RESUMEN
BACKGROUND: Mast cells (MCs) play a pivotal role in innate and adaptive immune responses. However, MCs are also involved in different pathologic conditions. Studies on the mechanisms that govern human MC functions are impeded by their limited and difficult recovery. Therefore, several research groups have developed protocols to culture human MCs from progenitor cells. These protocols vary with respect to culture duration and used maturation cytokines. How MCs obtained by different protocols differ in phenotype and functionality is currently unknown. OBJECTIVE: To compare different protocols for the generation of human MCs from peripheral blood progenitors. METHODS: Thirteen paired human MC cultures were investigated. MCs were cultured form CD34+ progenitors cells for 4 or 8 weeks and with or without the addition of IL-6. Phenotyping comprised staining for CD117, CD203c, FcεRI, MRGPRX2, CD300a and CD32. Functional studies included measurements of the up-regulation of CD63 and CD203c after allergen-specific cross-linking of sIgE/FcεRI complexes or ligation of MRGPRX2 with substance P and different drugs. RESULTS: Cell cultures for 4 weeks in the presence of IL-6 consistently yielded the highest numbers of MCs. MCs cultured for 8 weeks with IL-6 showed more autofluorescence significantly impeding correct analyses of FcεRI and CD32. The density of FcεRI and CD32 was comparable between the different culture conditions. MRGPRX2 expression was significantly higher in the 8 week cultures. The density of CD300a was increased in the cultures with IL-6. Cells cultured for 8 weeks were more responsive to MRGPRX2 activation. In contrast, the 4-weeks cultures with IL-6 showed significantly higher allergen-specific activation. CONCLUSION: Four weeks of culture with IL-6 are sufficient to generate sizeable numbers of human mast cells from blood progenitors, thereby enabling simultaneous exploration of allergen-specific sIgE/FcεRI cross-linking and non-specific activation via MRGPRX2.
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Interleucina-6/metabolismo , Mastocitos/inmunología , Cultivo Primario de Células/métodos , Células Cultivadas/inmunología , Células Cultivadas/metabolismo , Medios de Cultivo/metabolismo , Citometría de Flujo , Voluntarios Sanos , Humanos , Inmunofenotipificación , Mastocitos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Factores de TiempoRESUMEN
Polycyclic aromatic hydrocarbons (PAHs) are generated by the incomplete combustion of carbon. Exposures correlate with systemic immune dysfunction and overall immune suppression. Real-world exposures to PAHs are almost always encountered as mixtures; however, research overwhelmingly centers on isolated exposures to a single PAH, benzo[a]pyrene (B[a]P). Here, a human monocyte line (U937) was exposed to B[a]P, benz[a]anthracene (B[a]A), or a mixture of six PAHs (6-MIX) to assess the differential toxicity on monocytes. Further, monocytes were exposed to PAHs with and without CYP1A1 inhibitors during macrophage differentiation to delineate PAH exposure and PAH metabolism-driven alterations to the immune response. U937 monocytes exposed to B[a]P, B[a]A, or 6-MIX had higher levels of cellular health and growth not observed following equimolar exposures to other individual PAHs. PAH exposures during differentiation did not alter monocyte-derived macrophage (MDM) numbers; however, B[a]A and 6-MIX exposures significantly altered M1/M2 polarization in a CYP1A1-dependent manner. U937-MDM adherence was differentially suppressed by all three PAH treatments with 6-MIX exposed U937-MDM having significantly more adhesion than U937-MDM exposed to either individual PAH. Finally, 6-MIX exposures during differentiation reduced U937-MDM endocytic function significantly less than B[a]A exposed cells. Exposure to a unique PAH mixture during U937-MDM differentiation resulted in mixture-specific alterations of pro-inflammatory markers compared to individual PAH exposures. While subtle, these differences highlight the probability that using a model PAH, B[a]P, may not accurately reflect the effects of PAH mixture exposures. Therefore, future studies should include various PAH mixtures that encompass probable real-world PAH exposures for the endpoints under investigation.
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Benzo(a)Antracenos/toxicidad , Benzopirenos/toxicidad , Diferenciación Celular/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Hidrocarburos Policíclicos Aromáticos/toxicidad , Diferenciación Celular/inmunología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , HumanosRESUMEN
Keratinization provides tolerance to desiccation and mechanical durability. Loricrin, which is an epidermal thiol-rich protein, efficiently stabilizes terminally differentiated keratinocytes and maintains redox homeostasis. The discovery of the largely asymptomatic loricrin knockout (LKO) phenotype decades ago was rather unpredicted. Nevertheless, when including redox-driven, NF-E2-related factor 2-mediated backup responses, LKO mice provide opportunities for the observation of altered or "quasi-normal" homeostasis. Specifically, given that the tissue structure, as well as the local metabolism, transmits immunological signals, we sought to dissect the consequence of truncated epidermal differentiation program from immunological perspectives. Through a review of the aggregated evidence, we have attempted to generate an integrated view of the regulation of the peripheral immune system, which possibly occurs within the squamous epithelial tissue with truncated differentiation. This synthesis might not only provide insights into keratinization but also lead to the identification of factors intrinsic to the epidermis that imprint the immune effector function.
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Células Cultivadas/inmunología , Impresión Molecular/métodos , Piel/fisiopatología , Animales , Humanos , RatonesRESUMEN
Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and non-expensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001). Under the conditions of this study, the use of centrifugation in density gradients allow for the simultaneous purification of PBMCs and neutrophils, with high purity of both populations, from the same sample of blood. The isolation of monocytes could be subsequently achieved through two different methods, i.e. based on immunomagnetic columns or adherence. The preference between both methods would depend on the necessities of the experiment, the initial sample with high purity of monocytes or a final population of MDMs required.
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Recuento de Células/métodos , Separación Celular/métodos , Separación Celular/normas , Leucocitos Mononucleares/fisiología , Macrófagos/fisiología , Rumiantes/inmunología , Animales , Recuento de Células/normas , Diferenciación Celular , Células Cultivadas/inmunología , Células Dendríticas/inmunología , Cabras/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Leucocitos/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Monocitos/inmunología , Ovinos/inmunologíaRESUMEN
Adjuvant cytokine-induced killer (CIK) cell immunotherapy has shown potential in improving the prognosis of hepatocellular carcinoma (HCC) patients after curative resection. However, whether an individual could obtain survival benefit from CIK cell treatment remains unknown. In the present study, we focused on the characteristics of CIK cells and aimed to identify the best predictive biomarker for adjuvant CIK cell treatment in patients with HCC after surgery. This study included 48 patients with HCC treated with postoperative adjuvant CIK cell immunotherapy. The phenotype activity and cytotoxic activity of CIK cells were determined by flow cytometry and xCELLigence™ Real-Time Cell Analysis (RTCA) system, respectively. Correlation analysis revealed that the cytotoxic activity of CIK cells was significantly negative correlated with the percentage of CD3+ CD4+ cell subsets, but significantly positive correlated with CD3-CD56+ and CD3+ CD56+ cell subsets. Survival analysis showed that there were no significant associations between patients' prognosis and the phenotype of CIK cells. By contrast, there was statistically significant improvement in recurrence-free survival (RFS) and overall survival (OS) for patients with high cytotoxic activity of CIK cells as compared with those with low cytotoxic activity of CIK cells. Univariate and multivariate analyses indicated that CIK cell cytotoxicity was an independent prognostic factor for RFS and OS. In conclusion, a high cytotoxic activity of CIK cells can serve as a valuable biomarker for adjuvant CIK cell immunotherapy of HCC patients after surgery.
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Carcinoma Hepatocelular/terapia , Células Asesinas Inducidas por Citocinas/trasplante , Citotoxicidad Inmunológica , Inmunoterapia/métodos , Neoplasias Hepáticas/terapia , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/mortalidad , Técnicas de Cultivo de Célula , Células Cultivadas/inmunología , Células Cultivadas/trasplante , Terapia Combinada/métodos , Células Asesinas Inducidas por Citocinas/inmunología , Pruebas Inmunológicas de Citotoxicidad , Femenino , Citometría de Flujo , Hepatectomía , Humanos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Periodo Posoperatorio , Pronóstico , Análisis de Supervivencia , Trasplante Autólogo/métodosRESUMEN
Background: Viral infections are considered a major driving factor of chronic obstructive pulmonary disease (COPD) exacerbations and thus contribute to disease morbidity and mortality. Respiratory syncytial virus (RSV) is a frequently detected pathogen in the respiratory tract of COPD patients during an exacerbation. We previously demonstrated in a murine model that leukemia inhibitory factor (LIF) expression was increased in the lungs during RSV infection. Subduing LIF signaling in this model enhanced lung injury and airway hypersensitivity. In this study, we investigated lung LIF levels in COPD patient samples to determine the impact of disease status and cigarette smoke exposure on LIF expression. Materials and methods: Bronchoalveolar lavage fluid (BALF) was obtained from healthy never smokers, smokers, and COPD patients, by written informed consent. Human bronchial epithelial (HBE) cells were isolated from healthy never smokers and COPD patients, grown at the air-liquid interface and infected with RSV or stimulated with polyinosinic:polycytidylic acid (poly (i:c)). Mice were exposed to cigarette smoke daily for 6 months and were subsequently infected with RSV. LIF expression was profiled in all samples. Results: In human BALF, LIF protein was significantly reduced in both smokers and COPD patients compared to healthy never smokers. HBE cells isolated from COPD patients produced less LIF compared to never smokers during RSV infection or poly (i:c) stimulation. Animals exposed to cigarette smoke had reduced lung levels of LIF and its corresponding receptor, LIFR. Smoke-exposed animals had reduced LIF expression during RSV infection. Two possible factors for reduced LIF levels were increased LIF mRNA instability in COPD epithelia and proteolytic degradation of LIF protein by serine proteases. Conclusions: Cigarette smoke is an important modulator for LIF expression in the lungs. Loss of LIF expression in COPD could contribute to a higher degree of lung injury during virus-associated exacerbations.
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Líquido del Lavado Bronquioalveolar/inmunología , Fumar Cigarrillos , Factor Inhibidor de Leucemia/análisis , Nicotiana/efectos adversos , Enfermedad Pulmonar Obstructiva Crónica , Mucosa Respiratoria , Infecciones por Virus Sincitial Respiratorio , Humo/efectos adversos , Animales , Células Cultivadas/inmunología , Fumar Cigarrillos/inmunología , Fumar Cigarrillos/patología , Modelos Animales de Enfermedad , Humanos , Exposición por Inhalación , Ratones , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/patología , Brote de los SíntomasRESUMEN
Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II.
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Angiotensina II/farmacología , Anticuerpos Monoclonales Humanizados/inmunología , Hipertensión/inmunología , Activación de Linfocitos/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Análisis de Varianza , Animales , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , Distribución de Chi-Cuadrado , Modelos Animales de Enfermedad , Humanos , Hipertensión/tratamiento farmacológico , Hipertensión/fisiopatología , Riñón/efectos de los fármacos , Riñón/inmunología , Riñón/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Persona de Mediana Edad , Distribución Aleatoria , Valores de Referencia , Muestreo , Estadísticas no Paramétricas , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
Inflammasomes are immune complexes that induce an inflammatory response upon sensing of different stress signals. This effect is mainly mediated by activation and secretion of the proinflammatory cytokines proIL-1ß and -18. Here we report that infection of human primary keratinocytes with the double-stranded DNA viruses modified vaccinia virus Ankara (MVA) or herpes simplex virus type 1 (HSV-1)-induced secretion of mature IL-1ß and -18. This secretion was dependent on several inflammasome complexes; however, the absent in melanoma 2 (AIM2) inflammasome, which is activated by binding of double-stranded DNA, played the most important role. Whereas prestimulation of keratinocytes with IFN-γ moderately increased MVA-induced IL-1ß and IL-18 secretion, it was essential for substantial secretion of these cytokines in response to herpes simplex virus type 1 infection. IFN-γ partially restored HSV-1 suppressed proIL-1ß expression and was also required for inflammasome activation. Most importantly, IFN-γ strongly suppressed virus replication in keratinocytes in vitro and ex vivo, which was independent of inflammasome activation. Our results suggest that, similar to Herpesviridae infection in mice, HSV-1 replication in human skin is controlled by a positive feedback loop of keratinocyte-derived IL-1/IL-18 and IFN-γ expressed by immune cells.
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Células Cultivadas/virología , Herpesvirus Humano 1/inmunología , Inflamasomas/inmunología , Interleucina-18/inmunología , Queratinocitos/inmunología , Análisis de Varianza , Animales , Células Cultivadas/inmunología , Herpes Simple/inmunología , Herpes Simple/fisiopatología , Humanos , Inmunidad Innata/fisiología , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Queratinocitos/metabolismo , RatonesRESUMEN
Allopurinol, a first-line drug for treating gout and hyperuricemia, is one of the leading causes of severe cutaneous adverse reactions (SCARs). To investigate the molecular mechanism of allopurinol-induced SCAR, we enrolled 21 patients (13 Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) and 8 drug reaction with eosinophilia and systemic symptoms (DRESS)), 11 tolerant controls, and 23 healthy donors. We performed in vitro T-cell activation assays by culturing peripheral blood mononuclear cells (PBMCs) with allopurinol, oxypurinol, or febuxostat and measuring the expression of granulysin and IFN-γ in the supernatants of cultures. TCR repertoire was investigated by next-generation sequencing. Oxypurinol stimulation resulted in a significant increase in granulysin in the cultures of blood samples from SCAR patients (n=14) but not tolerant controls (n=11) or healthy donors (n=23). Oxypurinol induced T-cell response in a concentration- and time-dependent manner, whereas allopurinol or febuxostat did not. T cells from patients with allopurinol-SCAR showed no crossreactivity with febuxostat. Preferential TCR-V-ß usage and clonal expansion of specific CDR3 (third complementarity-determining region) were found in the blister cells from skin lesions (n=8) and oxypurinol-activated T-cell cultures (n=4) from patients with allopurinol-SCAR. These data suggest that, in addition to HLA-B*58:01, clonotype-specific T cells expressing granulysin upon oxypurinol induction participate in the pathogenesis of allopurinol-induced SCAR.
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Alopurinol/efectos adversos , Antígenos de Diferenciación de Linfocitos T/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Oxipurinol/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Alopurinol/farmacología , Estudios de Casos y Controles , Células Cultivadas/inmunología , Reacciones Cruzadas , Erupciones por Medicamentos/etiología , Erupciones por Medicamentos/inmunología , Ensayo de Inmunoadsorción Enzimática , Febuxostat , Femenino , Humanos , Interferón gamma/efectos de los fármacos , Interferón gamma/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Oxipurinol/farmacología , Valores de Referencia , Síndrome de Stevens-Johnson/etiología , Síndrome de Stevens-Johnson/inmunología , Tiazoles/efectos adversos , Tiazoles/farmacologíaRESUMEN
OBJECTIVE: Interleukin-26 (IL-26) is a member of the IL-10 cytokine family, first discovered based on its peculiar expression by virus-transformed T cells. IL-26 is overexpressed in chronic inflammation (rheumatoid arthritis and Crohn's disease) and induces proinflammatory cytokines by myeloid cells and some epithelial cells. We thus investigated the expression and potential role of IL-26 in chronic HCV infection, a pathology associated with chronic inflammation. DESIGN: IL-26 was quantified in a cohort of chronically HCV-infected patients, naive of treatment and its expression in the liver biopsies investigated by immunohistochemistry. We also analysed the ability of IL-26 to modulate the activity of natural killer (NK) cells, which control HCV infection. RESULTS: The serum levels of IL-26 are enhanced in chronically HCV-infected patients, mainly in those with severe liver inflammation. Immunohistochemistry reveals an intense IL-26 staining in liver lesions, mainly in infiltrating CD3+ cells. We also show that NK cells from healthy subjects and from HCV-infected patients are sensitive to IL-26. IL-26 upregulates membrane tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression on CD16- CD56(bright) NK cells, enabling them to kill HCV-infected hepatoma cells, with the same efficacy as interferon (IFN)-α-treated NK cells. IL-26 also induces the expression of the antiviral cytokines IFN-ß and IFN-γ, and of the proinflammatory cytokines IL-1ß and TNF-α by NK cells. CONCLUSIONS: This study highlights IL-26 as a new player in the inflammatory and antiviral immune responses associated with chronic HCV infection.
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Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/inmunología , Interferón-alfa/uso terapéutico , Interleucinas/sangre , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Antivirales/uso terapéutico , Biomarcadores/sangre , Biopsia con Aguja , Antígeno CD56/inmunología , Antígeno CD56/metabolismo , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , Citocinas/metabolismo , Femenino , Hepatitis C Crónica/sangre , Humanos , Inmunohistoquímica , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Masculino , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Índice de Severidad de la Enfermedad , Estadísticas no ParamétricasRESUMEN
PURPOSE: Gender-related differences in sex hormones might have a key role in the development of atherosclerosis though direct vascular effects of sex hormones are not yet well understood. Thus, the main purpose of this study was to compare the effects of sex hormones on inflammatory response in Human Umbilical Vein Endothelial Cells (HUVECs) obtained from both male and female donors. METHODS: We analyzed the expression of receptors and enzymes relevant to the action of androgens (AR, 5α-reductase 1 and 5α-reductase 2) and estrogens (ERα, ERß, and aromatase) in male and female HUVECs. Furthermore, we analyzed the effect of testosterone (T), 17ß-estradiol (E2), dihydrotestosterone (DHT), and several androgenic-anabolic steroids (AAS) on VCAM-1, ICAM-1, and E-selectin gene expression and on adhesion of U937 cells to TNF-α-stimulated male and female HUVECs. RESULTS: Our results reveal that in HUVECs, regardless of gender, the components involved in the androgen action pathway are predominant as compared to those of estrogen action pathway. In both HUVEC genders, the inflammatory effect of TNF-α was amplified by co-administration of T or DHT and several AAS frequently used in doping, while E2 had no effect. CONCLUSIONS: This is the first study analyzing, under identical culture conditions, the key components of sex hormone response in male and female HUVECs and the possible role of sex hormones in regulating the endothelial inflammatory response. The data obtained in our experimental system showed a pro-inflammatory effect of androgens, while conclusively excluding any protective effect for all the tested hormones.
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Dihidrotestosterona/metabolismo , Estradiol/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Inflamación/metabolismo , Testosterona/metabolismo , Células Cultivadas/inmunología , Células Cultivadas/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Inflamación/inmunología , MasculinoRESUMEN
Adult corneal epithelium is often exposed to environmental stress, injured and repaired by limbal stem cells. Injury of corneal epithelial layer leads to reduction of visual clarity and loss of vision. Recently it was shown that epithelial layer also contains stem cells. Obtaining cell culture of corneal epithelium will allow understanding mechanisms of cell behavior and differentiation, their metabolism and reaction on environmental stress in health and disease. Moreover, cultured corneal epithelial cells can be considered as a promising material for constructing bioartificial cornea. The aim of this study was to isolate cells of anterior corneal epithelium from human donor cornea and to study their morphological and functional characteristics in vitro. The results of our study showed the possibility of culturing epithelial cells in vitro. The observed changes in cell morphology, their flow growth character as well as active proliferation and up-regulation of mesenchymal markers expression, indicate, in our opinion, epithelial-mesenchymal transition taking place in long-lasting culture of human anterior corneal epithelial cells. The obtained cultures can be used for further studies of pathological processes taking place in cells during drugs testing or controlling the phototoxic effect of different types of emission.
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Células Cultivadas/citología , Epitelio Corneal/citología , Cultivo Primario de Células/métodos , Células Madre Adultas/citología , Células Madre Adultas/inmunología , Células Madre Adultas/metabolismo , Anciano , Diferenciación Celular , Células Cultivadas/inmunología , Células Cultivadas/metabolismo , Epitelio Corneal/inmunología , Epitelio Corneal/metabolismo , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismoRESUMEN
After antigen encounter by CD4(+) T cells, polarizing cytokines induce the expression of master regulators that control differentiation. Inactivation of the histone methyltransferase Ezh2 was found to specifically enhance T helper 1 (Th1) and Th2 cell differentiation and plasticity. Ezh2 directly bound and facilitated correct expression of Tbx21 and Gata3 in differentiating Th1 and Th2 cells, accompanied by substantial trimethylation at lysine 27 of histone 3 (H3K27me3). In addition, Ezh2 deficiency resulted in spontaneous generation of discrete IFN-γ and Th2 cytokine-producing populations in nonpolarizing cultures, and under these conditions IFN-γ expression was largely dependent on enhanced expression of the transcription factor Eomesodermin. In vivo, loss of Ezh2 caused increased pathology in a model of allergic asthma and resulted in progressive accumulation of memory phenotype Th2 cells. This study establishes a functional link between Ezh2 and transcriptional regulation of lineage-specifying genes in terminally differentiated CD4(+) T cells.
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Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/fisiología , Complejo Represivo Polycomb 2/fisiología , Subgrupos de Linfocitos T/citología , Células TH1/citología , Células Th2/citología , Animales , Asma/genética , Asma/inmunología , Asma/patología , Diferenciación Celular , Células Cultivadas/citología , Células Cultivadas/inmunología , Células Cultivadas/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Factor de Transcripción GATA3/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Memoria Inmunológica , Ensayos de Liberación de Interferón gamma , Linfocinas/biosíntesis , Linfocinas/genética , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/deficiencia , Complejo Represivo Polycomb 2/genética , Procesamiento Proteico-Postraduccional , Eliminación de Secuencia , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Maintenance of blood DC homeostasis is essential to preventing autoimmunity while controlling chronic infection. However, the ability of bacteremic pathogens to directly regulate blood DC homeostasis has not been defined. One such bacteremic pathogen, Porphyromonas gingivalis, is shown by our group to survive within mDCs under aerobic conditions and therein, metastasize from its oral mucosal niche. This is accompanied by expansion of the blood mDC pool in vivo, independently of canonical DC poietins. We presently know little of how this bacteremic pathogen causes blood DC expansion and the pathophysiological significance. This work shows that optimum differentiation of MoDCs from primary human monocytes, with or without GM-CSF/IL-4, is dependent on infection with P. gingivalis strains expressing the DC-SIGN ligand mfa-1. DC differentiation is lost when DC-SIGN is blocked with its ligand HIV gp120 or knocked out by siRNA gene silencing. Thus, we have identified a novel, noncanonical pathway of DC differentiation. We term these PDDCs and show that PDDCs are bona fide DCs, based on phenotype and phagocytic activity when immature and the ability to up-regulate accessory molecules and stimulate allo-CD4(+) T cell proliferation when matured. The latter is dependent on the P. gingivalis strain used to initially "educate" PDDCs. Moreover, we show that P. gingivalis-infected, conventional MoDCs become resistant to apoptosis and inflammatory pyroptosis, as determined by levels of Annexin V and caspase-8, -3/7, and -1. Taken together, we provide new insights into how a relatively asymptomatic bacteremia may influence immune homeostasis and promote chronic inflammation.
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Bacteriemia/inmunología , Proteínas Bacterianas/inmunología , Moléculas de Adhesión Celular/inmunología , Células Dendríticas/patología , Proteínas Fimbrias/inmunología , Lectinas Tipo C/inmunología , Porphyromonas gingivalis/fisiología , Receptores de Superficie Celular/inmunología , Aerobiosis , Anexina A5/inmunología , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Caspasas/fisiología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Diferenciación Celular , Supervivencia Celular , Células Cultivadas/inmunología , Células Cultivadas/patología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Proteínas Fimbrias/deficiencia , Proteínas Fimbrias/genética , Fimbrias Bacterianas/inmunología , Proteína gp120 de Envoltorio del VIH/farmacología , Homeostasis , Interacciones Huésped-Patógeno/inmunología , Humanos , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/genética , Monocitos/citología , Mucosa Bucal/inmunología , Mucosa Bucal/microbiología , Fagocitosis , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/inmunología , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genéticaRESUMEN
The present study demonstrates that RA has activity of an IgA switch factor and is more specific than TGF-ß1. RA independently caused only IgA switching, whereas TGF-ß1 caused IgA and IgG2b switching. We found that RA increased IgA production and that this was a result of its ability to increase the frequency of IgA-secreting B cell clones. Increased IgA production was accompanied by an increase of GLTα. RA activity was abrogated by an antagonist of the RAR. Additionally, RA affected intestinal IgA production in mice. Surprisingly, RA, in combination with TGF-ß1, notably enhanced not only IgA production and GLTα expression but also CCR9 and α4ß7 expression on B cells. These results suggest that RA selectively induces IgA isotype switching through RAR and that RA and TGF-ß have important effects on the overall gut IgA antibody response.
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Inmunoglobulina A/inmunología , Cambio de Clase de Inmunoglobulina/efectos de los fármacos , Factor de Crecimiento Transformador beta1/inmunología , Tretinoina/farmacología , Animales , Células Cultivadas/inmunología , Selección Clonal Mediada por Antígenos , Endotoxinas/toxicidad , Genes de Inmunoglobulinas , Inmunidad Mucosa/efectos de los fármacos , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/inmunología , Integrinas/biosíntesis , Integrinas/genética , Ganglios Linfáticos/inmunología , Mesenterio/inmunología , Ratones , Ratones Endogámicos BALB C , Ganglios Linfáticos Agregados/inmunología , Receptores CCR/biosíntesis , Receptores CCR/genética , Receptores Mensajeros de Linfocitos/efectos de los fármacos , Receptores Mensajeros de Linfocitos/inmunología , Receptores de Ácido Retinoico/fisiologíaRESUMEN
DCs orchestrate immune responses to infectious pathogens and disturbances in tissue integrity. Equipped with C-type lectins, DCs can respond to environmental changes in glycosylation. Many C-type lectins are capable of modulating TLR activation, thereby facilitating tailor-made immune reactions. Here, we investigated the signaling properties of the C-type lectin MGL and show that MGL engagement by agonistic antibodies or carbohydrate ligands couples to TLR signal transduction for increased IL-10 and TNF-α secretion by human monocyte-derived DCs. MGL triggering especially synergized with TLR2-induced pathways, leading to elevated IL-10 mRNA levels and enhanced TNF-α mRNA stability. In addition, MGL signaling promoted phosphorylation of the MAPK ERK and the transcription factor CREB. Whereas specific inhibitors of p90RSK blocked the MGL-induced cytokine secretion, AP-1 was not involved. Strikingly, NF-κB was only crucial for the IL-10 response and dispensable for TNF-α production. Together, our results demonstrate that MGL activation of the ERK-p90RSK-CREB axis converges with TLR2-induced pathways, thereby fine-tuning the DC maturation phenotype.
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Interleucina-10/metabolismo , Lectinas Tipo C/fisiología , Receptor Toll-Like 2/fisiología , Acetilgalactosamina/inmunología , Secuencia de Aminoácidos , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Antígenos Virales/inmunología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/inmunología , Células Cultivadas/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Interleucina-10/biosíntesis , Interleucina-10/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/inmunología , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Fragmentos de Péptidos/inmunología , Fosforilación/efectos de los fármacos , Poliovirus/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , Toxina Tetánica/inmunología , Receptor Toll-Like 2/biosíntesis , Receptor Toll-Like 2/genética , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Activation of innate pattern-recognition receptors promotes CD4+ T-cell-mediated autoimmune myocarditis and subsequent inflammatory cardiomyopathy. Mechanisms that counterregulate exaggerated heart-specific autoimmunity are poorly understood. METHODS AND RESULTS: Experimental autoimmune myocarditis was induced in BALB/c mice by immunization with α-myosin heavy chain peptide and complete Freund's adjuvant. Together with interferon-γ, heat-killed Mycobacterium tuberculosis, an essential component of complete Freund's adjuvant, converted CD11b(hi)CD11c(-) monocytes into tumor necrosis factor-α- and nitric oxide synthase 2-producing dendritic cells (TipDCs). Heat-killed M. tuberculosis stimulated production of nitric oxide synthase 2 via Toll-like receptor 2-mediated nuclear factor-κB activation. TipDCs limited antigen-specific T-cell expansion through nitric oxide synthase 2-dependent nitric oxide production. Moreover, they promoted nitric oxide synthase 2 production in hematopoietic and stromal cells in a paracrine manner. Consequently, nitric oxide synthase 2 production by both radiosensitive hematopoietic and radioresistant stromal cells prevented exacerbation of autoimmune myocarditis in vivo. CONCLUSIONS: Innate Toll-like receptor 2 stimulation promotes formation of regulatory TipDCs, which confine autoreactive T-cell responses in experimental autoimmune myocarditis via nitric oxide. Therefore, activation of innate pattern-recognition receptors is critical not only for disease induction but also for counterregulatory mechanisms, protecting the heart from exaggerated autoimmunity.
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Enfermedades Autoinmunes/fisiopatología , Células Dendríticas/metabolismo , Tolerancia Inmunológica/fisiología , Interferón gamma/fisiología , Miocarditis/fisiopatología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico/biosíntesis , Transducción de Señal , Linfocitos T Colaboradores-Inductores/patología , Receptor Toll-Like 2/fisiología , Animales , Enfermedades Autoinmunes/inmunología , Cardiomiopatía Dilatada/etiología , Cardiomiopatía Dilatada/inmunología , Cardiomiopatía Dilatada/prevención & control , Diferenciación Celular/efectos de los fármacos , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , Inducción Enzimática/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/efectos de la radiación , Tolerancia Inmunológica/inmunología , Ratones , Ratones Endogámicos , Ratones Noqueados , Monocitos/citología , Monocitos/efectos de los fármacos , Mycobacterium tuberculosis/inmunología , Miocarditis/inmunología , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Comunicación Paracrina , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad , Quimera por Radiación , Tolerancia a Radiación , Células del Estroma/enzimología , Células del Estroma/efectos de la radiación , Linfocitos T Colaboradores-Inductores/inmunología , Miosinas Ventriculares/inmunología , Miosinas Ventriculares/toxicidadRESUMEN
Patients with systemic lupus erythematosus (SLE) display reduced numbers and functions of invariant natural killer T (iNK T) cells, which are restored upon treatment with corticosteroids and rituximab. It is unclear whether the iNK T cell insufficiency is a consequence of disease or is a primary abnormality that precedes the onset of disease. To address this, we analysed iNK T cell function at different stages of disease development using the genetically lupus-susceptible NZB × NZW F1 (BWF(1)) model. We found that iNK T cell in-vivo cytokine responses to an iNK T cell ligand α-galactosylceramide (α-GalCer) were lower in BWF(1) mice than in non-autoimmune BALB/c and major histocompatibility complex (MHC)-matched NZB × N/B10.PL F1 mice, although iNK T cell numbers in the periphery were unchanged in BWF(1) mice compared to control mice. Such iNK T cell hyporesponsiveness in BWF(1) mice was detected at a young age long before the animals exhibited any sign of autoimmunity. In-vivo activation of iNK T cells is known to transactivate other immune cells. Such transactivated T and B cell activation markers and/or cytokine responses were also lower in BWF(1) mice than in BALB/c controls. Finally, we show that iNK T cell responses were markedly deficient in the NZB parent but not in NZW parent of BWF(1) mice, suggesting that BWF(1) might inherit the iNK T cell defect from NZB mice. Thus, iNK T cells are functionally insufficient in lupus-prone BWF(1) mice. Such iNK T cell insufficiency precedes the onset of disease and may play a pathogenic role during early stages of disease development in SLE.
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Lupus Eritematoso Sistémico/inmunología , Células T Asesinas Naturales/inmunología , Síntomas Prodrómicos , Animales , Antígenos CD1/inmunología , Células Cultivadas/inmunología , Células Cultivadas/metabolismo , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Femenino , Galactosilceramidas/inmunología , Humanos , Lupus Eritematoso Sistémico/etiología , Activación de Linfocitos , Cooperación Linfocítica , Recuento de Linfocitos , Linfocinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NZB , Células T Asesinas Naturales/metabolismoRESUMEN
In Argentina, more than 3 million people suffer from asthma, with numbers rising. When asthma patients acquire viral infections which, in turn, trigger the asthmatic response, they may develop subsequent bacterial infections, mainly by Streptococcus (S.) pneumoniae. This encapsulated Gram(+) bacterium has been considered historically a T cell-independent antigen. Nevertheless, several papers describe the role of T cells in the immune response to S. pneumoniae. We evaluated the response to S. pneumoniae and compared it to the response to Mycobacterium (M.) tuberculosis, a different type of bacterium that requires a T helper type 1 (Th1) response, in cells from atopic asthmatic children, to compare parameters for the same individual under exacerbation and in a stable situation whenever possible. We studied asthma patients and a control group of age-matched children, evaluating cell populations, activation markers and cytokine production by flow cytometry, and cytokine concentration in serum and cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). No differences were observed in γδ T cells for the same patient in either situation, and a tendency to lower percentages of CD4(+) CD25(hi) T cells was observed under stability. A significantly lower production of tumour necrosis factor (TNF)-α and a significantly higher production of interleukin (IL)-5 was observed in asthma patients compared to healthy individuals, but no differences could be observed for IL-4, IL-13 or IL-10. A greater early activation response against M. tuberculosis, compared to S. pneumoniae, was observed in the asthmatic patients' cells. This may contribute to explaining why these patients frequently acquire infections caused by the latter bacterium and not the former.
Asunto(s)
Asma/inmunología , Streptococcus pneumoniae/inmunología , Subgrupos de Linfocitos T/inmunología , Células Th2/inmunología , Adolescente , Androstadienos/uso terapéutico , Antiasmáticos/uso terapéutico , Antígenos Bacterianos/inmunología , Asma/tratamiento farmacológico , Vacuna BCG , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , Células Cultivadas/metabolismo , Niño , Citocinas/sangre , Femenino , Fluticasona , Humanos , Inmunofenotipificación , Interferón gamma/sangre , Leucocitos Mononucleares/inmunología , Activación de Linfocitos , Masculino , Mycobacterium tuberculosis/inmunología , Adulto JovenRESUMEN
BACKGROUND: It has been well recognised that a deficit of numbers and function of CD4(+)CD25(+)Foxp3(+)cells (Treg) is attributed to the development of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue; however, there are controversial data regarding the suppressive effect of Treg cells on the T-cell response in auto-immune diseases. Additionally, interleukin-17 (IL-17)-producing cells (Th17) have a pro-inflammatory role. The balance between Th17 and Treg may be essential for maintaining immune homeostasis and has long been thought as one of the important factors in the development/prevention of auto-immune diseases, inflammatory diseases, tumour and rejection of transplanted tissue, but their role in multiple trauma remains unclear. OBJECTIVE: This study aims to investigate whether an imbalance of Treg and Th17 effector cells is characteristic of rats suffering from multiple trauma. METHODS AND SUBJECTIVE: Sixty Sprague-Dawley (SD) rats were randomly divided into three groups. The control group (n=20, group I) no received procedures (normal). The sham group (n=20, group II) only received anaesthesia, cannulation and observation. The bilateral femoral shaft fractures with haemorrhagic shock groups (n=20, group III). Rats in groups II and III were killed at the end of 4h after models were established. Peripheral blood samples were collected for assessment of Treg cells, Th17 cells and cytokines (IL-17, IL-6, IL-2, transforming growth factor beta (TGF-ß)) and intestine tissue was collected for intestine histological analysis. RESULTS: We observed decreased Treg/Th17 ratios in CD4(+)T cells in rats with multiple trauma and a strong inverse correlation with disease activity (intestinal histological scores). CONCLUSION: We suggest a role for immune imbalance in the pathogenesis and development of multiple trauma. The alteration of the index of Treg/Th17 cells likely indicates the therapeutic response and progress in the clinic.