed an automated image analysis tool that simplifies and standardizes the biological interpretation of 3D HSC microenvironment images. The algorithm identifies HSCs and measures their localization relative to surrounding osteoblast cells and bone collagen. We demonstrate here the effectiveness, consistency, and accuracy of the proposed approach compared to current manual analysis and its wider applicability to analyze other 3D bone marrow components.
Asunto(s)
Médula Ósea/ultraestructura , Células Madre Hematopoyéticas/ultraestructura , Microscopía Intravital , Nicho de Células Madre , Humanos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
Lithium has been reported to inhibit opioid-induced properties. The present study examined the effect of acute and chronic administration of lithium chloride (LiCl) on morphine's biphasic modulation of susceptibility to pentylenetetrazole (PTZ)-induced clonic seizure in mice. We also examined the possible involvement of nitric oxide (NO) pathway in lithium effect. Both acute (0.1 and 1 mg/kg) and chronic (same doses, 21 consecutive days) administration of LiCl completely inhibited the anticonvulsant and proconvulsant effects of morphine (at doses 1 and 30 mg/kg, respectively). A very low and per se noneffective dose of LiCl (0.05 mg/kg) significantly inhibited both phases of morphine effect when administered concomitant with a noneffective low dose of naloxone (0.1 mg/kg). The NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME) at a per se noneffective dose of 0.3 mg/kg potentiated the inhibitory effects of low doses of LiCl (0.01 and 0.05 mg/kg) on both phases of morphine effect. l-arginine, a NO synthase substrate, at a per se noneffective dose of 30 mg/kg reversed the inhibitory effects of lithium (1 mg/kg). Lithium is capable of antagonizing both modulatory effects of morphine on seizure susceptibility even at relatively low doses. These inhibitory effects of lithium may also involve NO synthesis.
Asunto(s)
Cloruro de Litio/farmacología , Morfina/farmacología , Óxido Nítrico/metabolismo , Convulsiones/inducido químicamente , Convulsiones/metabolismo , Análisis de Varianza , Animales , Anticonvulsivantes/farmacología , Arginina/metabolismo , Convulsivantes/farmacología , Umbral Diferencial/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Masculino , Ratones , NG-Nitroarginina Metil Éster/farmacología , Antagonistas de Narcóticos , Pentilenotetrazol , Receptores Opioides/agonistas , Convulsiones/tratamiento farmacológico , Transducción de Señal/efectos de los fármacosRESUMEN
PURPOSE: To evaluate the effects of sex and estrus cycle on biphasic anticonvulsant and proconvulsant modulation of seizure threshold by morphine. METHODS: The threshold for the clonic seizures (CST) induced by acute intravenous administration of gamma-aminobutyric acid (GABA)-antagonist pentylenetetrazole (PTZ) was assessed in male and female mice. Estrus cycle was assessed by vaginal smears. The effect of removing circulating sex hormones was assessed by gonadectomy. RESULTS: At baseline, diestrus females had a higher CST compared with males and estrus females. Morphine at lower doses (0.5-3 mg/kg) had a significant anticonvulsant effect in males and estrus females compared with that in vehicle-treated controls, whereas female mice in diestrus phase showed a relative subsensitivity to this effect. Morphine at higher doses (30 and 60 mg/kg) significantly decreased CST in males and diestrus females, with less relative effect in estrus mice. In both phases, morphine exerted stronger effects in males compared with females. Ovariectomy brought the baseline CST to the male level and resulted in significant expression of both phases of morphine effect but did not abolish the sex difference in responsiveness to morphine. CONCLUSIONS: The biphasic modulation of seizure threshold is subject to both constitutive sex differences in sensitivity to morphine and hormonal fluctuations during the estrus cycle.