RESUMEN
The ribosome is a ribonucleoprotein complex found in all domains of life. Its role is to catalyze protein synthesis, the messenger RNA (mRNA)-templated formation of amide bonds between α-amino acid monomers. Amide bond formation occurs within a highly conserved region of the large ribosomal subunit known as the peptidyl transferase center (PTC). Here we describe the step-wise design and characterization of mini-PTC 1.1, a 284-nucleotide RNA that recapitulates many essential features of the Escherichia coli PTC. Mini-PTC 1.1 folds into a PTC-like structure under physiological conditions, even in the absence of r-proteins, and engages small molecule analogs of A- and P-site tRNAs. The sequence of mini-PTC 1.1 differs from the wild type E. coli ribosome at 12 nucleotides that were installed by a cohort of citizen scientists using the on-line video game Eterna. These base changes improve both the secondary structure and tertiary folding of mini-PTC 1.1 as well as its ability to bind small molecule substrate analogs. Here, the combined input from Eterna citizen-scientists and RNA structural analysis provides a robust workflow for the design of a minimal PTC that recapitulates many features of an intact ribosome.
Asunto(s)
Escherichia coli , Ribosomas , Humanos , Amidas , Escherichia coli/genética , Escherichia coli/metabolismo , Peptidil Transferasas/genética , Peptidil Transferasas/química , Ribosomas/metabolismo , ARN de Transferencia/metabolismoRESUMEN
S100A8 and S100A9 are myeloid cell-derived proteins that are elevated in several types of inflammatory lung disorders. Pro- and anti-inflammatory properties are reported and these proteins are proposed to activate TLR4. S100A8 and S100A9 can function separately, likely through distinct receptors but a systematic comparison of their effects in vivo are limited. Here we assess inflammation in murine lung following S100A9 and S100A8/A9 inhalation. Unlike S100A8, S100A9 promoted mild neutrophil and lymphocyte influx, possibly mediated in part, by increased mast cell degranulation and selective upregulation of some chemokine genes, particularly CXCL-10. S100 proteins did not significantly induce proinflammatory mediators including TNF-α, interleukin-1ß (IL-1ß), IL-6 or serum amyloid A3 (SAA3). In contrast to S100A8, neither preparation induced S100A8 or IL-10 mRNA/protein in airway epithelial cells, or in tracheal epithelial cells in vitro. Like S100A8, S100A9 and S100A8/A9 reduced neutrophil influx in acute lung injury provoked by lipopolysaccharide (LPS) challenge but were somewhat less inhibitory, possibly because of differential effects on expression of some chemokines, IL-1ß, SAA3 and IL-10. Novel common pathways including increased induction of an NAD+-dependent protein deacetylase sirtuin-1 that may reduce NF-κB signalling, and increased STAT3 activation may reduce LPS activation. Results suggest a role for these proteins in normal homeostasis and protective mechanisms in the lung.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Proteínas S100/metabolismo , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/patología , Animales , Líquido del Lavado Bronquioalveolar , Quimiocina CXCL10/metabolismo , Femenino , Regulación de la Expresión Génica , Inflamación/genética , Inflamación/patología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos , Pulmón/metabolismo , Pulmón/patología , Linfocitos/patología , Ratones Endogámicos BALB C , Infiltración Neutrófila , Fosforilación , Transducción de Señal/genéticaRESUMEN
The original centrally defining features of "Homo floresiensis" are based on bones represented only in the single specimen LB1. Initial published values of 380-mL endocranial volume and 1.06-m stature are markedly lower than later attempts to confirm them, and facial asymmetry originally unreported, then denied, has been established by our group and later confirmed independently. Of nearly 200 syndromes in which microcephaly is one sign, more than half include asymmetry as another sign and more than one-fourth also explicitly include short stature. The original diagnosis of the putative new species noted and dismissed just three developmental abnormalities. Subsequent independent attempts at diagnosis (Laron Syndrome, Majewski osteodysplastic primordial dwarfism type II, cretinism) have been hampered a priori by selectively restricted access to specimens, and disparaged a posteriori using data previously unpublished, without acknowledging that all of the independent diagnoses corroborate the patent abnormal singularity of LB1. In this report we establish in detail that even in the absence of a particular syndromic diagnosis, the originally defining features of LB1 do not establish either the uniqueness or normality necessary to meet the formal criteria for a type specimen of a new species. In a companion paper we present a new syndromic diagnosis for LB1.
Asunto(s)
Huesos , Fósiles , Hominidae , Animales , Hominidae/clasificación , Indonesia , Filogenia , ProbabilidadRESUMEN
Human skeletons from Liang Bua Cave, Flores, Indonesia, are coeval with only Homo sapiens populations worldwide and no other previously known hominins. We report here for the first time to our knowledge the occipitofrontal circumference of specimen LB1. This datum makes it possible to link the 430-mL endocranial volume of LB1 reported by us previously, later confirmed independently by other investigators, not only with other human skeletal samples past and present but also with a large body of clinical data routinely collected on patients with developmental disorders. Our analyses show that the brain size of LB1 is in the range predicted for an individual with Down syndrome (DS) in a normal small-bodied population from the geographic region that includes Flores. Among additional diagnostic signs of DS and other skeletal dysplasiae are abnormally short femora combined with disproportionate flat feet. Liang Bua Cave femora, known only for LB1, match interlimb proportions for DS. Predictions based on corrected LB1 femur lengths show a stature normal for other H. sapiens populations in the region.
Asunto(s)
Síndrome de Down/diagnóstico , Fósiles , Homeostasis , Hominidae , Animales , Cara/anatomía & histología , Indonesia , Esqueleto , Cráneo/anatomía & histologíaRESUMEN
S100A8 is considered proinflammatory by activating TLR4 and/or the receptor for advanced glycation end products. The aim was to investigate inflammatory effects of S100A8 in murine lung. S100A8 was administered to BALB/c mice by nasal inhalation and genes induced over a time-course assessed. LPS was introduced intranasally either alone or 2 h after pretreatment of mice with intranasal application of S100A8 or dexamethasone. A Cys(42)-Ala(42) mutant S100A8 mutant was used to assess whether S100A8's effects were via pathways that were dependent on reactive oxygen species. S100A8 induced IL-10 mRNA, and expression was apparent only in airway epithelial cells. Importantly, it suppressed acute lung injury provoked by LPS inhalation by suppressing mast-cell activation and induction of mediators orchestrating leukocyte recruitment, possibly by reducing NF-κB activation via an IκBα/Akt pathway and by downmodulating pathways generating oxidative stress. The Cys(42)-Ala(42) S100A8 mutant did not induce IL-10 and was less immunosuppressive, indicating modulation by scavenging oxidants. S100A8 inhibition of LPS-mediated injury was as potent, and outcomes were remarkably similar to immunosuppression by dexamethasone. We challenge the notion that S100A8 is an agonist for TLR4 or the receptor for advanced glycation end products. S100A8 induced IL-10 in vivo and initiates a feedback loop that attenuates acute lung injury.
Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Calgranulina A/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-10/genética , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Administración Intranasal , Animales , Antiinflamatorios/administración & dosificación , Western Blotting , Calgranulina A/genética , Análisis por Conglomerados , Dexametasona/administración & dosificación , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Inmunohistoquímica , Interleucina-10/metabolismo , Lipopolisacáridos/administración & dosificación , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Proteínas Mutantes/administración & dosificación , Proteínas Mutantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
Glucocorticoids are commonly used for treating asthma and its exacerbations but have well-recognised adverse effects and are not always effective. Few alternative treatments exist. Using a murine model of an acute exacerbation of asthma, we assessed the ability of ISU201, a novel protein drug, to suppress the inflammatory response when administered after induction of an exacerbation. Sensitised mice were chronically challenged with a low mass concentration of aerosolised ovalbumin, and then received a single moderate-level challenge to simulate an allergen-induced exacerbation. ISU201 was administered to mice 2 and 8 hours later, while pulmonary inflammation and expression of mRNA for chemokines and proinflammatory cytokines were assessed after 4, 12, and 24 hours. Relative to vehicle-treated controls, ISU201 suppressed accumulation of pulmonary neutrophils and eosinophils, while accelerating the decline in CXCL1, TNF-α, and IL-6 in lavage fluid and lung tissue. ISU201 significantly reduced peak expression of mRNA for the chemokines Cxcl9 and Cxcl10, the adhesion molecules Icam1 and Vcam1, and the proinflammatory cytokines Il1b, Il12p40, and Csf1. The ability of ISU201 to promote resolution of inflammation suggests that it may have potential as an alternative to glucocorticoids in the management of asthma, including when administered after the onset of an acute exacerbation.
Asunto(s)
Antígenos CD/uso terapéutico , Asma/tratamiento farmacológico , Asma/inmunología , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Pulmón/inmunología , Pulmón/metabolismo , Fragmentos de Péptidos/uso terapéutico , Animales , Asma/metabolismo , Quimiocina CXCL10/genética , Quimiocina CXCL9/genética , Modelos Animales de Enfermedad , Femenino , Técnicas para Inmunoenzimas , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Interleucina-1beta/genética , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genéticaRESUMEN
BACKGROUND: Sarcoidosis has often been termed an "immune paradox" as there is peripheral anergy to common recall antigens despite pronounced TH1-dominant inflammation at disease sites, such as the lung, with up-regulation of interferon γ, IL-27 and transcription factors. Peripheral blood may reflect the anergic state, while exhaled breath condensate (EBC) analysis may offer insights into the lung disease. METHODS: A cross-sectional study was conducted to investigate the expression of TH1 cytokines and transcription factors (IFNγ, IL-27 and T-bet) in the peripheral blood and/or EBC of sarcoidosis patients and healthy controls. Whole blood and EBC were collected from sarcoidosis patients and healthy controls. TH1 cytokine expression levels were then measured in peripheral blood mononuclear cells (PBMCs) and/or plasma and EBC using quantitative real-time PCR, ELISA and via Western blotting. RESULTS: Compared to healthy controls, PBMC IL-27 mRNA was higher in patients (p = 0.0019). There were no significant differences in plasma IL-27 protein between patients and controls (p = 0.20). T-bet mRNA and protein were lower (p = 0.010 and p = 0.0043, respectively) in patients compared to controls. There were no significant differences in PBMC IFNγ mRNA and protein expression (p = 0.68 and p = 0.74, respectively) nor in EBC IL-27 levels. CONCLUSIONS: Our data indicate that depressed T-bet mRNA and protein expression could contribute to the peripheral anergy in sarcoidosis and that IL-27 mRNA levels are elevated in the PBMC from those with sarcoidosis.
Asunto(s)
Factores Inmunológicos/biosíntesis , Interleucinas/biosíntesis , Sarcoidosis/metabolismo , Proteínas de Dominio T Box/biosíntesis , Células TH1/metabolismo , Adulto , Estudios Transversales , Femenino , Humanos , Factores Inmunológicos/inmunología , Interleucinas/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Sarcoidosis/inmunología , Proteínas de Dominio T Box/inmunología , Células TH1/inmunología , Adulto JovenRESUMEN
Lung cancer is the leading cause of cancer-related death worldwide. Increasing evidence indicates a critical role for chronic inflammation in lung carcinogenesis. S100A8 is a protein with reported pro- and anti-inflammatory functions. It is highly expressed in myeloid-derived suppressor cells (MDSC) that accumulate in the tumor microenvironment and abrogate effective anti-cancer immune responses. Mechanisms of MDSC-mediated immunosuppression include production of reactive oxygen species and nitric oxide, and depletion of L-arginine required for T cell function. Although S100A8 is expressed in MDSC, its role in the lung tumor microenvironment is largely unknown. To address this, mouse recombinant S100A8 was repeatedly administered intranasally to mice bearing orthotopic lung cancers. S100A8 treatment prolonged survival from 19 days to 28 days (p < 0.001). At midpoint of survival, whole lungs and bronchoalveolar lavage fluid (BALF) were collected and relevant genes/proteins measured. We found that S100A8 significantly lowered expression of cytokine genes and proteins that promote expansion and activation of MDSC in lungs and BALF from cancer-bearing mice. Moreover, S100A8 enhanced activities of antioxidant enzymes and suppressed production of nitrite to create a lung microenvironment conducive to cytotoxic lymphocyte expansion and function. In support of this, we found decreased MDSC numbers, and increased numbers of CD4+ T cells and natural killer T (NK-T) cells in lungs from cancer-bearing mice treated with S100A8. Ex-vivo treatment of splenocytes with S100A8 protein activated NK cells. Our results indicate that treatment with S100A8 may favourably modify the lung microenvironment to promote an effective immune response in lungs, thereby representing a new strategy that could complement current immunotherapies in lung cancer.
Asunto(s)
Calgranulina A , Neoplasias Pulmonares , Animales , Calgranulina A/genética , Calgranulina A/metabolismo , Pulmón/metabolismo , Ratones , Proteínas/metabolismo , Tórax , Microambiente TumoralRESUMEN
The mechanisms underlying the exaggerated distal airway inflammation and hyperresponsiveness that characterize acute exacerbations of asthma are largely unknown. Using BALB/c mouse experimental models, we demonstrated a potentially important role for alveolar macrophages (AM) in the development of an allergen-induced exacerbation of asthma. To induce features of airway inflammation and remodeling characteristic of mild chronic asthma, animals were systemically sensitized and exposed to low mass concentrations (≈3 mg/m(3)) of aerosolized ovalbumin for 30 minutes per day, 3 days per week, for 4 weeks. A subsequent single moderate-level challenge (≈30 mg/m(3)) was used to trigger an acute exacerbation. In chronically challenged animals, cytokine expression by AM was not increased, whereas after an acute exacerbation, AM exhibited significantly enhanced expression of proinflammatory cytokines, including interleukin (IL) 1ß, IL-6, CXCL-1, and tumor necrosis factor α. In parallel, there was a marked increase in the expression of several cytokines by CD4(+) T-lymphocytes, notably the Th2 cytokines IL-4 and IL-13. Importantly, AM from an acute exacerbation stimulated the expression of Th2 cytokines when cocultured with CD4(+) cells from chronically challenged animals, and their ability to do so was significantly greater than AM from either chronically challenged or naïve controls. Stimulation was partly dependent on interactions involving CD80/86. We conclude that in an acute exacerbation of asthma, enhanced cytokine expression by AM may play a critical role in triggering increased expression of cytokines by pulmonary CD4(+) T-lymphocytes.
Asunto(s)
Asma/inmunología , Linfocitos T CD4-Positivos/inmunología , Citocinas/metabolismo , Pulmón/inmunología , Macrófagos Alveolares/inmunología , Células Th2/inmunología , Alérgenos/inmunología , Animales , Asma/patología , Hiperreactividad Bronquial , Líquido del Lavado Bronquioalveolar , Linfocitos T CD4-Positivos/patología , Enfermedad Crónica , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Volumen Espiratorio Forzado , Inflamación/inmunología , Inflamación/patología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/fisiología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Th2/patologíaRESUMEN
The S100 calcium-binding proteins S100A8 and S100A9 are elevated systemically in patients with viral infections. The S100A8-S100A9 complex facilitated viral replication in human CD4(+) T lymphocytes latently infected with HIV-1- and S100A8-induced HIV-1 transcriptional activity. Mechanisms inducing the S100 genes and the potential source of these proteins following viral activation are unknown. In this study, we show that S100A8 was induced in murine macrophages, and S100A8 and S100A9 in human monocytes and macrophages, by polyinosinic:polycytidylic acid, a dsRNA mimetic. Induction was at the transcriptional level and was IL-10 dependent. Similar to LPS-induced S100A8, induction by dsRNA was dependent on p38 and ERK MAPK. Protein kinase R (PKR) mediates antiviral defense and participates in MyD88-dependent/independent signaling triggered by TLR4 or TLR3. Like IL-10, S100 induction by polyinosinic:polycytidylic acid and by LPS was inhibited by the specific PKR inhibitor 2-aminopurine, indicating a novel IL-10, PKR-dependent pathway. Other mediators such as IFN-beta, which synergized with dsRNA, may also be involved. C/EBPbeta bound the defined promoter region in response to dsRNA. S100A8 was expressed in lungs of mice infected with influenza virus and was maximal at day 8 with strong immunoreactivity in epithelial cells lining the airways and in mononuclear cells and declined early in the recovery phase, implying down-regulation by mediator(s) up-regulated during resolution of the infection. IL-10 is implicated in viral persistence. Since S100A8/S100A9 levels are likely to be maintained in conditions where IL-10 is raised, these proteins may contribute to viral persistence in patients infected by some RNA viruses.
Asunto(s)
Calgranulina A/genética , Regulación de la Expresión Génica/inmunología , Macrófagos/inmunología , Monocitos/inmunología , ARN Bicatenario/inmunología , Animales , Calgranulina A/metabolismo , Calgranulina B/inmunología , Calgranulina B/metabolismo , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Humanos , Macrófagos/virología , Ratones , Monocitos/virología , Análisis de Secuencia por Matrices de Oligonucleótidos , Poli I-C/inmunología , Infecciones por Virus ARN/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , eIF-2 Quinasa/inmunología , eIF-2 Quinasa/metabolismoRESUMEN
Macrophages, cytokines, and matrix metalloproteinases (MMP) play important roles in atherogenesis. The Ca(2+)-binding protein S100A12 regulates monocyte migration and may contribute to atherosclerosis by inducing proinflammatory cytokines in macrophages. We found significantly higher S100A12 levels in sera from patients with coronary artery disease than controls and levels correlated positively with C-reactive protein. S100A12 was released into the coronary circulation from ruptured plaque in acute coronary syndrome, and after mechanical disruption by percutaneous coronary intervention in stable coronary artery disease. In contrast to earlier studies, S100A12 did not stimulate proinflammatory cytokine production by human monocytes or macrophages. Similarly, no induction of MMP genes was found in macrophages stimulated with S100A12. Because S100A12 binds Zn(2+), we studied some functional aspects that could modulate atherogenesis. S100A12 formed a hexamer in the presence of Zn(2+); a novel Ab was generated that specifically recognized this complex. By chelating Zn(2+), S100A12 significantly inhibited MMP-2, MMP-9, and MMP-3, and the Zn(2+)-induced S100A12 complex colocalized with these in foam cells in human atheroma. S100A12 may represent a new marker of this disease and may protect advanced atherosclerotic lesions from rupture by inhibiting excessive MMP-2 and MMP-9 activities by sequestering Zn(2+).
Asunto(s)
Aterosclerosis/metabolismo , Enfermedad Coronaria/metabolismo , Proteínas S100/fisiología , Adulto , Anciano , Aterosclerosis/patología , Biomarcadores/metabolismo , Línea Celular Tumoral , Células Cultivadas , Enfermedad Coronaria/patología , Femenino , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/fisiología , Macrófagos/enzimología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Persona de Mediana Edad , Rotura Espontánea/enzimología , Rotura Espontánea/metabolismo , Rotura Espontánea/prevención & control , Proteínas S100/sangre , Proteína S100A12 , Zinc/fisiologíaRESUMEN
Chimeric Antigen Receptor (CAR) T-cell therapy, as an approved treatment option for patients with B cell malignancies, demonstrates that genetic modification of autologous immune cells is an effective anti-cancer regimen. Erythropoietin-producing Hepatocellular receptor tyrosine kinase class A2 (EphA2) is a tumour associated antigen expressed on a range of sarcomas, including paediatric osteosarcoma (OS) and Ewing sarcoma (ES). We tested human EphA2 directed CAR T cells for their capacity to target and kill human OS and ES tumour cells using in vitro and in vivo assays, demonstrating that EphA2 CAR T cells have potent anti-tumour efficacy in vitro and can eliminate established OS and ES tumours in vivo in a dose and delivery route dependent manner. Next, in an aggressive metastatic OS model we demonstrated that systemically infused EphA2 CAR T cells can traffic to and eradicate tumour deposits in murine livers and lungs. These results support further pre-clinical evaluation of EphA2 CAR T cells to inform the design of early phase clinical trial protocols to test the feasibility and safety of this immune cell therapy in paediatric bone sarcoma patients.
Asunto(s)
Neoplasias Óseas/terapia , Inmunoterapia Adoptiva/métodos , Receptor EphA2/inmunología , Linfocitos T/inmunología , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Terapia Molecular DirigidaRESUMEN
S100A8 and S100A9, highly expressed by neutrophils, activated macrophages, and microvascular endothelial cells, are secreted during inflammatory processes. Our earlier studies showed S100A8 to be an avid scavenger of oxidants, and, together with its dependence on IL-10 for expression in macrophages, we postulated that this protein has a protective role. S-nitrosylation is an important posttranslational modification that regulates NO transport, cell signaling, and homeostasis. Relatively few proteins are targets of S-nitrosylation. To date, no inflammation-associated proteins with NO-shuttling capacity have been identified. We used HPLC and mass spectrometry to show that S100A8 and S100A9 were readily S-nitrosylated by NO donors. S-nitrosylated S100A8 (S100A8-SNO) was the preferred nitrosylated product. No S-nitrosylation occurred when the single Cys residue in S100A8 was mutated to Ala. S100A8-SNO in human neutrophils treated with NO donors was confirmed by the biotin switch assay. The stable adduct transnitrosylated hemoglobin, indicating a role in NO transport. S100A8-SNO suppressed mast cell activation by compound 48/80; intravital microscopy was used to demonstrate suppression of leukocyte adhesion and extravasation triggered by compound 48/80 in the rat mesenteric microcirculation. Although S100A8 is induced in macrophages by LPS or IFN-gamma, the combination, which activates inducible NO synthase, did not induce S100A8. Thus, the antimicrobial functions of NO generated under these circumstances would not be compromised by S100A8. Our results suggest that S100A8-SNO may regulate leukocyte-endothelial cell interactions in the microcirculation, and suppression of mast cell-mediated inflammation represents an additional anti-inflammatory property for S100A8.
Asunto(s)
Antiinflamatorios no Esteroideos/inmunología , Calgranulina A/inmunología , Calgranulina B/inmunología , Depuradores de Radicales Libres/inmunología , Leucocitos/inmunología , Procesamiento Proteico-Postraduccional/inmunología , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/inmunología , Calgranulina A/química , Calgranulina B/química , Cisteína/química , Cisteína/inmunología , Células Endoteliales/inmunología , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología , Hemoglobinas/inmunología , Humanos , Inflamación/inmunología , Interferón gamma/farmacología , Interleucina-10/inmunología , Lipopolisacáridos/farmacología , Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Microcirculación/inmunología , Óxido Nítrico/inmunología , Óxido Nítrico Sintasa de Tipo II/inmunología , Oxidantes/inmunología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3'-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.
Asunto(s)
Dependovirus/genética , Vectores Genéticos/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Cápside/metabolismo , Línea Celular , Línea Celular Tumoral , Células Cultivadas , ADN Viral , Femenino , Fibroblastos , Técnicas de Transferencia de Gen , Terapia Genética , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas , Masculino , Ratones , Receptor EphB2 , Linfocitos T , Transducción Genética , TransgenesRESUMEN
Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.
Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively 'swarm' towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.
Asunto(s)
Quimiocina CCL3/inmunología , Quimiocina CCL4/inmunología , Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Movimiento Celular , Quimiocina CCL3/genética , Quimiocina CCL4/genética , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Neoplasias/genética , Neoplasias/fisiopatología , Transducción de Señal , Linfocitos T Citotóxicos/citologíaRESUMEN
Leukocyte immunoglobulin-like receptor A5 (LILRA5) belongs to a family of receptors known to regulate leukocyte activation. There are two membrane-bound and two soluble forms of LILRA5. The transmembrane LILRA5 contain a short cytoplasmic domain and a charged arginine residue within the transmembrane region. Cross-linking of LILRA5 on monocytes induced production of pro-inflammatory cytokines, suggesting that LILRA5 plays a role in inflammation. However, expression of LILRA5 in diseases with extensive inflammatory component is unknown. Rheumatoid arthritis (RA) is a chronic inflammatory synovitis characterized by unregulated activation of leukocytes leading to joint destruction. Here we demonstrate extensive LILRA5 expression on synovial tissue macrophages and in synovial fluid of patients with active RA but not in patients with osteoarthritis. We also show that LILRA5 associated with the common gamma chain of the FcR and LILRA5 cross-linking induced phosphorylation of Src tyrosine kinases and Spleen tyrosine kinase (Syk). Furthermore, LILRA5 induced selective production of pro-inflammatory cytokines as well as IL-10. LILRA5 mRNA and protein expression was tightly regulated by TNF-alpha, IL-10 and IFN-gamma. Increased expression of LILRA5 in rheumatoid tissue, together with its ability to induce key cytokines involved in RA, suggests that this novel receptor may contribute to disease pathogenesis.
Asunto(s)
Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Citocinas/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Receptores Inmunológicos/metabolismo , Membrana Sinovial/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/patología , Membrana Celular/inmunología , Células Cultivadas , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Macrófagos/citología , Masculino , Persona de Mediana Edad , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Membrana Sinovial/inmunologíaRESUMEN
BACKGROUND: HIV-1 penetrates the central nervous system, which is vital for HIV-associated dementia (HAD). But the role of cellular infiltration and activation together with HIV in the development of HAD is poorly understood. METHODS: To study activation and infiltration patterns of macrophages, CD8+ T cells in relation to HIV in diverse CNS areas of patients with and without dementia. 46 brain regions from two rapidly progressing severely demented patients and 53 regions from 4 HIV+ non-dementia patients were analyzed. Macrophage and CD8+ T cell infiltration of the CNS in relation to HIV was assessed using immuno-histochemical analysis with anti-HIV (P24), anti-CD8 and anti-CD68, anti-S-100A8 and granzyme B antibodies (cellular activation). Statistical analysis was performed with SPSS 12.0 with Student's t test and ANOVA. RESULTS: Overall, the patterns of infiltration of macrophages and CD8+ T cells were indiscernible between patients with and without dementia, but the co-localization of macrophages and CD8+ T cells along with HIV P24 antigen in the deeper midline and mesial temporal structures of the brain segregated the two groups. This predilection of infected macrophages and CD8+ T cells to the middle part of the brain was unique to both HAD patients, along with unique nature of provirus gag gene sequences derived from macrophages in the midline and mesial temporal structures. CONCLUSION: Strong predilection of infected macrophages and CD8+ T cells was typical of the deeper midline and mesial temporal structures uniquely in HAD patients, which has some influence on neurocognitive impairment during HIV infection.
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Complejo SIDA Demencia/inmunología , Encéfalo/patología , Linfocitos T CD8-positivos/virología , Macrófagos/virología , Complejo SIDA Demencia/patología , Adulto , Secuencia de Bases , Encéfalo/inmunología , Encéfalo/virología , Linfocitos T CD8-positivos/inmunología , Preescolar , Femenino , Proteína p24 del Núcleo del VIH/metabolismo , VIH-1/genética , VIH-1/inmunología , Humanos , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la PolimerasaRESUMEN
STUDY DESIGN: Nondestructive flexibility tests were performed in vitro, comparing multiple conditions of fixation in a single group of specimens. OBJECTIVE: To compare the biomechanical behavior of the lumbar spine in the intact condition, after implanting a novel motion stabilizer, and after implanting a rigid fixator. SUMMARY OF BACKGROUND DATA: Two specific scenarios that may benefit from dynamic lumbar stabilization are single-level moderate instability, where the stabilizing tissues are relatively incompetent, and juxta-level to fusion, where the last instrumented level requires intermediate stiffness ("topping off") to prevent transfer of high stresses from the stiffer fusion construct to the intact adjacent levels. Both scenarios were evaluated in vitro. METHODS: Seven human cadaveric L2-S1 segments were tested (1) intact, (2) after moderate destabilization, (3) after 2-level hybrid posterior fixation, consisting of bilateral dynamic pedicle screws at L4 interconnected with rigid rods to standard pedicle screws at L5 and S1, (4) after 2-level rigid fixation, (5) after 1-level (L4-L5) dynamic fixation, and (6) after 1-level rigid fixation. In each condition, angular range of motion (ROM) and sagittal instantaneous axis of rotation (IAR) were assessed. RESULTS: In 1-level constructs, dynamic hardware allowed 104% of intact ROM, whereas rigid hardware allowed 49% of intact ROM. Relative to the intact, the IAR was shifted significantly farther posterior by rigid 1-level instrumentation than by dynamic 1-level instrumentation. In 2-level constructs, the dynamic level allowed significantly greater ROM than the rigid level in all directions but allowed significantly less ROM than the intact level in all directions except axial rotation. CONCLUSION: Dynamic instrumentation shifted the IAR less than rigid instrumentation, providing more favorable kinematics. This dynamic stabilizer provided 1-level ROM that was close to intact ROM during all loading modes in vitro. In the topping-off construct, the dynamic segment allowed intermediate ROM to give balanced transitional flexibility. LEVEL OF EVIDENCE: N/A.
Asunto(s)
Tornillos Óseos , Inestabilidad de la Articulación/cirugía , Vértebras Lumbares/cirugía , Fusión Vertebral/instrumentación , Fenómenos Biomecánicos , Cadáver , Femenino , Humanos , Inestabilidad de la Articulación/fisiopatología , Vértebras Lumbares/fisiopatología , Masculino , Ensayo de Materiales , Persona de Mediana Edad , Rango del Movimiento Articular , Fusión Vertebral/métodos , Estrés MecánicoRESUMEN
Growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta (TGF-beta) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+-binding proteins and are now accepted as markers of inflammation. They are expressed by keratinocytes and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon gamma (IFNgamma), tumour-necrosis factor (TNF) and TGF-beta did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maximally after 12 h. The FGF-2 response was strongly enhanced and prolonged by heparin. Interleukin-1beta (IL-1beta) alone, or in synergy with FGF-2/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF-2 and IL-1beta was partially dependent on the mitogen-activated-protein-kinase pathway and dependent on new protein synthesis. FGF-2-responsive elements were distinct from the IL-1beta-responsive elements in the S100A8 gene promoter. FGF-2-/heparin-induced, but not IL-1beta-induced responses were significantly suppressed by TGF-beta, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8-positive fibroblast-like cells 2 and 4 days post injury; numbers declined by 7 days. Up-regulation of S100A8 by FGF-2/IL-1beta, down-regulation by TGF-beta, and its time-dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair.