RESUMEN
OBJECTIVE: Although the natural compound curcumin exerts antitumor properties in vitro, its clinical application is hampered due to rapid metabolism. Light exposure following curcumin application has been demonstrated to improve curcumin's bioavailability. Therefore, this investigation was directed towards evaluating whether light exposure in addition to curcumin application enhances curcumin's efficacy against bladder cancer cell adhesion and migration. MATERIALS AND METHODS: RT112, UMUC3, and TCCSUP cells were incubated with low curcumin concentrations (0.1-0.4 µg/ml) and then exposed to 1.65 J/cm2 visible light for 5 min. Controls remained untreated or were treated with curcumin or light alone. Cell adhesion to Human umbilical vein endothelial cells (HUVECs), to immobilized collagen or fibronectin and chemotactic behavior, integrin α and ß receptor expression with functional relevance, as well as focal adhesion kinase (total and phosphorylated FAK) were evaluated. RESULTS: Curcumin plus light, but neither curcumin nor light alone, significantly altered tumor cell adhesion and suppressed chemotaxis. Integrin α and ß subtypes were dissimilarly modified, depending on the cell line. Suppression of pFAK was noted in RT112 and UMUC3, but not in TCCSUP cells. The integrins α3, α5, and ß1 were involved in curcumin's regulation of adhesion and migration. Blocking studies revealed α3, α5, and ß1 to be associated with TCCSUP adhesion and migration, whereas α5 and ß1, but not α3 contributed to UMUC3 adhesion and migration. Integrin α5 and ß1 controlled RT112 chemotaxis as well, but only α5 was involved in the RT112 adhesion process. CONCLUSIONS: Combining curcumin with light exposure enhances curcumin's anti-tumor potential.
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Antineoplásicos/farmacología , Curcumina/farmacología , Luz , Fotoquimioterapia/métodos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Disponibilidad Biológica , Adhesión Celular/efectos de los fármacos , Adhesión Celular/efectos de la radiación , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Quimiotaxis/efectos de los fármacos , Quimiotaxis/efectos de la radiación , Curcumina/uso terapéutico , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Inhibitors of the mammalian target of rapamycin (mTOR) might become a novel tool to treat advanced prostate cancer. However, chronic drug exposure may trigger resistance, limiting the utility of mTOR inhibitors. METHODS: Metastatic potential of PC3 prostate cancer cells, susceptible (PC3(par)) or resistant (PC3(res)) to the mTOR-inhibitor RAD001 was investigated. Adhesion to vascular endothelium or immobilised collagen, fibronectin and laminin was quantified. Motility, migration and invasion were explored by modified Boyden chamber assay. Integrin α and ß subtypes were analysed by flow cytometry, western blotting and real-time PCR. Integrin-related signalling, EGFr, Akt, p70S6kinase and ERK1/2 activation were determined. RESULTS: Adhesion was reduced, whereas motility, migration and invasion were enhanced in PC3(res). The α2 and ß1 integrin subtypes were dramatically elevated, integrins α1 and α6 were lowered, whereas α5 was nearly lost in PC3(res). Activation of the Akt signalling pathway was strongly upregulated in these cells. Treating PC3(par) cells with RAD001 reduced motility, migration and invasion and deactivated Akt signalling. Blocking studies revealed that α2 and ß1 integrins significantly trigger the motile behaviour of the tumour cells. CONCLUSION: Chronic RAD001 treatment caused resistance development characterised by distinct modification of the integrin-expression profile, driving prostate cancer cells towards high motility.
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Movimiento Celular/efectos de los fármacos , Integrina alfa2/metabolismo , Integrina beta1/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Sirolimus/análogos & derivados , Serina-Treonina Quinasas TOR/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Everolimus , Humanos , Integrina alfa2/biosíntesis , Integrina beta1/biosíntesis , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/biosíntesisRESUMEN
BACKGROUND: The influence of the bisphosphonate zoledronic acid (ZA) on prostate cancer (PC) growth, adhesion and invasive behavior was investigated. METHODS: PC-3, DU-145 and LNCaP cells were treated with ZA, and tumor-cell growth was then investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Furthermore, tumor-cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins, as well as migratory properties of the cells, was evaluated. Integrin ß subtypes, integrin-dependent signaling, as well as cell-cycle regulating proteins, were analyzed by western blots. RESULTS: ZA dose-dependently reduced tumor-cell growth but did not impair tumor-endothelium and tumor-matrix interaction. However, ZA significantly inhibited tumor migration and invasive activity. Cyclin E was reduced by ZA in LNCaP and DU-145, and p21 was elevated in LNCaP cells. p27 was upregulated in all tumor cell lines, compared with the controls. ZA elevated ß1-integrin in PC-3 and diminished ß4-integrin in PC-3 and DU-145 cells. CONCLUSIONS: ZA inhibits PC growth and motility but does not influence the mechanical contact between tumor cells and the vascular wall.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Difosfonatos/farmacología , Imidazoles/farmacología , Neoplasias de la Próstata/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Neoplasias de la Próstata/patología , Transducción de Señal/efectos de los fármacos , Ácido ZoledrónicoRESUMEN
PURPOSE: Renal cell carcinoma (RCC) is highly resistant to chemotherapy and unresponsive to radio- and immunotherapy. Recently, we have documented that the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) in combination with low-dosed interferon (IFN)-alpha significantly inhibits RCC proliferation and adhesion in vitro and in vivo. The current study investigated the effects of these compounds on gene transcription of metastatic RCC cell line Caki-1 after 3 and 5 days exposure. METHODS: To evaluate the gene expression profiles of the RCC cells, we performed microarray analysis using Affymetrix GeneChip. Selected significant genes were further validated by Real Time PCR. RESULTS: Microarray revealed that VPA altered genes that are involved in cell growth, cell survival, immune response, cell motility and cell adhesion. Combination of VPA with IFN-alpha not only enhanced the effects on gene transcription but also resulted in the expression of novel genes, which were not induced by either VPA or IFN-alpha alone. Among the up-regulated genes were chemokines (CXCL10, CXCL11, CXCL16) and integrins (ITGA2, ITGA4, ITGA5, ITGA6, ITGA7). Genes encoding for adhesion molecules (NCAM1, ICAM1, VCAM1) were also modulated. Real Time PCR approved these findings. CONCLUSION: This data provides insight into the molecular mechanism of action of the combined treatment of VPA and IFN-alpha in RCC. Implications are that the combined application of VPA and IFN-alpha may represent a more efficient alternative to existing therapy options for RCC.
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Carcinoma de Células Renales/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Interferón-alfa/farmacología , Neoplasias Renales/genética , Ácido Valproico/farmacología , Carcinoma de Células Renales/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Factores Inmunológicos/farmacología , Neoplasias Renales/patología , Análisis por MicromatricesRESUMEN
OBJECTIVE: To evaluate the incidence of voiding dysfunction in older male renal transplant recipients. PATIENTS AND METHODS: Data for 103 patients aged 60 years or older (mean age, 65.7 years; group 1) who underwent transplantation at our center between January 1999 and August 2007 were compared with data for a group of 139 younger patients (mean age, 50.1 years; group 2) treated within the same time frame. RESULTS: Postoperatively, 28 group 1 recipients (27%) and 26 group 2 recipients (19%) experienced voiding dysfunction after removal of the transurethral catheter (P = .12). The most common cause was bladder outlet obstruction due to benign prostatic hyperplasia in 26 patients in group 1 (25%) and 17 patients in group 2 (12%) (P = .009). Bladder neck contracture, urethral stricture, and detrusor underactivity were diagnosed in the other patients. Transurethral resection of the prostate gland was performed in 21 group 1 patients (20%) and 14 group 2 patients (10%) (P = .02) at a mean of 31.1 and 29.5 days, respectively (P = .23) after transplantation. Surgical procedures were performed without complication, and symptoms did not recur postoperatively. CONCLUSIONS: Our data reveal a high incidence of voiding dysfunction in older male renal transplant recipients. High residual urine and urinary retention after renal transplantation may induce recurrent urinary tract infections, cause relevant complications, and seriously affect graft function. Recognizing the substantial effects of postoperative voiding dysfunction will enable optimum management of older kidney transplant recipients.
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Envejecimiento/fisiología , Trasplante de Riñón/efectos adversos , Obstrucción Uretral/etiología , Retención Urinaria/etiología , Urodinámica/fisiología , Anciano , Humanos , Masculino , Persona de Mediana Edad , Periodo Posoperatorio , Prostatectomía , Hiperplasia Prostática/epidemiología , Estudios Retrospectivos , Obstrucción Uretral/epidemiología , Obstrucción Uretral/cirugía , Obstrucción del Cuello de la Vejiga Urinaria/etiología , Obstrucción del Cuello de la Vejiga Urinaria/cirugía , Cateterismo Urinario , Retención Urinaria/epidemiologíaRESUMEN
BACKGROUND: Conventional therapeutic approaches to treat advanced renal cell carcinoma (RCC) are of limited benefit. Receptor tyrosine kinase inhibitors (RTKI) may open up novel treatment options. In the present study, the effects of the RTKI AEE788 on the growth and adhesion capacity of RCC cell lines were evaluated in vitro. MATERIALS AND METHODS: RCC cells were treated with AEE788, and alterations of tumor growth and tumor cell interaction with vascular endothelium or extracellular matrix proteins were analyzed. Furthermore, the addition of interferon alpha (IFNalpha) was investigated to see whether it may enhance the anti-tumoral potential of AEE788. RESULTS: AEE788 significantly blocked RCC cell growth and adhesion. Analysis of alpha- and beta-integrins revealed distinct alterations of the receptor expression profile and downregulation of integrin-dependent signaling. Growth-blocking effects were further enhanced when the AEE788-IFNalpha combination protocol was applied. In addition, downregulation of integrin-dependent signaling was more intense in the presence of a combination of AEE788 and IFNalpha than with AEE788 monotherapy. CONCLUSIONS: AEE788 exerts significant anti-tumoral properties, particularly when combined with IFNalpha. AEE788 may therefore be an encouraging compound to treat advanced RCC.
Asunto(s)
Carcinoma de Células Renales/patología , Adhesión Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Neoplasias Renales/patología , Purinas/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Humanos , Técnicas In Vitro , Cadenas alfa de Integrinas/metabolismo , Cadenas beta de Integrinas , Interferón alfa-2 , Interferón-alfa/farmacología , Proteínas Recombinantes , Transducción de Señal/efectos de los fármacosRESUMEN
The anti-epileptic drug valproic acid is also under trial as an anti-cancer agent due to its histone deacetylase (HDAC) inhibitory properties. However, the effects of valproic acid (VPA) are limited and concentrations required for exerting anti-neoplastic effects in vitro may not be reached in tumour patients. In this study, we tested in vitro and in vivo effects of two VPA-derivatives (ACS2, ACS33) on pre-clinical prostate cancer models. PC3 and DU-145 prostate tumour cell lines were treated with various concentrations of ACS2 or ACS33 to perform in vitro cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and to evaluate tumour cell adhesion to endothelial cell monolayers. Analysis of acetylated histones H3 and H4 protein expression was performed by western blotting. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. Tumour sections were assessed by immunohistochemistry for histone H3 acetylation and proliferation. ACS2 and ACS33 significantly up-regulated histone H3 and H4 acetylation in prostate cancer cell lines. In micromolar concentrations both compounds exerted growth arrest in PC3 and DU-145 cells and prevented tumour cell attachment to endothelium. In vivo, ACS33 inhibited the growth of PC3 in subcutaneous xenografts. Immunohistochemistry and western blotting confirmed increased histone H3 acetylation and reduced proliferation. ACS2 and ACS33 represent novel VPA derivatives with superior anti-tumoural activities, compared to the mother compound. This investigation lends support to the clinical testing of ACS2 or ACS33 for the treatment of prostate cancer.
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Inhibidores Enzimáticos/uso terapéutico , Inhibidores de Histona Desacetilasas , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Ácido Valproico/análogos & derivados , Ácido Valproico/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). In pursuit of alternative treatments for chemoresistant tumour cells, we tested the response of multidrug-resistant SKNSH and of vincristine (VCR)-, doxorubicin (DOX)-, or cisplatin (CDDP)-resistant UKF-NB-2, UKF-NB-3 or UKF-NB-6 NB tumour cell lines to valproic acid (VPA), a differentiation inducer currently in clinical trials. Drug resistance caused elevated NB adhesion (UKF-NB-2(VCR), UKF-NB-2(DOX), UKF-NB-2(CDDP), UKF-NB-3(VCR), UKF-NB-3(CDDP), UKF-NB-6(VCR), UKF-NB-6(CDDP)) to an endothelial cell monolayer, accompanied by downregulation of the adhesion receptor neural cell adhesion molecule (NCAM). Based on the UKF-NB-3 model, N-myc proteins were enhanced in UKF-NB-3(VCR) and UKF-NB-3(CDDP), compared to the drug naïve controls. p73 was diminished, whereas the p73 isoform deltaNp73 was upregulated in UKF-NB-3(VCR) and UKF-NB-3(CDDP). Valproic acid blocked adhesion of UKF-NB-3(VCR) and UKF-NB-3(CDDP), but not of UKF-NB-3(DOX), and induced the upregulation of NCAM surface expression, NCAM protein content and NCAM coding mRNA. Valproic acid diminished N-myc and enhanced p73 protein level, coupled with downregulation of deltaNp73 in UKF-NB-3(VCR) and UKF-NB-3(CDDP). Valproic acid also reverted enhanced adhesion properties of drug-resistant UKF-NB-2, UKF-NB-6 and SKNSH cells, and therefore may provide an alternative approach to the treatment of drug-resistant NB by blocking invasive processes.
Asunto(s)
Cisplatino/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Neuroblastoma/patología , Ácido Valproico/farmacología , Vincristina/farmacología , Antígenos de Superficie/química , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Antineoplásicos/farmacología , Adhesión Celular/efectos de los fármacos , Endotelio Vascular/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Invasividad Neoplásica , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
The branched-chain fatty acid valproate (valproic acid; VPA) displays antitumoral properties by blocking tumor growth, progression and invasion. Recent data have shown that VPA reduces the angiogenic activity of endothelial cells. The object of this study was to investigate whether endothelial modulation might also influence the level of chemotactic mediators. Endothelial cells were isolated from human umbilical cord veins (HUVEC) and treated with VPA-concentrations ranging from 0.125 mM to 1 mM. The mRNA level of CXC-chemokines was investigated by reverse transcriptase-polymerase chain reaction. The proliferative activity of HUVEC was measured as well. VPA evoked a striking increase in the neutrophil chemoattractants CXCL1, CXCL3, CXCL4, CXCL5 and a moderate increase in CXCL6 with maximal effects after a 3-day incubation period. Other CXC-chemokines and CXC-receptors remained unaffected. HUVEC growth was diminished time- and dose-dependently by VPA. We conclude that VPA treatment leads to alterations in the chemokine expression profile of endothelial cells. This might allow more neutrophils to reach the tumor area and trigger cytolysis.
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Antineoplásicos/farmacología , Quimiocinas CXC/biosíntesis , Células Endoteliales/efectos de los fármacos , Receptores de Quimiocina/biosíntesis , Ácido Valproico/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiocinas CXC/genética , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular , ARN Mensajero/biosíntesis , Receptores de Quimiocina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell-endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 microm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 microm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy.
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Neoplasias del Colon/patología , Inmunosupresores/farmacología , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Neoplasias del Colon/inmunología , Relación Dosis-Respuesta Inmunológica , Regulación hacia Abajo/efectos de los fármacos , Endotelio Vascular/inmunología , Humanos , Integrinas/efectos de los fármacos , Integrinas/metabolismo , Células Tumorales CultivadasRESUMEN
STUDY DESIGN: In vitro study on the effects of mycophenolate mofetil (MMF) on isolated human monocytes and endothelial cells. OBJECTIVES: Haematogenous macrophages play an essential role in the development of secondary damage following spinal cord injury (SCI), and there is evidence that the use of immunosuppressants such as MMF can reduce monocyte invasion and neuronal damage. SETTING: University Hospital for Orthopaedic Surgery, Frankfurt am Main, Germany. METHODS: The effects of MMF on the adhesion of human monocytes to human umbilical vein endothelial cells (HUVEC), monocyte binding to immobilised E-selectin, and monocyte expression of intercellular adhesion molecule (ICAM)-1, sialyl Lewis X (sLeX) and major histocompatibility complex (MHC)-II were studied. The binding of monocytes to E-selectin was examined by using purified and immobilised E-selectin fusion protein. Adhesion molecule expression was investigated by flow cytometry. RESULTS: The binding of monocytes to HUVEC was significantly reduced by 30.1% after treatment of monocytes with MMF (10 microg/ml), whereas the pretreatment of HUVEC with MMF did not result in significant changes in monocyte adhesion. MMF forcefully inhibited monocyte binding to immobilised E-selectin by 55.7%. Furthermore, MMF significantly inhibited the upregulation of ICAM-1- and MHC-II-expression on monocytes stimulated with either lipopolysaccharide or interferon-gamma, whereas the expression of sLeX was not impaired. Toxic effects were excluded by propidium-iodide staining and measurement of fluorescein-diacetate metabolism. CONCLUSION: MMF can downregulate important monocytic adhesion molecules and inhibits monocyte adhesion to endothelial cells, thus indicating that treatment with MMF could be beneficial after SCI. SPONSORSHIP: This study was supported by the DFG (Ha 2721/1-3), the Paul und Ursula Klein-Stiftung and the Stiftung Friedrichsheim.
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Moléculas de Adhesión Celular/metabolismo , Comunicación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Inmunosupresores/farmacología , Monocitos/efectos de los fármacos , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Supervivencia Celular , Células Cultivadas , Cromatografía Líquida de Alta Presión , Pruebas Inmunológicas de Citotoxicidad/métodos , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Selectina E/metabolismo , Células Endoteliales/metabolismo , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Humanos , Inmunohistoquímica/métodos , Técnicas In Vitro , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno Lewis X/metabolismo , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Propidio/metabolismo , Factores de Transcripción , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismoRESUMEN
OBJECTIVE: To evaluate the impact of passenger leukocytes in liver grafts on the rate of microchimerism induction after liver transplantation and to evaluate immunological changes thereafter based on serial donor-specific MLC's in these patients. METHODS: 26 orthotopic liver transplant recipients were prospectively evaluated for immunological changes based on the co-transplantation of donor-derived leukocytes. Intraoperatively harvested liver biopsies and peripheral blood-lymphocytes of liver transplant recipients were sampled at various time points. Donor spleen cells were obtained during organ procurement. RESULTS: HLA-PCR analysis demonstrated a stable pattern of microchimerism in 15 out of 26 patients. Microchimerism was detectable by PCR up to a mean of 7 weeks after transplantation, when chimerism in the peripheral blood became negative. Passenger donor leukocytes were present in all biopsies obtained during backtable preparation of the liver graft. For the 15 patients presenting microchimerism the rate of passenger leukocytes in the liver graft biopsies showed a mean of 155.8 leukocytes per mm 2 liver tissue (SD +/- 23.2 cells/mm2, range 121 to 217 cells per mm2 tissue). Otherwise patients without chimerism showed a mean of 90.4 passenger leukocytes per mm2 tissue (SD +/- 14.5 cells/mm2, range: 52 to 99 cells/mm2). Lymphocyte proliferation, determined by donor-specific "multiple" single-way- mixed-lymphocyte-cultures (dsmMLC) was reduced to a mean of 62.2 % of preoperative values (SD +/- 14.5 %, range 33 % to 88 %) in the 15 patients with stable microchimerism. Otherwise in the 11 patients without microchimerism dsmMLC results stayed at continuously higher levels with a mean of 106 % (SD +/- 13.4, range 92 % to 134 %). CONCLUSIONS: The results from these studies of microchimerism and lymphocyte reactivity after liver transplantation suggest that the co-transplantation of donor leukocytes plays an important and active role in the modulation of the host-immune system.
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Leucocitos , Trasplante de Hígado/métodos , Prueba de Cultivo Mixto de Linfocitos , Quimera por Trasplante/inmunología , Biopsia con Aguja , Estudios de Seguimiento , Antígenos HLA-DR/genética , Humanos , Leucocitos/inmunología , Leucocitos/patología , Hígado/patología , Trasplante de Hígado/inmunología , Trasplante de Hígado/patología , Activación de Linfocitos/inmunología , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Donantes de Tejidos , Tolerancia al Trasplante/inmunologíaRESUMEN
Over the past few years, hepatocyte transplantation has been considered as an alternative method for orthotopic liver transplantation for the treatment of various liver diseases. Beside curative approach for genetic metabolic deficiencies (familial hypercholesterolemia, hemophilia, etc.), it could be a useful tool for bridging the waiting period until an appropriate donor organ is obtained. In preclinical animal studies, hepatocytes injected intraperitoneally, intraportally or into the spleen settle down in the diseased liver. This enables genetic modification to correct inborn metabolic deficiencies and improves survival in acute liver failure. In 1992, the first clinical transplantation of isolated hepatocytes in 10 patients was performed. In 1998, Fox and coworkers described the successful transplantation of allogeneic liver cells in a child with Crigler-Najjar syndrome. Accomplished studies of Strom et al. resp. Bilir et al. of the same year proved the effectiveness of liver cell transplantation for transient treatment of acute liver failure. Prerequisite of this cell-based therapeutic strategy is a sufficient amount of highly differentiated hepatocytes, hence, a well established in-vitro cell-culture technique is necessary to yield a reproducible number of proliferating hepatocytes and to preserve the physiological cell function. This review discusses the different experimental approaches regarding the cultivation of human hepatocytes and also the use of alternative cell sources (like animal hepatocytes, immortalized cells of human origin, progenitor cells from fetal human liver/liver stem cells) for hepatocyte transplantation.
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Hepatocitos/trasplante , Fallo Hepático/cirugía , Errores Innatos del Metabolismo/cirugía , Animales , Células Cultivadas , Humanos , Fallo Hepático/genética , Errores Innatos del Metabolismo/genética , Resultado del TratamientoRESUMEN
Antiviral activity of the metal chelator ethylenediaminedisuccinic acid (EDDS) was examined in vitro against human cytomegalovirus (HCMV) wild type strains and strains that are resistant against ganciclovir (GCV) and cidofovir (HPMPC). EDDS inhibited the replication of wild-type as well as GCV- and HPMPC-resistant strains with a 50% effective concentration of 7.4-12 microg/ml. At concentrations of 100 microg/ml EDDS, unlike GCV or HPMPC, suppressed HCMV-induced up-regulation of intercellular adhesion molecule-1 (ICAM-1) and reduced T-cell adhesion to HCMV-infected cells in a monolayer adhesion model. In vitro EDDS inhibited murine cytomegalovirus (MCMV) replication (EC50 8.6 microg/ml) and caused in mice some protection against MCMV induced mortality at a non-toxic dose. Since immunopathological factors may play a significant role in HCMV disease it will be of interest to further study whether EDDS is effective in terms of modulation of inflammatory responses to HCMV infections.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antivirales/farmacología , Quelantes/farmacología , Citomegalovirus/efectos de los fármacos , Citosina/análogos & derivados , Etilenodiaminas/farmacología , Organofosfonatos , Succinatos/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Cidofovir , Citomegalovirus/inmunología , Citomegalovirus/fisiología , Citosina/farmacología , Farmacorresistencia Microbiana , Infecciones por Herpesviridae/tratamiento farmacológico , Infecciones por Herpesviridae/mortalidad , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Células Jurkat , Ratones , Ratones SCID , Muromegalovirus/efectos de los fármacos , Compuestos Organofosforados/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
Although the control of biliary ductular morphogenesis has received some attention particularly using isolated rat biliary epithelial cell models, the regulation of human bile duct formation is not well defined. In the present study, using a 3-dimensional culture model comprising primary human biliary epithelial cells (BECs) and coculture with primary human hepatocytes, we have sought to define the factors involved. We have shown that primary human BECs can be expanded on collagen gels in the absence of growth factors or serum. When plated in high density in double collagen gels, BECs established 3-dimensional structures that subsequently developed into well differentiated polarized luminal ducts. This morphogenic response occurred in the absence of hepatocyte growth factor (HGF) and epidermal growth factor. Strikingly, the addition of growth factors (in the presence of serum) resulted in loss of polarity although the cells retained growth responses to both factors. Coculture of BECs with autologous human hepatocytes enhanced the ability of low-density BECs to undergo ductulogenesis. This effect was mimicked by addition of conditioned medium from previous hepatocyte-BEC cocultures. These findings indicate that for human biliary ductular morphogenesis, epithelial cell-cell interactions are required but that mesenchymally derived factors such as HGF may not be important.
Asunto(s)
Conductos Biliares/citología , Técnicas Citológicas , Hepatocitos/fisiología , Conductos Biliares/fisiología , División Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Colágeno , Medios de Cultivo Condicionados , Medio de Cultivo Libre de Suero , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Células Epiteliales/fisiología , Geles , Factor de Crecimiento de Hepatocito/farmacología , HumanosRESUMEN
Several factors contribute to the maintenance of central nervous system immune privilege and astrocytes have been identified as a major source of immunomodulatory cytokines. To investigate whether hematogenous monocytes are immunologically deactivated by astrocyte-derived factors human monocytes were stimulated with lipopolysaccharide or interferon (IFN)-gamma and treated with the supernatant from pure astrocyte cultures, interleukin (IL)-4, IL-10, or with IL-1-receptor antagonist (1L-1-RA). Flow cytometry demonstrated that the supernatant from astrocyte cultures was the most potent agent in reducing the levels of major histocompatibility complex (MHC)-class-II- as well as intercellular adhesion molecule-1-expression, whereas IL-4, IL-10, and IL-1-RA had only marginal effects. The expression of leukocyte function antigen-1 and very late antigen-4 was not modulated by either factor. In conclusion, astrocytes seem to provide soluble factors that have the capacity to deactivate hematogenous monocytes.
Asunto(s)
Astrocitos/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Monocitos/metabolismo , Células Cultivadas , Regulación hacia Abajo , Citometría de Flujo , Humanos , Integrina alfa4beta1 , Integrinas/metabolismo , Interferón gamma/farmacología , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/farmacología , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Lipopolisacáridos/farmacología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Sialoglicoproteínas/metabolismoRESUMEN
Interaction of endothelial P-selectin with sialyl Lewis(x)-glycoprotein or P-selectin glycoprotein ligand (PSGL)-1 on leukocytes represents an early step in leukocyte recruitment. Redistribution of P-selectin to the endothelial cell surface occurs rapidly after challenge with several proinflammatory agents, for example, histamine, leucopterins, or lipopolysaccharide. We present evidence that prostaglandin E2 (PGE2) is an efficient inductor of surface P-selectin on cultured human umbilical vein endothelial cells (HUVEC). The increase in P-selectin-immunoreactivity coincided with redistribution of cytoplasmic P-selectin-reactive granulae to the endothelial cell surface, as visualized by confocal laser microscopic examination. CD4-T-cell adhesion to PGE2-stimulated HUVEC was also enhanced by a factor of 4, and blocking mAb directed against the binding site of P-selectin almost completely abrogated this increase in CD4-T-cell adhesion. In summary, our findings show that liberation of PGE2 is an important inductor of P-selectin surface expression on endothelial cells, resulting in enhanced recruitment of inflammatory cells.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Dinoprostona/farmacología , Endotelio Vascular/citología , Selectina-P/biosíntesis , Adhesión Celular , Células Cultivadas , Endotelio Vascular/metabolismo , Histamina/farmacología , Humanos , Acetato de Tetradecanoilforbol/farmacología , Venas Umbilicales/citologíaRESUMEN
Previously, experimental in vivo results showed that the productively and persistently human cytomegalovirus (HCMV)-infected neuroblastoma cell line UKF-NB-4AD169 exhibits a more malignant phenotype than the non-infected variant UKF-NB-4. To prove the assumption that enhanced malignancy may be due to enhanced invasive potential of the infected cells we studied interactions of both lines with monolayers of cultured endothelial cells. UKF-NB-4AD169 cells adhered to and transmigrated through endothelial monolayer to a significantly higher extent compared with UKF-NB4. Furthermore, the adhesion of UKF-NB-4AD169 but not of UKF-NB4 resulted in focal disruption of the monolayer integrity which facilitates tumor cell transmigration. Blocking antibodies directed against the beta1 integrin chain as well as beta1alpha5 on the tumor cells specifically inhibited adhesion in a concentration-dependent manner. When UKF-NB-4 were pretreated with a beta1 integrin activating antibody, focal disruption of the endothelial integrity also occurred. These findings lead us to suggest that HCMV infection activates beta1alpha5 in the host neuroblastoma cell which in turn enables these cells to tightly adhere to endothelial cells. In the presence of the protease inhibitor phenantroline, beta1alpha5-mediated adhesion was not impaired whereas UKF-NB4AD169-mediated endothelial monolayer permeabilization was dose dependently inhibited. We conclude that human cytomegalovirus infection contributes to augmented neuroblastoma invasiveness via adhesion of activated beta1alpha5 and subsequent matrix digestion by proteases.
Asunto(s)
Infecciones por Citomegalovirus/patología , Citomegalovirus , Neuroblastoma/patología , Receptores de Fibronectina/metabolismo , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/farmacología , Adhesión Celular/fisiología , Comunicación Celular/fisiología , Movimiento Celular/fisiología , Selectina E/análisis , Células Epiteliales/química , Células Epiteliales/citología , Células Epiteliales/virología , Citometría de Flujo , Humanos , Inmunofenotipificación , Molécula 1 de Adhesión Intercelular/análisis , Glicoproteínas de Membrana/análisis , Invasividad Neoplásica , Neuroblastoma/virología , Receptores de Fibronectina/análisis , Receptores de Fibronectina/inmunología , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/virología , Cordón Umbilical/citología , Molécula 1 de Adhesión Celular Vascular/análisisRESUMEN
BACKGROUND: Prostaglandin E2 (PGE2) is a powerful endogenous immune suppressant and interferes with various T-cell functions. However, it is not known in detail whether immunosuppressive drugs influence the PGE2-driven immune response in transplant patients. Therefore, we investigated the effect of several immunosuppressive compounds, in particular the novel drug mycophenolate mofetil (MMF), on endothelial PGE2 release. METHODS: Endothelial cells (HUVEC) were activated by either allogeneic CD4+ or CD8+ T cells, or by the cytokines interleukin-1 or gamma-interferon. Using an enzyme-linked immunosorbent assay, we analyzed PGE2 release of the activated HWEC in the presence of MMF, cyclosporine, or tacrolimus. As verapamil and mibefradil also possess immunosuppressive properties, they were included in the study as well. RESULTS: Activation of HUVEC with interleukin-1 or T cells resulted in a drastic accumulation of PGE2 in the supernatant. Cyclosporine or tacrolimus had no effect on PGE2 release. However, Ca2+ channel blockers, when applied at higher dosages, caused a significant increase in PGE2. Interestingly, MMF strongly diminished the PGE2 level in the cell culture supernatant in a concentration-dependent manner. CONCLUSION: The results demonstrate an inhibitory effect of MMF on PGE2 production, which may lower the benefits of the PGE2-triggered immune response after organ transplantation.
Asunto(s)
Citocinas/farmacología , Dinoprostona/biosíntesis , Endotelio Vascular/metabolismo , Inmunosupresores/farmacología , Ácido Micofenólico/análogos & derivados , Linfocitos T/fisiología , Células Cultivadas , Ciclosporina/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Ácido Micofenólico/farmacología , Tacrolimus/farmacologíaRESUMEN
BACKGROUND: Cyclosporine A (CsA) and tacrolimus prevent proliferation but not transendothelial migration of alloreactive lymphocytes into donor organs. As a result, serious adverse effects, such as nephrotoxicity and neurotoxicity, have been observed under CsA/tacrolimus therapy. The incorporation of new drugs with infiltration blocking properties might enhance the efficacy of the current immunosuppressive protocol, allowing lower CsA/tacrolimus dosage. Because Ca2+ plays a critical role in cell-cell interaction, the Ca2+-channel blocker verapamil might be a good cany. didate for supporting CsA/tacrolimus-based therapy. METHODS: A T-cell endothelial cell coculture model or immobilized immunoglobulin G globulin chimeras were employed to investigate how S- and R- verapamil interfere with the lymphocytic infiltration process. The expression and arrangement of membranous adhesion receptors and cytoskeletal F-actin filaments were analyzed by fluorometric method in the presence of. verapamil. RESULTS: Both verapamil enantiomers strongly inhibited lymphocyte infiltration. CD4+ and CD8+ T-cells were influenced to a similar extent with regard to horizontal locomotion (CD4+=CD8+), but to a different extent with regard to adhesion and penetration (CD4+ > CD8+). Moreover, penetration was blocked to a higher extent than was adhesion. ID50-values were 31 microM (CD4+-adhesion) and 11 microM (CD4+-penetration). Verapamil reduced P-selectin expression on endothelial cells and effectively down-regulated binding of T-cells to immobilized P-selectin immunoglobulin G globulins (ID50=4.4 microM; CD4+). A verapamil-induced reduction of intracellular F-actin in T-lymphocytes was proven to be mainly responsible for diminished cell locomotion. CONCLUSIONS: The prevention of CD4+ T-cell penetration by verapamil might argue for its use as an adjunct to CsA/tacrolimus-based immunosuppressive therapy.