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1.
Cell Physiol Biochem ; 54(1): 142-159, 2020 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-32028545

RESUMEN

BACKGROUND/AIMS: It is well established that oxidative stress and inflammation are common pathogenic features of retinal degenerative diseases. ITH12674 is a novel compound that induces the transcription factor Nrf2; in so doing, the molecule exhibits anti-inflammatory, and antioxidant properties, and affords neuroprotection in rat cortical neurons subjected to oxidative stress. We here tested the hypothesis that ITH12674 could slow the retinal degeneration that causes blindness in rd10 mice, a model of retinitis pigmentosa. METHODS: Animals were intraperitoneally treated with 1 or 10 mg/Kg ITH12674 or placebo from P16 to P30. At P30, retinal functionality and visual acuity were analyzed by electroretinography and optomotor test. By immunohistochemistry we quantified the photoreceptor rows and analyzed their morphology and connectivity. Oxidative stress and inflammatory state was studied by Western blot, and microglia reactivity was monitored by flow cytometry. The blood-brain barrier permeation of ITH12674 was evaluated using a PAMPA-BBB assay. RESULTS: In rd10 mice treated with 10 mg/Kg of the compound, the following changes were observed (with respect to placebo): (i) a decrease of vision loss with higher scotopic a- and b-waves; (ii) increased visual acuity; (iii) preservation of cone photoreceptors morphology, as well as their synaptic connectivity; (iv) reduced expression of TNF-α and NF-κB; (v) increased expression of p38 MAPK and Atg12-Atg5 complex; and (vi) decreased CD11c, MHC class II and CD169 positive cell populations. CONCLUSION: These data support the view that a Nrf2 inducer compound may arise as a new therapeutic strategy to combat retinal neurodegeneration. At present, we are chemically optimising compound ITH12674 with the focus on improving its neuroprotective potential in retinal neurodegenerative diseases.


Asunto(s)
Isotiocianatos/uso terapéutico , Melatonina/análogos & derivados , Factor 2 Relacionado con NF-E2/agonistas , Retinitis Pigmentosa/tratamiento farmacológico , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Isotiocianatos/química , Isotiocianatos/farmacología , Masculino , Melatonina/química , Melatonina/farmacología , Melatonina/uso terapéutico , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/metabolismo , Células Fotorreceptoras/efectos de los fármacos , Células Fotorreceptoras/patología , Retina/efectos de los fármacos , Retina/metabolismo , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Factor de Necrosis Tumoral alfa/metabolismo , Agudeza Visual/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
2.
Zhonghua Xin Xue Guan Bing Za Zhi ; 48(1): 61-65, 2020 Jan 24.
Artículo en Chino | MEDLINE | ID: mdl-32008297

RESUMEN

Objective: To investigate the feasibility of echocardiography-guided closed-chest repeated intraventricular blood sampling in mice, and to clarify the maximum blood volume that can be collected by this method, and whether the method can be used for long-term repeated blood collection in mice. Methods: Twenty-four male C57BL/6J mice (10-14 weeks old) were divided into the terminal experiment group (n=4, for investigating the maximum blood amount that could be sampled at one time), the repeated 0.5 ml blood collection group (n=10, sampling 0.5 ml whole blood each time, once every two days for consecutive 4 weeks), and the repeated 0.75 ml blood collection group (n=10, sampling 0.75 ml whole blood each time, once every two days for consecutive 4 weeks). High-frequency echocardiography was used to display the largest section of the left ventricle, guiding the insulin syringe needle through the thorax into the left ventricle for blood collection. In the repeated 0.5 ml blood collection group, echocardiography was used to detect the cardiac structure and function before blood collection, three minutes after blood collection, and one week after the last (the 14th) blood collection. Results: We successfully performed echocardiography-guided closed-chest intraventricular blood sampling, with an average operating time (88±19)s per mouse, and a maximum blood volume (1.43±0.11)ml per mouse. In the repeated 0.5 ml blood collection group, heart rate, left ventricular ejection fraction, left ventricular fractional shortening, left ventricular end-diastolic dimension and left ventricular posterior wall end-diastolic thickness remained uncganged before the first blood collection and after 4 weeks of repeated blood collection (all P>0.05). No death in the repeated 0.5 ml blood collection group. However, in the 0.75 ml blood collection group, two mice died before the end point. Conclusions: The echocardiography-guided closed-chest intraventricular blood sampling is a safe, minimally invasive, convenient and efficient method, and can be used repeatedly for long-term blood collection in mice.


Asunto(s)
Ecocardiografía , Ventrículos Cardíacos , Animales , Estudios de Factibilidad , Masculino , Ratones , Ratones Endogámicos C57BL , Función Ventricular Izquierda
3.
J Photochem Photobiol B ; 203: 111731, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31935633

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is a progressive and chronic inflammatory disease with a poor prognosis and very few available treatment options. Low-level laser therapy (LLLT) has been gaining prominence as a new and effective anti-inflammatory and immunomodulatory agent. Can lung inflammation and the airway remodeling be regulated by LLLT in an experimental model of IPF in C57Bl/6 mice? The present study investigated if laser attenuates cellular migration to the lungs, the airway remodeling as well as pro-fibrotic cytokines secretion from type II pneumocytes and fibroblasts. Mice were irradiated (780 nm and 30 mW) and then euthanized fifteen days after bleomycin-induced lung fibrosis. Lung inflammation and airway remodeling were evaluated through leukocyte counting in bronchoalveolar lavage fluid (BALF) and analysis of collagen in lung, respectively. Inflammatory cells in blood were also measured. For in vitro assays, bleomycin-activated fibroblasts and type II pneumocytes were irradiated with laser. The pro- and anti-inflammatory cytokines level in BALF as well as cells supernatant were measured by ELISA, and the TGFß in lung was evaluated by flow cytometry. Lung histology was used to analyze collagen fibers around the airways. LLLT reduced both migration of inflammatory cells and deposition of collagen fibers in the lungs. In addition, LLLT downregulated pro-inflammatory cytokines and upregulated the IL-10 secretion from fibroblasts and pneumocytes. Laser therapy greatly reduced total lung TGFß. Systemically, LLLT also reduced the inflammatory cells counted in blood. There is no statistical difference in inflammatory parameters studied between mice of the basal group and the laser-treated mice. Data obtained indicate that laser effectively attenuates the lung inflammation, and the airway remodeling in experimental pulmonary fibrosis is driven to restore the balance between the pro- and anti-inflammatory cytokines in lung and inhibit the pro-fibrotic cytokines secretion from fibroblasts.


Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Citocinas/metabolismo , Fibrosis Pulmonar Idiopática/radioterapia , Rayos Láser , Animales , Líquido del Lavado Bronquioalveolar/química , Células Cultivadas , Citocinas/análisis , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de la radiación , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Fibrosis Pulmonar Idiopática/patología , Terapia por Láser , Masculino , Ratones , Ratones Endogámicos C57BL , Regulación hacia Arriba/efectos de la radiación
4.
Cancer Immunol Immunother ; 69(2): 199-213, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31982939

RESUMEN

Neutrophils play a major role in tumor biology. Among other functions, neutrophils can release extracellular traps (NETs), mesh-like structures of decondensed chromatin fibers, in a process termed NETosis. Originally characterized as an antimicrobial mechanism, NETosis has been described in cancer, but cancer-related predisposition is not clear. In the current study, we investigated the predisposition of circulating neutrophils to release NETs in lung cancer and the impact of G-CSF on this function, comparing circulating neutrophils isolated from cancer patients to the LLC and AB12 mouse models. We find that neutrophils from both healthy donors and cancer patients display high NETotic potential, with 30-60% of cells undergoing NETosis upon PMA stimulation. In contrast, neutrophils isolated from tumor-bearing mice displayed only 4-5% NETotic cells, though significantly higher than naive controls (1-2%). Despite differential mechanisms of activation described, Ionomycin and PMA mainly triggered suicidal rather than vital NETosis. G-CSF secreting tumors did not increase NETotic rates in murine neutrophils, and direct G-CSF stimulation did not promote their NET release. In contrast, human neutrophils strongly responded to G-CSF stimulation resulting also in a higher response to PMA + G-CSF stimulation. Our data show clear differences in NETotic potentials between human and murine neutrophils. We do not find a predisposition of neutrophils to release NETs in lung cancer patients compared to healthy controls, whereas cancer may modulate neutrophils' NETotic potential in mice. G-CSF secreted from tumors differentially affects murine and human NETosis in cancer. These important differences should be considered in future studies of NETosis in cancer.


Asunto(s)
Trampas Extracelulares/fisiología , Neoplasias Pulmonares/inmunología , Neutrófilos/fisiología , Animales , Línea Celular Tumoral , Trampas Extracelulares/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Ionomicina/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Acetato de Tetradecanoilforbol/farmacología
5.
Life Sci ; 242: 117189, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31891724

RESUMEN

AIMS: Neointimal hyperplasia contributes to arterial restenosis after percutaneous transluminal coronary angioplasty or vascular surgery. Neointimal thickening after arterial injury is determined by inflammatory processes. We investigated the role of the innate immune receptor toll-like receptor 2 (TLR2) in neointima formation after arterial injury in mice. MATERIALS AND METHODS: Carotid artery injury was induced by 10% ferric chloride in C57Bl/6J wild type (WT), TLR2 deficient (B6.129-Tlr2tm1Kir/J, TLR2-/-) and WT mice treated with a TLR2 blocking antibody. 21 days after injury, carotid arteries were assessed histomorphometrically and for smooth muscle cell (SMC) content. To identify the contribution of circulating cells in mediating the effects of TLR2-deficiency, arterial injury was induced in WT/TLR2-/--chimeric mice and the paracrine modulation of bone marrow-derived cells from WT and TLR2-/- on SMC migration compared in vitro. KEY FINDINGS: TLR2-/- mice and WT mice treated with TLR2 blocking antibodies exhibited reduced neointimal thickening (23.7 ± 4.2 and 6.5 ± 3.0 vs. 43.1 ± 5.9 µm, P < 0.05 and P < 0.01), neointimal area (5491 ± 1152 and 315 ± 76.7 vs. 13,756 ± 2627 µm2, P < 0.05 and P < 0.01) and less luminal stenosis compared to WT mice (8.5 ± 1.6 and 5.0 ± 1.3 vs. 22.4 ± 2.2%, both P < 0.001n = 4-8 mice/group). The phenotypes of TLR2-/- vs. WT mice were completely reverted in WT/TLR2-/- bone marrow chimeric mice (5.9 ± 1.5 µm neointimal thickness, 874.2 ± 290.2 µm2 neointima area and 2.7 ± 0.6% luminal stenoses in WT mice transplanted with TLR2-/- bone marrow vs. 23.6 ± 5.1 µm, 3555 ± 511 µm2 and 12.0 ± 1.3% in WT mice receiving WT bone marrow, all P < 0.05, n = 6/group). Neointimal lesions of WT and WT mice transplanted with TLR2-/- bone marrow chimeric mice showed increased numbers of SMC (10.8 ± 1.4 and 12.6 ± 1.4 vs. 3.8 ± 0.9 in TLR2-/- and 3.5 ± 1.1 cells in WT mice transplanted with TLR2-/- bone marrow, all P < 0.05, n = 6). WT bone marrow cells stimulated SMC migration more than TLR2-deficient bone marrow cells (1.7 ± 0.05 vs. 1.3 ± 0.06-fold, P < 0.05, n = 7) and this effect was aggravated by TLR2 stimulation and diminished by TLR2 blockade (1.1 ± 0.03-fold after stimulation with TLR2 agonists and 0.8 ± 0.02-fold after TLR2 blockade vs. control treated cells defined as 1.0, P < 0.05, n = 7). SIGNIFICANCE: TLR2-deficiency on hematopoietic but not vessel wall resident cells augments vascular healing after arterial injury. Pharmacological blockade of TLR2 may thus be a promising therapeutic option to improve vessel patency after iatrogenic arterial injury.


Asunto(s)
Células de la Médula Ósea/metabolismo , Receptor Toll-Like 2/deficiencia , Cicatrización de Heridas , Animales , Arterias/lesiones , Trasplante de Médula Ósea , Traumatismos de las Arterias Carótidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/lesiones , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Neointima/metabolismo , Neointima/patología , Receptor Toll-Like 2/metabolismo
6.
Clin Sci (Lond) ; 134(2): 239-259, 2020 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-31943002

RESUMEN

Mitochondrial stress has been widely observed in diabetic kidney disease (DKD). Cyclophilin D (CypD) is a functional component of the mitochondrial permeability transition pore (mPTP) which allows the exchange of ions and solutes between the mitochondrial matrix to induce mitochondrial swelling and activation of cell death pathways. CypD has been successfully targeted in other disease contexts to improve mitochondrial function and reduced pathology. Two approaches were used to elucidate the role of CypD and the mPTP in DKD. Firstly, mice with a deletion of the gene encoding CypD (Ppif-/-) were rendered diabetic with streptozotocin (STZ) and followed for 24 weeks. Secondly, Alisporivir, a CypD inhibitor was administered to the db/db mouse model (5 mg/kg/day oral gavage for 16 weeks). Ppif-/- mice were not protected against diabetes-induced albuminuria and had greater glomerulosclerosis than their WT diabetic littermates. Renal hyperfiltration was lower in diabetic Ppif-/- as compared with WT mice. Similarly, Alisporivir did not improve renal function nor pathology in db/db mice as assessed by no change in albuminuria, KIM-1 excretion and glomerulosclerosis. Db/db mice exhibited changes in mitochondrial function, including elevated respiratory control ratio (RCR), reduced mitochondrial H2O2 generation and increased proximal tubular mitochondrial volume, but these were unaffected by Alisporivir treatment. Taken together, these studies indicate that CypD has a complex role in DKD and direct targeting of this component of the mPTP will likely not improve renal outcomes.


Asunto(s)
/metabolismo , Diabetes Mellitus Experimental/metabolismo , Enfermedades Renales/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Albuminuria/genética , Albuminuria/metabolismo , Animales , /genética , Ciclosporina/farmacología , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/metabolismo , Peróxido de Hidrógeno/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Enfermedades Renales/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , ATPasas de Translocación de Protón/metabolismo
7.
Nat Commun ; 11(1): 538, 2020 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-31988323

RESUMEN

Lymphatic endothelial cells (LECs) chemoattract naïve T cells and promote their survival in the lymph nodes, and can cross-present antigens to naïve CD8+ T cells to drive their proliferation despite lacking key costimulatory molecules. However, the functional consequence of LEC priming of CD8+ T cells is unknown. Here, we show that while many proliferating LEC-educated T cells enter early apoptosis, the remainders comprise a long-lived memory subset, with transcriptional, metabolic, and phenotypic features of central memory and stem cell-like memory T cells. In vivo, these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall, and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8+ T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Endoteliales/fisiología , Animales , Proliferación Celular , Células Endoteliales/inmunología , Perfilación de la Expresión Génica , Memoria Inmunológica , Ratones Endogámicos C57BL , Ratones Transgénicos
8.
Nat Commun ; 11(1): 524, 2020 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-31988324

RESUMEN

Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses can provide a context optimal for generating antigen-specific CD8 T cells, as they have natural tropism for dendritic cells, preeminent inducers of CD8 T cell immunity; elicit Th1-promoting inflammation; and lack interference with innate or adaptive immunity. However, notorious genetic instability and underlying neuropathogenicity has hampered poliovirus-based vector applications. Here we devised a strategy based on the polio:rhinovirus chimera PVSRIPO, devoid of viral neuropathogenicity after intracerebral inoculation in human subjects, for stable expression of exogenous antigens. PVSRIPO vectors infect, activate, and induce epitope presentation in DCs in vitro; they recruit and activate DCs with Th1-dominant cytokine profiles at the injection site in vivo. They efficiently prime tumor antigen-specific CD8 T cells in vivo, induce CD8 T cell migration to the tumor site, delay tumor growth and enhance survival in murine tumor models.


Asunto(s)
Linfocitos T CD8-positivos/fisiología , Células Dendríticas/inmunología , Poliovirus/inmunología , Animales , Vacunas contra el Cáncer , Vectores Genéticos/inmunología , Glioma/inmunología , Células HEK293 , Células HeLa , Humanos , Inmunidad Innata , Inmunoterapia/métodos , Interferón Tipo I/inmunología , Melanoma/inmunología , Ratones , Ratones Endogámicos C57BL , Poliovirus/genética
9.
Sichuan Da Xue Xue Bao Yi Xue Ban ; 51(1): 1-6, 2020 Jan.
Artículo en Chino | MEDLINE | ID: mdl-31950781

RESUMEN

Objective: To construct a recombinant Listeria ivanovii (LI) strain that expressed Mycobacterium tuberculosis (MTB) specific antigen protein as a novel multistage tuberculosis (TB) vaccine candidate, and evaluate the biosafety and immunogenicity in mouse model. Methods: T cell epitopes of four genes related to different stages of MTB infection were fused in series to form an antigen gene, i.e. the multistage antigen gene (named msv). Then msv was inserted into the targeting plasmid that contained LI homologous sequences. Recombinant LI strain was obtained by transfecting LI with targeting plasmid and screening the recombinant LI strain that carried msv in the genome after series of homologous gene recombination processes. The growth rate of the recombinant LI strain in vitro was observed and the expression of target protein was verified by Western blot. The 50% lethal dose (LD 50) of the recombinant strain to C57BL/6 mice was measured. Mice were intravenously inoculated with vaccine candidate in dose of 0.1×LD 50.The serum alanine aminotransferase (ALT) levels, bacterial load in organs, and organ pathological sections before and 1, 2, 3, 5, 7, 14 d after vaccination were used to evaluate the safety of vaccine candidate strain. To analyze the immunogenicity of vaccine candidate strain, mice were intravenously inoculated with LI- msv, LI, and NS respectively. Nine days post immunization, the spleens were isolated under sterile conditions and splenocytes were collected and stimulated. Lyphocytes which secret specific cytokines, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2 were analyzed by flow cytometry. Results: A recombinant strain named LI- msv which was capable of expressing the multistage TB antigen protein was successfully constructed. The LD 50 value of LI- msv for C57BL/6 mice (i.v.) was 3.3×10 8 CFU. After intravenously immunized the mice, this strain mainly multiplied in the liver and spleen, and was cleared at 7 d post innoculation. Such infection process caused transient pathological damages of the liver and spleen. Results of flow cytometry showed specific IFN-γ + CD4 + and IFN-γ + CD8 +T lymphocytes were successfully induced in LI -msv immunized mice spleen lymphocytes. The frequency of IFN-γ positive CD4 + and CD8 +T cells was significantly higher than those of vector control group and NS control group ( P<0.005). Additionally, the frequency of specific TNF-α + CD4 + T cell in LI -msv immunized group was significantly higher than that of vector control ( P<0.01) and NS control group ( P<0.005), and TNF-α + CD8 + T cell frequency obviously increased than NS control group ( P<0.005). Conclusions: A novel multistage TB vaccine candidate expressing TB multistage antigen based on LI was successfully constructed. This vaccine candidate is safe and can induce specific cellular immune response to some extent. It is promising to be further studied as a candidate vaccine against tuberculosis.


Asunto(s)
Antígenos Bacterianos , Listeria , Mycobacterium tuberculosis , Vacunas contra la Tuberculosis , Tuberculosis/prevención & control , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Inmunidad Celular/inmunología , Listeria/genética , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Tuberculosis/inmunología , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/normas
10.
Immunity ; 52(1): 151-166.e6, 2020 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-31924474

RESUMEN

In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation.


Asunto(s)
Granzimas/inmunología , Neoplasias/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Proteínas de Dominio T Box/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/trasplante , Traslado Adoptivo , Animales , Línea Celular Tumoral , Humanos , Interferón gamma/inmunología , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/citología , Microambiente Tumoral/inmunología
11.
Immunity ; 52(1): 136-150.e6, 2020 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-31940267

RESUMEN

Effector CD8+ T cells are important mediators of adaptive immunity, and receptor-ligand interactions that regulate their survival may have therapeutic potential. Here, we identified a subset of effector CD8+ T cells that expressed the inhibitory fragment crystallizable (Fc) receptor FcγRIIB following activation and multiple rounds of division. CD8+ T cell-intrinsic genetic deletion of Fcgr2b increased CD8+ effector T cell accumulation, resulting in accelerated graft rejection and decreased tumor volume in mouse models. Immunoglobulin G (IgG) antibody was not required for FcγRIIB-mediated control of CD8+ T cell immunity, and instead, the immunosuppressive cytokine fibrinogen-like 2 (Fgl2) was a functional ligand for FcγRIIB on CD8+ T cells. Fgl2 induced caspase-3/7-mediated apoptosis in Fcgr2b+, but not Fcgr2b-/-, CD8+ T cells. Increased expression of FcγRIIB correlated with freedom from rejection following withdrawal from immunosuppression in a clinical trial of kidney transplant recipients. Together, these findings demonstrate a cell-intrinsic coinhibitory function of FcγRIIB in regulating CD8+ T cell immunity.


Asunto(s)
Apoptosis/inmunología , Linfocitos T CD8-positivos/inmunología , Fibrinógeno/inmunología , Receptores de IgG/inmunología , Adulto , Anciano , Animales , Caspasa 3/inmunología , Caspasa 7/inmunología , Línea Celular Tumoral , Femenino , Fibrinógeno/genética , Rechazo de Injerto/inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunosupresión , Masculino , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptores de IgG/genética , Adulto Joven
12.
Plast Reconstr Surg ; 145(2): 420-431, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31985635

RESUMEN

BACKGROUND: Secondary lymphedema is a refractory disease, for which adipose-derived stem cells have shown some therapeutic potential. However, the mechanism of this action remains poorly understood. METHODS: The authors identified podoplanin-expressing adipose-derived stem cells, which allowed them to divide adipose-derived stem cells into podoplanin-positive and podoplanin-negative groups that they characterized in vitro. The authors then used a mouse hindlimb model for lymphedema to trace the fate of podoplanin-positive, podoplanin-negative, and unsorted adipose-derived stem cells in vivo. RESULTS: When induced in culture, podoplanin-positive cells were noted to up-regulate the expression of lymphatic endothelial cell markers, including LYVE-1, and assumed a cobblestone morphology. In addition, a substantial increase in lymphangiogenic cytokines was detected in the podoplanin-positive supernatant. The above findings were largely absent from the podoplanin-negative and unsorted groups. In the mouse model, the implanted cells relieved the limb lymphedema by promoting lymphangiogenesis, with the podoplanin-positive group showing the most significant effect. Immunocolocalization further revealed that the podoplanin-positive cells incorporated into lymphatic vessels were positive for LYVE-1. CONCLUSIONS: These data demonstrated that actions by means of both paracrine and differentiation pathways were involved in the adipose-derived stem cell-mediated therapeutic effects. The podoplanin-positive cells possessed lymphatic paracrine and differentiation abilities and may represent lymphatic endothelial cell precursor cells. The podoplanin-negative cells, which constitute a considerable proportion of the adipose-derived stem cells, may play an important paracrine role by secreting mesenchymal stem cell-related factors.


Asunto(s)
Linfangiogénesis/fisiología , Vasos Linfáticos/fisiología , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/fisiología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/fisiología , Femenino , Proteínas Fluorescentes Verdes , Linfedema/fisiopatología , Ratones Endogámicos C57BL , Ratones Transgénicos , Neovascularización Fisiológica/fisiología , Fenotipo
13.
Invest Ophthalmol Vis Sci ; 61(1): 3, 2020 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-31995154

RESUMEN

Purpose: The purpose of this study was to investigate the expression of death ligands in the lacrimal glands (LGs), identify upstream factors that regulate their expression, and determine the functional roles of these factors in the pathogenesis of dry eye disease (DED). Methods: For DED experiment, ex vivo coculture system with LG and in vivo murine model using a controlled environment chamber were utilized. C57BL/6 mice and hypoxia-inducible factor (HIF)-1α conditional knockout (CKO) mice were used. Immunohistochemical staining, polymerase chain reaction, and immunoblotting were performed to determine levels of death ligands including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in DED-induced LGs. Additionally, acinar cell and CD45+ cell apoptosis was determined with neutralizing TRAIL treatment. Results: Desiccating stress significantly increased HIF-1α expression in LG-acinar cells. Furthermore, HIF-1α deficiency significantly enhanced the infiltration of CD45+ inflammatory cells in LG and induced LG-acinar cell death. Meanwhile, only TRAIL expression was increased in DED-LG, but abrogated in HIF-1α CKO. Interestingly, the main source of TRAIL was the CD45- LG-acinar cells, but not CD45+ immune cells after DED induction. Using ex vivo coculture system, we confirmed LG-induced apoptosis of immune cells via HIF-1α-mediated TRAIL secretion following DED. Consistent with ex vivo, the insufficiency of HIF-1α and TRAIL enhanced recruitment of inflammatory cells to the LG and subsequently exacerbated ocular surface damage in DED mice. Conclusions: Our findings offer novel insight into the regulatory function of acinar cell-derived TRAIL in limiting inflammatory damage and could be implicated in the development of potential therapeutic strategies for DED.


Asunto(s)
Dacriocistitis/metabolismo , Síndromes de Ojo Seco/metabolismo , Regulación de la Expresión Génica/fisiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Aparato Lagrimal/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Animales , Apoptosis , Técnicas de Cocultivo , Dacriocistitis/patología , Modelos Animales de Enfermedad , Síndromes de Ojo Seco/patología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Immunoblotting , Etiquetado Corte-Fin in Situ , Aparato Lagrimal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Reacción en Cadena en Tiempo Real de la Polimerasa
14.
Life Sci ; 242: 117239, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31901444

RESUMEN

AIMS: Reactive oxygen species (ROS) and pro-inflammatory cytokines play a critical role in organ damage induced by ethanol consumption. Interleukin (IL)-10 maintain tissue homeostasis through restriction of excessive inflammatory responses and inhibition of ROS generation. These responses limit unnecessary tissue damage in the cardiorenal system. We hypothesized that IL-10 would limit the deleterious effects induced by ethanol consumption in the cardiorenal system. MATERIALS AND METHODS: Male C57BL/6J wild-type (WT) or IL10-deficient mice (IL-10-/-) were treated with ethanol (20% v/v) for 6 weeks. KEY FINDINGS: IL-10 deficiency was associated with an increased mortality rate. Ethanol consumption decreased plasma levels of IL-10 in WT mice. Increased levels of IL-6 were detected in the aorta from IL-10-deficient mice, but not WT mice. No alterations in the levels of urea, creatinine, sodium, potassium or creatine kinase (CK)-MB were found after treatment with ethanol. Augmented concentration of thiobarbituric acid reactive substances (TBARS) was found in the left ventricle (LV) of IL-10-deficient mice, but not WT mice. Increased levels of superoxide anion (O2-) were found in the renal cortex of both WT and IL-10-deficient mice. Renal cortex from WT mice chronically treated with ethanol showed decreased levels of H2O2. No changes in the expression of Nox1, Nox4 or catalase were found in the renal cortex from ethanol-treated mice. SIGNIFICANCE: IL-10 limited the production of ROS and the synthesis of pro-inflammatory cytokines induced by ethanol in the cardiorenal system. These findings provided novel evidence that IL-10 counteracted the initial mechanisms whereby ethanol induces its cardiorenal damages.


Asunto(s)
Etanol/efectos adversos , Corazón/efectos de los fármacos , Interleucina-10/metabolismo , Riñón/efectos de los fármacos , Lesión Renal Aguda/inducido químicamente , Animales , Western Blotting , Forma MB de la Creatina-Quinasa/sangre , Creatinina/sangre , Interleucina-10/sangre , Interleucina-10/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Potasio/sangre , Sodio/sangre , Urea/sangre
15.
Zhonghua Yan Ke Za Zhi ; 56(1): 32-40, 2020 Jan 11.
Artículo en Chino | MEDLINE | ID: mdl-31937061

RESUMEN

Objective: To investigate the role and mechanism of microglial activation in the process of retinal ganglion cell (RGC) death in the oxygen-glucose deprivation/reperfusion (OGD/R) model which mimicked retinal ischemia/reperfusion injury in vitro. Methods: Experimental study. Primary RGCs from C57BL/6 mice and BV2 microglia were co-cultured or cultured alone. The OGD/R model was established in vitro (reoxygenation time was set to 6 h, 24 h, 36 h, 48 h). BV2 microglial activation was assessed by immunofluorescence staining of ionized calcium binding adapter molecule 1 (iba1), and the survival rate of RGCs was detected by the Cell Counting Kit-8. The apoptosis rate of RGC was detected by using apoptosis detection kit. The levels of Toll-like receptor-4 (TLR4) and Nod-like receptor family pyrin domain containing protein 3 (NLRP3) in BV2 cells were detected by PCR, Western-blot and immunofluorescence staining. The activity of caspase-8 in BV2 cells was detected by the CaspGLOW Kit, and the content of interleukine-1ß (IL-1ß) in the supernatant was detected by enzyme linked immunosorbent assay. After the corresponding pathways were blocked by TLR4 small interfering RNA (siRNA) transfection or caspase-8 inhibitor, the expression changes of TLR4 and NLRP3, the activity of caspase-8, and the difference of IL-1ß content could be observed as well as the activity of RGCs co-cultured with BV2. Statistical analysis was performed using analysis of variance. Results: Under co-culture of RGC and BV2 cells, cellular immunofluorescence detection showed that the expression of iba1 in BV2 cells increased, which indicated BV2 cells were activated significantly in the OGD/R model. In the OGD/R model, the apoptosis rate of RGC co-cultured with BV2 cells (71.1%±3.2%) was significantly higher than that of RGC cultured alone (35.1%±1.8%) (t=10.10, P<0.01). Cellular immunofluorescence detection showed that the expression of TLR4 and NLRP3 in BV2 cells in the OGD/R model increased significantly when BV2 cells were cultured alone, and their mRNA levels increased significantly with prolongation of reoxygenation time (F=64.45, 72.74; P<0.01), and peaked at OGD/R 24 h (TLR4 mRNA, relative ratio to control was 2.83±0.23; NLRP3 mRNA, relative ratio to control was 3.12±0.27). Caspase-8 activity also increased with prolonged reoxygenation time, the difference was statistically significant (F=93.57, P<0.01), and peaked at OGD/R 24 h (relative ratio to control was 2.92±0.31). After transfection of BV2 cells with TLR4 siRNA, its caspase-8 activity was significantly inhibited, but using caspase-8 inhibitor did not affect the up-regulation of TLR4 expression in BV2 cells. However, the mature IL-1ß secreted by BV2 cells exposed to OGD/R was significantly reduced by using caspase-8 inhibitor (from 3.52±0.55 to 1.39±0.37, t=7.19, P<0.01), meanwhile, the expression of NLRP3 was also significantly decreased after caspase-8 inhibitor pretreatment (from 2.79±0.23 to 1.37±0.19, t=9.37, P<0.01). In the OGD/R model, the activity of RGC cells co-cultured with TLR4 siRNA-transfected BV2 cells was 74.5%±1.2%, and the activity of RGC cells co-cultured with BV2 cells treated with caspase-8 inhibitor was 62.8%±1.5%, those were both higher than that of RGC cells co-cultured with untreated BV2 cells (36.7%±0.3%), and the difference was statistically significant (t=11.60, 6.83; both P<0.01). Conclusion: TLR4-caspase-8-NLRP3 inflammasome pathway is activated in microglia exposed to OGD/R, resulting in the production of IL-1ß, thereby contributing to the death of RGCs. (Chin J Ophthalmol, 2020, 56: 32-40).


Asunto(s)
Caspasa 8/metabolismo , Muerte Celular , Inflamasomas/metabolismo , Microglía/metabolismo , Células Ganglionares de la Retina/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Caspasa 8/genética , Línea Celular , Inflamasomas/genética , Ratones , Ratones Endogámicos C57BL , Hipertensión Ocular , Daño por Reperfusión , Receptor Toll-Like 4/genética
16.
Adv Exp Med Biol ; 1232: 401-408, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31893437

RESUMEN

Parkinson's disease, a progressive neurodegenerative disease, is caused by the loss of dopaminergic neurons in the substantia nigra (SN). It is characterized by the formation of intracytoplasmic Lewy bodies that are primarily composed of the protein alpha-synuclein (α-syn), along with dystrophic neurites. Acupuncture stimulation results in an enhanced survival of dopaminergic neurons in the SN in Parkinsonism animal models. We investigated the role of acupuncture in inhibiting the increase in α-syn expression that is related to dopaminergic cell loss in the SN in a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinsonism mouse model. In this model, acupuncture stimulation at GB34 and LR3 attenuated the decrease in tyrosine hydroxylase in the SN. Moreover, acupuncture stimulation attenuated the increase in α-syn in SN. Acupuncture stimulation also maintained the phosphorylated α-syn on serine 129 at levels similar to the control group. Our findings indicate that the MPTP-mediated increase in α-syn, and the acupuncture-mediated inhibition of the increase in α-syn, may be responsible for the neuroprotective effects of acupuncture in the SN following damage induced by MPTP.


Asunto(s)
Terapia por Acupuntura , Trastornos Parkinsonianos , Sustancia Negra , alfa-Sinucleína , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/patología , Ratones , Ratones Endogámicos C57BL , Fármacos Neuroprotectores , Trastornos Parkinsonianos/inducido químicamente , Sustancia Negra/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , alfa-Sinucleína/metabolismo
17.
J Agric Food Chem ; 68(3): 779-787, 2020 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-31894986

RESUMEN

The chain length of fructan determines its different physiological effects. This study is to explore the effects of low-performance inulin [LPI, degree of polymerization (DP) ≤ 9] and high-performance inulin (HPI, DP ≥ 23) on obesity-associated liver injury of high-fat diet (HFD) feeding mice and its underlying mechanism. Eight weeks of supplementation of C57BL/6J mice with HPI, relative to LPI (p < 0.05), caused the more efficient improvement against the HFD-induced liver insulin resistance through activating IRS1/PI3K/Akt pathway and reduced protein expressions of inflammatory factors nuclear factor-kappaB (NF-κB) and interleukin-6 (IL-6) in the liver. HPI exhibited the more positive effects on liver steatosis by inhibiting acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and sterol regulatory element binding protein 1 (SREBP1) in comparison with LPI (p < 0.05). HPI also increased acetic acid, propionic acid, and butyric acid levels in the colon of HFD-fed mice (p < 0.05). Compared to LPI, HPI feeding of HFD-fed mice led to the more effective decrease in the Firmicutes abundance from 72.1% to 34.5%, but a more significant increase in the Bacteroidetes population from 19.8 to 57.1% at the phyla level, and increased the abundance of Barnesiella, Bacteroides, and Parabacteroides at the genus level (p < 0.05). Depending on DP, HPI exerts the more positive regulation on liver injury and gut microbiota dysfunction than LPI.


Asunto(s)
Disbiosis/tratamiento farmacológico , Microbioma Gastrointestinal/efectos de los fármacos , Inulina/administración & dosificación , Inulina/química , Hígado/lesiones , Obesidad/tratamiento farmacológico , Animales , Bacterias/clasificación , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/aislamiento & purificación , Dieta Alta en Grasa/efectos adversos , Suplementos Dietéticos/análisis , Disbiosis/genética , Disbiosis/metabolismo , Disbiosis/microbiología , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , FN-kappa B/genética , FN-kappa B/metabolismo , Obesidad/genética , Obesidad/metabolismo , Obesidad/microbiología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Polimerizacion
18.
J Exp Med ; 217(4)2020 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-31985756

RESUMEN

In a forward genetic screen of N-ethyl-N-nitrosourea (ENU)-induced mutant mice for aberrant immune function, we identified mice with a syndromic disorder marked by growth retardation, diabetes, premature death, and severe lymphoid and myeloid hypoplasia together with diminished T cell-independent (TI) antibody responses. The causative mutation was in Pdia6, an essential gene encoding protein disulfide isomerase A6 (PDIA6), an oxidoreductase that functions in nascent protein folding in the endoplasmic reticulum. The immune deficiency caused by the Pdia6 mutation was, with the exception of a residual T cell developmental defect, completely rescued in irradiated wild-type recipients of PDIA6-deficient bone marrow cells, both in the absence or presence of competition. The viable hypomorphic allele uncovered in these studies reveals an essential role for PDIA6 in hematopoiesis, but one extrinsic to cells of the hematopoietic lineage. We show evidence that this role is in the proper folding of Wnt3a, BAFF, IL-7, and perhaps other factors produced by the extra-hematopoietic compartment that contribute to the development and lineage commitment of hematopoietic cells.


Asunto(s)
Linfocitos/inmunología , Células Mieloides/inmunología , Proteína Disulfuro Isomerasas/inmunología , Animales , Factor Activador de Células B/inmunología , Línea Celular , Femenino , Células HEK293 , Hematopoyesis/inmunología , Humanos , Interleucina-7/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Proteína Wnt3A/inmunología
19.
Plast Reconstr Surg ; 145(2): 348e-359e, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31985636

RESUMEN

BACKGROUND: The authors developed a noncontact low-frequency ultrasound device that delivers high-intensity mechanical force based on phased-array technology. It may aid wound healing because it is likely to be associated with lower risks of infection and heat-induced pain compared with conventional ultrasound methods. The authors hypothesized that the microdeformation it induces accelerates wound epithelialization. Its effects on key wound-healing processes (angiogenesis, collagen accumulation, and angiogenesis-related gene transcription) were also examined. METHODS: Immediately after wounding, bilateral acute wounds in C57BL/6J mice were noncontact low-frequency ultrasound- and sham-stimulated for 1 hour/day for 3 consecutive days (10 Hz/90.6 Pa). Wound closure (epithelialization) was recorded every 2 days as the percentage change in wound area relative to baseline. Wound tissue was procured on days 2, 5, 7, and 14 (five to six per time point) and subjected to histopathology with hematoxylin and eosin and Masson trichrome staining, CD31 immunohistochemistry, and quantitative polymerase-chain reaction analysis. RESULTS: Compared to sham-treated wounds, ultrasound/phased-array-treated wounds exhibited significantly accelerated epithelialization (65 ± 27 percent versus 30 ± 33 percent closure), angiogenesis (4.6 ± 1.7 percent versus 2.2 ± 1.0 percent CD31 area), and collagen deposition (44 ± 14 percent versus 28 ± 13 percent collagen density) on days 5, 2, and 5, respectively (all p < 0.05). The expression of Notch ligand delta-like 1 protein (Dll1) and Notch1, which participate in angiogenesis, was transiently enhanced by treatment on days 2 and 5, respectively. CONCLUSIONS: The authors' noncontact low-frequency ultrasound phased-array device improved the wound-healing rate. It was associated with increased early neovascularization that was followed by high levels of collagen-matrix production and epithelialization. The device may expand the mechanotherapeutic proangiogenesis field, thereby helping stimulate a revolution in infected wound care.


Asunto(s)
Piel/lesiones , Terapia por Ultrasonido/métodos , Cicatrización de Heridas/fisiología , Heridas y Traumatismos/terapia , Animales , Colágeno/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica/fisiología , Piel/metabolismo , Heridas y Traumatismos/metabolismo , Heridas y Traumatismos/patología
20.
Toxicol Lett ; 319: 213-224, 2020 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-31783120

RESUMEN

The upregulated α-synuclein (α-syn) and Tau co-occur in methamphetamine (METH) abusers' brains. Here, we designed experiments mainly to investigate whether α-syn and Tau interact in METH exposure. We detected the expression of α-syn, total Tau, and phosphorylation of Tau at Serine 396 (pSer396 Tau) under in vitro and in vivo conditions after METH exposure to determine the co-occurrence of α-syn and Tau. We also explored the effect of α-syn or Tau on one another by silencing and knocking-out one of them in METH treatment. We found that METH increased the α-syn, total Tau, and pSer396 Tau protein level in SH-SY5Y cells, primary cultured neurons, and in mice brains. In additional, reducing α-syn level can relieve and even normalize the pSer396 Tau and total Tau overexpression after treatment of METH. Furthermore, knocking out Tau can effectively inhibit METH induced overexpression of α-syn in mice brains. Finally, knocking out α-syn or Tau can effectively reduce METH-induced neurotoxicity in mice brains. This research could provide potential therapeutic approaches targeting the vicious circle between α-syn and Tau in METH abusers and patients with neurodegenerative disorders.


Asunto(s)
Estimulantes del Sistema Nervioso Central/toxicidad , Metanfetamina/toxicidad , Síndromes de Neurotoxicidad/genética , Síndromes de Neurotoxicidad/metabolismo , alfa-Sinucleína/biosíntesis , Proteínas tau/biosíntesis , Animales , Conducta Animal/efectos de los fármacos , Línea Celular , Silenciador del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Síndromes de Neurotoxicidad/psicología , Cultivo Primario de Células , ARN Interferente Pequeño , alfa-Sinucleína/genética , Proteínas tau/genética , Proteínas tau/metabolismo
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