RESUMEN
There is a need to adapt cell bioassays to 384-well and 1536-well formats instead of the traditional 96-well format as high-throughput screening (HTS) demands increase. However, the sensitivity and performance of the bioassay must be re-verified in these higher micro-well plates, and verification of cell health must also be HT (high-throughput). We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances (EASs). This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay with which to normalize luciferase activity of cell lysates without requiring any transfer of the cell lysates. Here we demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays. Additionally, we demonstrate via the fluorescamine assay improved interpretation of luciferase activity in wells along the edge of the plate (the so-called "edge effect"), thereby increasing usable wells to the entire plate, not just interior wells.
Asunto(s)
Disruptores Endocrinos/toxicidad , Fluorescamina/análisis , Colorantes Fluorescentes/análisis , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Reporteros/efectos de los fármacos , Luciferasas/metabolismo , Activación Transcripcional/efectos de los fármacos , Antineoplásicos/farmacología , Bioensayo , Neoplasias de la Mama/inducido químicamente , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Sistema Libre de Células/efectos de los fármacos , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Femenino , Fluorescamina/química , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Antagonistas de Hormonas/farmacología , Humanos , Cinética , Límite de Detección , Luciferasas/genética , Proteínas Recombinantes/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Cardiac progenitor cells (CPCs) have emerged as potential therapy to improve cardiac repair and prevent damage in cardiac diseases. CPCs are a promising cell source for cardiac therapy as they can generate all cardiovascular lineages in vitro and in vivo. Originating from the heart itself, CPCs may be destined to activate endogenous repair mechanisms. These CPCs release paracrine molecules that are able to stimulate cardiac repair mechanisms, including stimulation of vessel formation and inhibition of cardiomyocyte apoptosis. In addition to proteins and growth factors, CPCs release extracellular membrane vesicles, such as exosomes, which have gained increasing interest in recent years. Exosomal-derived miRNAs have been indicated to play an important role in these processes. Hereby, CPC exosomes can be considered as potential off-the-shelf therapeutics, as they are able to stimulate the regenerative capacity of the heart by increasing vessel density and lowering apoptosis of cardiomyocytes.
Asunto(s)
Enfermedades Cardiovasculares/cirugía , Sistema Libre de Células/trasplante , Exosomas/trasplante , Miocardio/patología , Miocitos Cardíacos/patología , Regeneración , Células Madre/patología , Animales , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Exosomas/genética , Exosomas/metabolismo , Exosomas/patología , Regulación de la Expresión Génica , Humanos , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Comunicación Paracrina , Recuperación de la Función , Transducción de Señal , Células Madre/metabolismoRESUMEN
Cardiovascular diseases are the number one cause of death globally with an estimated 7.4 million people dying from coronary heart disease. Studies have been conducted to identify the therapeutic utility of exosomes in many diseases, including cardiovascular diseases. It has been demonstrated that exosomes are immune modulators, can be used to treat cardiac ischemic injury, pulmonary hypertension and many other diseases, including cancers. Exosomes can be used as a biomarker for disease and cell-free drug delivery system for targeting the cells. Many studies suggest that exosomes can be used as a cell-free vaccine for many diseases. In this chapter, we explore the possibility of future therapeutic potential of exosomes in various cardiovascular diseases.
Asunto(s)
Enfermedades Cardiovasculares/cirugía , Sistema Libre de Células/trasplante , Exosomas/trasplante , Miocardio/patología , Regeneración , Medicina Regenerativa/tendencias , Animales , Biomarcadores/metabolismo , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Enfermedades Cardiovasculares/fisiopatología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Difusión de Innovaciones , Portadores de Fármacos , Sistemas de Liberación de Medicamentos/tendencias , Exosomas/metabolismo , Exosomas/patología , Predicción , Terapia Genética/tendencias , Humanos , Miocardio/metabolismo , Recuperación de la FunciónRESUMEN
BACKGROUND: Pancreatic cancer has a 5-year survival rate of only 5-7%. Difficulties in detecting pancreatic cancer at early stages results in the high mortality and substantiates the need for additional diagnostic tools. Surgery is the only curative treatment and unfortunately only possible in localized tumours. A diagnostic biomarker for pancreatic cancer will have a major impact on patient survival by facilitating early detection and the possibility for curative treatment. DNA promoter hypermethylation is a mechanism of early carcinogenesis, which can cause inactivation of tumour suppressor genes. The aim of this study was to examine promoter hypermethylation in a panel of selected genes from cell-free DNA, as a diagnostic marker for pancreatic adenocarcinoma. METHODS: Patients with suspected or biopsy-verified pancreatic cancer were included prospectively and consecutively. Patients with chronic/acute pancreatitis were included as additional benign control groups. Based on an optimized accelerated bisulfite treatment protocol, methylation-specific PCR of a 28 gene panel was performed on plasma samples. A diagnostic prediction model was developed by multivariable logistic regression analysis using backward stepwise elimination. RESULTS: Patients with pancreatic adenocarcinoma (n = 95), chronic pancreatitis (n = 97) and acute pancreatitis (n = 59) and patients screened, but negative for pancreatic adenocarcinoma (n = 27), were included. The difference in mean number of methylated genes in the cancer group (8.41 (95% CI 7.62-9.20)) vs the total control group (4.74 (95% CI 4.40-5.08)) was highly significant (p < 0.001). A diagnostic prediction model (age >65, BMP3, RASSF1A, BNC1, MESTv2, TFPI2, APC, SFRP1 and SFRP2) had an area under the curve of 0.86 (sensitivity 76%, specificity 83%). The model performance was independent of cancer stage. CONCLUSIONS: Cell-free DNA promoter hypermethylation has the potential to be a diagnostic marker for pancreatic adenocarcinoma and differentiate between malignant and benign pancreatic disease. This study brings us closer to a clinical useful diagnostic marker for pancreatic cancer, which is urgently needed. External validation is, however, required before the test can be applied in the clinic. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02079363.
Asunto(s)
Adenocarcinoma/diagnóstico , Biomarcadores de Tumor/genética , Metilación de ADN , Neoplasias Pancreáticas/diagnóstico , Regiones Promotoras Genéticas , Adenocarcinoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Sistema Libre de Células/patología , Detección Precoz del Cáncer , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Modelos Teóricos , Neoplasias Pancreáticas/genética , Estudios ProspectivosRESUMEN
The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Bioprótesis , Células Progenitoras Endoteliales/inmunología , Glicoproteínas/inmunología , Prótesis Valvulares Cardíacas , Péptidos/inmunología , Válvula Pulmonar/citología , Antígeno AC133 , Línea Celular , Separación Celular/métodos , Sistema Libre de Células/inmunología , Sistema Libre de Células/patología , Humanos , Válvula Pulmonar/inmunologíaRESUMEN
Molecular biological analysis of nucleic acids in blood or other bodily fluids (i.e. liquid biopsy analyses) may supplement the pathologists' diagnostic armamentarium in a reasonable way-particularly in cancer precision medicine. Within the field of oncology, liquid biopsy can potentially be used to monitor tumor burden in the blood and to early detect emerging resistance in the course of targeted cancer therapies. An already approved application of liquid biopsy is the detection of epidermal growth factor receptor (EGFR) driver mutations in blood samples of lung cancer patients in those cases where no tissue biopsy is available. However, there is still currently considerable insecurity associated with blood-based DNA analytic methods that must be solved before liquid biopsy can be implemented for broader routine application in the diagnosis of cancer. In this article, the current state of development of liquid biopsy in molecular diagnostics from a pathology point of view is presented.
Asunto(s)
Biopsia/métodos , Sistema Libre de Células/patología , Análisis Citogenético/métodos , ADN de Neoplasias/análisis , Neoplasias/patología , Patología Molecular/métodos , Medicina de Precisión , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Humanos , Pulmón/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
It is proposed that an acellular natural osteochondral scaffold will provide a successful repair material for the early intervention treatment of cartilage lesions, to prevent or slow the progression of cartilage deterioration to osteoarthritis. Here, we investigated the efficacy of methods for the decellularisation of bovine osteochondral plugs. The plugs were subject to four freeze/thaw cycles followed by two cycles of washes in hypotonic solution and low concentration (0.1% w/v) sodium dodecyl sulphate with protease inhibitors. Plugs were treated with nuclease (DNase and RNase) treatment followed by sterilization in peracetic acid. Full tissue decellularisation was achieved as confirmed by histological analysis and DNA quantification, however the resultant acellular matrix had reduced glycosaminoglycan content which led to an increased percent deformation of cartilage. Furthermore, the acellular scaffold was not reproducibly biocompatible. Additional terminal washes were included in the process to improve biocompatibility, however, this led to visible structural damage to the cartilage. This damage was found to be minimised by reducing the cut edge to cartilage area ratio through decellularisation of larger cuts of osteochondral tissue.
Asunto(s)
Materiales Biocompatibles/síntesis química , Bioprótesis , Cartílago Articular/química , Cartílago Articular/crecimiento & desarrollo , Andamios del Tejido , Animales , Sistema Libre de Células/química , Sistema Libre de Células/patología , Fuerza Compresiva , Diseño de Equipo , Análisis de Falla de Equipo , Dureza , Ensayo de Materiales , PorcinosAsunto(s)
Sistema Libre de Células/patología , ADN de Neoplasias/genética , Técnicas de Diagnóstico Molecular , Neoplasias/genética , Neoplasias/terapia , Células Neoplásicas Circulantes/metabolismo , Medicina de Precisión/métodos , Biopsia , Humanos , Neoplasias/diagnóstico , Células Neoplásicas Circulantes/patología , Valor Predictivo de las Pruebas , Pronóstico , Factores de Riesgo , Carga TumoralRESUMEN
Small intestine submucosa (SIS) has emerged as one of a number of naturally derived extracellular matrix (ECM) biomaterials currently in clinical use. In addition to clinical applications, ECM materials form the basis for a variety of approaches within tissue engineering research. In our preliminary work it was found that SIS can be consistently and reliably made into tubular scaffolds which confer certain potential advantages. Given that decellularization protocols for SIS are applied to sheet-form SIS, it was hypothesized that a tubular-form SIS would behave differently to pre-existing protocols. In this work, tubular SIS was produced and decellularized by the conventional peracetic acid-agitation method, peracetic acid under perfusion along with two commonly used detergent-perfusion protocols. The aim of this was to produce a tubular SIS that was both adequately decellularized and possessing the mechanical properties which would make it a suitable scaffold for oesophageal tissue engineering, which was one of the goals of this work. Analysis was carried out via mechanical tensile testing, DNA quantification, scanning electron and light microscopy, and a metabolic assay, which was used to give an indication of the biocompatibility of each decellularization method. Both peracetic acid protocols were shown to be unsuitable methods with the agitation-protocol-produced SIS, which was poorly decellularized, and the perfusion protocol resulted in poor mechanical properties. Both detergent-based protocols produced well-decellularized SIS, with no adverse mechanical effects; however, one protocol emerged, SDS/Triton X-100, which proved superior in both respects. However, this SIS showed reduced metabolic activity, and this cytotoxic effect was attributed to residual reagents. Consequently, the use of SIS produced using the detergent SD as the decellularization agent was deemed to be the most suitable, although the elimination of the DNase enzyme would give further improvement.
Asunto(s)
Fraccionamiento Celular/instrumentación , Sistema Libre de Células/patología , Esófago/citología , Esófago/crecimiento & desarrollo , Mucosa Intestinal/citología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Bioprótesis , Fraccionamiento Celular/métodos , Sistema Libre de Células/trasplante , Análisis de Falla de Equipo , Mucosa Intestinal/trasplante , Intestino Delgado/citología , Intestino Delgado/trasplante , Diseño de Prótesis , Porcinos , Resistencia a la Tracción , Ingeniería de Tejidos/instrumentaciónRESUMEN
Uterus transplantation (UTx) may be the only possible curative treatment for absolute uterine factor infertility, which affects 1 in every 500 females of fertile age. We recently presented the 6-month results from the first clinical UTx trial, describing nine live-donor procedures. This routine involves complicated surgery and requires potentially harmful immune suppression to prevent rejection. However, tissue engineering applications using biomaterials and stem cells may replace the need for a live donor, and could prevent the required immunosuppressive treatment. To investigate the basic aspects of this, we developed a novel whole-uterus scaffold design for uterus tissue engineering experiments in the rat. Decellularization was achieved by perfusion of detergents and ionic solutions. The remaining matrix and its biochemical and mechanical properties were quantitatively compared from using three different protocols. The constructs were further compared with native uterus tissue composition. Perfusion with Triton X-100/dimethyl sulfoxide/H2O led to a compact, weaker scaffold that showed evidence of a compromised matrix organization. Sodium deoxycholate/dH2O perfusion gave rise to a porous scaffold that structurally and mechanically resembled native uterus better. An innovative combination of two proteomic analyses revealed higher fibronectin and versican content in these porous scaffolds, which may explain the improved scaffold organization. Together with other important protocol-dependent differences, our results can contribute to the development of improved decellularization protocols for assorted organs. Furthermore, our study shows the first available data on decellularized whole uterus, and creates new opportunities for numerous in vitro and in vivo whole-uterus tissue engineering applications.
Asunto(s)
Órganos Artificiales , Fraccionamiento Celular/instrumentación , Sistema Libre de Células/patología , Ingeniería de Tejidos/métodos , Andamios del Tejido , Útero/citología , Útero/crecimiento & desarrollo , Animales , Bioprótesis , Fraccionamiento Celular/métodos , Sistema Libre de Células/trasplante , Análisis de Falla de Equipo , Femenino , Diseño de Prótesis , Ratas , Ratas Endogámicas Lew , Ingeniería de Tejidos/instrumentación , Útero/trasplanteRESUMEN
In vivo studies of implanted acellular biological scaffolds in experimental animals have shown constructive remodeling mediated by anti-inflammatory macrophages. Little is known about the human macrophage response to such biomaterials, or the nature of the signaling mechanisms that govern the macrophage phenotype in this environment. The cellular events at the interface of a tissue and implanted decellularized biomaterial were examined by establishing a novel ex vivo tissue culture model in which surgically excised human urinary tract tissue was combined with porcine acellular bladder matrix (PABM). Evaluation of the tissue-biomaterial interface showed a time-dependent infiltration of the biomaterial by CD68(+) CD80(-) macrophages. The migration of CD68(+) cells from the tissue to the interface was accompanied by maturation to a CD163(hi) phenotype, suggesting that factor(s) associated with the biomaterial or the wound edge was/were responsible for the active recruitment and polarization of local macrophages. Glucocorticoid receptor (GR) and peroxisome proliferator activated receptor gamma (PPARγ) signaling was investigated as candidate pathways for integrating inflammatory responses; both showed intense nuclear labeling in interface macrophages. GR and PPARγ activation polarized peripheral blood-derived macrophages from a default M1 (CD80(+)) toward an M2 (CD163(+)) phenotype, but PPARγ signaling predominated, as its antagonism blocked any GR-mediated effect. Seeding on PABM was effective at polarizing peripheral blood-derived macrophages from a default CD80(+) phenotype on glass to a CD80(-) phenotype, with intense nuclear localization of PPARγ. These results endorse in vivo observations that the infiltration of decellularized biological scaffolds, exemplified here by PABM, is pioneered by macrophages. Thus, it appears that natural factors present in PABM are involved in the active recruitment and polarization of macrophages to a CD163(+) phenotype, with activation of PPARγ identified as the candidate pathway. The harnessing of these natural matrix-associated factors may be useful in enhancing the integration of synthetic and other natural biomaterials by polarizing macrophage activation toward an M2 regulatory phenotype.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Matriz Extracelular/metabolismo , Activación de Macrófagos/fisiología , Macrófagos/metabolismo , PPAR gamma/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Glucocorticoides/metabolismo , Andamios del Tejido , Animales , Bioprótesis , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Células Cultivadas , Matriz Extracelular/química , Humanos , Porcinos , Vejiga Urinaria/química , Vejiga Urinaria/metabolismoRESUMEN
BACKGROUND: Chondral defects of the articular surface are a common condition that can lead to osteoarthritis if not treated. Therapy of this condition is a topic of constant debate and a variety of chondral repair strategies are currently used. One strategy involves implantation of a cell-free matrix of type I collagen (COL1), to provide a scaffold for chondrocyte migration and proliferation and extracellular matrix production. Although several studies have suggested that chondrocytes can move, to the best of our knowledge there is still no proof of chondrocyte occurrence in a former cell-free scaffold for articular cartilage repair in humans. CASE PRESENTATION: An 18-year-old male patient underwent arthroscopic surgery of the knee for patellar instability and a chondral defect of the femoral condyle. Clinical outcome scores were recorded pre-operatively, after 6 weeks and after 6, 12, 24 and 36 months. MRI was recorded after 6 weeks and after 6, 12, 24 and 36 months postoperatively. At 42 months after implantation of a cell-free type I collagen matrix and reconstruction of the medial patellofemoral ligament, the patient was again treated arthroscopically for a tear of the medial meniscus of the same knee. A biopsy of the previous chondral defect was taken during arthroscopy for histological examination. CONCLUSION: In addition to good clinical and radiological results reported for cell-free scaffolds for cartilage repair in several other studies, transformation of the scaffold could be observed during re-arthroscopy for the meniscal tear. Histological examination of the specimen revealed articular cartilage with vital chondrocytes and a strong staining reaction for type II collagen (COL II), but no reaction for type I collagen staining. This might indicate a complete transformation of the scaffold and supports the theory that cell free scaffolds could support cell migration. Although the cell source remains unclear, migrating chondrocytes from the periphery remain a possibility.
Asunto(s)
Artroscopía/métodos , Enfermedades de los Cartílagos/cirugía , Condrocitos/trasplante , Adolescente , Enfermedades de los Cartílagos/metabolismo , Enfermedades de los Cartílagos/patología , Movimiento Celular/fisiología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Condrocitos/citología , Condrocitos/metabolismo , Colágeno Tipo I/administración & dosificación , Colágeno Tipo I/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Resultado del TratamientoRESUMEN
B cells are important in the pathogenesis of multiple sclerosis (MS) and some of the effects are not dependent on maturation of B cells into immunoglobulin (Ig) producing plasmablasts and plasma cells. B cells present antigen, activate T cells, and are involved in immunoregulation and cytokine secretion. To determine if B cells from MS patients secrete products that have deleterious effects on glial cells not mediated by Ig, and to compare effects with secretory products of normal controls (NC), we isolated B cells from 7 patients with relapsing remitting MS (RRMS) and 4 NC. B cells were cultured alone or after stimulation with CD40 ligand (CD40L), CD40L+cross-linking of the B cell antigen receptor (xBCR) and CD40L+xBCR+stimulation of toll like receptor 9 (TLR9). Supernatants were harvested and incubated with mixed central nervous system (CNS) neonatal rat glial cells. Supernatants from unstimulated NC B cells induced on average death of 7% (range 0-24%) of differentiated oligodendrocytes (OL); in contrast, supernatants from unstimulated B cells from RRMS patients induced death of 57% (range 35-74%) of OL. Supernatants of stimulated B cells from NC did not increase the minimal OL death whereas stimulation of B cells from RRMS had variable results compared to unstimulated B cells. Supernatants from both NC and RRMS induced microglial enlargement and loss of normal resting bipolar morphology. OL death did not correlate with levels of tumor necrosis alpha (TNF-α), lymphotoxin alpha (LT-α), interleukin 6 (IL-6), IL-10, transforming growth factor beta 1 (TGF-ß1) or any combination or ratio of these cytokines. Analysis of 26 supernatants from NC and RRMS patients failed to detect IgM. There were very low levels of IgG in 8 of the 26 supernatants, and no correlation between of OL death and presence or absence of IgG. Sera used in both the B cell and glial cell cultures were heated, which inactivates complement. The effects of B cell supernatants on OL could be direct and/or indirect involving either microglia and/or astrocytes. The identity of the toxic factor(s) is as yet unknown. Thus we have demonstrated that B cells from patients with RRMS but not NC secrete one or more factors toxic to OL. It is possible that such factors produced by peripheral blood B cells when within the CNS could contribute to demyelination in MS patients.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Muerte Celular/inmunología , Esclerosis Múltiple Recurrente-Remitente/inmunología , Esclerosis Múltiple Recurrente-Remitente/patología , Oligodendroglía/inmunología , Oligodendroglía/patología , Animales , Animales Recién Nacidos , Subgrupos de Linfocitos B/patología , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Células Cultivadas , Humanos , Microglía/inmunología , Microglía/patología , Esclerosis Múltiple Recurrente-Remitente/metabolismo , Oligodendroglía/citología , RatasRESUMEN
Traumatic brain injury (TBI) is a major cause of morbidity and mortality, and early predictors of neurological outcomes are of great clinical importance. Cell free DNA (CFD), a biomarker used for the diagnosis and monitoring of several diseases, has been implicated as a possible prognostic indicator after TBI. The purpose of this study was to determine the pattern and timing of CFD levels after TBI, and whether a relationship exists between the level of CFD and brain edema and neurological outcomes. Thirty-nine Sprague-Dawley rats were randomly assigned to two groups: rats in group 1 (sham group) were anesthetized and had a scalp incision without TBI, and rats in group 2 were anesthetized and had a scalp incision with TBI, which was induced by using a weight drop model that causes diffuse brain injury. A neurological severity score (NSS) was assessed at 1, 24, and 48 h after TBI. CFD was measured via blood samples drawn at t=0 (baseline), 12, 24, 48, 72, and 120 h after TBI. At 48 h after TBI, brain edema was determined in a subgroup of 11 rats by calculating the difference between rats' wet and dry brain weight. The significance of comparisons between and within groups (CFD levels, brain water content, and NSS) were determined using the Kruskal-Wallis, Mann-Whitney and Student t test. The correlation between CFD levels and the NSS, as well as between CFD levels and the extent of brain edema, was calculated using the Spearman and Pearson tests, respectively. Compared with baseline levels, the CFD levels in rats subjected to TBI were significantly increased at 24 and 48 h after TBI (p<0.01 and p<0.05, respectively). A positive correlation was demonstrated between CFD levels 24 h following TBI and the extent of brain edema (r=0.63, p<0.05), as well as between CFD levels and the NSS (r=0.79, p<0.005). In this study, we demonstrated an increase in CFD levels after TBI, as well as a correlation between CFD levels and brain edema and NSS. CFD levels may provide a quick, reliable, and simple prognostic indicator of neurological outcome in animals after TBI. Its role in humans has not been clearly elucidated, but has potentially significant clinical implications.
Asunto(s)
Daño Encefálico Crónico/sangre , Edema Encefálico/sangre , Lesiones Encefálicas/sangre , ADN/sangre , Animales , Biomarcadores/sangre , Daño Encefálico Crónico/diagnóstico , Daño Encefálico Crónico/patología , Edema Encefálico/diagnóstico , Edema Encefálico/patología , Lesiones Encefálicas/diagnóstico , Lesiones Encefálicas/patología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Modelos Animales de Enfermedad , Masculino , Valor Predictivo de las Pruebas , Ratas , Ratas Sprague-DawleyRESUMEN
In response to cell injury, caused, for example, by trauma, several processes must be initiated simultaneously to achieve an acute inflammatory response designed to prevent sustained tissue damage and infection and to restore and maintain tissue homeostasis. Detecting cell injury is facilitated by the fact that damaged cells release intracellular molecules not normally present in the extracellular space. However, potential underlying mechanisms for the recognition of endogenous danger signals released upon cell injury have yet to be elucidated. In this study, we demonstrate that mast cells, potent promoters of acute inflammation, play a key role in responding to cell injury by recognizing IL-33 released from necrotic structural cells. In an in vitro model of cell injury, this recognition was shown to involve the T1/ST2 receptor and result in the secretion of proinflammatory leukotrienes and cytokines by mouse mast cells. Remarkably, of all of the components released upon necrosis, our results show that IL-33 alone is a key component responsible for initiating proinflammatory responses in mast cells reacting to cell injury. Our findings identify IL-33 as a key danger signal released by necrotic structural cells capable of activating mast cells, thus providing novel insights concerning the role of mast cells as sensors of cell injury.
Asunto(s)
Interleucinas/metabolismo , Mastocitos/inmunología , Mastocitos/patología , Animales , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Células Cultivadas , Citocinas/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interleucina-33 , Interleucinas/deficiencia , Interleucinas/genética , Leucotrienos/metabolismo , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NecrosisRESUMEN
A key event in the pathogenesis of prion diseases is the conversion of the normal cellular isoform of the prion protein into the disease-associated isoform, but the mechanisms operating in this critical event are not yet fully understood. A number of novel approaches have recently been developed to study factors influencing this process. One of these, the protein misfolding cyclical amplification (PMCA) technique, has been used to explore defined factors influencing the conversion of cellular prion protein in a cell-free model system. Although initially developed in animal models, this technique has been increasingly applied to human prion diseases. Recent studies have focused on the role of different isoforms of the disease-associated human prion protein and the effects of the naturally occurring polymorphism at codon 129 in the human prion protein gene on the conversion process, improving our understanding of the interaction between host and agent factors that influence the wide range of phenotypes in human prion diseases. This technique also allows a greatly enhanced sensitivity of detection of disease-associated prion protein in human tissues and fluids, which is potentially applicable to disease screening, particularly for variant Creutzfeldt-Jakob disease. The PMCA technique can also be used to model human susceptibility to a range of prions of non-human origin, which is likely to prove of considerable future interest as more novel and potentially pathogenic prion diseases are identified in animal species that form part of the human food chain.
Asunto(s)
Sistema Libre de Células , Enfermedades por Prión/diagnóstico , Enfermedades por Prión/patología , Priones/química , Priones/metabolismo , Animales , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Humanos , Modelos Animales , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Enfermedades por Prión/genética , Priones/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. The genesis of these diseases is believed to be associated with the route (mucosa versus blood) and mode (cell-free versus cell-associated) of primary infection as well as the modulation of dendritic cell (DC) functions. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Moloney murine leukemia virus to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-diphtheria toxin receptor transgenic mouse model system that permits conditional transient depletion of CD11c(+) DCs. We infected these transgenic mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free but not cell-associated HTLV-1 in both CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA in the non-CD4 fraction. Moreover, depletion of DCs significantly dampened the cellular immune response (IFN-gamma(+)CD8(+) T cells) against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of adult T cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis after the initial infection.
Asunto(s)
Antígeno CD11c/genética , Células Dendríticas/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Péptidos y Proteínas de Señalización Intercelular/genética , Leucaféresis , Paraparesia Espástica Tropical/inmunología , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Línea Celular , Sistema Libre de Células/inmunología , Sistema Libre de Células/patología , Sistema Libre de Células/virología , Células Dendríticas/patología , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/virología , Factor de Crecimiento Similar a EGF de Unión a Heparina , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucaféresis/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Paraparesia Espástica Tropical/metabolismo , Paraparesia Espástica Tropical/virologíaRESUMEN
Exacerbation of disease in systemic lupus erythematosus (SLE) is associated with bacterial infection. In conventional dendritic cells (cDCs), the TLR4 ligand bacterial LPS induces IFN-beta gene expression but does not induce IFN-alpha. We hypothesized that when cDCs are primed by cytokines, as may frequently be the case in SLE, LPS would then induce the production of IFN-alpha, a cytokine believed to be important in lupus pathogenesis. In this study we show that mouse cDCs and human monocytes produce abundant IFN-alpha following TLR4 engagement whether the cells have been pretreated either with IFN-beta or with a supernatant from DCs activated by RNA-containing immune complexes from lupus patients. This TLR4-induced IFN-alpha induction is mediated by both an initial TRIF-dependent pathway and a subsequent MyD88-dependent pathway, in contrast to TLR3-induced IFN-alpha production, which is entirely TRIF-dependent. There is also a distinct requirement for IFN regulatory factors (IRFs), with LPS-induced IFN-alpha induction being entirely IRF7- and partially IRF5-dependent, in contrast to LPS-induced IFN-beta gene induction which is known to be IRF3-dependent but largely IRF7-independent. This data demonstrates a novel pathway for IFN-alpha production by cDCs and provides one possible explanation for how bacterial infection might precipitate disease flares in SLE.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Interferón-alfa/biosíntesis , Interferón beta/fisiología , Lipopolisacáridos/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Células Cultivadas , Humanos , Inmunoglobulina G/farmacología , Interferón-alfa/fisiología , Ligandos , Lipopolisacáridos/farmacología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Transducción de Señal/inmunología , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genéticaRESUMEN
Dendritic cells (DCs) are APCs that play an essential role by bridging innate and adaptive immunity. DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is one of the major C-type lectins expressed on DCs and exhibits high affinity for nonsialylated Lewis (Le) glycans. Recently, we reported the characterization of oligosaccharide ligands expressed on SW1116, a typical human colorectal carcinoma recognized by mannan-binding protein, which is a serum C-type lectin and has similar carbohydrate-recognition specificities as DC-SIGN. These tumor-specific oligosaccharide ligands were shown to comprise clusters of tandem repeats of Lea/Leb epitopes. In this study, we show that DC-SIGN is involved in the interaction of DCs with SW1116 cells through the recognition of aberrantly glycosylated forms of Lea/Leb glycans on carcinoembryonic Ag (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1). DC-SIGN ligands containing Lea/Leb glycans are also highly expressed on primary cancer colon epithelia but not on normal colon epithelia, and DC-SIGN is suggested to be involved in the association between DCs and colorectal cancer cells in situ by DC-SIGN recognizing these cancer-related Le glycan ligands. Furthermore, when monocyte-derived DCs (MoDCs) were cocultured with SW1116 cells, LPS-induced immunosuppressive cytokines such as IL-6 and IL-10 were increased. The effects were significantly suppressed by blocking Abs against DC-SIGN. Strikingly, LPS-induced MoDC maturation was inhibited by supernatants of cocultures with SW1116 cells. Our findings imply that colorectal carcinomas affecting DC function and differentiation through interactions between DC-SIGN and colorectal tumor-associated Le glycans may induce generalized failure of a host to mount an effective antitumor response.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/inmunología , Neoplasias Colorrectales/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/patología , Glicoesfingolípidos/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Antígenos CD/metabolismo , Antígeno CA-19-9 , Antígeno Carcinoembrionario/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Sistema Libre de Células/inmunología , Sistema Libre de Células/metabolismo , Sistema Libre de Células/patología , Técnicas de Cocultivo , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Glicosilación , Humanos , Antígenos del Grupo Sanguíneo de Lewis , Ligandos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Monocitos/metabolismo , Monocitos/patología , Células U937RESUMEN
Acellular nerve allografts have been explored as an alternative to nerve autografting. It has long been recognized that there is a distinct limit to the effective length of conventional acellular nerve grafts, which must be overcome for many grafting applications. In rodent models nerve regeneration fails in acellular nerve grafts greater than 2 cm in length. In previous studies we found that nerve regeneration is markedly enhanced with acellular nerve grafts in which growth-inhibiting chondroitin sulfate proteoglycan was degraded by pretreatment with chondroitinase ABC (ChABC). Here, we tested if nerve regeneration can be achieved through 4-cm acellular nerve grafts pretreated with ChABC. Adult rats received bilateral sciatic nerve segmental resection and repair with a 4 cm, thermally acellularized, nerve graft treated with ChABC (ChABC graft) or vehicle-treated acellularized graft (Control graft). Nerve regeneration was examined 12 weeks after implantation. Our findings confirm that functional axonal regeneration fails in conventional long acellular grafts. In this condition we found very few axons in the distal host nerve, and there were marginal signs of sciatic nerve reinnervation in few (2/9) rats. This was accompanied by extensive structural disintegration of the distal graft and abundant retrograde axonal regeneration in the proximal nerve. In contrast, most (8/9) animals receiving nerve repair with ChABC grafts showed sciatic nerve reinnervation by direct nerve pinch testing. Histological examination revealed much better structural preservation and axonal growth throughout the ChABC grafts. Numerous axons were found in all but one (8/9) of the host distal nerves and many of these regenerated axons were myelinated. In addition, the amount of aberrant retrograde axonal growth (originating near the proximal suture line) was markedly reduced by repair with ChABC grafts. Based on these results we conclude that ChABC treatment substantially increases the effective length of acellular nerve grafts.