Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
1.
Stem Cell Res Ther ; 15(1): 171, 2024 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-38886860

RESUMEN

BACKGROUND: There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac). METHODS: We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks. RESULTS: The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone. CONCLUSIONS: The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications.


Asunto(s)
Reactores Biológicos , Células Madre Pluripotentes Inducidas , Macrófagos , Organoides , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Organoides/citología , Organoides/metabolismo , Diferenciación Celular , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/instrumentación , Hematopoyesis
2.
Cell ; 187(2): 390-408.e23, 2024 01 18.
Artículo en Inglés | MEDLINE | ID: mdl-38157855

RESUMEN

We describe a human lung disease caused by autosomal recessive, complete deficiency of the monocyte chemokine receptor C-C motif chemokine receptor 2 (CCR2). Nine children from five independent kindreds have pulmonary alveolar proteinosis (PAP), progressive polycystic lung disease, and recurrent infections, including bacillus Calmette Guérin (BCG) disease. The CCR2 variants are homozygous in six patients and compound heterozygous in three, and all are loss-of-expression and loss-of-function. They abolish CCR2-agonist chemokine C-C motif ligand 2 (CCL-2)-stimulated Ca2+ signaling in and migration of monocytic cells. All patients have high blood CCL-2 levels, providing a diagnostic test for screening children with unexplained lung or mycobacterial disease. Blood myeloid and lymphoid subsets and interferon (IFN)-γ- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-mediated immunity are unaffected. CCR2-deficient monocytes and alveolar macrophage-like cells have normal gene expression profiles and functions. By contrast, alveolar macrophage counts are about half. Human complete CCR2 deficiency is a genetic etiology of PAP, polycystic lung disease, and recurrent infections caused by impaired CCL2-dependent monocyte migration to the lungs and infected tissues.


Asunto(s)
Proteinosis Alveolar Pulmonar , Receptores CCR2 , Niño , Humanos , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Proteinosis Alveolar Pulmonar/genética , Proteinosis Alveolar Pulmonar/diagnóstico , Receptores CCR2/deficiencia , Receptores CCR2/genética , Receptores CCR2/metabolismo , Reinfección/metabolismo
3.
J Immunother Cancer ; 11(12)2023 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-38135346

RESUMEN

BACKGROUND: Macrophages have recently become attractive therapeutics in cancer immunotherapy. The potential of macrophages to infiltrate and influence solid malignancies makes them promising targets for the chimeric antigen receptor (CAR) technology to redirect their stage of polarization, thus enhancing their anticancer capacities. Given the emerging interest for CAR-macrophages, generation of such cells so far mainly depends on peripheral blood monocytes, which are isolated from the respective donor prior to genetic manipulation. This procedure is time-intensive and cost-intensive, while, in some cases, insufficient monocyte amounts can be recovered from the donor, thus hampering the broad applicability of this technology. Hence, we demonstrate the generation and effectiveness of CAR-macrophages from various stem cell sources using also modern upscaling technologies for next generation immune cell farming. METHODS: Primary human hematopoietic stem and progenitor cells and induced pluripotent stem cells were used to derive anti-CD19 CAR-macrophages. Anticancer activity of the cells was demonstrated in co-culture systems, including primary material from patients with leukemia. Generation of CAR-macrophages was facilitated by bioreactor technologies and single-cell RNA (scRNA) sequencing was used to characterize in-depth response and behavior of CAR-macrophages. RESULTS: Irrespective of the stem-cell source, CAR-macrophages exhibited enhanced and antigen-dependent phagocytosis of CD19+ target cancer cells with increased pro-inflammatory responses. Phagocytic capacity of CAR-macrophages was dependent on target cell CD19 expression levels with superior function of CAR-macrophages against CD19+ cancer cell lines and patient-derived acute lymphocytic leukemia cancer cells. scRNA sequencing revealed CAR-macrophages to be distinct from eGFP control cells after co-culture with target cells, which includes the activation of pro-inflammatory pathways and upregulation of chemokines and cytokines associated with adaptive immune cell recruitment, favoring the repolarization of CAR-macrophages to a pro-inflammatory state. Taken together, the data highlight the unique features of CAR-macrophages in combination with the successful upscaling of the production pipeline using a three-dimensional differentiation protocol and intermediate scale bioreactors. CONCLUSION: In summary, our work provides insights into the seminal use and behavior of CAR-macrophages which are derived from various sources of stem cells, while introducing a unique technology for CAR-macrophage manufacturing, all dedicated to the clinical translation of CAR-macrophages within the field of anticancer immunotherapies.


Asunto(s)
Células Madre Pluripotentes Inducidas , Leucemia , Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores de Antígenos de Linfocitos T , Células Madre Pluripotentes Inducidas/metabolismo , Linfocitos T , Leucemia/terapia , Macrófagos/metabolismo
4.
Biomedicines ; 10(5)2022 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-35625934

RESUMEN

Nephrotoxic drugs can cause acute kidney injury (AKI) and analgesic nephropathy. Diclofenac is potentially nephrotoxic and frequently prescribed for pain control. In this study, we investigated the effects of single and repetitive oral doses of diclofenac in the setting of pre-existing subclinical AKI on the further course of AKI and on long-term renal consequences. Unilateral renal ischemia-reperfusion injury (IRI) for 15 min was performed in male CD1 mice to induce subclinical AKI. Immediately after surgery, single oral doses (100 mg or 200 mg) of diclofenac were administered. In a separate experimental series, repetitive treatment with 100 mg diclofenac over three days was performed after IRI and sham surgery. Renal morphology and pro-fibrotic markers were investigated 24 h and two weeks after the single dose and three days after the repetitive dose of diclofenac treatment using histology, immunofluorescence, and qPCR. Renal function was studied in a bilateral renal IRI model. A single oral dose of 200 mg, but not 100 mg, of diclofenac after IRI aggravated acute tubular injury after 24 h and caused interstitial fibrosis and tubular atrophy two weeks later. Repetitive treatment with 100 mg diclofenac over three days aggravated renal injury and caused upregulation of the pro-fibrotic marker fibronectin in the setting of subclinical AKI, but not in sham control kidneys. In conclusion, diclofenac aggravated renal injury in pre-existing subclinical AKI in a dose and time-dependent manner and already a single dose can cause progression to chronic kidney disease (CKD) in this model.

5.
Am J Physiol Renal Physiol ; 319(4): F563-F570, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32799675

RESUMEN

Acute kidney injury (AKI) frequently complicates major surgery and can be associated with hypertension and progress to chronic kidney disease, but reports on blood pressure normalization in AKI are conflicting. In the present study, we investigated the effects of an angiotensin-converting enzyme inhibitor, enalapril, and a soluble epoxide hydrolase inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), on renal inflammation, fibrosis, and glomerulosclerosis in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. Male CD1 mice underwent unilateral IRI for 35 min. Blood pressure was measured by tail cuff, and mesangial matrix expansion was quantified on methenamine silver-stained sections. Renal perfusion was assessed by functional MRI in vehicle- and TPPU-treated mice. Immunohistochemistry was performed to study the severity of AKI and inflammation. Leukocyte subsets were analyzed by flow cytometry, and proinflammatory cytokines were analyzed by quantitative PCR. Plasma and tissue levels of TPPU and lipid mediators were analyzed by liquid chromatography mass spectrometry. IRI resulted in a blood pressure increase of 20 mmHg in the vehicle-treated group. TPPU and enalapril normalized blood pressure and reduced mesangial matrix expansion. However, inflammation and progressive renal fibrosis were severe in all groups. TPPU further reduced renal perfusion on days 1 and 14. In conclusion, early antihypertensive treatment worsened renal outcome after AKI by further reducing renal perfusion despite reduced glomerulosclerosis.


Asunto(s)
Lesión Renal Aguda/tratamiento farmacológico , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glomerulonefritis/prevención & control , Hipertensión/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Piperidinas/farmacología , Daño por Reperfusión/tratamiento farmacológico , Lesión Renal Aguda/etiología , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Antihipertensivos/toxicidad , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Enalapril/farmacología , Inhibidores Enzimáticos/toxicidad , Epóxido Hidrolasas/antagonistas & inhibidores , Fibrosis , Mesangio Glomerular/efectos de los fármacos , Mesangio Glomerular/patología , Mesangio Glomerular/fisiopatología , Glomerulonefritis/etiología , Glomerulonefritis/patología , Glomerulonefritis/fisiopatología , Hipertensión/etiología , Hipertensión/fisiopatología , Masculino , Ratones , Compuestos de Fenilurea/toxicidad , Piperidinas/toxicidad , Daño por Reperfusión/complicaciones , Daño por Reperfusión/fisiopatología
6.
Am J Physiol Renal Physiol ; 314(5): F881-F892, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29357437

RESUMEN

Renal ischemia-reperfusion injury (IRI) is a severe complication of major surgery and a risk factor for increased morbidity and mortality. Here, we investigated mechanisms that might contribute to IRI-induced progression to chronic kidney disease (CKD). Acute kidney injury (AKI) was induced by unilateral IRI for 35 min in CD1 and C57BL/6 (B6) mice. Unilateral IRI was used to overcome early mortality. Renal morphology, NGAL upregulation, and neutrophil infiltration as well as peritubular capillary density were studied by immunohistochemistry. The composition of leukocyte infiltrates in the kidney after IRI was investigated by flow cytometry. Systemic blood pressure was measured with a tail cuff, and renal perfusion was quantified by functional magnetic resonance imaging (fMRI). Mesangial matrix expansion was assessed by silver staining. Following IRI, CD1 and B6 mice developed similar morphological signs of AKI and increases in NGAL expression, but neutrophil infiltration was greater in CD1 than B6 mice. IRI induced an increase in systemic blood pressure of 20 mmHg in CD1, but not in B6 mice; and CD1 mice also had a greater loss of renal perfusion and kidney volume than B6 mice ( P < 0.05). CD1 mice developed substantial interstitial fibrosis and decreased peritubular capillary (PTC) density by day 14 while B6 mice showed only mild renal scarring and almost normal PTC. Our results show that after IRI, CD1 mice develop more inflammation, hypertension, and later mesangial matrix expansion than B6 mice do. Subsequently, CD1 animals suffer from CKD due to impaired renal perfusion and pronounced permanent loss of peritubular capillaries.


Asunto(s)
Lesión Renal Aguda/complicaciones , Hipertensión/etiología , Riñón/irrigación sanguínea , Circulación Renal , Insuficiencia Renal Crónica/etiología , Daño por Reperfusión/complicaciones , Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Animales , Velocidad del Flujo Sanguíneo , Presión Sanguínea , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibrosis , Mesangio Glomerular/patología , Hipertensión/metabolismo , Hipertensión/patología , Hipertensión/fisiopatología , Inmunohistoquímica , Riñón/metabolismo , Riñón/patología , Lipocalina 2/metabolismo , Imagen por Resonancia Magnética , Masculino , Ratones Endogámicos C57BL , Infiltración Neutrófila , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/fisiopatología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Factores de Tiempo
7.
Eur Radiol ; 28(1): 44-50, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28710580

RESUMEN

OBJECTIVES: To evaluate T1 mapping as a non-invasive, functional MRI biomarker in patients shortly after solid organ transplantation to detect acute postsurgical kidney damage and to correlate T1 times with renal function. METHODS: 101 patients within 2 weeks after solid organ transplantation (49 kidney transplantation, 52 lung transplantation) and 14 healthy volunteers were examined by MRI between July 2012 and April 2015 using the modified Look-Locker inversion recovery (MOLLI) sequence. T1 times in renal cortex and medulla and the corticomedullary difference were compared between groups using one-way ANOVA adjusted for multiple comparison with the Tukey test, and T1 times were correlated with renal function using Pearson's correlation. RESULTS: Compared to healthy volunteers T1 times were significantly increased after solid organ transplantation in the renal cortex (healthy volunteers 987 ± 102 ms; kidney transplantation 1299 ± 101 ms, p < 0.001; lung transplantation 1058 ± 96 ms, p < 0.05) and to a lesser extent in the renal medulla. Accordingly, the corticomedullary difference was diminished shortly after solid organ transplantation. T1 changes were more pronounced following kidney compared to lung transplantation, were associated with the stage of renal impairment and significantly correlated with renal function. CONCLUSIONS: T1 mapping may be helpful for early non-invasive assessment of acute kidney injury and renal pathology following major surgery such as solid organ transplantation. KEY POINTS: • Renal cortical T1 relaxation times are prolonged after solid organ transplantation. • Cortical T1 values increase with higher stages of renal function impairment. • Corticomedullary difference decreases with higher stages of renal function impairment. • Renal cortical T1 relaxation time and corticomedullary difference correlate with renal function. • T1 mapping may be helpful for non-invasive assessment of post-operative renal pathology.


Asunto(s)
Lesión Renal Aguda/diagnóstico , Trasplante de Riñón/efectos adversos , Riñón/patología , Trasplante de Pulmón/efectos adversos , Imagen por Resonancia Magnética/métodos , Complicaciones Posoperatorias/diagnóstico , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad
8.
J Pharm Pharmacol ; 69(9): 1125-1135, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28573734

RESUMEN

OBJECTIVES: IL-17A contributes to acute kidney injury and fibrosis. Therefore, we asked whether IL-17A deficiency or treatment with a IL-17A blocking antibody impacts severe renal ischaemia reperfusion injury (IRI) and the progression to chronic kidney disease (CKD). METHODS: IL-17A-deficient and wild-type (WT) mice underwent transient unilateral renal pedicle clamping for 45 min to induce IRI and subsequent renal fibrosis. Furthermore, a neutralizing anti-IL-17A antibody (mAb) was injected into WT mice before induction of renal IRI intravenously. On days 1, 7 and 21, inflammation, fibrosis, leukocyte infiltration and pro-inflammatory and pro-fibrotic cytokine expression were assessed in kidneys using histology, qPCR and flow cytometry. KEY FINDINGS: IL-17A was significantly increased after renal IRI in WT kidneys. Levels of pro-inflammatory (MCP-1) cytokine and pro-fibrotic (collagen 1α1, fibronectin) transcripts were similar in the experimental groups studied. IL-17A deficiency had no effect on renal T-cell influx or the number, inflammatory phenotype, or spatial distribution of macrophages. Similarly, administration of an IL-17A blocking antibody did not attenuate inflammation. CONCLUSIONS: Despite the effects of IL-17 in other inflammation models, neither genetic IL-17A deficiency nor treatment with an IL-17A blocking antibody attenuated IRI and progression to CKD. We conclude that in severe renal IRI IL-17A is not crucially involved in disease progression.


Asunto(s)
Lesión Renal Aguda/fisiopatología , Interleucina-17/genética , Insuficiencia Renal Crónica/prevención & control , Daño por Reperfusión/fisiopatología , Lesión Renal Aguda/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibrosis , Citometría de Flujo , Inflamación/inmunología , Inflamación/fisiopatología , Interleucina-17/antagonistas & inhibidores , Interleucina-17/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Insuficiencia Renal Crónica/inmunología , Daño por Reperfusión/inmunología
9.
PLoS One ; 12(3): e0173248, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28319118

RESUMEN

PURPOSE: The purpose was to characterize acute kidney injury (AKI) in C57BL/6 (B6)- and 129/Sv (Sv)-mice by noninvasive measurement of renal perfusion and tissue edema using functional MRI. METHODS: Different severities of AKI were induced in B6- and Sv-mice by renal ischemia reperfusion injury (IRI). Unilateral clamping of the renal pedicle for 35 min (moderate AKI) or 45 min (severe AKI) was done. MRI (7-Tesla) was performed 1, 7 and 28 days after surgery using a flow alternating inversion recovery (FAIR) arterial spin labeling (ASL) sequence. Maps of perfusion and T1-relaxation time were calculated. Relative MRI-parameters of the IRI kidney compared to the contralateral not-clipped kidney were compared between AKI severities and between mouse strains using unpaired t-tests. In addition, fibrosis was assessed by Masson Trichrome and collagen IV staining. RESULTS: After moderate AKI relative perfusion impairment was significantly higher in B6- than in Sv-mice at d7 (55±7% vs. 82±8%, p<0.05) and d28 (76±7% vs. 102±3%, p<0.01). T1-values increased in the early phase after AKI in both mouse strains. T1-increase was more severe after prolonged ischemia times of 45 min compared to 35 min in both mouse strains, measured in the renal cortex and outer stripe of outer medulla. Kidney volume loss (compared to the contralateral kidney) occurred already after 7 days but proceeded markedly towards 4 weeks in severe AKI. Early renal perfusion impairment was predictive for later kidney volume loss. The progression to chronic kidney disease (CKD) in the severe AKI model was similar in both mouse strains as revealed by histology. CONCLUSION: Quantification of renal perfusion and tissue edema by functional MRI allows characterization of strain differences upon AKI. Renal perfusion impairment was stronger in B6- compared to Sv-animals following moderate AKI. Prolonged ischemia times were associated with more severe perfusion impairment and edema formation in the early phase and progression to CKD within 4 weeks of observation.


Asunto(s)
Lesión Renal Aguda/diagnóstico por imagen , Modelos Animales de Enfermedad , Riñón/diagnóstico por imagen , Imagen por Resonancia Magnética , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Animales , Progresión de la Enfermedad , Edema/diagnóstico por imagen , Edema/patología , Edema/fisiopatología , Fibrosis/diagnóstico por imagen , Fibrosis/patología , Fibrosis/fisiopatología , Inmunohistoquímica , Riñón/patología , Riñón/fisiopatología , Macrófagos/patología , Macrófagos/fisiología , Masculino , Tamaño de los Órganos , Daño por Reperfusión , Índice de Severidad de la Enfermedad , Especificidad de la Especie , Factores de Tiempo
10.
Kidney Int ; 89(6): 1253-67, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27142955

RESUMEN

Chronic exposure to commercial glucose-based peritoneal dialysis fluids during peritoneal dialysis induces peritoneal membrane damage leading to ultrafiltration failure. In this study the role of protein kinase C (PKC) α in peritoneal membrane damage was investigated in a mouse model of peritoneal dialysis. We used 2 different approaches: blockade of biological activity of PKCα by intraperitoneal application of the conventional PKC inhibitor Go6976 in C57BL/6 wild-type mice and PKCα-deficient mice on a 129/Sv genetic background. Daily administration of peritoneal dialysis fluid for 5 weeks induced peritoneal upregulation and activation of PKCα accompanied by epithelial-to-mesenchymal transition of peritoneal mesothelial cells, peritoneal membrane fibrosis, neoangiogenesis, and macrophage and T cell infiltration, paralleled by reduced ultrafiltration capacity. All pathological changes were prevented by PKCα blockade or deficiency. Moreover, treatment with Go6976 and PKCα deficiency resulted in strong reduction of proinflammatory, profibrotic, and proangiogenic mediators. In cell culture experiments, both treatment with Go6976 and PKCα deficiency prevented peritoneal dialysis fluid-induced release of MCP-1 from mouse peritoneal mesothelial cells and ameliorated transforming growth factor-ß1-induced epithelial-to-mesenchymal transition and peritoneal dialysis fluid-induced MCP-1 release in human peritoneal mesothelial cells. Thus, PKCα plays a crucial role in the pathophysiology of peritoneal membrane dysfunction induced by peritoneal dialysis fluids, and we suggest that its therapeutic inhibition might be a valuable treatment option for peritoneal dialysis patients.


Asunto(s)
Carbazoles/uso terapéutico , Soluciones para Diálisis/efectos adversos , Inhibidores Enzimáticos/uso terapéutico , Glucosa/efectos adversos , Diálisis Peritoneal/efectos adversos , Fibrosis Peritoneal/prevención & control , Proteína Quinasa C-alfa/antagonistas & inhibidores , Animales , Línea Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/fisiología , Transición Epitelial-Mesenquimal , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peritoneo/citología , Peritoneo/patología , Cultivo Primario de Células , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba
11.
PLoS One ; 10(10): e0139147, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26440517

RESUMEN

Epidemiologic studies show a correlation between the dietary intake of food polyphenols and beneficial health effects. Several in vitro studies indicate that the anti-inflammatory potential of polyphenols is, at least in part, mediated by a modulation of the enzymes of the arachidonic acid cascade, such as the prostaglandin forming cyclooxygenases (COXs). Evidence that this mode of action can be transferred to the situation in vivo is scarce. This study characterized effects of a subset of polyphenols on COX-2 expression and activity in vitro and compared the potency with known drugs. Next, the in vivo relevance of the observed in vitro effects was tested. Enzyme assays and incubations of polyphenols with the cancer cell line HCA-7 and lipopolysaccharide (LPS) stimulated primary monocytes support the hypothesis that polyphenols can effect COX-2 expression and activity in vitro. The effects were most pronounced in the monocyte assay for wogonin, apigenin, resveratrol and genistein with IC50 values of 1.5 µM, 2.6 µM, 2.8 µM and 7.4 µM. However, these values are 100- to 1000-fold higher in comparison to those of the known pharmaceuticals celecoxib, indomethacin and dexamethasone. In an animal model of LPS induced sepsis, pretreatment with polyphenols (i. p. 100 mg/kg bw) did not result in decreased plasma or tissue prostaglandin levels, whereas the positive control celecoxib effectively attenuated LPS induced prostaglandin formation. These data suggest that despite the moderate potency in vitro, an effect of polyphenols on COX-2 during acute inflammation is unlikely, even if a high dose of polyphenols is ingested.


Asunto(s)
Ciclooxigenasa 2/metabolismo , Polifenoles/farmacología , Animales , Antiinflamatorios/farmacología , Apigenina/farmacología , Celecoxib/farmacología , Línea Celular Tumoral , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Genisteína/farmacología , Humanos , Indometacina/farmacología , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Resveratrol , Estilbenos/farmacología
12.
Am J Physiol Renal Physiol ; 308(12): F1444-51, 2015 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-25925250

RESUMEN

Delayed graft function (DGF) after kidney transplantation is not uncommon, and it is associated with long-term allograft impairment. Our aim was to compare renal perfusion changes measured with noninvasive functional MRI in patients early after kidney transplantation to renal function and allograft histology in biopsy samples. Forty-six patients underwent MRI 4-11 days after transplantation. Contrast-free MRI renal perfusion images were acquired using an arterial spin labeling technique. Renal function was assessed by estimated glomerular filtration rate (eGFR), and renal biopsies were performed when indicated within 5 days of MRI. Twenty-six of 46 patients had DGF. Of these, nine patients had acute rejection (including borderline), and eight had other changes (e.g., tubular injury or glomerulosclerosis). Renal perfusion was significantly lower in the DGF group compared with the group with good allograft function (231 ± 15 vs. 331 ± 15 ml·min(-1)·100 g(-1), P < 0.001). Living donor allografts exhibited significantly higher perfusion values compared with deceased donor allografts (P < 0.001). Renal perfusion significantly correlated with eGFR (r = 0.64, P < 0.001), resistance index (r = -0.57, P < 0.001), and cold ischemia time (r = -0.48, P < 0.01). Furthermore, renal perfusion impairment early after transplantation predicted inferior renal outcome and graft loss. In conclusion, noninvasive functional MRI detects renal perfusion impairment early after kidney transplantation in patients with DGF.


Asunto(s)
Funcionamiento Retardado del Injerto/patología , Funcionamiento Retardado del Injerto/fisiopatología , Supervivencia de Injerto/fisiología , Trasplante de Riñón , Riñón/fisiopatología , Imagen por Resonancia Magnética , Adulto , Anciano , Femenino , Tasa de Filtración Glomerular/fisiología , Rechazo de Injerto/patología , Humanos , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/métodos , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Perfusión/métodos , Trasplante Homólogo/efectos adversos , Trasplante Homólogo/métodos
13.
Biochem Biophys Res Commun ; 337(1): 209-15, 2005 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-16182248

RESUMEN

Hypoxia-inducible factor-1 (HIF-1), which consists of oxygen-sensitive HIF-1alpha and constitutively expressed HIF-1beta subunits, activates transcription of genes encoding proteins that mediate adaptive responses to reduced oxygen availability. The mouse inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS) functions as a negative regulator in HIF-mediated gene expression. In this report, we cloned the human orthologs of the mouse IPAS gene, IPASH1 and IPASH2, to further study the regulatory mechanism of HIF-1 by the IPAS proteins. The human IPAS proteins inhibited the transactivation function of HIF-1alpha under hypoxic conditions. In addition, human IPAS proteins blocked the hypoxia-induced VEGF expression and inhibited cell migration and tube formation of human umbilical vein endothelial cells. Interestingly, both HIF-1alpha and HIF-1beta interacted with the IPAS proteins. Collectively, these results suggest that human IPAS proteins inhibit angiogenesis by binding to and inhibiting HIF-1alpha and HIF-1beta.


Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Translocador Nuclear del Receptor de Aril Hidrocarburo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Hipoxia de la Célula , Línea Celular , Movimiento Celular , Proteínas de Unión al ADN/metabolismo , Endotelio Vascular/citología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ratones , Datos de Secuencia Molecular , Neovascularización Fisiológica , Receptores de Hidrocarburo de Aril/metabolismo , Proteínas Represoras/genética , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Factor A de Crecimiento Endotelial Vascular/biosíntesis
14.
J Biol Chem ; 279(16): 16050-6, 2004 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-14761963

RESUMEN

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase family member that plays a central role in cytokine- and stress-induced apoptosis by activating c-Jun N-terminal kinase and p38 signaling cascades. ASK1-induced apoptotic activity is up-regulated by two cellular factors, Daxx and TRAF2, through direct protein-protein interactions. Daxx and TRAF2 are death receptor-associated proteins in Fas and tumor necrosis factor-alpha pathways, respectively. Recent studies suggest that calcium signaling may regulate ASK1 pathway. Here we report that human D53L1, a member of the tumor protein D52 family involved in cell proliferation and calcium signaling, up-regulates the ASK1-induced apoptosis. The human D53L1 physically interacts with the C-terminal regulatory domain of ASK1 and promotes ASK1-induced apoptotic activity by activating caspase signaling in mammalian cells. In luciferase reporter assays, hD53L1 activates c-Jun N-terminal kinase-mediated transactivation in the presence of ASK1. Expression of hD53L1 enhances autophosphorylation and kinase activity of ASK1 but has no effect on ASK1 oligomerization that is necessary for kinase activity and on binding of ASK1 to MKK6, a downstream factor of ASK1. Taken together, these results suggest that activation of ASK1 by hD53L1 may provide a novel mechanism for ASK1 regulation.


Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales , Apoptosis/fisiología , Línea Celular , Proteínas Co-Represoras , Activación Enzimática , Humanos , MAP Quinasa Quinasa Quinasa 5 , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Proteínas/metabolismo , Factor 2 Asociado a Receptor de TNF
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA